Computer-aided identi f i cation of protein targets of four polyphenols in Alzheimer’s disease(AD)and validation in a mouse ADmodel

Chaoyun Li,Ping Meng,Benzheng Zhang,Hong Kang,Hanli Wen,Hermann Schluesener,Zhiwei Cao,Zhiyuan Zhang,✉

1 Institute of Pathology and Neuropathology,University of Tuebingen,Tuebingen D-72076,Germany;

2 Department of Pathology,Nanjing Medical University,Nanjing,Jiangsu 211166,China;

3 School of Life Sciences and Technology,Tongji University,Shanghai 200092,China.

Abstract Natural polyphenols are a large class of phytochemicals with neuroprotective effects.Four polyphenolic compounds:hesperidin,icariin,dihydromyricetin and baicalin were selected to evaluate their effectson Alzheimer’s disease(AD).We analyzed by an inverse docking procedure(INVDOCK)the potential protein targets of these polyphenolswithin the KEGGADpathway.Consequently,their therapeutic effectswereevaluated and compared in a transgenic APP/PS1 mousemodel of AD.Thesepolyphenolsweredocked to several targets,including APP,BACE,PSEN,IDE,CASP,calpain and TNF-α,suggesting potential in vivo activities.Five month old transgenic mice were treated with thesepolyphenols.Icariin and hesperidin restored behavioral de f i citsand ameliorated Aβdepositsin both thecortex and hippocampuswhilebaicalin and dihydromyricetin showed no substantial effects.Our f i ndingssuggest that hesperidin and icariin could be considered potential therapeutic candidates of human AD.

Keywords:Alzheimer's disease,polyphenol,INVDOCK,cerebral amyloidosis,behavioral de f i cit

Introduction

Alzheimer’s disease(AD)is now the most common form of dementia among the elderly population,accounting for more than half of cases in clinical series and at autopsy[1].It is clinically characterized by progressive cognitive deterioration,neuropsychiatric and behavioral symptoms.AD pathogenesis is supposed to be triggered by the accumulation of amyloid beta(Aβ)in brain parenchyma[2].

Natural polyphenols are a large class of phytochemical compoundsthatarecomposed of aromatic ringsand one or more phenolic rings.They may interact with the aromatic residuepresent in amyloidogenic proteinsand then inhibit the self-assembly process resulting into amyloid f i bril formation[3].Baicalin,icariin,dihydromyricetin(DHM)and hesperidin are drug components of Traditional Chinese Medicine(TCM).Baicalin isone of themain bioactive f l avoneglucuronidesderived from Scutellariabaicalensis Georgi,which isoneof themost popular traditional medical herbs in Asia[4].Icariin is themostactive compound of Epimedium species,which have been used for more than one thousand years to treat chronic nephritis,osteoporosis,asthma,cardiovascular problems and hepatitis in East Asia.Hoveniadulcis,the premier anti-hangover herbal medicine,was listed in Tang Materia Medica,China’s f i rst pharmacopeia published in the year of 659.DHM,also known as Ampelopsin,is a natural supplement derived from Hoveniadulcis.Hesperidin is a naturally occurring bio f l avonoid found abundantly in citrus fruits,like Citrus sinensis and Citrus reticulate[5].A ll of these compounds have pleiotropic biological properties,including anti-oxidant,anti-in f l ammatory,anti-hypotensive,anti-microbial and anti-carcinogenic activities.Moreover,all of them exert little adverse effect,have low or no cytotoxicity,and could be able to cross the blood-brain barrier(BBB).They mighthavepotential in the treatment of neuroin f l ammatory and neurodegenerative diseases.

Identi f i cation of AD-related proteins that directly interact w ith drugs is of great importance for understanding anti-AD activities.Therefore,we tried to predict potential target proteins of these four polyphenols by an inverse docking procedure(INVDOCK).INVDOCK has been employed to conduct computerautomated inverse-docking searches of every entries in the Protein Data Bank(PDB)database to identify potential protein targets of a small molecule[6].It has been successfully used in identifying protein targets of bioactive ingredients for explaining the molecular mechanisms underlying their biological activities[7].Further,the compounds were analyzed in a transgenic AD mouse model.

M aterials and methods

Inverse docking

An inverse docking procedure,INVDOCK,hasbeen introduced to selectproteinswhich directly bind to these four polyphenols[7].INVDOCK has a built-in biomolecular cavity database derived from PDB entries.For thepurposeof saving computing time,asubsetof cavity database including only human proteins presented in“A lzheimer’s disease pathway”from Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway database[8],rather than the whole cavity database resulting from all PDB entries,was employed to run INVDOCK.The energy score for each docked conformation(compound and cavity)was calculated,aiming to determ ine if one protein could be bound by the compound.Only those docked conformation w ith interaction energy belowΔEthreshold,which is derived from empirical calculation,was supposed as binding targets of this polyphenol compound[6].

Transgenic m ice

Heterozygous male transgenic APP/PS1-(21)mice were obtained from Prof.M.Jucker[9]and were bred w ith female w ild-type C57BL/6J m ice.A ll offspring were characterized by PCR genotyping and kept as previously described[10].The experiments were approved by Regional Adm inistrative Council of Tuebingen(HF2/11)and licensed according to The German Animal Welfare Act(TierSchG)of 2006.

M aterials

Baicalin,icariin,DHM and hesperidin(all＞98%)were purchased from Huike Botanical Development Co.,Ltd(Xi’an,China),MR Natural Product Co.,Ltd.(Xi’an,China),Lyphar Biotech Co.,Ltd(Xi’an,China)and Tokyo Chem istry Co.,Ltd(Tokyo,Japan),respectively.A ll of them were suspended in 1%carboxymethylcellulose(CMC,Blanose®,Hercules-Aqualon,Düsseldorf,Germany)and administered orally in a dose of 100 mg/kg(12.5 mg/m L).Control mice received the same volume of CMC.

Treatment

Because it wasnot possible to obtain so many miceat a time,seven groupsof transgenic mice(n=six for each group)wereutilized for three sequentially experimental treatments.All micewere f i vemonthsold and treated by daily gavage for ten days.In experimentⅠ,baicalin group received 100 mg/kg of baicalin;in experimentⅡ,icariin group received 100 mg/kg of icariin and DHM group were treated by 100 mg/kg of DHM;in experimentⅢ,hesperidin group received 100 mg/kg of hesperidin.In each experiment,gender-,age-and bodyweight matched transgenic m ice,as control(controlⅠ,controlⅡand controlⅢgroup),received the same volume of 1%CMC dissolved in water.

Immunohistochem istry

Polyphenol-treated and control mice were sacri f i ced by CO2euthanasia after the ten day treatment.Brain tissues were processed and stained by immunohistochemistry as described previously[9],w ith antibodies againstβ-amyloid(1:100;Abcam,Cambridge,UK)for Aβdeposits.

After immunostaining,tissuesectionswereexamined by light m icroscopy(Nikon Cool scope,Nikon,Düsseldorf,Germany).Aβplaques in cortex and hippocampus were counted by a certain diameter and clear deposition for plaques.Then,the percentages of areasw ith speci f i c immune-reactivity(IR)in the cortex and hippocampus were analyzed using the software Meta Morph Of f l ine 7.1(Molecular Devices,Toronto,Canada).

Nest-building assay

Nest-building assay was performed as reported previously[11].It was used to determine the potential changesof af f i liativesocial behavior of transgenic mice following treatment.The presence and quality of the nest construction was evaluated on a three-point scale by three independent observers blinded to treatment categories.

Social interaction assay

The social interaction assay was performed according to previousstudies[12-13]with minor modi f i cations.Asa broad screen of activities,theresident-intruder assay was video-recorded to quantify the independent and interactive behaviors of control and polyphenol-treated mice as a resident in the absence(f i rst 15 minutes)and presence(second 15 minutes)of an intruder mouse,combined with analysisof movement to evaluateoverall activity level and overt neurobiological differences.By playing back these videotapes,behavioral events of resident mice were counted by three independent observers blinded to group assignment.The mouse movements were tracked and the positions recorded for each framein thecomputing environment Java,using the ICY software[14].Then,the total distance traveled per transgenic mouse could be easily calculated using the mouse behavior analysis software developed in our lab.

Statistical analysis

Data are showed as medians and quartiles for continuous data.The differences of behavioral events,plaque counts or IR area percentages between baicalin and control groups,or hesperidin and control groups were analyzed by exact nonparametric Mann-Whitney U test.All of the parameters described above among icariin,DHM and their control groups,were compared using the Kruskal-Wallis test,followed by post-hoc tests.Statistical signi f i cance was de f i ned as P values≤0.05.All statistical analyses were performed with the GraphPad Prism 5.0 Software(GraphPad Software Inc.,San Diego,CA,USA).

Results

Candidate target proteins of baicalin,icariin,DHM and hesperidin

For selecting potential targets of baicalin,DHM,icariin and hesperidin,INVDOCK was adopted to analyze proteins of the“Alzheimer’s disease pathway”from Kyoto Encyclopedia of Genes and Genomes(KEGG)database[8,15].Finally,18(baicalin),12(icariin),17(DHM)and 16(hesperidin)proteins were predicted as potential AD-related targets by virtual docking(Fig.1-4,all targetshighlighted by red boxes).As illustrations,models of hesperidin binding to BACE1 and TNF-αare shown in Fig.5.

Effects of four polyphenols on Aβaccumulation in brains of APP/PS1 mice

The data presented above suggest that all four polyphenols,with only minor differences,have similar binding patternsand might havesimilar anti-AD effects by modulating amyloidosis.To make an in vivo comparison of drug effects,we used a transgenic APP/PS1 mouse model,which is a widely applied animal model of cerebral amyloidosis.Weevaluated the potential therapeutic effects of the four polyphenols on Aβdeposition and behavioral dysfunction.

In the f i ve month old transgenic APP/PS1 mice,amyloid plaquesweredistributed throughoutthecortex,some of them were small with dense core plaques and somewerelarger plaqueswith a dense coreincluding a large halo of diffuse amyloid(Fig.6A,C).In the hippocampus,plaquedensity wasdistinctly lower(Fig.6B,D).

Polyphenolsweregiven daily to miceby gavagefor a period of ten days.The mice were then killed and the effect on amyloid plaque was quanti f i ed by immunostaining forβ-amyloid.In the cortex of baicalin-and DHM-treated mice,the plaque numbersand IRwere all not signi f i cant changed as compared to their respective controls[Plaque number:experimentⅠ,controlⅠ=163.5(144.8-185.8),baicalin=154.0(122.8-165.8),P＞0.05,Fig.7A;experimentⅡ,controlⅡ=149.0(128.5-197.8),DHM=136.0(117.5-165.8),P＞0.05,Fig.7C.IR area:experiment I,controlⅠ=0.82%(0.77%-0.96%),baicalin=0.63%(0.47%-0.89%),P＞0.05,Fig.7E;experimentⅡ,controlⅡ=0.67%(0.56%-0.77%),DHM=0.58%(0.50-0.74),P≤0.05].Fromhippocampus,similar dataof plaquenumbersand IRareaswererecorded[plaque number:experimentⅠ,controlⅠ=21.0(16.5-31.8),baicalin=19.0(14.3-26.3),P＞0.05,Fig.7A;experimentⅡ,controlⅡ=21.5(16.8-29.8),DHM=15.5(9.5-21.3),P＞0.05,Fig.7C.IR area:experimentⅠ,controlⅠ=0.69%(0.56%-0.87%),baicalin=0.57%(0.45%-0.79%),P＞0.05,Fig.7C;experimentⅡ,controlⅡ=0.48%(0.44%-0.85%),DHM=0.42%(0.39%-0.64%),P＞0.05,Fig.7E].Our previous results[16]showed a signi f i cant reduction in plaque numbers in the cortex of controlⅢ=0.48%(0.34%-0.70%),hesperidin=0.24%(0.16%-0.41%),P≤0.05,Fig.7F].icariin-and hesperidin-treated mice[experimentⅡ,controlⅡ=149.0(128.5-197.8),icariin=100.5(61.8-130.3),P≤0.05,Fig.7C;experimentⅢ,controlⅢ=157.0(147.0-169.3),hesperidin=126.0(103.0-142.0),P≤0.05,Fig.7E].In thehippocampus,thedifferencein the plaque numbers between icariin group and control group was statistically signi f i cant[experimentⅡ,controlⅡ=21.5(16.8-29.8),icariin=11.5(4.8-15.5),P≤0.05,Fig.7C].After ten days of icariin and hesperidin administration,the AβIR areas were signi f i cantly reduced in the cortex[experimentⅡ,controlⅡ=0.67%(0.56%-0.77%),icariin=0.16%(0.11%-0.38%),P≤0.05,Fig.7D;experimentⅢ,controlⅢ=0.72% (0.58%-0.87%),hesperidin=0.41%(0.34%-0.47%),P≤0.05,Fig.7F];in the hippocampus of hesperidin-treated mice,the AβIR areas were also signi f i cantly reduced[experimentⅢ,

Fig.1 Distribution of target proteinsof baicalin in the“Alzheimer’sdiseasepathway”.Potential targetsof baicalin arehighlighted by red boxes.

Remediation of af f iliative behavior impairment of APP/PS1 mice(nest construction assay)

Nest building is one type of af f i liative,social behavior which is of great importance to the survival of an animal.Our previous studies showed that the nesting ability of the transgenic mice was impaired in comparison to normal mice[10],which might be the result of anxiety(neophobia),hypolocomotion,and/or a reduction in normal chewing tendencies[17].

Prior to treatment,nest building performance of all groups was not signi f i cantly different(Fig.8A,B and C).After ten days of treatment(day 11),the nesting scores of baicalin-and DHM-treated remained unchanged[experimentⅠ,controlⅠ=1.50(1.00-1.63),baicalin=1.00(1.00-2.00),P＞0.05,Fig.8A;experimentⅡ,controlⅡ=1.50(1.00-1.75),DHM=2.00(1.38-2.00),P＞0.05,Fig.8B],while nests built by icariin-and hesperidin-treated mice were of improved quality asindicated by signi f i cant differences in nesting scores[experimentⅡ,controlⅡ=1.50(1.00-1.75),icariin=2.00(2.00-2.63),P≤0.05,Fig.8B;experimentⅢ,controlⅢ=1.50(1.00-1.63),hesperidin=2.25(1.50-2.50),P≤0.05,Fig.8C][16,18].Moreover,the baicalin-and DHM-treated control transgenic mice investigated and slightly chewed on paper towels.They did not really destruct the paper towels like they did just ten days ago,however paper towels were found all over the cage grouped and nongrouped away from any corners.In sharp contrast,relatively immediate chewing and tearing of the paper towels was observed in icariin-and hesperidin-treated mice;the paper towels were torn into pieces and grouped into a corner.

Fig.2 Distribution of target proteins of icariin within the“Alzheimer’sdisease pathway”.Potential targets icariin are highlighted by red boxes.

Remediation of social interaction impairment of APP/PS1 mice(resident-intruder assay)

In the social interaction assay,no matter before or after treatment,the distances traveled were not statistically signi f i cant between treatment and their respective control groups(data not shown)[18].

Two unfamiliar mice placed in the same cage will often display high levels of snif f i ng,following,grooming,rearing at the other mouse,sitting or lying next to theother mouseand so on.Theresident-intruder sessionswerevideotaped and de f i ned behavioral events were counted.Prior to treatment,the difference ininteractive behavior counts between groups was not statistically signi f i cant(Fig.8 D,E and F).Following ten days of treatment,interactive behavior events in baicalin-,icariin-and DHM-treated mice were not signi f i cantly different compared to vehicle-treated mice[experimentⅠ,controlⅠ=8.00(7.00-11.00),baicalin=11.00(9.25-14.00),P＞0.05,Fig.8D;experimentⅡ,controlⅡ=12.00(8.75-13.25),icariin=16.00(12.50-19.25),DHM=10.50(8.50-15.25),P＞0.05,Fig.8E].Hesperidin-treated arm showed a signi f i cant higher frequency of interactive behaviors as compared to control mice[experimentⅢ,controlⅢ=8.00(4.50-12.50),hesperidin=17.00(12.00-39.50),P≤0.05,Fig.8F][18].

Fig.3 Distribution of target proteinsof dihydromyricetin within the“Alzheimer’sdiseasepathway”.Potential targetsdihydromyricetin are highlighted by red boxes.

Because of compensation,the frequencies of independent behaviors in all polyphenol-treated mice were higher compared to control mice.However,the differences were not statistically signi f i cant(data not shown).At the same time,there was no difference in control groups between day one and day eleven(data not shown).

Discussion

Epidemiological evidences suggest that regular consumption of vegetables and fruits with a high polyphenol content could reduce the incidence of some age-associated neurological diseases[19].These f i ndingsaresupported by in vivo modelsof neurological disorders,revealing that polyphenol-rich plant extracts have neuroprotective effects and can even reversed cognitive de f i cits[20].Notably,in prevention and treatment of neurodegeneration,polyphenols have gathered much interest and effects at different sites within the known AD pathways.

Fig.4 Distribution of target proteins of hesperidin in the“Alzheimer’s disease pathway”.Potential targets of hesperidin are highlighted by red boxes.

Fig.5 Illustration of hesperidin molecule docked in BACE1 and TNF-αby INVDOCK program.A:Docking model of the complex hesperidin-BACE1.B:Docking model of thecomplex hesperidin-TNF-α.Thehesperidin moleculeisdisplayed asball and stick,theprotein as ribbon model.

Virtual drug screening isapowerful approach to gain anoverview of complex drug-protein interaction[21].We haveused this approach to study polyphenol binding to proteins of the“Alzheimer’s disease pathway”from Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway database.Surprisingly,baicalin,DHM,icariin and hesperidin were docked to several proteins of this pathway with a very similar pattern of potential target proteins.Some of these targets were anticipated,like binding toβ-amyloid.APPis a long protein formed by up to 771 amino acids.It isproduced in largequantities in neurons and is metabolized very rapidly.According to the“amyloid hypothesis”,Aβpeptides are produced through the endoproteolysis of APP[22].The enzymes that cleave APP have been extensively characterized.BACE1,a transmembrane aspartic protease,makes the f i rst cut in APP,and produces the secreted sAPPβectodomain and the membrane-bound C-terminal fragment C99.Neuronsfrom BACE1-/-micedo not produce Aβ,which con f i rms that BACE1 is the principalβsecretase in neurons and suggests focusing on the design of therapeutics to inhibit BACE1 activity[23].γsecretase is a multicomponent protease consisting of four integral membrane proteins,with presenilin-1(PS1)or presenilin-2(PS2)as the catalytic components[24].C99 is cleaved by theγ-secretase complex to release Aβ40and Aβ42.γ-secretase inhibitors(GSIs),such as CHF5074,strongly inhibit Aβproduction by modulating the preferred site ofγ-secretase cleavage[25].ADAM10,the physiologically relevant,constitutiveα-secretase,cuts APP within the Aβdomain,thus preventing Aβformation.ADAM10 activation by SIRT1 in amousemodel of AD signi f i cantly attenuated Aβdeposition and cognitive de f i cits[26].Aβin turn can be degraded by proteases such as insulin degrading enzyme(IDE)and neprilysin(NEP),which are remarkably enhanced by ApoE.The transgenic overexpression of IDE or NEP in neurons signi f i cantly reduces Aβlevels and plaque associated with AD pathology[27].Caspase-8 is involved in presenilin/γsecretase activation and Aβproduction in apoptosis.Blocking of caspase activity with caspase-8 inhibitor z-IETD-fmk reduced Aβproduction in H4 cell line[28].APP-binding proteins are also involved in Aβproduction and brain development:APP-BP1 may inhibit Aβ production by interacting with PS1 under physiological conditions and overexpression of APP-BP1 in neurons causing apoptosis[29];the Fe65 proteins transmit an APP-dependent signal important for neuronal positioning in thedeveloping cortex and overexpression of Fe65 inducesadramatic increasein Aβsecretion[30];not only is GAPD a key enzyme in cellular energy production,but it also plays an important role in neuronal apoptosis[31].In addition,during the progression of AD pathology,Aβaccumulation initiates a chronic in f l ammatory response in the cerebral cortex which is considered to gradually exacerbate the disease[32].

Fig.6 Hesperidin reduced cerebral amyloidosisin transgenic APP/PS1 mice.A-D:Brain coronal sections from APP/PS1 micetreated by hesperidin or vehicle.Representative images of Aβimmuno-reactivity show the reduction of amyloid plaques in the mouse brains following treatment by hesperidin.In the cortex and hippocampus of mice treated by hesperidin(C&D),fewer and smaller Aβplaques were found,compared to the control groups(A&B).

Fig.7 Effect of four polyphenolson cerebral amyloidosisof transgenic APP/PS1 mice.Brain coronal sectionsfrom APP/PS1 micetreated by baicalin,icariin,DHM,hesperidin or vehicle.A&B:The differences in the number and IRarea of amyloid plaques between baicalin-treated and control micewerenot statistically signi f i cant.C:Thenumber of amyloid plaquesin thecortex and hippocampusof icariin-treated micewas signi f i cantly lowered,but plaquenumber in the brains between DHM-treated micewas not signi f i cantly different from controls.D:Percentages of AβIRarea in sections of the cortex from icariin-and DHM-treated mice were obviously reduced as compared to the control mice.E:The number of plaques in the cortex of hesperidin-treated mice was signi f i cantly reduced.Though the amyloid plaque count in the hippocampus of mice treated by hesperidin was lower as compared to the control group,the difference was not statistically signi f i cant.F:Aβimmuno-reactive areas in the cortex and hippocampus of hesperidin-treated mice were obviously decreased.*P＜0.05 compared to the control group.

Neuroin f l ammation withactivation of microglial cells is a much debated issue in AD research,but the KEGG pathway is only listing IL-1βand TNF-α.Thus,we limited our approach to the cytokines and,very unexpectedly,observed docking of all four polyphenols to TNF-α.TNF-αthought to play a crucial role in the self-propagation of neuroin f l ammation,brain development and normal behaviors[33].Non-steroidal antiin f l ammatory drugs(NSAIDs),such as R-Flurbiprofen,PMX205,and CNI-1493,have been claimed to affect thein f l ammatory processby reducing TNF-αlevelsand to be bene f i cial in the treatment of AD[34].Thus,this interesting issuecertainly warrantsfurther investigation.

Fig.8 Effect of four polyphenols on impaired nesting ability and resident-intruder assay.APP/PS1 mice were treated for ten days with hesperidin(or CMC,control)by gavage.Nest construction wasexplored with paper towel material using athree-point scale.A:No matter prior to or after treatment,the differencein the nesting scores between thebaicalin and control group was not signi f i cant.B:Prior to treatment,there wereno obviousdifferencesamong theicariin-,DHM-treated and control mice.After ten daysof treatment,thedifferencein thenesting scores among the three groups were statistically signi f i cant as multiple comparisons showed that the nesting score of the icariin group was notably higher than the score of the control group,while there was no signi f i cant difference between the DHM and the control group.C:No signi f i cant differences between the hesperidin and control group scores were observed right at the beginning of treatment,namely on day one.By day eleven,the nest building score was signi f i cantly higher in mice treated by hesperidin,compared to the control mice.D&E:In the baicalin-,icariin-,and DHM-treated group,the interactive event counts were not signi f i cantly different from their respective control counterparts.F:The difference in the number of interactive events between hesperidin-treated and control mice was not statistically signi f i cant at day one.After ten daysof treatment,thedifferencein thenumber of interactiveeventsbetween thehesperidin-treated and control group wasobviously signi f i cant.*P＜0.05 compared to the control group.

The proteins described above are all potential targets of these four polyphenols(Fig.1-5).It had been suggested that baicalin,DHM,icariin and hesperidin might produce anti-AD effects by modulating amyloidosis.Interestingly,the biggest difference among these four binding patterns is that calpain protein being the potential target of DHM and baicalin rather than icariin and hesperidin.Itistempting to speculatethatthismight be one reason for the observed different in vivo effects.Calpain activation promotes BACE1 expression,APP expression and amyloid plaque formation[35].Several calpain inhibitions,such as E64,improved synaptic and memory de f i cits produced by Aβin APP/PS1 mice[36].Thus,calpain isaclinically relevant target and potential differential binding of polyphenols merits further investigation.

Although baicalin and DHM were reported to exert neuroprotective effects in several other in vivo and in vitro studies,they could improve neither thebehavioral dysfunction nor pathology.In addition to considering results from our docking experiments to explain differences between disease reducing icariin and hesperidin and non-effective baicalin and DHM,it should be considered that the in vivo situation is complex.For example,although baicalin can cross the BBB,the permeability is not high.For instance,after injection of a dose of 24 mg/kg,the concentration of baicalin in cerebrospinal f l uid(CSF)wasmerely 27%of that in serum[37].In contrast,Youdim et al.’s study reported that the apparent permeability to cross the in vitro BBB model was higher for the citrus f l avonoids,especially hesperitin and their more polar glucuronidated conjugates[38].In addition,after oral administration of icariin at the dose of 100 mg/kg,the absolute availability was12.02%in rats[39];whilethisparameter wasmuch lower(4.84%)in rats after giving 100 mg/kg of baicalin by gavage[40].Moreover,the excreted amount of icariin from urine,faeces and bile was very small,and the accumulated amount for 24 hours was merely 1.99%,12.83%and 0.066%of the oral administration dosage(100 mg/kg)[39];in contrast,the excreted amount of DHM was a little higher as about 29.0%of the dose(100 mg/kg)was eliminated via faeces[41].Thus,other factors will certainly affect the therapeutic effects of some polyphenols in animal models of AD.

In summary,molecular docking revealed a potential interaction of polyphenols with several proteins of the AD pathway.Some of the proteins were known targets of polyphenols,othersarepotential novel targets.Thus,the docking procedure revealed many very interesting and previously unknown targets.It further shows that it might be dif f i cult to pin-down polyphenol effects interacting with only a single protein,but maybe the promiscuous binding to several proteins will result into a complex net effect.

In vivo data supports the anti-amyloidosis effects of some polyphenols.Considering the patterns of in silico binding,the only discriminator between effective and non-effective polyphenols is the binding to calpain.While this is a noteworthy result,it should be considered that additional pharmacological factors will certainly contribute to in vivo effects.

Acknowledgments

The authors would like to thank Prof.M.Jucker for providing male transgenic APP/PS1-(21)mice.The study was supported by the China Scholarship Council(CSC)and the Deutscher Akademischer Austausch-Dienst(DAAD).Theauthorsdeclarethereisno con f l ict of interest.