Amyloid β-but not Tau-induced neurotoxicity is suppressed by Manuka honey via HSP-16.2 and SKN-1/Nrf2 pathways in an in vivo model of Alzheimer’s disease

Food & Func ion PAPER Ci e his: Food Func ., 2022, 13, 11185 Recei ed 21s June 2022, Accep ed 29 h Sep embe 2022 DOI: 10.1039/d2 o01739c sc.li/ ood- unc ion Amyloid β-bu no Tau-induced neu o oxici y is supp essed by Manuka honey ia HSP-16.2 and SKN-1/N 2 pa hways in an in i o model o Alzheime ’s disease Ma ía D. Na a o-Ho al, † a Jose M. Rome o-Má quez, † a Ped o Muñoz-Olle o, a Vic o ia Jiménez-T igo, a Adelaida Es eban-Muñoz, b Kilian Tu usaus, c,d F ancesca Giampie i, c,e Mau izio Ba ino, c, ,g C is ina Sánchez-González, a,h Lo enzo Ri as-Ga cía, a,h Juan Llopis, a,h Tama a Y. Fo bes-He nández* a and José L. Quiles * a,c Alzheime ’s is a ch onic degene a i e disease o he cen al ne ous sys em conside ed he leading cause o demen ia in he wo ld. I is cha ac e ized by wo e iopa hological e en s ela ed o oxida i e s ess: he agg ega ion o β-amyloid pep ide and he o ma ion o neu ofib illa y angles o hype phospho yla ed Tau p o ein in he b ain. The incidence o his disease inc eases wi h age and has been associa ed wi h inadequa e li es yles. Some na u al compounds ha e been shown o imp o e he hallma ks o he disease. Howe e , despi e i s po en ial, he e is no scien ific e idence abou Manuka honey (MH) in his ega d. In he p esen wo k we e alua ed he effec o MH on he oxici y induced by Aβagg ega ion and Tau in a Caeno habdi is elegans model. Ou esul s demons a ed ha MH was able o imp o e indica o s o oxi- da i e s ess and delayed Aβ-induced pa alysis in he AD model CL4176 h ough HSP-16.2 and SKN-1/ NRF2 pa hways. Ne e heless, i s suga con en impai ed he indica o s o locomo ion (an indica o o au neu o oxici y) in bo h he ansgenic s ain BR5706 and in he wild- ype N2 wo ms. 1. In oduc ion Alzheime ’s disease (AD) is a p og essi e neu odegene a i e disease o he cen al ne ous sys em, cha ac e ized by accumula ion o amyloid-β(Aβ) pep ide and abno mal s ands o hype phospho yla ed p o ein Tau ( angles) in he b ain. 1–3 These modi ica ions a e accompanied by neu onal loss 2,4 along wi h synap ic and b ain issue damage 2,5 esul ing in memo y impai men , 1,6 dep ession, diso ien a ion, poo judg- men and, in la e s ages, speaking and walking difficul ies. 5 I is es ima ed ha AD causes be ween 60–80% o all demen ia cases, 5 ep esen ing an impo an economic bu den wo ld- wide. 4 Cu en ly, mos o he app o ed d ugs o he ea men o he illness only alle ia e symp oms, bu do no modi y he unde lying biology o Alzheime ’s o al e he cou se o he disease. In addi ion, mos o hese d ugs usually a e accompanied by side effec s including nausea, headache, 5 insomnia and hepa o oxici y. 3 Only one o he FDA-app o ed d ugs, aducanumab, can educe Aβplaques in he b ain. 5 Hence, he sea ch o new d ug candida es o igh AD is s ill an a ac i e esea ch a ea. In he las yea s, se e al s udies ha e shown ha bioac i e compounds om na u al p oduc s possess neu op o ec i e p ope ies and a e capable o elie ing AD symp oms, indica ing ha na u al sou ce-based d ugs could be a alid al e na i e in his he apeu ic a ea. 3,4,7 In ha sense, Manuka honey (MH), a much app ecia ed honey de i ed om he Lep ospe mum scopa ium ee (My aceae amily), ypical in New Zealand, eas e n Aus alia and he Medi e anean egion, has shown excellen biological ac i i ies including an ibac e ial, an ioxidan , wound-healing †These au ho s con ibu ed equally o his manusc ip . a Depa men o Physiology, Ins i u e o Nu i ion and Food Technology “José Ma aix Ve dú”, Biomedical Resea ch Cen e, Uni e si y o G anada, A milla, A da. del Conocimien o s.n., 18100 A milla, Spain. E-mail: [email p o ec ed], o [email p o ec ed] b Depa men o Nu i ion and B oma ology, Uni e si y o G anada, 18071 G anada, Spain c Resea ch G oup on Foods, Nu i ional Biochemis y and Heal h, Uni e sidad Eu opea del A lán ico, Isabel To es, 21, 39011 San ande , Spain d Uni e sidad In e nacional Ibe oame icana, 24560 Campeche, Mexico e Depa men o Biochemis y, Facul y o Sciences, King Abdulaziz Uni e si y, Jeddah, Saudi A abia Depa men o Clinical Sciences, Poly echnic Uni e si y o Ma che, Ancona, 60131, I aly g In e na ional Join Resea ch Labo a o y o In elligen Ag icul u e and Ag i-p oduc s P ocessing, Jiangsu Uni e si y, Zhenjiang, China h Spo and Heal h Resea ch Cen e, Uni e si y o G anada, C/.Menéndez Pelayo 32, 18016 A milla, G anada, Spain This jou nal is © The Royal Socie y o Chemis y 2022 Food Func ., 2022, 13,11185–11199 | 11185 Open Access A icle. Published on 07 Oc obe 2022. Downloaded on 11/7/2022 9:46:08 AM. This a icle is licensed unde a C ea i e Commons A ibu ion 3.0 Unpo ed Licence. View A icle Online View Jou nal | View Issue and an ip oli e a i e p ope ies as well as a e y in e es ing physicochemical composi ion. 8,9,10–12 Howe e , o he bes o ou knowledge, he e is no e idence on i s neu op o ec i e po en ial. On he o he hand, he nema ode Caeno habdi is elegans (C. elegans), due o i s sho li espan and i s limi ed numbe o neu ons and ac able gene ics, ep esen s an ideal sys em o s udy aging and age- ela ed diseases, such as AD. 1,2,13 Likewise, he e a e mul iple ansgenic s ains, speci ically designed o examine he Aβplaque accumula ion and angle- caused neu o oxici y. 2,6,13 In he p esen s udy, we in es iga ed he effec s o MH on Aβagg ega ion and au neu o oxici y in C. elegans AD models. 2. Ma e ials & me hods 2.1 Honey samples and eagen s MH was bough in a local supe ma ke in Spain a e impo a- ion in o Eu ope by he Comp oi s e Compagnies. All eagen s we e pu chased om Sigma-Ald ich (S Louis, Missou i, USA), Me ck (Da ms ad , Ge many), The mo Fishe (Wal ham, Massachuse s, USA) o Roche (Basel, Swi ze land). 2.2 MH cha ac e iza ion 2.2.1 To al phenolic compounds (TPC) and o al la onoids con en (TFC) de e mina ion. TPC o MH we e de e mined by he Folin-Ciocal eu me hod, 14 while i s la onoid con en was e alua ed acco ding o he p o ocol desc ibed by Na a o- Ho al e al. 15 2.2.2 To al an ioxidan capaci y (TAC). TAC o MH was e al- ua ed by e ic educing an ioxidan powe (FRAP), olox equi- alen an ioxidan capaci y (TEAC) and 2,4-DNP; 2,2-diphenyl- 1-pic ylhyd azyl (DPPH) me hods, as p e iously epo ed by ou g oup. 15–17 In all cases, he abso bance was measu ed in a Neo2 mic opla e eade (Bio ek, Winooski, Ve mon , U.S.A.) and he esul s we e exp essed as μM o T olox equi alen s (TE) pe g am (g) o honey. 2.2.3 Ex ac ion, iden i ica ion, and quan i ica ion o phe- nolic compounds. The ex ac ion o phenolic compounds was pe o med acco ding o A in e al. 11 Fo hei iden i ica ion a SYNAPT G2 HDMS Q-TOF sys em (Wa e s, Mild o d, USA) equipped wi h a dual elec osp ay ioniza ion p obe was used, ollowing he p o ocol epo ed by Es eban-Muñoz e al. 18 UPLC sepa a ion was pe o med using an ACQUITY UPLC ™ HSS T3 2.1 × 100 mm, 1.8 mm column and he p og am o ch oma og aphy was se wi h a bina y g adien consis ing o (A) wa e wi h 0.5% ace ic acid and (B) ace oni ile, as ollows: ini ial 0.0 min 5% (B), 15.0 min 95% (B); 15.1 min 5% (B); and 18.0 min 5% (B) and he low a e was 0.4 mL min −1 . Phenolic compounds we e iden i ied h ough he MassLynx V4 so wa e (Wa e s Labo a o y In o ma ics, Mild o d, USA) by compa ing he ions and molecula agmen s ob ained wi h p e ious da a. The quan i ica ion was ca ied ou by an ACQUITY UPLC I-Class Sys em (Wa e s, Mississauga, ON, Canada) equipped wi h a mass spec ome e Wa e s XEVO TQ-XS wi h ioniza ion pe o med by UniSp ay (US), acco ding o he me hod desc ibed by Sánchez-He nández e al. 19 A g adien consis ing o (A) wa e and (B) me hanol wi h 0.1% [ / ] ace ic acid o 25 min a a low a e o 0.4 mL min −1 was se as ollows: 0.0 min 5% B, 15–15.10 min 95% B and 15.10–25 min 5% B. An ACQUITY UPLC ™HSS T3 1.8 mm column was used. Quan i ica ion o he phenolic compounds was achie ed by compa ing he e en ion imes o peaks and agmen a ion da a ob ained, wi h hose o he phenolic compound s an- da ds. Measu emen s in Mul iple eac ion moni o ing was used. 2.3 C. elegans s ains and main enance condi ions Wild- ype N2 B is ol C. elegans, he ansgenic s ains CL802 [smg-1(cc546) I; ol-6(su1006) II], CL4176 (d Is27 [myo-3p:: A-Be a (1–42)::le -851 3′UTR) + ol-6(su1006)] X), CF1553 (mu1s84[pAD76 (sod-3::GFP) + ol-6(su1006)]), CL2166 (d Is19 [(pAF15)gs -4p:GFP:NLS] III), LD1 (ldIs7 [skn-1b/c::GFP + ol-6 (su1006)]), TJ375 (gpIs1[hsp-16.2::GFP]), OS3062 [myo-2p::hs - 1 + hsp-16.2::GFP + hsp-16.41::GFP + ol-6(su1006)], TJ356 (zIs356[da -16p::da -16a/b::GFP + ol-6 (su1006)]), BR5706 (bkIs10 [aex-3p:hTau V337M + myo-2p:GFP]) and E. coli OP50 we e ob ained om he Caeno habdi is Gene ics Cen e (Minneapolis, MI, USA). All wo ms we e g own and main ained on nema ode g ow h medium (NGM)-aga pla es seeded wi h a lawn o E. coli OP50 a 20 °C o 16 °C, depending on he s ain’s cha ac e is ics o he expe imen design. To ob ain age-synch onized nema odes, he bleaching p o- ocol was used. B ie ly, g a id he maph odi es we e exposed o an alkaline bleach solu ion (0.5 N NaOH in 20% NaOCl) and igo ously s i ed o six minu es ollowed by cen i uga ion a 1280g× 2 minu es. Se led eggs we e hen esuspended and washed o h ee imes wi h M9 medium. A e ha , eggs we e seeded on esh NGM aga pla es wi h a lawn o E. coli OP50 and allowed o ha ch. Wo ms we e allowed o ma u e un il he desi ed la al s age, changing he pla es e e y 2–3days. 2.4 Le hali y es To assess acu e le hali y, N2 B is ol wo ms synch onized a L4 la al s age we e placed on NGM pla es con aining 0, 25, 50, 100, 200 o 300 mg mL −1 o MH o 24 h a 20 °C in he absence o E. coli OP50. A e ha ime, nema odes we e sco ed as li e o dead by using a Mo ic dissec ing mic oscope (Mo ic Inc., L d, Hong Kong, China). Wo ms ha did no espond o epea ed s imuli in lic ed wi h a pla inum wi e we e conside ed dead. A leas 30 wo ms we e used pe g oup and he expe imen was epea ed a leas h ee imes. Resul s we e exp essed as su i al a e. Fo he subsequen expe i- men s a non-le hal submaximal concen a ion (100 mg mL −1 ) o MH was used. 2.5 Pha yngeal pumping assay and g ow h e alua ion Egg-synch onized N2 wo ms we e placed on NGM pla es seeded wi h E. coli OP50 and con aining o no 100 mg mL −1 MH and hen incuba ed a 20 °C. Nine y-six hou s la e , Pape Food & Func ion 11186 |Food Func .,2022,13, 11185–11199 This jou nal is © The Royal Socie y o Chemis y 2022 Open Access A icle. Published on 07 Oc obe 2022. Downloaded on 11/7/2022 9:46:08 AM. This a icle is licensed unde a C ea i e Commons A ibu ion 3.0 Unpo ed Licence. View A icle Online wo ms we e ans e ed o esh pla es and he numbe o con- ac ions pe minu e o he pha ynx e minal bulb was coun ed using a Mo ic mic oscope (Mo ic Inc. L d Hong Kong, China). A he end, wo ms we e collec ed by washing wi h M9 medium and analyzed h ough a Mul iRange la ge pa icle low cy ome e Bioso e (Union Biome ica. Massachuse s, USA) o de e mine he ime o ligh (TOF) which is indica i e o he leng h. A leas 10 wo ms we e used pe g oup o pha - yngeal pump coun ing and abou 100 nema odes o de e mine he size. Bo h expe imen s we e pe o med in iplica e. 2.6 Eggs e ili y assay L4 s age synch onized N2 wo ms we e indi idually placed in 24 well NGM pla es seeded wi h E. coli OP50 and con aining o no 100 mg mL −1 MH. A e 24 h a 20 °C, he laid eggs we e coun ed by using a Mo ic dissec ing mic oscope (Mo ic Inc., L d, Hong Kong, China) and he wo ms we e mo ed o a esh pla e o dis inguish om he offsp ing. This p ocedu e was epea ed e e y single day un il egg-laying s opped. A leas 15 wo ms we e used pe g oup and he expe imen was epea ed a leas h ee imes. Resul s we e exp essed as he mean o he o al numbe o eggs pe g oup. 2.7 Oxida i e s ess esis ance assays To assess oxida i e s ess esis ance, he in acellula eac i e oxygen species (ROS) le els in L4 s age-synch onized N2 wo ms we e measu ed by using he 2′,7′-dichlo o luo escein diace a e (DCFDA) assay, as p e iously desc ibed by Na a o- Ho al e al. 15 B ie ly, eggs-synch onized emb yos we e placed on NGM pla es seeded wi h E. coli OP50 con aining o no 100 mg mL −1 MH o 48 h a 20 °C. Nex , young-adul wo ms we e collec ed and washed h ee imes wi h M9 medium in o de o emo e bac e ia and we e exposed (excep con ol g oup) o 2.5 μM AAPH (2,2′-azobis-2-amidinop opane dihy- d ochlo ide) o 15 min o induce oxida i e s ess. Wo ms we e u he washed wi h M9 medium and di ec ly incuba ed wi h he luo ogenic dye ( inal concen a ion, 25 μM) o 2 h. The luo escence was measu ed by a Mul iRange la ge pa icle low cy ome e Bioso e (Exci a ion wa eleng h 490 nm, emission wa eleng h 510 nm) (Union Biome ica. Massachuse s, USA). Resul s we e exp essed as he mean o he luo escence in en- si y no malized by TOF. A leas 200 nema odes we e used pe g oup and he expe imen s we e epea ed in iplica e. 2.8 Pa alysis es T ansgenic s ain CL4176 and he non-pa alyzable con ol s ain CL802 we e synch onized as desc ibed abo e in he main enance condi ions sec ion and placed on NGM pla es seeded wi h E. coli OP50 con aining o no 100 mg mL −1 MH. Wo ms we e incuba ed o 48 h a 16 °C and hen ans e ed o a 25 °C incuba o o s imula e he Aβ-induced pa alysis. A e 20 h, 25 nema odes o each g oup we e picked o esh NGM pla es (main aining empe a u e condi ions) and sco ed o pa alysis e e y 2 h un il 32 h a e he empe a u e ise. Nema odes we e conside ed pa alyzed when hey did no espond o epea ed s imuli in lic ed wi h a pla inum wi e o i hey only mo ed he head wi hou showing a ull body wa e. Resul s we e exp essed as pe cen age o non-pa alyzed wo ms. The expe imen was pe o med in iplica e. 2.9 β-Amyloid agg ega es isualiza ion Wo ms o he pa alyzable s ain CL4176 and he nega i e con ol s ain CL802 we e g own as desc ibed abo e o he pa alysis assay. A e 26 h o incuba ion a 16 °C, wo ms we e ixed wi h a 4% pa a o maldehyde/M9 medium solu ion (pH 7.4) o 24 h and subsequen ly pe meabilized wi h a solu ion con aining 5% esh β-me cap oe hanol, 125 mM T is and 1% T i on X-100 (pH 7.4) o ano he 24 h a 37 °C. Aβ-agg ega es we e s ained wi h 0.125% Thio la in T (Sigma-Ald ich, S Louis, MI, USA) in 50% e hanol o 30 min and we e obse ed by using a Nikon epi- luo escence mic oscope (Eclipse Ni, Nikon, Tokyo, Japan). Abou 20–30 images pe g oup we e acqui ed a 40× magni ica ion using he GFP il e wi h a Nikon DS-Ri2 came a (Tokyo, Japan). 2.10 RNA In e e ence (RNAi) Assay Supe oxide dismu ase (SOD)-2 and 3, hea shock p o ein 16.2 (HSP-16.2), ansc ip ion ac o skinhead-1 (SKN-1) and daue o ma ion (DAF-16) gene exp ession was knocked down by eeding CL4176 wo ms wi h E. coli s ain HT115 ca ying he speci ic dsRNA. B ie ly, L3/L4 s age-synch onized CL4176 wo ms we e ans e ed o RNAi pla es con aining 1 mM iso- p opyl β-D-1- hiogalac opy anoside (IPTG) and 25 μgmL −1 ca - benicillin and incuba ed o 48–72 h. Once hey eached he e ile age, ano he synch oniza ion was ca ied ou and he eggs/emb yos ob ained we e placed on esh RNAi pla es con- aining o no 100 mg mL −1 MH and we e allowed o ma u e o L4 young la ae. The esul ing adul wo ms we e used o he pa alysis assay as p e iously desc ibed. 2.11 G een luo escence p o ein (GFP)- epo e ansgenic s ains Egg-synch onized wo ms o he ansgenic s ains CF1553, CL2166, LD1, OS3062, TJ375, and TJ356 exp essing he GFP epo e SOD-3, glu a hione S- ans e ase 4 (GST-4), SKN-1, hea -shock ansc ip ion ac o 1 (HSF-1), HSP-16.2 and DAF-16, espec i ely, we e g own in NGM pla es seeded wi h E. coli OP50 con aining o no 100 mg mL −1 MH o 48 h a 20 °C. Nex , 20–30 wo ms pe g oup we e immobilized wi h sodium azide (1 mM) on a slide and subjec ed o imaging using a Nikon epi- luo escence mic oscope (Eclipse Ni, Nikon, Tokyo, Japan). The o al GFP luo escence was analyzed using he NIS-Elemen s BR so wa e (Nikon, Tokyo, Japan). The sub- cellula DAF-16 dis ibu ion in he TJ356 s ain was analyzed by classi ying he exp ession/loca ion pa e ns in o h ee ca - ego ies: cy osolic, in e media e, and nuclea ; each o which was assigned a alue (1, 2, 3) o ca y ou a semi-quan i ica ion. The expe imen s we e epea ed a leas h ee imes. 2.12 Locomo ion beha io analysis Egg-synch onized emb yos o wild ype-N2 and BR5706 s ains we e placed on NGM pla es seeded wi h E. coli OP50 con ain- Food & Func ion Pape This jou nal is © The Royal Socie y o Chemis y 2022 Food Func .,2022,13, 11185–11199 | 11187 Open Access A icle. Published on 07 Oc obe 2022. Downloaded on 11/7/2022 9:46:08 AM. This a icle is licensed unde a C ea i e Commons A ibu ion 3.0 Unpo ed Licence. View A icle Online ing o no diffe en ea men s (100 mg mL −1 MH, 100 mg mL −1 a i icial honey (AH) o 232 µg mL −1 o MH hyd oalco- holic ex ac (ME) o 72 h a 20 °C. Nex , abou 20–30 wo ms pe g oup we e o ced o swim by placing hem in a d op o M9 medium in he cen e o a slide. Wo ms mo emen was acked, eco ded and analyzed by using a Wo mLab Imaging Sys em (MBF Bioscience, Willis on, Ve mon , EE. UU). The wa eleng h, swimming speed and ac i i y we e analyzed as ep esen a i e pa ame e s o locomo i e beha io . The expe i- men was ca ied ou a leas in iplica e. 2.13 S a is ical analysis S a is ical analysis was pe o med by SPSS 24.0 so wa e (IBM, A monk, NY, USA). Da a we e subjec ed o one-way analysis o a iance (ANOVA) ollowed by Tukey’s hones ly signi ican diffe ence (HSD) pos hoc es o compa e s a is ical diffe ence among mul iple g oups o o -s uden es o pai wise da a se s. A p< 0.05 was conside ed s a is ically signi ican . Da a a e exp essed as mean ± SEM om a leas h ee independen expe imen s. 3. Resul s 3.1 MH cha ac e iza ion A o al o 31 compounds we e iden i ied in MH (Table 1) and despi e he limi o quan i ica ion o some o hem, i was possible o quan i y que ce in, que ce in-3-O-glucopy anoside, ch ysan hemin, epica echin, apigenin, na ingenin, m-couma- ic acid, e ulic acid, osma inic acid, ellagic acid and o- anil- lin (Table 2). Na ingenin (295.3 ± 4.7 ppb) and o- anillin (373.5 ± 5.6 ppb) we e he majo compounds. Hyd oxybenzaldehydes ep esen ed 45.14% o he quan i ied Table 1 Phenolic compounds iden ified in MH Compounds CAS numbe Ion mode [M] +/− (m/z) Re . Fla anones Na ingenin 67604- 48-2 + 153 20–23 −151 Pinocemb in 480-39-7 + 151 23–26 −255 Saku ane in 2957-21- 3+ 133 25 and 26 −287 Fla ones Apigenin 520-36-5 + 153 22–25 and 27 −117 Ch ysin 480-40-0 + 143 21 and 23–28 −253 Lu eolin 491-70-3 + 153 20 and 22–28 −133 Nepe in 520-11-6 −315 29 Fla onols Galangin 548-83-4 −213 23–28 Iso hamne in 480-19-3 −199 20, 25, 26 and 28 Kaemp e ol 520-18-3 + 153 20 and 22–27 −151 Que ce in 117-39-5 −151 20–27 Que ce in 3-O- u inoside 153-18-4 + 609 20, 22–25 and 27 Rhamne in 90-19-7 −315 25, 26 and 28 Iso la onoids Biochanin A 491-80-5 + 283 27 −268 Daidzein 486-66-8 + 253 23 and 27 −209 Fo monone in 485-72-3 −252 27 Genis ein 446-72-0 + 269 27 −181 Hyd oxybenzaldehydes Sy ingaldehyde 134-96-3 + 181 28 and 30 Vanillin 121-33-5 −151 30 and 31 Hyd oxycouma ins Scopole in 92-61-5 −190 32 Hyd oxybenzoic acids Sy ingic acid 530-57-4 + 197 20, 22, 28, 31 and 33 4-Hyd oxybenzoic acid 99-96-7 + 137 22, 23, 26–28, 30 and 33 Vanillic acid 121-34-6 −152 22, 23, 27, 30 and 33 Hyd oxycinnamic acids 3-Caffeoylquinic acid 906-33-2 −353 25 5-Caffeoylquinic acid 327-97-9 −353 23, 31 and 34 Caffeic acid 331-39-5 + 179 20–22, 24–28, 30 and 35−135 Cinnamic acid 621-82-9 −147 25 and 33 Fe ulic acid 1135-24- 6+ 193 20–24, 26, 27, 30, 32, 33 and 35 −134 − p-Couma ic acid 501-98-4 + 163 20, 22, 23, 25–27, 30, 32 and 35 −119 − Hyd oxyphenylace ic acids 4-Hyd oxyphenylace ic acid 156-38-7 −107 22 and 23 Phenylace ic acid 103-82-2 −107 27 Table 2 Quan ifica ion o he phenolic compounds iden ified in MH Phenolic compounds Mean (ppb) ± SD Fla onols Que ce in 52.6 ± 1.4 Fla onol glycosides Ru in <LOQ Kaemp e ol-3-O-glucoside <LOQ Que ce in-3-O-glucopy anoside 0.6 ± 0.2 An hocyanins Ch ysan hemin 0.3 ± 0.0 Fla anols Epica echin 4.5 ± 0.4 Fla ones Apigenin 1.6 ± 0.4 Fla anones Na ingenin 295.3 ± 4.7 Iso la ones Glyci ein <LOQ Fo monone in <LOQ Phenolic acids m-Couma ic acid 16 ± 1.0 Fe ulic acid 6.3 ± 0.1 Rosma inic acid 3.5 ± 0.2 Ellagic acid 73.2 ± 6.1 Hyd oxybenzaldehydes o-Vanillin 373.5 ± 5.6 To al phenolic con en 827.4 LOQ: limi o quan i ica ion. Da a a e means (n= 3). Pape Food & Func ion 11188 |Food Func ., 2022, 13, 11185–11199 This jou nal is © The Royal Socie y o Chemis y 2022 Open Access A icle. Published on 07 Oc obe 2022. Downloaded on 11/7/2022 9:46:08 AM. This a icle is licensed unde a C ea i e Commons A ibu ion 3.0 Unpo ed Licence. View A icle Online compounds, while la onoids and phenolic acids ep esen ed 42.78% and 11.97%, espec i ely (Table 2). Acco ding o he colo ime ic me hods, he TPC o MH was 0.47 ± 0.03 mg GAE g −1 , while i s TFC was 0.05 ± 0.01 mg CAE g −1 . Likewise, i s an ioxidan capaci y was 2.65 ± 0.10 μM T olox eq. pe g, 1.40 ± 0.13 μM T olox eq. pe g and 4.58 ± 0.22 μM T olox eq. pe g acco ding o he FRAP, ABTS and DPPH de e mina ion me hods, espec i ely. The yield o he hyd oalcoholic ex ac ion o phenolic compounds was 2.32 ± 0.42 mg g −1 o honey (Table 3). 3.2 Sho e m oxici y o MH The sho - e m oxici y o MH in C. elegans was e alua ed h ough diffe en assays, including 24 h le hali y, pha yngeal pumping, egg-laying ( e ili y) and g ow h. As shown in Fig. 1A, no s a is ically signi ican diffe ences (p< 0.05) we e ound be ween he con ol g oup and wo ms ea ed wi h inc easing concen a ions (up o 300 mg mL −1 )o MHin e ms o su i al pe cen age. Likewise, MH supplemen a ion did no affec he numbe o pumps pe minu e o he wo m’s e ili y, unde s ood as hei spawning capaci y. Howe e , wo ms ea ed wi h 100 mg mL −1 MH we e sligh ly smalle (p< 0.05) (Fig. 1B). 3.3 Effec s o MH agains AAPH-induced oxida i e s ess An escala ion in endogenous ROS le els accele a es he aging p ocess and, in u n, he nema odes abili y o deal wi h ha m ul s imuli dec eases d as ically o e ime. To e alua e he effec s o MH agains AAPH-induced s ess, we measu ed in a- cellula ROS le els by DCF-DA me hod. As shown in Fig. 2, nema odes ea ed wi h MH p esen ed a g ea e esis ance agains hese s ess condi ions. MH comple ely p e en ed he inc ease in ROS obse ed in he s essed g oup as no signi ican diffe ence (p< 0.05) was ound compa ed o he con ol. 3.4 Effec s o MH agains Aβ-induced pa alysis Aβagg ega ion is conside ed one o he majo pa hological causes o AD. To e alua e whe he MH amelio a ed he Aβ-induced oxici y, we used he ansgenic C. elegans s ain CL4176, which exp esses he human Aβpep ide in body wall muscle cells ollowing empe a u e upshi om 16 o 25 °C, esul ing in a pa alysis pheno ype. As shown in Fig. 3A, non- ea ed CL4176 wo ms exhibi ed almos comple e pa alysis a e 34 h o he empe a u e upshi , while 100 mg mL −1 MH ea men signi ican ly (p< 0.05) delayed his p ocess. In he MH- ea ed g oup, no signi ican diffe ences (p< 0.05) we e obse ed wi h espec o he non-pa alyzable s ain CL802 (nega i e con ol) up o 30 h a e empe a u e upshi . A he end poin o he analysis, he pa alyzed wo ms ep esen ed only 25% o he en i e popula ion. In addi ion, MH signi i- can ly educed Aβdeposi s in he ail egions o CL4176 wo ms as e idenced by hio la in T s aining (Fig. 3B). To u he elucida e he molecula mechanisms unde lying he p o ec i e effec s o MH on Aβ oxici y, we epea ed he pa alysis es in CL4176 wo ms silencing some genes by RNAi, including sod-2and sod-3,hsp-16.2,skn-1,andda -16.34ha e he empe a u e upshi , RNAi o sod-3and da -16 had no effec on he pe cen age o non-pa alyzed wo ms compa ed o he Table 3 Phy ochemical cha ac e iza ion, an ioxidan capaci y and yield o MH Pa ame e Mean ± SEM To al phenolic con en (mg GAE g −1 ) 0.47 ± 0.03 To al la onoids con en (mg CAE g −1 ) 0.05 ± 0.01 FRAP (μMTEg −1 ) 2.65 ± 0.10 DPPH (μMTEg −1 ) 1.40 ± 0.13 TEAC (μMTEg −1 ) 4.58 ± 0.22 Yield a 2.32 ± 0.42 a mg o hyd oalcoholic ex ac ob ained pe g o honey. FRAP: e ic educing an ioxidan powe ; DPPH: 2,2-diphenyl1-pic yl-hyd azyl- hyd a e; TEAC: olox equi alen an ioxidan capaci y; mg GAE g −1 :mg gallic acid equi alen ; mg CAE g −1 : mg ca echin equi alen ; TE: olox equi alen . Fig. 1 Sho - e m oxici y o Manuka honey in he N2 wild ype s ain. (A) 24 h le hali y. (B) Pha yngeal pumping, egg-laying ( e ili y) and g ow h. Wo ms we e ea ed wi h he indica ed concen a ion o MH o 24 h. Da a a e exp essed as mean ± SEM o h ee independen expe imen s (n= 3). As e isk ma ks (*) indica e s a is ically significan diffe ences (p< 0.05) compa ed o con ol (non- ea ed wo ms). CTL: con ol; MH: Manuka honey. Food & Func ion Pape This jou nal is © The Royal Socie y o Chemis y 2022 Food Func ., 2022, 13, 11185–11199 | 11189 Open Access A icle. Published on 07 Oc obe 2022. Downloaded on 11/7/2022 9:46:08 AM. This a icle is licensed unde a C ea i e Commons A ibu ion 3.0 Unpo ed Licence. View A icle Online MH- ea ed g oup. In con as , RNAi o hsp-16.2 and skn-1 showed a signi ican ly lowe % o non-pa alyzed wo ms, which indica es ha he e was a educ ion in he p o ec i e effec s o MH on Aβ-induced pa alysis, sugges ing ha hese genes a e likely implica ed in he p o ec i e effec s o MH (Fig. 4). 3.5 Effec s o MH on SOD-3::GFP, GST-4::GFP, SKN-1::GFP, HSP-16.2::GFP and DAF-16::GFP exp ession To deepen he comp ehension o he effec s o MH on he genes knocked down in he expe imen s desc ibed abo e, we used se e al ansgenic s ains ca ying he GFP ansc ip ional epo e s o in e es . A ma ked diminu ion (p< 0.05) in he exp ession o HSP-16.2::GFP in TJ375 wo ms ea ed wi h MH (by 34.41%) as well as in he exp ession o SOD-3::GFP in CF1553 nema odes (by 16.30%) was ound. No signi ican effec s om MH addi ion (p< 0.05) we e obse ed on he exp ession o GFP om he p omo e s o GST-4, SKN-1 and HSF-1 compa ed o con ol. Also, MH did no induce he ans- loca ion o DAF-16 om he cy oplasm o nuclei (Fig. 5A and B). Fig. 2 Effec s o Manuka honey on he eac i e oxygen species (ROS) p oduc ion. Wo ms we e p e- ea ed wi h MH a 100 mg mL −1 o 24 h and hen exposed o AAPH (2.5 mM) o 15 min. Da a a e exp essed as mean ± SEM o h ee independen expe imen s (n= 3). As e isk ma ks (*) indica e s a is ically significan diffe ences (p< 0.05) compa ed o con ol (non- ea ed wo ms). CTL: con ol; MH: Manuka honey; AAPH: 2,20-Azobis (2-me hylp opionamidine) dihyd ochlo ide. Fig. 3 Effec s o Manuka honey on Aβ-induced pa alysis. (A) Wo m pa alysis o e ime. Da a a e exp essed as mean ± SEM o h ee independen expe imen s (n= 3). Fo each ime, diffe en supe sc ip le e s among g oups indica e s a is ically significan diffe ences (p< 0.05). (B) Rep esen a i e images o Thiofla in T agg ega es a e 30 h o empe a u e shi ing. Aβagg ega es a e indica ed wi h whi e a ows (40× magnifi- ca ion; scale ba , 100 µm). CTL_802: non-pa alyzable s ain CL802 (nega i e con ol); CTL_4176: non- ea ed wo ms o he pa alyzable s ain CL4176 (posi i e con ol); MH (100 mg mL −1 )_4176: wo ms o he pa alyzable s ain CL4176 ea ed wi h 100 mg mL −1 o Manuka honey. Pape Food & Func ion 11190 |Food Func ., 2022, 13, 11185–11199 This jou nal is © The Royal Socie y o Chemis y 2022 Open Access A icle. Published on 07 Oc obe 2022. Downloaded on 11/7/2022 9:46:08 AM. This a icle is licensed unde a C ea i e Commons A ibu ion 3.0 Unpo ed Licence. View A icle Online 3.6 Effec s o MH on Tau P o eo oxici y Along wi h Aβplaques, he p esence o au p o ein agg ega es is ano he hallma k o AD. Al hough he mechanisms o au- associa ed neu odegene a ion emain unclea , agg ega ion o his p o ein is hough o be oxic o neu ons. The e o e, he effec s o MH on he ansgenic s ain BR5706 we e also e alu- a ed. BR5706 is cha ac e ized by accele a ed agg ega ion o in- soluble Tau, esul ing in se e e de elopmen al de ec s o he ne ous sys em, impai ed locomo ion, and slowed g ow h. Wo ms ea ed wi h 100 mg mL −1 MH showed a signi ican dec ease (p< 0.05) in swimming speed (by 70.76%), wa e- leng h (by 21.68%) and ac i i y (by 39.32%) compa ed o he con ol g oup (Fig. 6A and B). T ying o ind an explana ion o hese esul s, he con ibu ion o suga s o he assayed pa a- me e s was e alua ed. Fo ha , wo addi ional expe imen al g oups we e included: (i) wo ms ea ed wi h 232 µg mL −1 o a hyd oalcoholic ex ac (ME) only con aining he phenolic com- pounds o MH and (ii) wo ms ea ed wi h 100 mg mL −1 o an a i icial honey (AH). AH was p epa ed by dissol ing 22.0 g o mal ose, 4.4 g o suc ose, 98.5 g glucose and 119.1 g o uc- ose in 50 mL o de-ionized wa e . 9 As i can be app ecia ed in Fig. 6A and B, no signi ican diffe ences (p< 0.05) we e ound be ween he ME- ea ed g oup and he con ol g oup, while AH addi ion showed he same wo sening effec s as MH, leading o 39.43-, 71.21- and 70.51- old diminu ion o he swimming speed, wa eleng h, and ac i i y, espec i ely, compa ed o he con ol. To u he de e mine whe he he obse ed effec s we e di ec ly ela ed o Tau p o ein agg ega ion, he mobili y assay was pe o med in he wild ype N2 s ain (Fig. 7A and B). Also in his case, MH dec eased swimming speed, wa eleng h, and ac i i y by 54.92, 28.96 and 22.32%, espec i ely, compa ed o non- ea ed nema odes. Conside ing hese da a, i can be sugges ed ha he suga con en o MH affec s he gene al mobili y o C. elegans and no necessa ily he agg ega ion o he Tau p o ein. Howe e , u u e analyzes a e needed o co o- bo a e his hypo hesis. 4. Discussion Despi e he ac ha MH is a na u al p oduc wi h ema kable biological p ope ies bo h in i o 8–11,36–38 and in i o, 39,40 li le is known abou i s neu op o ec i e po en ial. Conside ing i s abili y o sca enge ee adicals and he implica ion o oxi- da i e s ess in he pa hogenesis o age- ela ed diseases such as AD, we decided o in es iga e he possible effec s o MH in alle ia ing Aβand Tau-induced oxici y in C. elegans models o AD. The quali a i e p o ile o MH phenolic compounds de e - mined by UPLC-TOF-MS/MS was simila o ha ound by o he au ho s. 8,9,11,41,42 Ne e heless, we we e unable o quan i y some o he mos ep esen a i e compounds epo ed o his honey, such as sy ingic acid, 11 me hyl sy inga e, 9,41,42 pino- cemb in and pinobanksin. 8,42 I is well known ha he ex ac- ion, conse a ion, and de ec ion me hods may in luence hese esul s. Rega ding he TPC and TFC, he alues ob ained (0.47 ± 0.03 mg GAE g −1 and 0.05 ± 0.01 mg CAE g −1 , espec - i ely) we e sligh ly lowe han hose epo ed by Y.-Z. Zhang e al., 43 Gośliński e al., 44 Ma shall e al., 45 and Anand e al. 46 Howe e , he TAC alues (2.65 ± 0.10 μMTEg −1 by FRAP; 1.40 ± 0.13 μMTEg −1 by TEAC and 4.58 ± 0.22 μMTEg −1 by DPPH) we e qui e simila o hose epo ed by Sadia A in e al. 10 and Ma shall e al. 45 Besides ha , 100 mg mL −1 MH did no show sho e m oxici y in he C. elegans nema odes as demons a ed by he esul s o he le hali y, pha yngeal pumping and egg-laying ( e ili y) assays. Nex , we also e alua ed he effec s o MH agains AAPH-induced oxida i e s ess, since mul iple lines o e idence indica e ha ROS o e p oduc ion and consequen ly, an oxida i e s ess escala ion, no only con ibu es o he aging p ocess 4,47 bu is also one o he main ac o s in ol ed in he ini ia ion and p og ession o neu odegene a i e diseases. 48 In he pa icula case o AD, oxida i e s ess is ela ed o Aβ agg ega ion 4,15 and he phospho yla ion and polyme iza ion o Tau, wo hallma ks o he disease. 4 He e, we demons a ed ha MH inc eased esis ance o s ess condi ions as e ealed by he DCF-DA me hod. In simila s udies, echinacoside, a phenyle hanoid glycoside om Cis anche dese icola, 47 ca no- sic acid, 3 cannabidiol, 48 gennianchun, a Chinese he bal o mula 7 and caffeic acid 4 educed ROS le els in he wild- ype C. elegans s ain. In addi ion o he ROS heo y o AD, he e a e mul iple hypo heses abou AD e iology, including he (i) au p o ein hype phospho yla ion hypo hesis, (ii) mi ochond ial unc ion hypo hesis, (iii) choline gic hypo hesis and (i ) amyloid cascade hypo hesis. 3 Al hough he opic is he subjec o deba e, he amyloid cascade hypo hesis, which sugges s ha Fig. 4 Effec s o diffe en RNAi (sod-3,hsp-16.2,skn-1,sod-2and da - 16) on he Aβ-induced pa alysis a e 34 h o induc ion. Wo ms o he pa alyzable s ain CL4176 ea ed wi h MH (100 mg mL −1 ) we e used in all g oups. Da a a e exp essed as mean ± SEM o h ee independen expe imen s (n= 3). As e isk ma ks (*) indica e s a is ically significan diffe ences (p< 0.05) compa ed o MH (wo ms non-exposed o RNAi and ea ed wi h MH). MH: Manuka honey. Food & Func ion Pape This jou nal is © The Royal Socie y o Chemis y 2022 Food Func ., 2022, 13, 11185–11199 | 11191 Open Access A icle. Published on 07 Oc obe 2022. Downloaded on 11/7/2022 9:46:08 AM. This a icle is licensed unde a C ea i e Commons A ibu ion 3.0 Unpo ed Licence. View A icle Online Aβdeposi ion igge s a se ies o e en s (mic oglial ac i a ion, in lamma o y esponse, eac i e as ocy osis) 49 ha lead o neu onal dys unc ion, is he mos widely accep ed. 3,7,49,50 Consequen ly, alle ia ing Aβaccumula ion has been con- side ed one o he mos p omising he apeu ic app oaches o AD. 48 To e alua e whe he MH was able o delay he Aβ-induced neu o oxici y, we conduc ed a pa alysis es in he ansgenic s ain CL4176, which has been widely used o s udy his p ocess because o i s abili y o exp ess human Aβpep ide in nema ode body wall muscle and de elop a p og essi e and ep oducible pa alysis pheno ype. 2,6,13,51 Ou esul s showed ha MH signi ican ly delayed he pa alysis o CL4176 wo ms caused by Aβagg ega ion in esponse o he empe a u e upshi and educed Aβdeposi s as e idenced by hio la in T s aining. I would be in e es ing, o u u e esea ch, o assess whe he MH, in addi ion o educing he agg ega ion o he Aβ pep ide, is capable o educing Aβoligome s, since i is well known ha hey a e mo e oxic han Aβmonome s and ha e been ela ed o memo y loss and se e i y o demen ia 13,52 Aβ oligome s may damage neu ons by in e e ing wi h neu on- o- neu on communica ion a synapses. 5 Many o he na u al com- pounds, such as be aine, 49 Ginkgo biloba ex ac , 7 gennian- chun, 7 Side i is sca dica ex ac s, 53 ucoidan, 54 e go hioneine, 55 ondoside A, 56 spe midine, 57 essen ial Oil o Aco us a a ino- wii Scho , 58 allophycocyanin, 50 caffeic acid, 4 s awbe y ex ac , 15 ca nosic acid, 3 scu ella ein, 59 oleu opein, 17 hyd oxy y osol, 16,59 and ed-bee be alain pigmen s, 60 also p o ec C. elegans nema odes agains Aβ-induced oxici y. Mos o he s udies e alua ing he mechanism unde lying he effec o na u al compounds agains Aβ-induced pa alysis Fig. 5 Effec s o Manuka honey on GFP- epo e ansgenic s ains. (A) Quan ifica ion o he GFP exp ession in he diffe en ansgenic ansc ip- ional epo e s ains wi h o wi hou MH ea men (100 mg mL −1 ). Da a a e exp essed as mean ± SEM o h ee independen expe imen s (n= 3). As e isk ma ks (*) indica e s a is ically significan diffe ences (p< 0.05) compa ed o con ol (non- ea ed wo ms). (B) Rep esen a i e images o each ansgenic s ain (CF1553/SOD-3::GFP s ain, CL2166/GST-4::GFP s ain, LD1/SKN-1::GFP s ain, OS3062/HSF::GFP s ain, TJ375/HSP-16.2::GFP s ain, TJ356/DAF-16::GFP s ain); (40× magnifica ion; scale ba , 100 µm). CTL: con ol; MH: Manuka honey. Pape Food & Func ion 11192 |Food Func ., 2022, 13, 11185–11199 This jou nal is © The Royal Socie y o Chemis y 2022 Open Access A icle. Published on 07 Oc obe 2022. Downloaded on 11/7/2022 9:46:08 AM. This a icle is licensed unde a C ea i e Commons A ibu ion 3.0 Unpo ed Licence. View A icle Online in C. elegans, ocus on pa hways ela ed o oxida i e s ess, hea shock esponse, insulin signaling cascade and edox homeos asis. 51 Hence, we knocked down he exp ession o SOD-2 and SOD-3, HSP-16.2, SKN-1, and DAF-16 genes by RNAi echnology and epea ed he pa alysis es in he CL4176 s ain. DAF-16 is he only homologue o he o khead box an- sc ip ion ac o s class O (FOXO) in C. elegans and he main ou pu o he insulin/insulin-like g ow h ac o 1 (IGF-1) sig- naling (IIS) pa hway. The e o e, i plays a undamen al ole in he egula ion o g ow h, aging, me abolism, de elopmen , ep oduc ion, s ess esis ance and immuni y. 7,52,61,62 DAF-16 ac i a ion inc eases he exp ession o mul iple genes in ol ed in he an ioxidan esponse, including he me allo hionein homologue (m l-1), mi ochond ial supe oxide dismu ase (sod- 3), he ca alase genes c l-1and c l-2and some small hea - shock p o ein (HSPs) genes. 7,62–64 Mo eo e , he esponse agains oxida i e s ess is also media ed by he join ac ion o o he ac o s such as SKN-1, SMK-1 (a egula o y subuni o he PP4 p o ein phospha ase complex) and HSF-1. 62 Up o he p esen , i has been epo ed ha se e al compounds ex end li espan and educe Aβ-induced oxici y in C. elegans ia DAF-16, as o example oyal jelly, 65 monascin, 66 dianxian- ning, 52 gengnianchun, 7 phospha idylcholine, 64 Zijuan Pu’e ea ex ac , 67 Hibiscus sabda iffaL. ex ac , 68 s awbe y ex ac , 15 oleu opein, 17 Mo inga oleí e a ex ac , 69 and hyd oxy- y osol. 16 Howe e , unde ou expe imen al condi ions, DAF-16 and SOD-3 RNAi ailed o delay he Aβ-induced pa alysis, indi- ca ing ha MH a enua ion o neu o oxici y is no media ed by hese pa hways. On he con a y, SKN-1 and HSP-16.2 we e equi ed o MH p o ec i e effec s. Zhu e al. 70 also demon- s a ed ha a ose essen ial oil was p o ec i e in a C. elegans model o Aβ- oxici y h ough SKN-1 ac i a ion bu independen o DAF-16. O he compounds ha ha e been shown o amelio- a e Aβ-induced oxici y h ough he SKN-1 pa hway a e Hibiscus sabda iffaL. ex ac , 68 Panax No oginseng saponins, 71 s awbe y ex ac , 15 and C a oxylum o mosum ex ac . 72 SKN-1 is a ansc ip ion ac o ha can ac in pa allel o DAF-16, ha can also inhibi he Aβ-induced oxici y by p o- mo ing he exp ession o s ess esponsi e genes. 52 This an- Fig. 6 Effec s o Manuka honey on Tau-induced al e ed locomo i e beha io al pheno ype. The BR5706 ansgenic s ain was used in all g oups. (A) Swimming speed, wa eleng h and ac i i y. Da a a e exp essed as mean ± SEM o h ee independen expe imen s (n= 3). Diffe en supe sc ip le e s o he same se o da a indica e s a is ically significan diffe ences (P< 0.05) compa ed o con ol (non- ea ed wo ms). (B) Image sequence illus a - ing how he nema odes mo e om le o igh in he expe imen al g oups. Images we e ob ained om ame-by- ame acking da a p o ided by he Wo mlab sys em/so wa e. CTL: con ol; MH: Manuka honey, ME: Manuka honey hyd oalcoholic ex ac , AH: a ificial honey. Food & Func ion Pape This jou nal is © The Royal Socie y o Chemis y 2022 Food Func ., 2022, 13, 11185–11199 | 11193 Open Access A icle. Published on 07 Oc obe 2022. Downloaded on 11/7/2022 9:46:08 AM. This a icle is licensed unde a C ea i e Commons A ibu ion 3.0 Unpo ed Licence. View A icle Online