APPLIED MICROBIAL AND CELL PHYSIOLOGY

Red mold rice extract represses amyloid beta peptide-inducedneurotoxicity via potent synergism of anti-inflammatoryand antioxidative effect

Chun-Lin Lee & Jyh-Jye Wang & Tzu-Ming Pan

Received: 24 February 2008 /Revised: 27 March 2008 /Accepted: 29 March 2008 /Published online: 26 April 2008# Springer-Verlag 2008

Abstract Amyloid -peptide (A), a risk of Alzheimer'sdisease (AD), causes cell death by inflammation andoxidative stress. Red mold rice (RMR) fermented byMonascus species is regarded as cholesterol-loweringfunctional food in virtue of the metabolite monacolin Kidentified as lovastatin. In addition, RMR is also demon-strated to express antioxidation because of multiple anti-oxidants. Therefore, this study focuses on the synergism ofRMR against A neurotoxicity and compares the effectbetween lovastatin and RMR including monacolin K andother functional metabolites. In this study, RE 568, anethanol extract of RMR produced by strain Monascuspurpureus NTU 568, is used to protect PC12 cell againstA40 neurotoxicity. All tests contain the treatments withlovastatin or RE 568 including equal monacolin K levels inorder to compare the effect and investigate whether othermetabolites of RE 568 provide potent assistance againstA40 neurotoxicity. In the results, monacolin K repressesA40 neurotoxicity via repressing small G-protein-mediatedinflammation, and other metabolites of RE 568 also exhibitpotent antioxidative ability against A-induced oxidative

stress. Importantly, stronger effects on repressing the A40-induced cell death, inflammation, and oxidative stress areperformed by RE 568 than that by the equal levels oflovastatin, which results from a potent synergism made up ofmonacolin K, antioxidants, and anti-inflammatory agents.The present study is the first report to demonstrate the potentsynergistic protection of RMR against A40 neurotoxicity,which would cause RMR to be developed as potential andnovel functional food for the prophylaxis of AD pathogenesis.

Keywords Alzheimer's disease . Amyloid .Monascus .

Monacolin K . Anti-inflammatory . Antioxidative

Introduction

Monascus-fermented rice known as the red mold rice(RMR) or red yeast rice has been used as a food colorantand spice and developed as the popular commercial capsuleincluding 0.51.0-g red mold rice powder in Asia and theUS. In the current study, monacolin K identified aslovastatin can be produced by Monascus species undersolid and submerged culture (Endo 1979). Monacolin Kproven as a 3-hydroxy-3-methylglutaryl-coenzyme A(HMG-CoA) reductase inhibitor can effectively block denovo cholesterol biosynthesis (Endo 1979). Monascus-fermented product was gradually applied as the hypolipidemicfunctional food because it includes high monacolin Kconcentration (Lee et al. 2006a). Besides monacolin K,Monascus-fermented product including many functionalsecondary metabolites has been gradually found in currentstudies such as azaphilone pigments (ex. monascin,ankaflavin, rubropunctatin, monascorburin, rubropunctamine,and monascorburamine) exhibiting anti-inflammation, -aminobutyric acid known as the neurotransmitter and hypo-

Appl Microbiol Biotechnol (2008) 79:829841DOI 10.1007/s00253-008-1480-8

C.-L. LeeR&D Division, Sunway Biotechnology Company Limited,Taipei, Taiwan

J.-J. WangDepartment of Biotechnology, Tajen University,Ping Tung, Taiwan

T.-M. Pan (*)Institute of Microbiology and Biochemistry,National Taiwan University,1, Sec. 4, Roosevelt Road,Taipei, Taiwan10617e-mail: tmpan@ntu.edu.tw

tensive agent, and a group of antioxidants including di-merumic acid, tannin, phenol, and unsaturated fatty acids(Akihisa et al. 2005; Aniya et al. 2000; Lee et al. 2006b; Suet al. 2003). In addition, our previous studies proved thatMonascus-purpureus-NTU-568-fermented red mold rice wasable to perform functional protection involved in hypo-lipidemic effect, antioxidation, and anti-inflammation (Lee etal. 2006a, b, 2007).

Alzheimer's disease (AD) known as the most commontype of senile dementia is a progressive neurodegenerativecondition characterized as the senile plaque of amyloid -peptide (A) fibrils in brain (Lee et al. 2006d). In vivooxidation and free radical would cause the formation of Afibrils, and further, A-fibril-induced oxidative stress andinflammatory response would occur and result in theformation of more A fibrils. The risk factor A and highlevels of nitric oxide (NO), reactive oxygen species (ROS),and free radicals would be accumulated repetitiously andlead to more serious AD pathogenesis (Esposito et al. 2006;Sastre et al. 2006).

Statins has been applied to reverse the A-inducedcytotoxin via the robust anti-inflammatory effect in micro-glia model (Cordle et al. 2005). The anti-inflammatoryeffects exhibited by statins are postulated to result from theblockade of isoprenoid intermediates in cholesterol prophy-laxis of AD pathogenesis (Liao and Laufs 2005). Isopren-oid intermediates result in the activation of small G-protein,which is regarded as the essential action for inflammatorysignaling cascades (Liao and Laufs 2005). In addition,epidemiological studies suggested that treatment withcholesterol-lowering drugs could reduce the risk of devel-oping AD (Rockwood et al. 2002; Wolozin et al. 2000).Therefore, statins are potential drug for the prophylaxis ofAD pathogenesis, but more and more researches areconcerned that long-term and overdose treatment withstatins cause many side effects such as myopathy, liverand renal function impairment, hypothyroidism, and diabe-tes mellitus (Iskra et al. 2005). However, RMR has beenproven to include high monacolin K levels and successfullymediates cholesterol and isoprenoid biosynthesis pathway(Lee et al. 2006a). Importantly, RMR used as a functionalfood for the prophylaxis of cardiovascular disease has beenregarded as a safe and healthful traditional food andpopularly accepted in Asia.

Although lovastatin has been proven to attenuate A40-induced neurotoxicity, RMR is different to lovastatinbecause it includes other functional compounds. In additionto monacolin K, multiple compounds including dimerumicacid, azaphilones, furanoisophthalides, tannin, phenol,sterol, and monounsaturated fatty acid have been reportedto exhibit anti-inflammatory and antioxidative effects(Akihisa et al. 2005; Aniya et al. 2000; Su et al. 2003). Inthe prophylaxis of AD pathogenesis, both antioxidative and

anti-inflammatory ability are regarded as the key factorsagainst A40-induced neurotoxicity. Therefore, monacolinK and other functional compounds may provide potent anti-inflammatory and antioxidative ability against A40-induced neurotoxicity. This study would further evaluatewhether the synergism performs more potent effect againstA40-induced neurotoxicity than lovastatin drug or not. Ifthe supposition is tenable, RMR will deserve to bedeveloped as potential and novel functional food for theprophylaxis of AD pathogenesis.

Materials and methods

Materials

The PC12 cell line was obtained from BioresourceCollection and Research Center (60048; BCRC, Hsinchu,Taiwan). Roswell Park Memorial Institute (RPMI) 1640medium, fecal bovine serum, and horse serum werepurchased from Gibco BRL (Grand Island, NY, USA) andthe A 140 from Tocris Bioscience (Ellisville, MO, USA).A 140 was aggregated at 37C for 7 days in order toprepare fibrillar A40. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA),ethylenediaminetetraacetic acid (EDTA), dimethyl sulfoxide(DMSO), 2,2-diphenyl-1-picrylhydrazyl (DPPH), geranyl-geranyl pyrophosphate, Greiss reagent, nitro blue tetrazolium(NBT), superoxide dismutase (SOD), sodium dodecyl sulfate(SDS), ferricytochrome c, vitamin C, cholesterol, mevalonate,monoclonal inducible nitric oxide synthase (iNOS) antibody,and monoclonal-actin antibody were purchased from Sigma(St. Louis, MO, USA). Enhanced chemiluminescence (ECL)substrate was purchased from Millipore Co. (Billerica,MA, USA).

Preparation of ethanol extract of red mold rice

The long-grain rice (Ipomoea batatas) was purchased froma local supermarket in Taiwan to be used for red moldmetabolites production under solid-state cultivation. Thestrains (M. purpureus NTU 560, 563, 564, 568, and CA)were used to prepare the red mold rice according to ourprevious study (Su et al. 2003). After fermentation, thecrushed and dried red mold rice (5 g) was further extractedby 95% ethanol of 50 ml at 37C for 24 h and then driedunder vacuum. The dried extract yield was 10% of redmold rice. The dried ethanol extract was resolved to a finalconcentration of 1 mg/ml by distilled water. RE 560, RE563, RE 564, RE 568, or RE CA is prepared from theextract of the red mold rice of M. purpureus NTU 560, 563,564, 568, and CA, respectively.

830 Appl Microbiol Biotechnol (2008) 79:829841

Determination of the monacolin K concentration

Red mold rice (1 g) was extracted with 50 ml ethanol at 37Cfor 24 h and then evaporated to dryness under vacuum.Ethanol extract was resolved to a final concentration of1 mg/ml by 95% ethanol. The filtrate was then filtered with0.45-m pore size filter and analyzed by high-performanceliquid chromatography (PU2089 plus, Jasco Co., Tokyo,Japan). Chromatographic separation was conducted on a C18column (25 cm4.6 mm i.d., 5 m, Discovery@, Bellefontia,PA, USA). Acetonitrilewatertrifluoroacetate (55:45:0.05,vol/vol/vol) was used as the mobile phase. The eluent waspumped at a flow rate of 1.0 ml/min. Monacolin K wasdetected by ultraviolet detector (UV2075 plus, Jasco Co.) setat 238 nm (Lee et al. 2006c).

Cell culture

Neuronally differentiated PC12 cells were maintained inRPMI 1640 medium, supplemented with 5% fetal bovineserum, 10% inactive horse serum, 2 mM glutamine, 50 U/mlpenicillin, and 50 mg/ml streptomycin in a humifiedincubator aerated with 95% air and 5% CO2 at 37C.

Evaluation of the antioxidative ability

The DPPH radical scavenging method proposed by Aniyaet al.( 1999) was modified as follows: The reaction mixtureconsisted of 1 ml of 0.1 mM DPPH in ethanol, 0.95 ml of0.05-M TrisHCl buffer (pH 7.4), 1 ml of ethanol, and50 l of sample or deionized water (control). Theabsorbance of the mixture was measured at 517 nm exactly30 min after adding the extract (Aniya et al. 1999). Thereducing power was determined using the method proposedby Oyaizu (1986). Conjugate diene inhibition was deter-mined using the method proposed by Lingnert et al. (1979).

MTT assay for cell viability

PC12 cell viability was measured using the MTT reductionassay. MTT is reduced by viable cells to form blue formazancrystals, and inhibition of this reaction is representative ofcellular redox alterations that could lead to neurotoxicity(Pollack et al. 1995). PC12 cells (3106 cells) were platedinto 96-well plates in a total volume of 100 l of culturemedium and then cultured for 24 h at 37C, 5% CO2. Afterthe pretreatment period, culture medium were removedand changed to serum-free RPMI 1640 medium. Cellswere stimulated with fibrillar A40, fibrillar A40 andmevalonate, fibrillar A40 and cholesterol, or fibrillar A40and GGpp and then pretreated with lovastatin or RE (RE568, RE 560, RE 563, RE 564, RE CA) for 48 h. MTT wasadded to the culture medium to yield a final concentration of

0.5 mg/ml. This mixture was incubated for 2 h at 37C, 5%CO2. After incubation, the cell medium was removed, and100 l of DMSO was added to dissolve the formed formazancrystals at room temperature for 10 min in the dark. Theabsorbance at 570 nm was measured and used to calculatethe cell viability.

Cholesterol measurement

PC12 cells (3106 cells) were plated into six-well platesin a total volume of 2 ml of culture medium and thencultured with 95% air and 5% CO2 at 37C for 24 h. Afterthe pretreatment period, culture media were removed andchanged to serum-free RPMI 1640 medium containingmevalonate or cholesterol, and then cells were treated withlovastatin or RE 568 for 48 h. Cholesterol concentrationswere determined using a cholesterol assay kit (RandoxLaboratories Ltd., Antrim, UK). Briefly, cells wereremoved from medium and lysed with lysis buffer (1%Triton X-100, 20 mM Tris, pH 7.5, 100 mM NaCl, 40 mMNaF, 0.2% SDS, 0.5% deoxycholate, 1 mM EDTA, 1 mMEGTA, and 1 mM Na3VO4) at 4C for 30 min. After that,cell lysates were incubated with cholesterol esterase,cholesterol oxidase, horseradish peroxidase, 4-aminoanti-pyrine, and phenol red at 37C for 10 min. The amount ofcholesterol was determined indirectly by measuringabsorbance at 500 nm. In addition, Bradford method wasused to normalize the cholesterol levels for proteinconcentration.

iNOS immunoblotting

PC12 cells (3106 cells) were plated into six-well plates ina total volume of 2 ml of 2% culture medium and thencultured with 95% air and 5% CO2 at 37C for 24 h. Afterthe pretreatment period, cells were stimulated with fibrillarA40, fibrillar A40 and mevalonate, fibrillar A40 andcholesterol, or fibrillar A40 and GGpp, and then pre-treated with lovastatin or RE 568 for 24 h. Cells werecollected and lysed with lysis buffer. Lysates weresonicated for 10 s and cleared by centrifugation(16,000g, 15 min, 4C). Protein concentration wasdetermined by the Bradford method. The samples (50 g)were separated on 10% SDS-polyacrylamide gel electro-phoresis gels and transferred to polyvinylidene fluoridemembranes. After blocking in a gelatinNET solution, blotswere incubated with anti-iNOS antibodies (1:1,000) atroom temperature for 1 h. Then, bands were incubatedwith antimouse horseradish-peroxidase-conjugated second-ary antibodies (1:200,000) at room temperature for 1 hand visualized by ECL substrate with UVP AutoChemiImage system (UVP Inc., Upland, CA, USA). Proteinloading was evaluated by antiactin antibody (1:3,000).

Appl Microbiol Biotechnol (2008) 79:829841 831

Band intensities were quantified using UVP LabWork 4.5software (UVP Inc.).

NO assay

PC12 cells (3106 cells) were plated into six-well plates ina total volume of 2 ml of culture medium and then culturedwith 95% air and 5% CO2 at 37C for 24 h. After thepretreatment period, cells were stimulated with fibrillarA40, fibrillar A40 and mevalonate, fibrillar A40 andcholesterol, or fibrillar A40 and GGpp and then pretreatedwith lovastatin or RE 568 for 24 h. One hundred microlitersof cell medium were collected and assayed for NO levelswith the Griess reagent (Green et al. 1982). The release ofNO was determined indirectly by measuring the absorbanceat 540 nm. Triplicate measurements were obtained for eachsample. In addition, 100 l was used for an MTT assay tonormalize the Griess values for cell viability and number.

Reactive oxygen species assay

PC12 cells (3106 cells) were plated into six-well plates ina total volume of 2 ml of 2% culture medium and thencultured for 24 h at 37C, 95% air and 5% CO2. After thepretreatment period, culture media were removed andchanged to serum-free RPMI 1640 medium includingNBT (final concentration, 1 mg/ml). Cells were stimulatedwith fibrillar A40, fibrillar A40 and mevalonate, fibrillarA40 and cholesterol, or fibrillar A40 and GGpp and thentreated with lovastatin or RE 568 for 2 h at 37C, 5% CO2.Cells were collected and lysed using lysis buffer and briefsonication (10 s). Aliquots of the samples were added to96-well plates, and NBT reduction was measured byabsorbance at 550 nm in triplicate.

Measurement of oxidative stress

The amount of superoxide produced was determined bymeasuring SOD-inhibitable reduction of cytochrome c. Cellcultures grown in 96-well plates were exposed to eachtreatment or vehicle in 150 l of treatment medium. Fiftymicroliters of ferricytochrome c (160 mM) in treatmentmedium with and without 800 U/ml SOD was added. Thecultures were incubated for 15 min at 37C, 95% air and5% CO2. Afterwards, the absorbance at 550 nm was readwith a microplate spectrophotometer. The catalase activityof cell lysate was determined as previously described(Beers and Sizer 1952). Cells were harvested and sonicatedin 0.05-M sodium phosphate buffer (pH 7.4) containing0.1% Triton X-100, and the supernatant was used for thecatalase assay. Catalase activity was monitored by disap-pearance of 10 mM H2O2 by measuring the changes inabsorbance at 240 nm for 3 min. SOD activity was

measured using SOD assay kit (Ransod, Randox Laborato-ries Ltd., Antrim, UK).

Western blot quantification and statistical analysis

The density of iNOS Western blot bands was determinedusing a UVP AutoChemi Image system (UVP Inc.). Banddensities were normalized for protein loading by compar-ison with actin band densities. Three experiments werepooled to create graphical representations. Data wereexpressed as meansSE (n=3) and evaluated for significantdifferences with one-way analysis of variance followed byDuncan's multiple-range test. Differences were consideredsignificant at p

environment. Therefore, monacolin K can be regarded asthe functional active ingredient of RE inhibiting A-induced cytotoxin. However, it is not absolute relationshipbetween monacolin K concentration and neuroprotectionbecause monacolin K should not be the only functionalactive ingredient according to the result of Table 1. RE 568containing 47.5 ng/ml monacolin K would gain higher cellviability at 98% than 50 ng/ml lovastatin at 83%. Inaddition, both RE 564 and RE CA (5,000 ng/ml) includinglower monacolin K levels at 30.1 ng/ml and 29.1 ng/mlwould gain higher cell viability than RE 568. These resultscertainly evidence that although monacolin K can beregarded as the major functional active ingredient, the othermetabolites may potently assist monacolin K in rescuingcell viability from toxic fibrils A40.

RE 568 represses A neurotoxicity via blockadingisoprenoid pathway

As proven by previous studies, A neurotoxicity formicroglia or neuron is sensitively modulated by cholesteroland isoprenoid pathway (Cordle et al. 2005). As shown inthe above-mentioned results, monacolin K is an importantcompound of RE to protect PC12 cell against the A-induced cytotoxin. Monacolin K known as a HMG-CoA

Table 1 The effect of lovastatin or RE from various Monascus strainsprotecting PC12 cells viability against A40-induced cytotoxin

Sample Monacolin Kconcentration(ng/ml medium)

Sampleconcentration(ng/ml medium)

Cellviability (%)

Control 0 6218.3Lovastatin 50 50 8313.4Lovastatin 500 500 10614.7RE 568 4.75 500 8511.5RE 568 47.5 5,000 9826.7RE 560 50 651.63RE 560 500 692.90RE 560 5,000 681.09RE 563 0.216 50 6612.6RE 563 2.165 500 809.25RE 563 21.7 5,000 958.41RE 564 0.301 50 9218.2RE 564 3.007 500 12020.3RE 564 30.1 5,000 1247.80RE CA 0.291 50 1006.80RE CA 2.905 500 11410.0RE CA 29.1 5,000 13737.1

Fig. 1 Effects of RE 568 andlovastatin on the A40-inducedneurotoxicity in PC12 cells.A40-induced cell death wereevaluated with microscopicanalysis (ac) and MTT assay(d). PC12 cells (3106) weretreated with increasing concen-tration of RE 568 or lovastatinin the serum-free medium(100 l) containing 1.2 MA40 fibrils for 48 h. Micro-scopic analysis and MTT assaywere carried out after treatment.The damaged and dead cells arepresented as the arrowed cells inthe graph. The data are themeansSE expressed as per-centage of control values (n=3).Statistical comparison was madeusing ANOVA, followed byDuncan's test. *p

reductase inhibitor would blockade the biosynthesis path-way of cholesterol and isoprenoids (Endo 1979). Therefore,cholesterol-lowering effect can be regarded as an ability ofRE blockading isoprenoid pathway. Figure 2 clearlyexpresses a significant downregulation of cholesterol levelsby RE 568 supplementation in PC12 cells. Even thoughstatins have been proven to repress A-induced cytotoxinvia the blockade of mevalonate biosynthesis (Cordle andLandreth 2005), the mechanism of repressing A neuro-toxicity with RE comprising complex substances cannot bedirectly imitated by that of ultrapure lovastatin. Toinvestigate whether RE can provide more potent protectionagainst mevalonate-raised A neurotoxicity than lovastatinor not is important. The results were shown in Fig. 3.Mevalonate supplementation would strengthen the A-induced neurotoxicity, but cholesterol supplementationwould not (Fig. 3a,b). This result indicates that theisoprenoid pathway is the key pathway for increasing A-induced neurotoxicity. Lovastatin is expected to repress theraised A neurotoxicity, but the protective ability issensitive to be reversed and become slight by themevalonate supplementation. Importantly, the defect wouldnot occur by RE 568 treatment. A significant differencebetween the inhibitory effect of mevalonate-raised Aneurotoxicity by RE 568 and that by lovastatin is clearlyshown in Fig. 3b. RE 568 can protect PC12 cell against2.5 mM mevalonate-raised A neurotoxicity and signifi-cantly rescue the cell viability from 29% to 68%, but therescue by lovastatin would be completely reversed bymevalonate supplementation.

Isoprenoid GGpp is an important factor to mediate theactivity of small G-protein involved in A-induced inflam-

matory response. Therefore, GGpp is used to substitute formevalonate, so as to directly activate small G-protein. Asshown in Fig. 3c, GGpp directly acts as a more robuststimulator in A-induced cytotoxin, which is difficult to berescued by RE 568 or lovastatin. This result certainlydemonstrates that monacolin K of RE 568 protects PC12cell against A neurotoxicity via blockade of isoprenoidsbiosynthesis. Inflammatory response and oxidative stressare both regarded as the key and serious damage for thereason of A-induced cytotoxin. The mechanism of statinsagainst A-induced cytotoxin is due to the inactivation ofisoprenoid-mediated small G-protein and indirectly attenu-ated formation of proinflammatory cytokine such asinterleukin-1 (IL-1), tumor necrosis factor, and IL-6(Cordle and Landreth 2005). Therefore, anti-inflammationand antioxidation should be the most important abilityagainst A neurotoxicity. RE was repeatedly proven toperform more potent protection against A neurotoxicitythan lovastatin in this study. We suppose other substancesof RE must provide some potent abilities against Aneurotoxicity in addition to monacolin K. Because of thisdoubtful point, RE and lovastatin should be evaluated fortheir protective ability against A-induced inflammationand oxidative stress, respectively.

The effect of RE repressing A-inducedinflammatory response

Many researches indicated that A would cause neuron,microglia, and astrocytes to release inflammatory factorsincluding iNOS with the subsequent production of NO(Cordle and Landreth 2005). In this study, RE 568 is usedto test the effect of repressing A40-induced iNOSexpression. As shown in the result of immunoblotting(Fig. 4a), iNOS expression of PC12 cell can be stronglystimulated by A40, and RE 568 exhibits a dose-dependentability to downregulate the A-induced iNOS expression.According to the results of Fig. 3 and above foregoingsupposition, monacolin K of RE 568 act as a mediator ofisoprenoids pathway to rescue PC12 cells viability in A40environment, which may result from the anti-inflammationmechanism as statins. However, more potent protectionagainst A neurotoxicity performed by RE probably resultsfrom more potent anti-inflammatory ability performed bythe synergism of monacolin K and other metabolites. Thissuspicion can be solved in Figs. 4 and 5. Figure 4bindicates NO production of PC12 cells would be increasedby twofold under A40-rich environment, but that can besignificantly reversed after RE 568 treatment. However,only a slight anti-inflammatory effect is shown in lovastatintreatment. Figure 4c also indicates that lovastatin is unableto resist the A-induced NO production after mevalonatesupplementation. Although mevalonate also reverses the

Fig. 2 Cholesterol-lowering effect of RE 568 and lovastatin in PC12cells. Cholesterol levels of PC12 cells (3106) raised with or without5 mM mevalonate were treated by RE 568 or lovastatin for 48 h. Aftertreatment, cholesterol levels were measured by cholesterol assay kitand normalized by total protein concentration. The data are the meansSE expressed as percentage of control values (n=3). Statisticalcomparison was made using ANOVA, followed by Duncan's test. *p /JPEG2000ColorACSImageDict > /JPEG2000ColorImageDict > /AntiAliasGrayImages false /DownsampleGrayImages true /GrayImageDownsampleType /Bicubic /GrayImageResolution 150 /GrayImageDepth -1 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict > /GrayImageDict > /JPEG2000GrayACSImageDict > /JPEG2000GrayImageDict > /AntiAliasMonoImages false /DownsampleMonoImages true /MonoImageDownsampleType /Bicubic /MonoImageResolution 600 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict > /AllowPSXObjects false /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile (None) /PDFXOutputCondition () /PDFXRegistryName (http://www.color.org?) /PDFXTrapped /False /SyntheticBoldness 1.000000 /Description >>> setdistillerparams> setpagedevice