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
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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
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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-
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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).
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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.
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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.
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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.
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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.
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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.
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