Oil Palm Phenolics Inhibit the In Vitro Aggregation of...

Research ArticleOil Palm Phenolics Inhibit the In Vitro Aggregation of120573-Amyloid Peptide into Oligomeric Complexes

Robert P Weinberg 1 Vera V Koledova1 Hyeari Shin1 Jennifer H Park1 Yew Ai Tan2

Anthony J Sinskey 3 Ravigadevi Sambanthamurthi2 and ChoKyun Rha 1

1Biomaterials Science and Engineering Laboratory Massachusetts Institute of Technology Cambridge MA 02139 USA2Malaysian Palm Oil Board No 6 Persiaran Institusi Bandar Baru Bangi 43000 Kajang Selangor Malaysia3Department of Biology Massachusetts Institute of Technology Cambridge MA 02139 USA

Correspondence should be addressed to ChoKyun Rha ckrhamitedu

Received 17 August 2017 Revised 23 November 2017 Accepted 7 December 2017 Published 31 January 2018

Academic Editor Francesco Panza

Copyright copy 2018 Robert P Weinberg 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

Alzheimerrsquos disease is a severe neurodegenerative disease characterized by the aggregation of amyloid-120573 peptide (A120573) into toxicoligomers which activate microglia and astrocytes causing acute neuroinflammation Multiple studies show that the solubleoligomers of A12057342 are neurotoxic and proinflammatory whereas the monomers and insoluble fibrils are relatively nontoxic Weshow that A12057342 aggregation is inhibited in vitro by oil palm phenolics (OPP) an aqueous extract from the oil palm tree (Elaeisguineensis) The data shows that OPP inhibits stacking of 120573-pleated sheets which is essential for oligomerization We demonstratethe inhibition of A12057342 aggregation by (1)mass spectrometry (2) Congo Red dye binding (3) 2D-IR spectroscopy (4) dynamic lightscattering (5) transmission electron microscopy and (6) transgenic yeast rescue assay In the yeast rescue assay OPP significantlyreduces the cytotoxicity of aggregating neuropeptides in yeast genetically engineered to overexpress these peptidesThe data showsthat OPP inhibits (1) the aggregation of A120573 into oligomers (2) stacking of 120573-pleated sheets and (3) fibrillar growth and coalescenceThese inhibitory effects prevent the formation of neurotoxic oligomers and hold potential as a means to reduce neuroinflammationand neuronal death and thereby may play some role in the prevention or treatment of Alzheimerrsquos disease

1 Introduction

Alzheimerrsquos disease (AD) is an aging-associated progres-sive debilitating neurodegenerative disorder of the brainwhose pathogenesis is associated with the aggregation ofthe amyloid-120573 peptide (A12057342A12057340) [1ndash5] The A12057342A12057340aggregates form soluble oligomers and insoluble fibrillardeposits extraneuronally resulting in the activation of mul-tiple pathophysiological processes with substantial loss ofsynapses and neurons [6ndash8] Current evidence stronglysupports the role of misfolded proteins forming amyloidaggregates playing a role in the pathogenesis of severaldiseases includingAlzheimerrsquos Parkinsonrsquos andHuntingtonrsquosdiseases as well as type II diabetes mellitus [9ndash12]

The amyloid-120573 aggregates into soluble neurotoxic oligom-ers [13ndash16] in awell-characterized fibril oligomerization proc-ess eventually forming stacked beta-sheet-rich fibrils [17ndash22]

The amyloid-120573 fibrils have a common parallel in-registerstacking organization based on the beta-pleated sheet stack-ing of the amyloid-120573 peptide [16 23ndash26] Whereas themonomers are relatively nontoxic the neurotoxic oligomersmay induce pathogenic pores in neurons [27 28] createabnormal calcium ion fluxes [29] altering intracellular sig-naling pathways including MAPK along with increasedoxidative stress [29ndash32] and also activate microglia andastrocytes to an acute inflammatory state releasing suchcytokines as TNF-120572 IL-1b and IFN-120574 [33 34]

The A120573 peptide-induced neuroinflammatory state resultsin synaptic loss and neuronal death in the brain regionsessential for cognitive function including the hippocampusentorhinal cortex and cerebral cortex [35] This pathologyresults in the loss of memories personalities and cognitivefunction resulting in dementia

HindawiInternational Journal of Alzheimerrsquos DiseaseVolume 2018 Article ID 7608038 12 pageshttpsdoiorg10115520187608038

2 International Journal of Alzheimerrsquos Disease

A120573 is a 4 kDa peptide with several isoforms themost common being a 40- and 42-amino acid peptide(A12057340A12057342) [35] The A120573 is a 39ndash44 amino acid peptide[17 36ndash40] which is endoproteolytically cleaved from a cellsurface protein the amyloid precursor protein (APP) by 120572- 120573- and 120574-secretases [41] At a critical molar concentrationA120573 undergoes an extensively studied 3-phase accretion intofibrils involving a nucleation-elongation process lag phaseexponential growth and plateau phase [5 42ndash54] The A120573fibrils have a parallel in-register structure [26]The A120573 fibrilsand the oligomerization process [55ndash58] have been wellcharacterized by multiple physicochemical measurementsincluding transmission electron microscopy (TEM) [59] X-ray diffraction [60 61] mass spectrometry (MS) [62ndash64]2D infrared spectroscopy (2D-IR) circular dichroism (CD)Congo Red dye binding (CR) Thioflavin-T fluorescence(TTf) [65 66] SDS-gel electrophoresis (SDS-PAGE) [67]atomic force microscopy (AFM) [68 69] gel filtration [7071] fluorescence resonance energy transfer (FRET) [7273] dynamic light scattering (DLS) [74] Fourier-transforminfrared spectroscopy (FTIR) and nuclear magnetic reso-nance imaging (NMR) [75] The soluble spherical oligomeraggregates measure approximately 2ndash20 nm [68 69 76 77]by hydrodynamic radius (DLS) in aqueous solution

Initially the amyloid-120573 peptide forms dimers in aqueoussolution following a lag phase involving a nucleation event[70 71] The gradual accumulation of dimers leads to theformation of higher order aggregates [78] The molecularweights of these higher order aggregates range between 105and 106 Daltons (Da) [71 79] comprising spherical particleswith an average diameter of 3 nm [59 76 77] with an averageof 24 A120573monomers per particleThe aggregation into higherorder oligomers requires a minimum critical concentrationof 25 120583M A120573 Following further incubation the sphericalparticles coalesce to form curvilinear fibrils which have acharacteristic bearded appearance called protofibrils [59 6869 78] In a continuing process of coalescence the growingmature A120573 fibrils consume the spherical aggregates andprotofibrils which disappear from solution [80]

The neurotoxic oligomers are A120573 aggregates which do notprecipitate at 100000timesg centrifugation [81ndash83]These solubleA120573 oligomers correlate better with AD symptomatology anddementia than the fibrillar A120573 [84 85] There are cognitivelynormal persons with higher numbers of fibrillar A120573 plaquesThere is a poor correlation between the presence of fibrillaramyloid deposits and dementia symptomatology [35] Themodified ldquoamyloid hypothesisrdquo of AD states that the solubleA120573 oligomers are neurotoxic but the fibrillar A120573 deposits arenot toxic [86ndash89]Multiple studies of A120573 oligomers show thatthey are toxic for neurons in vitro [86 90 91]

2 Materials and Methods

21 Preparation of Oil Palm Phenolics The preparation of oilpalm phenolics (OPP) has been previously described [92 93]OPP is an aqueous solution extracted from the fruit of thepalm oil tree (Elaeis guineensis) Following harvesting theoil palm fruit is mechanically crushed and squeezed underhigh pressure steam The biphasic filtrate is then decanted

to separate the top oil layer from the bottom aqueous layerThis aqueous layer is now termed ldquooil palm phenolicsrdquo andcontains such phytochemicals as polyphenols flavonoidsphenolic acids shikimate oligosaccharides and metal ions

22 A12057342 Amyloid Peptide The A12057342 was purchased as alyophilized powder comprising 025mg of purified A12057342from Millipore (Waltham Massachusetts USA) Then the250mcg A12057342 was initially dissolved in 50 microliters ofDMSO (dimethyl sulfoxide) This 50-microliter solution wasthenmixed with 950microliters of phosphate-buffered saline(PBS 10mM pH 74)This solution was aliquoted and frozenat minus20∘C Assays were carried out at a final concentration of50 120583MA12057342 in PBS (pH 74) A12057342 aggregation was effectedby incubating the solution at 30∘C without stirring for theindicated time periods

23 Congo Red Dye Binding Assay The Congo Red dyeworking solution was prepared as a 20 120583M Congo Red(Sigma Aldrich) in phosphate-buffered saline (PBS pH 74)The Congo Red dye binding assays were performed with aTECAN Spectrafluor Plus instrument using 96-well plasticmicrotiter plates (BDFalcon)The stock 50120583MA12057342 solutionwas diluted with PBS to a final concentration of 10120583MA12057342Then 50 120583L of this 10 120583M A12057342 solution was added to eachof the wells of the 96-well microtiter plate Following this50 120583L of OPP at the appropriate concentrations was added toeach well Finally 6 120583L of the 20120583M Congo Red dye solutionwas added to each well The TECAN Spectrafluor was setto take six repeat optical absorption readings at each timeinterval of 15 minutes with absorption readings taken at thewavelengths of 492 nm and 540 nm The optical absorptionreadings at these two wavelength allowed calculation of theA12057342 aggregates because of the known spectral shift whichoccurs when Congo Red is bound to aggregated A12057342 (see(1) below) The TECAN Spectrafluor was set for incubationof plates at 30∘C with time intervals set at optical absorptionmeasurements taken every 15 minutes

Aggregated A12057342 may be calculated by the followingequation

119862119887 =11986054147800minus11986040338100 (1)

where 119862119887 represents the amount of Congo Red dye bound toaggregated peptide 119860541 is the optical absorption measuredat the wavelength of 541 nm 119860403 is the optical absorp-tion measured at the wavelength of 403 nm 478000 is theextinction coefficient at 541 nm and 38100 is the extinctioncoefficient at 403 nm

24 Thioflavin-T Fluorescence Assay Although much workhas been done with Thioflavin-T to study the kinetics ofaggregation and fibrillization we are limited in the use of thisdye because of the very strong fluorescent signal emitted bythe oil palm phenolics at the wavelength of 535 nm followingexcitation at a wavelength of 450 nm The OPP fluorescentsignal is 4-5 times greater than that of Thioflavin-T bound toaggregates Therefore the OPP signal completely masks anydetectable signal fromThioflavin-T

International Journal of Alzheimerrsquos Disease 3

25 Mass Spectrometry by MALDI-TOF Ab42 MALDI-TOF mass spectrometric analyses were performed using aMicroflex mass spectrometer (Bruker Daltonics BillericaMA USA) equipped with a pulsed nitrogen laser operatingat 337 nm Small oligomer positive ion spectra were acquiredin linear mode over an119898119911 range from 2000 to 50000 usinga 20-kV accelerating voltage and a 150-ns delay extractiontime The spectrum for each spot was obtained by averagingthe results of 200 laser shots The analysis was performed byspotting on the target plate 10 ul of the sample mixed with anequal volume of the matrix solution 10mgml sinapinic acid(Sigma Aldrich) in CH3CNH2O (50 50 vv) containing01 (vv) trifluoroacetic acid (Sigma Aldrich) The samplewas prepared in the following way 10microliters was C4 Zip-Tipped eluted in 1 microliter of 70 acetonitrile mixed with1 microliter of matrix spotted and allowed to air dry

26 Transmission Electron Microscopy (TEM) Copper gridswith Formvar carbon coating (400meshes Ted Pella) wereglow discharged for 20 seconds and 5120583l of amyloid samplewas placed on the grids for 5 minutes The excess sample onthe grids was blotted off using filter paper The grids werethen floated onto a drop of filtered 15 uranyl acetate (SigmaAldrich) for 45 seconds Then the sample grids are placedunder a JEOL 1200 SX Transmission Electron Microscope(TEM) and digital photomicrographs were taken using theAMT 16000 camera system

27 2D FTIR Assay Similar to NMR spectroscopy two-dimensional infrared spectroscopy (2D-IR) provides impor-tant information on the secondary structure of polypeptidesincluding the quantification of alpha-helices and beta-pleatedsheets at high sensitivity as developed in the MIT laboratoryof Professor Andrei Tokmakoff [94ndash98] This secondarystructural information on the polypeptide is obtained byspreading the observed spectral information onto two fre-quency axes and correlating the observed frequency of initialvibrational excitation (1205961) with the final observed detectionfrequency (1205963)

Tokmakoff has demonstrated that the observed frequen-cies of the diagonal peaks on such a plot correspond to thevibrational transitionswithin the sample and cross-peaks canonly be observed when two vibrational modes are coupled(ie if the modes reside within the same structure or ifthere is energy transfer between two vibrations) Thus ina 2DIR spectrum each positive diagonal peak is accom-panied by a negative appearing below the diagonal thesenegative peaks are from vibrational transitions involving two-quantum states and contain information related to the anhar-monicities of the individualmodes Correlation 2DIR spectrawere acquired using a Fourier-transform 2D-IR spectrometerdescribed in detail elsewhere

Amyloid-beta sampleswere prepared inD2O at a concen-tration of 10mgml andbuffered to a final pHof 74 in a 10mMdeuterated phosphate and held between CaF2 windows ina 50 um path-length cell Spectra were collected in theperpendicular (ZZYY) polarization geometry to enhance theintensity of the cross-peak

28 Dynamic Light Scattering (DLS) In order to determinethe size of the A12057342 peptide aggregates in the presence andabsence of OPP dynamic light scattering (DLS) measure-ments were performed Dynamic light scattering (DLS) wasperformed with a DynaPro Plate Reader (Wyatt TechnologySanta Barbara California) using Wyatt optically transparent96-well microtiter plates The sensitivity of the DynaProPlate Reader is able to measure protein aggregates with ahydrodynamic radius between 05 and 1000 nm and weight-average molar masses between 1 and 1000 kDa Incubationtemperature of the plates was maintained at 30∘C duringaggregation studies

Themeasured hydrodynamic radius (Rh) was the averageof 50 measurements The mean Rh and polydispersity (Pd)were estimated on the basis of an autocorrelation analysis ofscattered light intensity based on the translational diffusioncoefficient from the Stokes-Einstein equation

119877ℎ =119896119879

6120587120578119863 (2)

where 119877ℎ is the hydrodynamic radius (nm) 119896 is Boltzmannrsquosconstant 119879 is the absolute temperature (K) 120578 is the viscosityof water and 119863 is the translational diffusion coefficient [99]The stock 50120583M A12057342 solution was diluted with PBS to afinal concentration of 10120583MA12057342 Then 50 120583L of this 10 120583MA12057342 solution was added to each of the wells of the 96-wellmicrotiter plate Following this 50120583L of the diluted OPP wasadded to eachwell at concentrations between 0 and 8mcgml(0 2 4 6 and 8)

29 Transgenic Yeast Rescue Assay Yeast cells were genet-ically engineered using transduced nucleic acid sequencesto overexpress neuropeptides in the Lindquist lab at MITas previously described [100ndash103] Yeast cells were grownin rich media (YPD) or in synthetic media lacking uraciland containing 2 glucose (SD-Ura) raffinose (SRaf-Ura)or galactose (SGal-Ura) Gateway entry clones contain-ing the full-length neuropeptides (120573-amyloid 120572-synucleinHuntingtin and TDP-43) in the vector pDONR221 wereobtained from Invitrogen A Gateway LR reaction wasused to shuttle each neuropeptide into Gateway-compatibleyeast expression vectors (pAG vectors httpwwwaddgeneorgkitslindquist-yeast-gateway) To generate C-terminallyGFP-tagged neuropeptide constructs a two-step PCR pro-tocol was used to amplify the neuropeptide sequence with-out a stop codon and incorporate the Gateway attB1 andattB2 sites along with a Kozak consensus sequence Result-ing PCR products were shuttled into pDONR221 using aGateway BR reaction The entry clones (120573-amyloidnostop120572-synucleinnostop Huntingtinnostop and TDP-43nostop) werethen used in LR reactions with pAG426Gal-ccdB-GFP togenerate the 2120583m neuropeptide-GFP fusion constructsTo generate the integrating neuropeptide-GFP constructthe neuropeptidenostop entry clone was used in an LRreaction with pAG306Gal-ccdB-GFP Two-micron plas-mid constructs (eg pAG426Gal-TDP-43-GFP) were trans-formed into BY4741 (MATa his3 leu2 met15 ura3) The


neuropeptide-GFP integrating strain was generated by lin-earizing pAG306Gal-neuropeptide-GFP by Bsm1 restrictiondigest followed by transformation in the w303 strain (MATacan1-100 his3-1115 leu2-3112 trp1-1 ura3-1 and ade2-1)The hsp104Δ and rnq1Δ strains are deletion mutants (genedisrupted by KanMX4) obtained from the haploid deletioncollection (Invitrogen)

Yeast procedures were performed according to standardprotocols The PEGlithium acetate method was used totransform yeast with plasmid DNA For spotting assays yeastcells were grown overnight at 30∘C in liquid media contain-ing raffinose (SRaf-Ura) until they reached log or mid-logphase Cultures were then normalized for OD600 seriallydiluted and spotted onto synthetic solid media containingglucose or galactose lacking uracil and were grown at 30∘Cfor 2-3 days

As the yeast grow synthesis of increasing levels of theneuropeptide resulted in the formation of cytotoxic aggre-gates which severely limited yeast growth eventually killingthe yeast unless a rescue drug was administered whichwould prevent the aggregated neuropeptide cytotoxicity Thetransgenic yeast growth data is expressed as percentagegrowth relative to the control strain which accounts for somevalues being greater than 100

210 Statistical Analysis of Data All data were presentedas mean plusmn standard deviation from 3 independent mea-surements The statistical analysis was made by performingone-way ANOVA for the 3 independent determinationsSignificance of results was determined as 119901 le 001 [unlessotherwise stated]

3 Results

31 Congo Red Dye Binding The Congo Red binding assayis one of the earliest methods developed to measure theaggregation of A12057342 The Congo Red assay is based upon aspectral shift which occurs in the absorption of Congo Redat 2 different reference wavelengths when the Congo Red isbound to beta amyloid peptide monomers versus aggregatesThe aggregation data in Figure 1 is normalized with themaximum inhibition of oligomerization set equal to 100inhibition

As seen in Figure 1 increasing concentrations of OPPresult in an increased inhibition of A12057342 aggregation We seethat the concentration-inhibition relationship is somewhatlinear between OPP concentrations of 10 and 70 120583gml Alsoit appears that maximum aggregation inhibition occurs at anOPP concentration of 70120583gml

Based on the above OPP inhibition of A12057342 aggregationthe IC50 (50 inhibitory concentration) is 324120583gml OPP

32 Dynamic Light Scattering In Figure 2 we see thatincreasing the concentration of OPP results in prolongingthe lag phase of aggregation Generally the exponential phaseof aggregate growth has a similar sigmoidal curve at theconcentrations testedThe inhibition of A12057342 aggregation byOPP reveals both a dose-dependent and a time-dependentcomponent

Inhi

bitio

n of

-a

myl

oid

aggr

egat

e siz

e (

)

2 4 6 8 1000

10

20

30

40

50

60

70

80

90

100

OPP concentration (gml)

y = 13991x + 4755

R2 = 09907

Figure 1 Spectral compensated curve for Congo Red dye bindingDose-dependent inhibition of A12057342 aggregation by OPP Equation(1) was used to quantify the aggregates from the spectral shiftbetween wavelengths 403 nm and 541 nm IC50 = 324 120583gml

Perc

ent o

f max

imum

pep

tide

aggr

egat

ion

()

0102030405060708090

100

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 160Time (hours)

4 gml OPP2 gml OPP0 gml OPP

8 gml OPP6 gml OPP

Figure 2 DLS kinetics of A120573 aggregation inhibition by OPP Thisfigure reveals both the dose-dependent and time-dependent effectsof inhibitory effects of OPP on A12057342 aggregation Doses varybetween 0 and 8 120583gml OPP with kinetics studied over a 16-hourperiod of time

33 Mass Spectrometry In Figure 3(a) in the absence ofOPP we see A12057342 aggregate peaks representing dimerstrimers tetramers hexamers and septamers In Figure 3(b)in the presence of OPP only the monomeric A120573 is seen atMW of 4500Da The mass spectroscopy data clearly showsthat the presence of OPP will inhibit the aggregation andpolymerization of beta amyloid peptide At a concentrationof 10 120583gml OPP only the monomeric A12057342 is present insolution

34 Tabulation of Mass Spectrometric Data Pooled from 5MALDI-TOF Runs In Figure 4 we see the pooled data for 5mass spectrometric runs In the absence of OPP (0mcgml)we see that there are 3 species of the peptide found themonomer the dimer and the trimer At a concentrationof 09 120583gml OPP we see that only 2 species are foundthe monomer and the dimer Then from a concentration of


12826911

17145485

2150770925799855

1141627

1660915

1228183

2150771

Inte

nsity

(au

)

15000 20000 25000 30000 35000 40000 45000100000

1000

2000

3000

(mz)

(a)

Inte

nsity

(au

)

30000 40000 500002000010000

0

200

400

600

800

1000

(b)

Figure 3 Mass spectrometric display of A12057342 aggregate size (a) A12057342 in PBS (negative control) (b) A12057342 with 10 120583gml OPP (a) shows thepresence of monomers dimers trimers tetramers and pentames (b) Shows the presence of only monomers

13513

9014

4530

0

9022

4510

0 0

4534

0 0

4511

0 0

4546

0 0

4510

0 09 90 900 4500 9000OPP concentration (gml)

-Amyloid trimer-Amyloid dimer-Amyloid monomer

0

2000

4000

6000

8000

10000

12000

14000

16000

-A

myl

oid

pept

ide M

W (D

a)

Figure 4 MALDI-TOF mass spectrometric data on A12057342 aggrega-tion pooled from 5 runs In the absence of OPP (0120583gml) A12057342dimers and trimers form At an OPP concentration of 09120583gmlonly dimers will form At 90120583gml and higher only monomers existand no dimers nor trimers are formed

90 120583gml up to 9mgml we see that only themonomeric formof the beta amyloid peptide is found

35 2D-IR Determinations of Secondary Polypeptide Struc-ture Figure 5 represents the amide-I two-dimensional IR

correlation spectra of the A12057342 showing the frequency plotsin the amide-I region where the beta-sheets are character-ized by the presence of two peaks centered near 1620 and1680 cmminus1 whose individual amide oscillators vibrate in-phase perpendicular or parallel to the 120573-strands respectivelyThe splitting between these modes at this frequency is relatedto the size of the folded 120573-sheet In a primarily beta-sheetprotein the corresponding cross-peaks give a characteristicZ-shape to the spectrum Here the focus is primarily on thecross-peak centered at [1205961 1205963] = [1620 1680] cmminus1 whoseamplitude is indicative of the total amount of beta-sheetpresent in the sample

Three 2D-IR spectra are shown in Figure 5 these corre-spond to the amyloid-beta sample incubated for a period of 1hour (a) and 10 hours (b) at 37∘C in the absence of OPP andat 10 hours in the presence of 10 120583gml OPP (c)

The spectra in (a) and (b) show that the cross-peak at[1205961 1205963] = [1620 1680] cmminus1 (indicated by a red arrow)increases in amplitude over this period This is indicativeof an overall increase in beta-sheet content of the samplewe ascribe this increase in amplitude to growth of the beta-fibrils in the sample Longer incubation times do not affectamplitude of the cross-peak (data not shown)

The sample incubated with 10 120583gml OPP (c) shows avery small cross-peak even after incubation for 10 hoursFurthermore the diagonal peaks associated with beta-sheetbecome significantly broader and there is an increase in signalnear the 1650 cmminus1 region a part of the spectrum associatedwith helical and random-coil conformations suggesting thatOPP induces disorder within the secondary structure of theamyloids and therefore prevents the formation of beta-fibrils

36 A12057342 Electron Micrographs from Transmission ElectronMicroscopy (TEM) In the transgenic yeast assay the pres-ence of 10120583gml OPP rescues the growth of the 120573-amyloid-producing yeast from 20 (withoutOPP) to 40 (withOPP)


Without OPP

1550

1600

1650

1700

1750

1600 1650 1700 17501550

-sheet

1 hour

(a)

Without OPP

1550

1600

1650

1700

1750

1600 1650 1700 17501550

10 hours

Enlarging -Sheet

(b)

With OPP

1550

1600

1650

1700

1750

1600 1650 1700 17501550

10 hours

No -sheet seen

(c)

Figure 5 Amide-I two-dimensional correlation spectra of the A12057342 samples incubated at 37∘C without OPP at 1-hour (a) and at 10-hour (b)incubation A12057342 sample incubated at 37∘C with 10 120583gml OPP at 10 hours (c) The diagonal peak at 1672 cmminus1 indicated by a blue arrow isdue to tetrafluoroacetic acid (TFA) present in the sample The cross-peak near [1205961 1205963] = [1620 1680] cmminus1

For the Huntingtin-producing yeast 10 120583gml OPP rescuesthe yeast growth from25 (withoutOPP) to 60 (withOPP)For the TDP-43-producing yeast 10 120583gml OPP rescues theyeast growth from 25 (without OPP) to 190 (with OPP)We see that there is no significant change of growth forthe 120572-synuclein-producing yeast with or without OPP Theserelative growth percentages compare the transgenic yeastgrowth to growth of control strains (Figure 7)

4 Discussion

Since the discovery that the beta amyloid peptide (A120573) is theprimary constituent of the fibrils found in the extraneuronal

senile neuritic plaques in the brains of Alzheimerrsquos patientsthis peptide has played a central role in Alzheimerrsquos researchIt is now believed that the soluble oligomers are neurotoxicand the mature fibrils of A120573 peptide are not neurotoxicper se Some speculate that the mature amyloid fibrils mayserve as a reservoir of soluble oligomers of A120573 There isexperimental evidence that larger aggregates may reversiblyrelease smaller aggregates following oligomerization Thesoluble A120573 oligomers are defined as what remains in aqueoussolution following the high-speed centrifugation of brainextracts The 2 most common human isoforms of A120573 areA12057340 [40 amino acids in length] and A12057342 [42 amino acidsin length] A12057342 peptide is known to be more fibrillogenic


100 nm

(a)

100 nm

(b)

Figure 6 TEM of 120573-amyloid (a) 120573-Amyloid (PBS) (b) 120573-amyloid (10 120583gml OPP) (a) shows the large A12057342 aggregates and meshes (sizegreater than 10 nm) which form in the absence of OPP (b) shows the smaller aggregates which are more sparse which form in the presenceof 10 120583gml OPP

than A12057340 so we have carried out these aggregation studieswith A12057342

The data presented herein show that OPP significantlyinhibits the oligomerization of A12057342 in part by preventingbeta-sheet folding of the peptide Furthermore OPP alsoreduces the cytotoxicity of aggregated neuropeptides ofA12057342 Huntingtin and TDP-43 peptides in transgenic yeast

The Congo Red data shows a linear relationship betweenthe degree of aggregation inhibition and the concentrationof OPP over the range of 10ndash70120583gml OPP There is 50inhibition of A120573 aggregation at an OPP concentration of324 120583gml OPP (IC50) (Figure 1) Dynamic light scatteringshows successive prolongation of the lag phase at higherconcentrations of OPP (Figure 2) The phase of exponentialfibrillar growth shows a lower slope at higher concentrationsof OPP Mass spectrometry shows distinct peaks for themonomer dimer trimer tetramer pentamer hexamer andseptamer in A12057342 solution without OPP (Figure 3(a)) Massspectrometry shows a single low-molecular weight peak inthe A12057342 solution with OPP (Figure 3(b)) The data formultiple mass spectrometry runs are pooled in Figure 4The A12057342 trimers only form reproducibly in the absence ofOPP A12057342 dimers form at a concentration of 09mcgmlOPP and in the absence of OPP Only A12057342 monomers areconsistently seen atOPP concentrations of 90 900 4500 and9000mcgml

2D-IR spectroscopy shows a developing beta-sheet signalas early as 1 hour with significant increase in size of thatsignal by 10 hours in the absence of OPP (Figure 5) Atan OPP concentration of 10mcgml no beta-sheet signalis seen after 10 hours of incubation Following 20 hours ofincubation at 30∘C in the presence of 10120583gmlOPP few smallaggregates of A12057342 can be visualized using transmissionelectronmicroscopy (TEM) (Figure 6(b)) However multiplelarge aggregates of A12057342 can be visualized by TEM whenthe A12057342 was incubated in the presence of 15 120583gml OPP(Figure 6(a))

In the transgenic yeast assay the presence of 10 120583gmlOPP rescues the growth of the 120573-amyloid-producing yeastfrom 20 (without OPP) to 40 (with OPP) For theHuntingtin-producing yeast 10 120583gml OPP rescues the yeastgrowth from 25 (without OPP) to 60 (with OPP) Forthe TDP-43-producing yeast 10 120583gml OPP rescues the yeast

growth from 25 (without OPP) to 190 (with OPP) Wesee that there is no significant change of growth for the120572-synuclein-producing yeast with or without OPP Theserelative growth percentages compare the transgenic yeastgrowth to growth of control strains

A12057340 and A12057342 are the most common human isoformsof the peptide A12057340 is the more common isoform butA12057342 is more fibrillogenic aggregates more quickly andappears to be the more toxic isoform especially in the dimerstate A120573 has little toxicity while in the monomeric stateBased upon neurotoxicity data in vitro animal models andclinical observations in patients with Alzheimerrsquos diseaseone therapeutic goal consists of inhibiting the formation ofsoluble A120573 dimers thus nullifying the neurotoxic effect ofthese dimers and should be effective in preventing andortreating Alzheimerrsquos disease Statistical studies on patientswith AD have shown that the cortical levels of soluble A120573correlate well with both the extent of synaptic loss and theseverity of the clinical symptoms [81 104 105] Multiplestudies have shown that the soluble assemblies of oligomericA120573 are the neurotoxic species which cause the cognitivelosses of Alzheimerrsquos disease [16 23ndash26] These oligomericA120573 (oA120573) have also been called A120573-derived diffusible ligands(ADDLs) Recent research has shown that the oA120573ADDLsplay a key role in cognitive decline [27 28]

The ldquoamyloid hypothesisrdquo that small soluble oligomers ofA120573 underlie the key phenotypic characteristics of Alzheimerrsquosdisease is supported by experimental data showing that thesesmall soluble oligomers may (1) cause synaptic loss anddecreased dendritic spine density (2) cause hyperphosphory-lation of tau proteins with resulting intraneuronal neurofib-rillary tangles and collapse of the neuritic cytoskeleton and(3) cause memory impairment and cognitive losses in theabsence of amyloid plaques

The experimental evidence presented in this paper showsthat oil palm phenolics derived from the palm oil plant(Elaeis guineensis) have inhibitory effects on the process ofaggregation and polymerization of beta amyloid peptide invitro This inhibition may cause the beta amyloid peptideto remain in the soluble monomeric state and facilitate theclearance of the peptide from the brain via the normalphysiologic mechanisms Several pharmaceutical companiesare currently developing drugs which inhibit the aggregation


HiToxInToxNoTox

(a)

-Amyloid -Synuclein Huntingtin TDP-43

Perc

ent r

elat

ive g

row

th (

)

0

50

100

150

200

250

+ ００

minus ００

(b)

Figure 7 (a) Transgenic yeast which overexpresses the TDP-43 peptide NoTox = low expression TDP-43 IntTox = intermediate expressionTDP-43 aggregation seen as clumps of GFP-TDP43 HiTox = high expression TDP-43 in larger clumps and increased death of yeastArrows demarcate the inclusion granules of TDP-43 (Susan Lindquist MIT) (b) OPP significantly reduces the cytotoxicity of the aggregatedneuropeptides in the transgenic yeast rescue assay Bar heights represent percent growth relative to control strain OPP doubles the growthfor 120573-amyloid-producing yeast from 20 (without OPP) to 40 (with OPP) more than doubles the growth for Huntingtin-producing yeastfrom 25 (without OPP) to 60 (with OPP) OPP increases the growth of TDP-43-producing yeast more than 6-fold from 25 (withoutOPP) to 190 (with OPP) There is no significant change in the growth of the 120572-synuclein-producing yeast with or without OPP OPP dosewas 10 120583gml for the 4 peptide-producing transgenic yeasts

and oligomerization of A12057342 Preventing the formation ofneurotoxic oligomers may represent a potential preventive ortherapeutic strategy in the treatment of Alzheimerrsquos disease

Thus the inhibitory effects of OPP on A12057342 aggregationmay lead to the development of a potential drug for the

prevention andor treatment of Alzheimerrsquos disease Ournext step consists in identifying isolating and purifyingthat component(s) of the oil palm phenolics which hasthese inhibitory and antifibrillogenic effects on beta amyloidpeptide


5 Conclusions

This is a study of the aggregation of 120573-amyloid peptide invitro and the inhibitory effects of OPP on this aggregationThe study led to the following experimental results

(1) Oil palm phenolics inhibit 120573-amyloid monomersfrom aggregating into dimers trimers or largeraggregates as shown by the observed reduction inaggregate size as measured by Congo Red bindingmass spectrometry dynamic light scattering 2D-IRspectroscopy and transmission electron microscopy

(2) Oil palm phenolics prolong the lag phase in thenucleation-elongation process of A12057342 oligomeriza-tion as observed by dynamic light scattering with ashift of the aggregation curves to the right

(3) Oil palm phenolics inhibit the folding of the beta-pleated sheet as observed in the 2D infrared spec-troscopy

(4) Fifty percent (50) of the maximum aggregate sizeis observed at an OPP concentration of 324120583gml(IC50) by Congo Red dye binding

(5) At an OPP concentration greater than or equal to90 120583gml only monomers of A12057342 are observed bymass spectrometry No dimers trimers or higheroligomers are observed at this concentration orhigher

(6) The cytotoxicity of aggregated neuropeptides isgreatly reduced byOPP at a concentration of 10120583gmlin transgenic yeast which overexpresses these pep-tides

The current scientific literature shows that the solubleA12057342 oligomers are neurotoxic and cause extensive patho-logic changes in neurons decrease dendritic spine densityand cause depression of long-term potentiation in neuronsand enhancement of long-term depression Therefore theproperties of the oil palm phenolics to inhibit the formationof oligomers may hold promise for the medical treatmentandor prevention of Alzheimerrsquos disease

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This research is supported by the government of Malaysiathrough its Malaysian Palm Oil Board (MPOB) SelangorMalaysia with funding to Professor ChoKyun Rha Massspectrometry was performed on a Microflex at the Biopoly-mers andProteomicsCore Facility at theDavidHKoch Insti-tute for Integrative Cancer Research at MIT The MALDI-TOF data included herein comes from this apparatus Col-laboration for the IR spectroscopic studies is supportedby grants from the NSF to Professor Andrei TokmakoffGrants nos CHE-0616575 andCHE-0911107The authors alsowish to acknowledge the collaboration with Professor Susan

Lindquist and the transgenic yeast studies done by postdocCatherine McLellan Finally this manuscript benefitted fromthe graphical expertise of Ms Kirsten Schneider

References

[1] R Wetzel ldquoKinetics and thermodynamics of amyloid fibrilassemblyrdquoAccounts of Chemical Research vol 39 no 9 pp 671ndash679 2006

[2] A M Morris M A Watzky and R G Finke ldquoProteinaggregation kinetics mechanism and curve-fitting A reviewof the literaturerdquo Biochimica et Biophysica Acta (BBA) - Proteinsand Proteomics vol 1794 no 3 pp 375ndash397 2009

[3] R M Murphy ldquoKinetics of amyloid formation and membraneinteraction with amyloidogenic proteinsrdquo Biochimica et Bio-physica Acta (BBA) - Biomembranes vol 1768 no 8 pp 1923ndash1934 2007

[4] C Frieden ldquoProtein aggregation processes In search of themechanismrdquo Protein Science vol 16 no 11 pp 2334ndash2344 2007

[5] S I A Cohen M Vendruscolo C M Dobson and T P JKnowles ldquoFrom macroscopic measurements to microscopicmechanisms of protein aggregationrdquo Journal of Molecular Biol-ogy vol 421 no 2-3 pp 160ndash171 2012

[6] D J Selkoe ldquoThe molecular pathology of Alzheimerrsquos diseaserdquoNeuron vol 6 no 4 pp 487ndash498 1991

[7] J Hardy ldquoThe Alzheimer family of diseases Many etiologiesone pathogenesisrdquo Proceedings of the National Acadamy ofSciences of the United States of America vol 94 no 6 pp 2095ndash2097 1997

[8] DMHoltzman J CMorris andAMGoate ldquoAlzheimers dis-ease the challenge of the second centuryrdquo Science TranslationalMedicine vol 3 2011

[9] I Hajimohammadreza V E R Anderson J B Cavanagh et alldquo120573-amyloid precursor protein fragments and lysosomal densebodies are found in rat brain neurons after ventricular infusionof leupeptinrdquo Brain Research vol 640 no 1-2 pp 25ndash32 1994

[10] S Waelter A Boeddrich R Lurz et al ldquoAccumulation ofmutant huntingtin fragments in aggresome-like inclusion bod-ies as a result of insufficient protein degradationrdquo MolecularBiology of the Cell (MBoC) vol 12 no 5 pp 1393ndash1407 2001

[11] M Bucciantini E Giannoni F Chiti et al ldquoInherent toxicity ofaggregates implies a common mechanism for protein misfold-ing diseasesrdquo Nature vol 416 no 6880 pp 507ndash511 2002

[12] J Bendiske and B A Bahr ldquoLysosomal activation is a com-pensatory response against protein accumulation and associ-ated synaptopathogenesismdashan approach for slowing alzheimerdiseaserdquo Journal of Neuropathology amp Experimental Neurologyvol 62 no 5 pp 451ndash463 2003

[13] D M Walsh and D J Selkoe ldquoA120573 oligomers a decade ofdiscoveryrdquo Journal of Neurochemistry vol 101 no 5 pp 1172ndash1184 2007

[14] S T Ferreira M N N Vieira and F G De Felice ldquoSolubleprotein oligomers as emerging toxins in Alzheimerrsquos and otheramyloid diseasesrdquo IUBMB Life vol 59 no 4-5 pp 332ndash3452007

[15] E Monsellier and F Chiti ldquoPrevention of amyloid-like aggrega-tion as a driving force of protein evolutionrdquo EMBO Reports vol8 no 8 pp 737ndash742 2007

[16] D Eisenberg and M Jucker ldquoThe amyloid state of proteins inhuman diseasesrdquo Cell vol 148 no 6 pp 1188ndash1203 2012


[17] GGGlenner andCWWong ldquoAlzheimerrsquos disease andDownrsquossyndrome sharing of a unique cerebrovascular amyloid fibrilproteinrdquo Biochemical and Biophysical Research Communica-tions vol 122 no 3 pp 1131ndash1135 1984

[18] T L S Benzinger D M Gregory T S Burkoth et al ldquoPropa-gating structure of Alzheimerrsquos 120573-amyloid((10-35)) is parallel 120573-sheet with residues in exact registerrdquo Proceedings of the NationalAcadamy of Sciences of the United States of America vol 95 no23 pp 13407ndash13412 1998

[19] O N Antzutkin R D Leapman J J Balbach and RTycko ldquoSupramolecular structural constraints on Alzheimerrsquos120573-amyloid fibrils from electron microscopy and solid-statenuclear magnetic resonancerdquo Biochemistry vol 41 no 51 pp15436ndash15450 2002

[20] J J Balbach A T Petkova N A Oyler et al ldquoSupramolecularstructure in full-length Alzheimerrsquos 120573-amyloid fibrils Evidencefor a parallel 120573-sheet organization from solid-state nuclearmagnetic resonancerdquo Biophysical Journal vol 83 no 2 pp1205ndash1216 2002

[21] M Torok S Milton R Kayed et al ldquoStructural and dynamicfeatures of Alzheimerrsquos A120573 peptide in amyloid fibrils studied bysite-directed spin labelingrdquoThe Journal of Biological Chemistryvol 277 no 43 pp 40810ndash40815 2002

[22] ADer-Sarkissiant C C Jao J Chen andR Langen ldquoStructuralorganization of 120572-synuclein fibrils studied by site-directed spinlabelingrdquo The Journal of Biological Chemistry vol 278 no 39pp 37530ndash37535 2003

[23] L C Serpell M Sunde and C C Blake ldquoThe molecular basisof amyloidosisrdquo Cellular andMolecular Life Sciences vol 53 no12 p 871

[24] R Kodali and R Wetzel ldquoPolymorphism in the intermediatesand products of amyloid assemblyrdquo Current Opinion in Struc-tural Biology vol 17 no 1 pp 48ndash57 2007

[25] O S Makin and L C Serpell ldquoStructures for amyloid fibrilsrdquoFEBS Journal vol 272 no 23 pp 5950ndash5961 2005

[26] M Margittai and R Langen ldquoFibrils with parallel in-registerstructure constitute a major class of amyloid fibrils Molecularinsights from electron paramagnetic resonance spectroscopyrdquoQuarterly Reviews of Biophysics vol 41 no 3-4 pp 265ndash2972008

[27] B L Kagan YHirakura R Azimov RAzimova andM-C LinldquoThe channel hypothesis of Alzheimerrsquos disease Current statusrdquoPeptides vol 23 no 7 pp 1311ndash1315 2002

[28] B Caughey and P T Lansbury Jr ldquoProtofibrils pores fibrilsand neurodegeneration separating the responsible proteinaggregates from the innocent bystandersrdquo Annual Review ofNeuroscience vol 26 pp 267ndash298 2003

[29] M P Mattson ldquoOxidative stress perturbed calcium homeosta-sis and immune dysfunction in Alzheimerrsquos diseaserdquo Journal ofNeuroVirology vol 8 no 6 pp 539ndash550 2002

[30] B Kaltschmidt M Uherek B Volk P A Baeuerle andC Kaltschmidt ldquoTranscription factor NF-120581B is activated inprimary neurons by amyloid 120573 peptides and in neurons sur-rounding early plaques from patients with Alzheimer diseaserdquoProceedings of the National Acadamy of Sciences of the UnitedStates of America vol 94 no 6 pp 2642ndash2647 1997

[31] W R Markesbery and J M Carney ldquoOxidative alterations inAlzheimerrsquos diseaserdquo Brain Pathology vol 9 no 1 pp 133ndash1461999

[32] J Josepha B Shukitt-Hale N A Denisova A Martin GPerry and M A Smith ldquoCopernicus revisited Amyloid beta

in Alzheimerrsquos diseaserdquo Neurobiology of Aging vol 22 no 1 pp131ndash146 2001

[33] C Behl and B Moosmann ldquoAntioxidant neuroprotection inAlzheimerrsquos disease as preventive and therapeutic approachrdquoFree Radical BiologyampMedicine vol 33 no 2 pp 182ndash191 2002

[34] P L McGeer and E G McGeer ldquoAnti-inflammatory drugs inthe fight against Alzheimerrsquos diseaserdquo Annals of the New YorkAcademy of Sciences vol 777 pp 213ndash220 1996

[35] R D Terry E Masliah and L A Hansen ldquoThe neuropathologyof Alzheimer disease and the structural basis of its cognitivealterationsrdquo in Alzheimer Disease R D Terry R Katzman KL Bick and and S S Sisodia Eds pp 187ndash206 LippincottWilliams and Wilkins Philadelphia Penn USA 1999

[36] C L Masters G Simms N A Weinman G Multhaup B LMcDonald and K Beyreuther ldquoAmyloid plaque core proteinin Alzheimer disease and Down syndromerdquo Proceedings of theNational Acadamy of Sciences of the United States of Americavol 82 no 12 pp 4245ndash4249 1985

[37] T Wisniewski M Lalowski E Levy M R F Marques andB Frangione ldquoThe amino acid sequence of neuritic plaqueamyloid from a familial Alzheimerrsquos disease patientrdquo Annals ofNeurology vol 35 no 2 pp 245-246 1994

[38] C W Wong V Quaranta and G G Glenner ldquoNeuriticplaques and cerebrovascular amyloid in Alzheimer disease areantigenically relatedrdquo Proceedings of the National Acadamy ofSciences of the United States of America vol 82 no 24 pp 8729ndash8732 1985

[39] D LMiller I A Papayannopoulos J Styles et al ldquoPeptide com-positions of the cerebrovascular and senile plaque core amyloiddeposits of Alzheimerrsquos diseaserdquo Archives of Biochemistry andBiophysics vol 301 no 1 pp 41ndash52 1993

[40] D J Selkoe C R Abraham M B Podlisny and L K DuffyldquoIsolation of Low-Molecular-Weight Proteins from AmyloidPlaque Fibers in Alzheimerrsquos Diseaserdquo Journal of Neurochem-istry vol 46 no 6 pp 1820ndash1834 1986

[41] D J Selkoe ldquoAlzheimers disease genes proteins and therapyrdquoPhysiological Reviews vol 81 pp 741ndash766 2001

[42] C B AndersenH YagiMManno et al ldquoBranching in amyloidfibril growthrdquo Biophysical Journal vol 96 no 4 pp 1529ndash15362009

[43] T P J Knowles D A White A R Abate et al ldquoObservationof spatial propagation of amyloid assembly from single nucleirdquoProceedings of the National Acadamy of Sciences of the UnitedStates of America vol 108 no 36 pp 14746ndash14751 2011

[44] F Ferrone ldquoAnalysis of protein aggregation kineticsrdquo Methodsin Enzymology vol 309 pp 256ndash274 1999

[45] T P J Knowles C AWaudby G L Devlin et al ldquoAn analyticalsolution to the kinetics of breakable filament assemblyrdquo Sciencevol 326 no 5959 pp 1533ndash1537 2009

[46] S I A Cohen M Vendruscolo M E Welland C M DobsonEM Terentjev and T P J Knowles ldquoNucleated polymerizationwith secondary pathways I Time evolution of the principalmomentsrdquoThe Journal of Chemical Physics vol 135 no 6 article065105 2011

[47] S I A Cohen M Vendruscolo C M Dobson and T P JKnowles ldquoNucleated polymerisation in the presence of pre-formed seed filamentsrdquo International Journal of MolecularSciences vol 12 no 9 pp 5844ndash5852 2011

[48] S B Padrick and A D Miranker ldquoIslet amyloid phase parti-tioning and secondary nucleation are central to the mechanismof fibrillogenesisrdquo Biochemistry vol 41 no 14 pp 4694ndash47032002


[49] F A Ferrone J Hofrichter H R Sunshine and W A EatonldquoKinetic studies on photolysis-induced gelation of sickle cellhemoglobin suggest a newmechanismrdquoBiophysical Journal vol32 no 1 pp 361ndash380 1980

[50] T Medkour F Ferrone F Galacteros and P Hannaert ldquoThedouble nucleation model for sickle cell haemoglobin polymer-ization Full integration and comparison with experimentaldatardquo Acta Biotheoretica vol 56 no 1-2 pp 103ndash122 2008

[51] A M Ruschak and A D Miranker ldquoFiber-dependent amyloidformation as catalysis of an existing reaction pathwayrdquo Proceed-ings of the National Acadamy of Sciences of the United States ofAmerica vol 104 no 30 pp 12341ndash12346 2007

[52] C J Roberts ldquoNon-native protein aggregation kineticsrdquoBiotechnology and Bioengineering vol 98 no 5 pp 927ndash9382007

[53] J M Andrews and C J Roberts ldquoA Lumry-Eyring nucleatedpolymerizationmodel of protein aggregation kinetics 1 Aggre-gation with pre-equilibrated unfoldingrdquoThe Journal of PhysicalChemistry B vol 111 no 27 pp 7897ndash7913 2007

[54] RWetzel ldquoFor proteinmisassembly itrsquos the rsquoIrsquo decaderdquoCell vol86 no 5 pp 699ndash702 1996

[55] D R Booth M Sunde V Bellotti et al ldquoInstability unfoldingand aggregation of human lysozyme variants underlying amy-loid fibrillogenesisrdquoNature vol 385 no 6619 pp 787ndash793 1997

[56] F Chiti P Webster N Taddei et al ldquoDesigning conditionsfor in vitro formation of amyloid protofilaments and fibrilsrdquoProceedings of the National Acadamy of Sciences of the UnitedStates of America vol 96 no 7 pp 3590ndash3594 1999

[57] D Canet M Sunde A M Last et al ldquoMechanistic studies ofthe folding of human lysozyme and the origin of amyloidogenicbehavior in its disease-related variantsrdquo Biochemistry vol 38no 20 pp 6419ndash6427 1999

[58] L A Morozova-Roche J Zurdo A Spencer et al ldquoAmyloidfibril formation and seeding by wild-type human lysozyme andits disease-related mutational variantsrdquo Journal of StructuralBiology vol 130 no 2-3 pp 339ndash351 2000

[59] J D Harper and P T Lansbury Jr ldquoModels of amyloid seedingin Alzheimerrsquos disease and scrapie mechanistic truths andphysiological consequences of the time-dependent solubility ofamyloid proteinsrdquo Annual Review of Biochemistry vol 66 pp385ndash407 1997

[60] L C Serpell M Sunde and C C Blake ldquoThe molecular basisof amyloidosisrdquo Cellular andMolecular Life Sciences vol 53 no12 pp 871ndash887

[61] M Sunde L C Serpell M Bartlam P E Fraser M B Pepysand C C F Blake ldquoCommon core structure of amyloid fibrilsby synchrotron X-ray diffractionrdquo Journal of Molecular Biologyvol 273 no 3 pp 729ndash739 1997

[62] M Moniatte F G Van Der Goot J T Buckley F Pattusand A Van Dorsselaer ldquoCharacterisation of the heptamericpore-forming complex of the Aeromonas toxin aerolysin usingMALDI-TOF mass spectrometryrdquo FEBS Letters vol 384 no 3pp 269ndash272 1996

[63] C Bleiholder N F Dupuis T Wyttenbach and M T BowersldquoIon mobilityg-mass spectrometry reveals a conformationalconversion from random assembly to 120573-sheet in amyloid fibrilformationrdquo Nature Chemistry vol 3 no 2 pp 172ndash177 2011

[64] R Beveridge Q Chappuis C Macphee and P Barran ldquoMassspectrometry methods for intrinsically disordered proteinsrdquoAnalyst vol 138 no 1 pp 32ndash42 2013

[65] H LeVine III ldquo441015840-dianilino-111015840-binaphthyl-551015840-disulfonateReport on non-120573-sheet conformers of Alzheimerrsquos peptide 120573(1-40)rdquo Archives of Biochemistry and Biophysics vol 404 no 1 pp106ndash115 2002

[66] H LeVine ldquoThioflavine T interaction with syntheticAlzheimerrsquos disease 120573-amyloid peptides detection of amyloidaggregation in solutionrdquo Protein Science vol 2 no 3 pp404ndash410 1993

[67] D Burdick B Soreghan M Kwon et al ldquoAssembly andaggregation properties of synthetic Alzheimerrsquos A4120573 amyloidpeptide analogsrdquo The Journal of Biological Chemistry vol 267no 1 pp 546ndash554 1992

[68] J D Harper C M Lieber and P T Lansbury Jr ldquoAtomicforce microscopic imaging of seeded fibril formation andfibril branching by the Alzheimerrsquos disease amyloid-120573 proteinrdquoChemistry amp Biology vol 4 no 12 pp 951ndash959 1997

[69] J D Harper S S Wong C M Lieber and P T Lansbury JrldquoObservation of metastable A120573 amyloid protofibrils by atomicforce microscopyrdquo Chemistry amp Biology vol 4 no 2 pp 119ndash125 1997

[70] C Hilbich B Kisters-Woike J Reed C L Masters and KBeyreuther ldquoAggregation and secondary structure of syntheticamyloid 120573A4 peptides of Alzheimerrsquos diseaserdquo Journal of Molec-ular Biology vol 218 no 1 pp 149ndash163 1991

[71] B Soreghan J Kosmoski and C Glabe ldquoSurfactant propertiesof Alzheimerrsquos A120573 peptides and the mechanism of amyloidaggregationrdquo The Journal of Biological Chemistry vol 269 no46 pp 28551ndash28554 1994

[72] W Garzon-Rodriguez M Sepulveda-Becerra S Milton and CG Glabe ldquoSoluble amyloid A120573-(1-40) exists as a stable dimerat low concentrationsrdquoThe Journal of Biological Chemistry vol272 no 34 pp 21037ndash21044 1997

[73] W Garzon-Rodriguez A Vega M Sepulveda-Becerra et al ldquoAconformation change in the carboxyl terminus of AlzheimerrsquosA120573(1-40) accompanies the transition from dimer to fibril asrevealed by fluorescence quenching analysisrdquo The Journal ofBiological Chemistry vol 275 no 30 pp 22645ndash22649 2000

[74] D M Walsh A Lomakin G B Benedek M M Condron andD B Teplow ldquoAmyloid 120573-protein fibrillogenesis Detection of aprotofibrillar intermediaterdquoThe Journal of Biological Chemistryvol 272 no 35 pp 22364ndash22372 1997

[75] M Kamihira A Naito S Tuzi A Y Nosaka and H SaitoldquoConformational transitions and fibrillation mechanism ofhuman calcitonin as studied by high-resolution solid-state 13CNMRrdquo Protein Science vol 9 no 5 pp 867ndash877 2000

[76] M Anguiano R J Nowak and P T Lansbury Jr ldquoProtofibrillarislet amyloid polypeptide permeabilizes synthetic vesicles by apore-like mechanism that may be relevant to type II diabetesrdquoBiochemistry vol 41 no 38 pp 11338ndash11343 2002

[77] H A Lashuel D Hartley B M Petre T Walz and P TLansbury Jr ldquoNeurodegenerative disease amyloid pores frompathogenic mutationsrdquo Nature vol 418 no 6895 p 291 2002

[78] D M Hartley D M Walsh C P Ye et al ldquoProtofibrillarintermediates of amyloid 120573-protein induce acute electrophys-iological changes and progressive neurotoxicity in corticalneuronsrdquoThe Journal of Neuroscience vol 19 no 20 pp 8876ndash8884 1999

[79] A Lomakin D B Teplow D A Kirschner and G B BenedekildquoKinetic theory of fibrillogenesis of amyloid 120573-proteinrdquo Pro-ceedings of the National Acadamy of Sciences of the United Statesof America vol 94 no 15 pp 7942ndash7947 1997


[80] R Kayed E Head J L Thompson et al ldquoCommon structureof soluble amyloid oligomers implies common mechanism ofpathogenesisrdquo Science vol 300 no 5618 pp 486ndash489 2003

[81] Y Gong L Chang K L Viola et al ldquoAlzheimerrsquos disease-affected brain Presence of oligomeric A120573 ligands (ADDLs)suggests a molecular basis for reversible memory lossrdquo Proceed-ings of the National Acadamy of Sciences of the United States ofAmerica vol 100 no 18 pp 10417ndash10422 2003

[82] Y-M Kuo M R Emmerling C Vigo-Pelfrey et al ldquoWater-soluble A120573 (N-40 N-42) oligomers in normal and Alzheimerdisease brainsrdquoThe Journal of Biological Chemistry vol 271 no8 pp 4077ndash4081 1996

[83] M Pitschke R Prior M Haupt and D Riesner ldquoDetection ofsingle amyloid120573-protein aggregates in the cerebrospinal fluid ofAlzheirnerrsquos patients by fluorescence correlation spectroscopyrdquoNature Medicine vol 4 no 7 pp 832ndash834 1998

[84] L-F Lue Y-M Kuo A E Roher et al ldquoSoluble amyloid 120573peptide concentration as a predictor of synaptic change inAlzheimerrsquos diseaserdquo The American Journal of Pathology vol155 no 3 pp 853ndash862 1999

[85] C A McLean R A Cherny F W Fraser et al ldquoSoluble pool ofA120573 amyloid as a determinant of severity of neurodegenerationin Alzheimerrsquos diseaserdquo Annals of Neurology vol 46 no 6 pp860ndash866 1999

[86] W L Klein G A Krafft and C E Finch ldquoTargeting small A120573oligomers the solution to an Alzheimerrsquos disease conundrumrdquoTrends in Neurosciences vol 24 no 4 pp 219ndash224 2001

[87] A Y Hsia E Masliah L McConlogue et al ldquoPlaque-independent disruption of neural circuits inAlzheimerrsquos diseasemousemodelsrdquo Proceedings of the National Acadamy of Sciencesof the United States of America vol 96 no 6 pp 3228ndash32331999

[88] J Hardy and D J Selkoe ldquoThe amyloid hypothesis ofAlzheimerrsquos disease progress and problems on the road totherapeuticsrdquo Science vol 297 no 5580 pp 353ndash356 2002

[89] M A Westerman D Cooper-Blacketer A Mariash et al ldquoTherelationship between A120573 and memory in the Tg2576 mousemodel of Alzheimerrsquos diseaserdquoThe Journal of Neuroscience vol22 no 5 pp 1858ndash1867 2002

[90] D M Walsh I Klyubin J V Fadeeva M J Rowan and D JSelkoe ldquoAmyloid-120573 oligomers their production toxicity andtherapeutic inhibitionrdquo Biochemical Society Transactions vol30 no 4 pp 552ndash557 2002

[91] MD Kirkitadze G Bitan andD B Teplow ldquoParadigm shifts inAlzheimerrsquos disease and other neurodegenerative disorders theemerging role of oligomeric assembliesrdquo Journal of NeuroscienceResearch vol 69 no 5 pp 567ndash577 2002

[92] R Sambanthamurthi Y Tan K Sundram et al ldquoOil palmvegetation liquor A new source of phenolic bioactivesrdquo BritishJournal of Nutrition vol 106 no 11 pp 1655ndash1663 2011

[93] US Patent No 7387802 issued in 2008[94] Z Ganim S C Hoi AW Smith L P Deflores K C Jones and

A Tokmakoff ldquoAmide I two-dimensional infrared spectroscopyof proteinsrdquo Accounts of Chemical Research vol 41 no 3 pp432ndash441 2008

[95] H S Chung M Khalil and A Tokmakoff ldquoProtein denaturingstudied with 2D IR and vibrational echo spectroscopy Equilib-rium and temperature-jump measurementsrdquo Biophysical Jour-nal pp 526andash526a 2004

[96] H S Chung and A Tokmakoff ldquoVisualization and characteri-zation of the infrared active amide i vibrations of proteinsrdquoThe

Journal of Physical Chemistry B vol 110 no 6 pp 2888ndash28982006

[97] N Demirdoven C M Cheatum H S Chung M Khalil JKnoester and A Tokmakoff ldquoTwo-dimensional infrared spec-troscopy of antiparallel 120573-sheet secondary structurerdquo Journal ofthe American Chemical Society vol 126 no 25 pp 7981ndash79902004

[98] M Khalil N Demirdoven and A Tokmakoff ldquoCoherent 2D IRspectroscopy Molecular structure and dynamics in solutionrdquoThe Journal of Physical Chemistry A vol 107 no 27 pp 5258ndash5279 2003

[99] J T Edward ldquoMolecular volumes and the Stokes-Einsteinequationrdquo Journal of Chemical Education vol 47 no 4 pp 261ndash270 1970

[100] B S Johnson J M McCaffery S Lindquist and A D GitlerldquoA yeast TDP-43 proteinopathymodel Exploring themoleculardeterminants of TDP-43 aggregation and cellular toxicityrdquoProceedings of the National Acadamy of Sciences of the UnitedStates of America vol 105 no 17 pp 6439ndash6444 2008

[101] S Krobitsch and S Lindquist ldquoAggregation of huntingtin inyeast varies with the length of the polyglutamine expansionand the expression of chaperone proteinsrdquo Proceedings of theNational Acadamy of Sciences of the United States of Americavol 97 no 4 pp 1589ndash1594 2000

[102] S Treusch S Hamamichi J L Goodman et al ldquoFunc-tional links between A120573 toxicity endocytic trafficking andAlzheimerrsquos disease risk factors in yeastrdquo Science vol 334 no6060 pp 1241ndash1245 2011

[103] D F Tardiff M L Tucci K A Caldwell G A Caldwell andS Lindquist ldquoDifferent 8-hydroxyquinolines protect models ofTDP-43 protein 120572-synuclein and polyglutamine proteotoxicitythrough distinct mechanismsrdquoThe Journal of Biological Chem-istry vol 287 no 6 pp 4107ndash4120 2012

[104] J-C Dodart K R Bales K S Gannon et al ldquoImmunizationreverses memory deficits without reducing brain A120573 burden inAlzheimerrsquos disease modelrdquo Nature Neuroscience vol 5 no 5pp 452ndash457 2002

[105] I Klyubin D MWalsh C A Lemere et al ldquoAmyloid 120573 proteinimmunotherapy neutralizes A120573 oligomers that disrupt synapticplasticity in vivordquo Nature Medicine vol 11 no 5 pp 556ndash5612005

Stem Cells International

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Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom


A120573 is a 4 kDa peptide with several isoforms themost common being a 40- and 42-amino acid peptide(A12057340A12057342) [35] The A120573 is a 39ndash44 amino acid peptide[17 36ndash40] which is endoproteolytically cleaved from a cellsurface protein the amyloid precursor protein (APP) by 120572- 120573- and 120574-secretases [41] At a critical molar concentrationA120573 undergoes an extensively studied 3-phase accretion intofibrils involving a nucleation-elongation process lag phaseexponential growth and plateau phase [5 42ndash54] The A120573fibrils have a parallel in-register structure [26]The A120573 fibrilsand the oligomerization process [55ndash58] have been wellcharacterized by multiple physicochemical measurementsincluding transmission electron microscopy (TEM) [59] X-ray diffraction [60 61] mass spectrometry (MS) [62ndash64]2D infrared spectroscopy (2D-IR) circular dichroism (CD)Congo Red dye binding (CR) Thioflavin-T fluorescence(TTf) [65 66] SDS-gel electrophoresis (SDS-PAGE) [67]atomic force microscopy (AFM) [68 69] gel filtration [7071] fluorescence resonance energy transfer (FRET) [7273] dynamic light scattering (DLS) [74] Fourier-transforminfrared spectroscopy (FTIR) and nuclear magnetic reso-nance imaging (NMR) [75] The soluble spherical oligomeraggregates measure approximately 2ndash20 nm [68 69 76 77]by hydrodynamic radius (DLS) in aqueous solution

Initially the amyloid-120573 peptide forms dimers in aqueoussolution following a lag phase involving a nucleation event[70 71] The gradual accumulation of dimers leads to theformation of higher order aggregates [78] The molecularweights of these higher order aggregates range between 105and 106 Daltons (Da) [71 79] comprising spherical particleswith an average diameter of 3 nm [59 76 77] with an averageof 24 A120573monomers per particleThe aggregation into higherorder oligomers requires a minimum critical concentrationof 25 120583M A120573 Following further incubation the sphericalparticles coalesce to form curvilinear fibrils which have acharacteristic bearded appearance called protofibrils [59 6869 78] In a continuing process of coalescence the growingmature A120573 fibrils consume the spherical aggregates andprotofibrils which disappear from solution [80]

The neurotoxic oligomers are A120573 aggregates which do notprecipitate at 100000timesg centrifugation [81ndash83]These solubleA120573 oligomers correlate better with AD symptomatology anddementia than the fibrillar A120573 [84 85] There are cognitivelynormal persons with higher numbers of fibrillar A120573 plaquesThere is a poor correlation between the presence of fibrillaramyloid deposits and dementia symptomatology [35] Themodified ldquoamyloid hypothesisrdquo of AD states that the solubleA120573 oligomers are neurotoxic but the fibrillar A120573 deposits arenot toxic [86ndash89]Multiple studies of A120573 oligomers show thatthey are toxic for neurons in vitro [86 90 91]

2 Materials and Methods

21 Preparation of Oil Palm Phenolics The preparation of oilpalm phenolics (OPP) has been previously described [92 93]OPP is an aqueous solution extracted from the fruit of thepalm oil tree (Elaeis guineensis) Following harvesting theoil palm fruit is mechanically crushed and squeezed underhigh pressure steam The biphasic filtrate is then decanted

to separate the top oil layer from the bottom aqueous layerThis aqueous layer is now termed ldquooil palm phenolicsrdquo andcontains such phytochemicals as polyphenols flavonoidsphenolic acids shikimate oligosaccharides and metal ions

22 A12057342 Amyloid Peptide The A12057342 was purchased as alyophilized powder comprising 025mg of purified A12057342from Millipore (Waltham Massachusetts USA) Then the250mcg A12057342 was initially dissolved in 50 microliters ofDMSO (dimethyl sulfoxide) This 50-microliter solution wasthenmixed with 950microliters of phosphate-buffered saline(PBS 10mM pH 74)This solution was aliquoted and frozenat minus20∘C Assays were carried out at a final concentration of50 120583MA12057342 in PBS (pH 74) A12057342 aggregation was effectedby incubating the solution at 30∘C without stirring for theindicated time periods

23 Congo Red Dye Binding Assay The Congo Red dyeworking solution was prepared as a 20 120583M Congo Red(Sigma Aldrich) in phosphate-buffered saline (PBS pH 74)The Congo Red dye binding assays were performed with aTECAN Spectrafluor Plus instrument using 96-well plasticmicrotiter plates (BDFalcon)The stock 50120583MA12057342 solutionwas diluted with PBS to a final concentration of 10120583MA12057342Then 50 120583L of this 10 120583M A12057342 solution was added to eachof the wells of the 96-well microtiter plate Following this50 120583L of OPP at the appropriate concentrations was added toeach well Finally 6 120583L of the 20120583M Congo Red dye solutionwas added to each well The TECAN Spectrafluor was setto take six repeat optical absorption readings at each timeinterval of 15 minutes with absorption readings taken at thewavelengths of 492 nm and 540 nm The optical absorptionreadings at these two wavelength allowed calculation of theA12057342 aggregates because of the known spectral shift whichoccurs when Congo Red is bound to aggregated A12057342 (see(1) below) The TECAN Spectrafluor was set for incubationof plates at 30∘C with time intervals set at optical absorptionmeasurements taken every 15 minutes

Aggregated A12057342 may be calculated by the followingequation

119862119887 =11986054147800minus11986040338100 (1)

where 119862119887 represents the amount of Congo Red dye bound toaggregated peptide 119860541 is the optical absorption measuredat the wavelength of 541 nm 119860403 is the optical absorp-tion measured at the wavelength of 403 nm 478000 is theextinction coefficient at 541 nm and 38100 is the extinctioncoefficient at 403 nm

24 Thioflavin-T Fluorescence Assay Although much workhas been done with Thioflavin-T to study the kinetics ofaggregation and fibrillization we are limited in the use of thisdye because of the very strong fluorescent signal emitted bythe oil palm phenolics at the wavelength of 535 nm followingexcitation at a wavelength of 450 nm The OPP fluorescentsignal is 4-5 times greater than that of Thioflavin-T bound toaggregates Therefore the OPP signal completely masks anydetectable signal fromThioflavin-T









119877ℎ =119896119879

6120587120578119863 (2)








3 Results





Inhi

bitio

n of

-a

myl

oid

aggr

egat

e siz

e (

)

2 4 6 8 1000

10

20

30

40

50

60

70

80

90

100


y = 13991x + 4755

R2 = 09907


Perc

ent o

f max

imum

pep

tide

aggr

egat

ion

()

0102030405060708090

100

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 160Time (hours)


8 gml OPP6 gml OPP





12826911

17145485

2150770925799855

1141627

1660915

1228183

2150771

Inte

nsity

(au

)

15000 20000 25000 30000 35000 40000 45000100000

1000

2000

3000

(mz)

(a)

Inte

nsity

(au

)

30000 40000 500002000010000

0

200

400

600

800

1000

(b)


13513

9014

4530

0

9022

4510

0 0

4534

0 0

4511

0 0

4546

0 0

4510



0

2000

4000

6000

8000

10000

12000

14000

16000

-A

myl

oid

pept

ide M

W (D

a)










Without OPP

1550

1600

1650

1700

1750

1600 1650 1700 17501550

-sheet

1 hour

(a)

Without OPP

1550

1600

1650

1700

1750

1600 1650 1700 17501550

10 hours

Enlarging -Sheet

(b)

With OPP

1550

1600

1650

1700

1750

1600 1650 1700 17501550

10 hours

No -sheet seen

(c)



4 Discussion




100 nm

(a)

100 nm

(b)












HiToxInToxNoTox

(a)


Perc

ent r

elat

ive g

row

th (

)

0

50

100

150

200

250

+ ００

minus ００

(b)






5 Conclusions











Acknowledgments



References



















































































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



























119877ℎ =119896119879

6120587120578119863 (2)








3 Results





Inhi

bitio

n of

-a

myl

oid

aggr

egat

e siz

e (

)

2 4 6 8 1000

10

20

30

40

50

60

70

80

90

100


y = 13991x + 4755

R2 = 09907


Perc

ent o

f max

imum

pep

tide

aggr

egat

ion

()

0102030405060708090

100

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 160Time (hours)


8 gml OPP6 gml OPP





12826911

17145485

2150770925799855

1141627

1660915

1228183

2150771

Inte

nsity

(au

)

15000 20000 25000 30000 35000 40000 45000100000

1000

2000

3000

(mz)

(a)

Inte

nsity

(au

)

30000 40000 500002000010000

0

200

400

600

800

1000

(b)


13513

9014

4530

0

9022

4510

0 0

4534

0 0

4511

0 0

4546

0 0

4510



0

2000

4000

6000

8000

10000

12000

14000

16000

-A

myl

oid

pept

ide M

W (D

a)










Without OPP

1550

1600

1650

1700

1750

1600 1650 1700 17501550

-sheet

1 hour

(a)

Without OPP

1550

1600

1650

1700

1750

1600 1650 1700 17501550

10 hours

Enlarging -Sheet

(b)

With OPP

1550

1600

1650

1700

1750

1600 1650 1700 17501550

10 hours

No -sheet seen

(c)



4 Discussion




100 nm

(a)

100 nm

(b)












HiToxInToxNoTox

(a)


Perc

ent r

elat

ive g

row

th (

)

0

50

100

150

200

250

+ ００

minus ００

(b)






5 Conclusions











Acknowledgments



References



















































































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of
























3 Results





Inhi

bitio

n of

-a

myl

oid

aggr

egat

e siz

e (

)

2 4 6 8 1000

10

20

30

40

50

60

70

80

90

100


y = 13991x + 4755

R2 = 09907


Perc

ent o

f max

imum

pep

tide

aggr

egat

ion

()

0102030405060708090

100

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 160Time (hours)


8 gml OPP6 gml OPP





12826911

17145485

2150770925799855

1141627

1660915

1228183

2150771

Inte

nsity

(au

)

15000 20000 25000 30000 35000 40000 45000100000

1000

2000

3000

(mz)

(a)

Inte

nsity

(au

)

30000 40000 500002000010000

0

200

400

600

800

1000

(b)


13513

9014

4530

0

9022

4510

0 0

4534

0 0

4511

0 0

4546

0 0

4510



0

2000

4000

6000

8000

10000

12000

14000

16000

-A

myl

oid

pept

ide M

W (D

a)










Without OPP

1550

1600

1650

1700

1750

1600 1650 1700 17501550

-sheet

1 hour

(a)

Without OPP

1550

1600

1650

1700

1750

1600 1650 1700 17501550

10 hours

Enlarging -Sheet

(b)

With OPP

1550

1600

1650

1700

1750

1600 1650 1700 17501550

10 hours

No -sheet seen

(c)



4 Discussion




100 nm

(a)

100 nm

(b)












HiToxInToxNoTox

(a)


Perc

ent r

elat

ive g

row

th (

)

0

50

100

150

200

250

+ ００

minus ００

(b)






5 Conclusions











Acknowledgments



References



















































































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















12826911

17145485

2150770925799855

1141627

1660915

1228183

2150771

Inte

nsity

(au

)

15000 20000 25000 30000 35000 40000 45000100000

1000

2000

3000

(mz)

(a)

Inte

nsity

(au

)

30000 40000 500002000010000

0

200

400

600

800

1000

(b)


13513

9014

4530

0

9022

4510

0 0

4534

0 0

4511

0 0

4546

0 0

4510



0

2000

4000

6000

8000

10000

12000

14000

16000

-A

myl

oid

pept

ide M

W (D

a)










Without OPP

1550

1600

1650

1700

1750

1600 1650 1700 17501550

-sheet

1 hour

(a)

Without OPP

1550

1600

1650

1700

1750

1600 1650 1700 17501550

10 hours

Enlarging -Sheet

(b)

With OPP

1550

1600

1650

1700

1750

1600 1650 1700 17501550

10 hours

No -sheet seen

(c)



4 Discussion




100 nm

(a)

100 nm

(b)












HiToxInToxNoTox

(a)


Perc

ent r

elat

ive g

row

th (

)

0

50

100

150

200

250

+ ００

minus ００

(b)






5 Conclusions











Acknowledgments



References



















































































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















Without OPP

1550

1600

1650

1700

1750

1600 1650 1700 17501550

-sheet

1 hour

(a)

Without OPP

1550

1600

1650

1700

1750

1600 1650 1700 17501550

10 hours

Enlarging -Sheet

(b)

With OPP

1550

1600

1650

1700

1750

1600 1650 1700 17501550

10 hours

No -sheet seen

(c)



4 Discussion




100 nm

(a)

100 nm

(b)












HiToxInToxNoTox

(a)


Perc

ent r

elat

ive g

row

th (

)

0

50

100

150

200

250

+ ００

minus ００

(b)






5 Conclusions











Acknowledgments



References



















































































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















100 nm

(a)

100 nm

(b)












HiToxInToxNoTox

(a)


Perc

ent r

elat

ive g

row

th (

)

0

50

100

150

200

250

+ ００

minus ００

(b)






5 Conclusions











Acknowledgments



References



















































































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















HiToxInToxNoTox

(a)


Perc

ent r

elat

ive g

row

th (

)

0

50

100

150

200

250

+ ００

minus ００

(b)






5 Conclusions











Acknowledgments



References



















































































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















5 Conclusions











Acknowledgments



References



















































































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















































































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















Oil Palm Phenolics Inhibit the In Vitro Aggregation of...

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