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Beneficial Effects of Sideritis scardica and Cichorium spinosum against Amyloidogenic Pathway and Tau Misprocessing in Alzheimer’s Disease Neuronal Cell Culture Models


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Background: Natural products are a significantly underutilized source of potential treatments against human disease. Alzheimer's disease (AD) is a prime example of conditions that could be amenable to such treatments as suggested by recent findings. Objective: Aiming to identify novel potentially therapeutic approaches against AD, we assessed the effects of Cichorium spinosum and Sideritis scardica extracts, both distinct components of the Mediterranean diet. Methods/results: After the detailed characterization of the extracts' composition using LC-HRMS methods, they were evaluated on two AD neuronal cell culture models, namely the AβPP overexpressing SH-SY5Y-AβPP and the hyperphosphorylated tau expressing PC12-htau. Initially their effect on cell viability of SH-SY5Y and PC12 cells was examined, and subsequently their downstream effects on AβPP and tau processing pathways were investigated in the SH-SY5Y-AβPP and PC12-htau cells. We found that the S. scardica and C. spinosum extracts have similar effects on tau, as they both significantly decrease total tau, the activation of the GSK3β, ERK1 and/or ERK2 kinases of tau, as well as tau hyperphosphorylation. Furthermore, both extracts appear to promote AβPP processing through the alpha, non-amyloidogenic pathway, albeit through partly different mechanisms. Conclusions: These findings suggest that C. spinosum and S. scardica could have a notable potential in the prevention and/or treatment of AD, and merit further investigations at the in vivo level.
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Journal of Alzheimer’s Disease xx (20xx) x–xx
DOI 10.3233/JAD-170862
IOS Press
Beneficial Effects of Sideritis scardica
and Cichorium spinosum against
Amyloidogenic Pathway and Tau
Misprocessing in Alzheimer’s Disease
Neuronal Cell Culture Models
Ioanna Chalatsaa,2, Demetrios A. Arvanitisb,2, Eleni V. Mikropoulouc, Athina Giaginia,
Zeta Papadopoulou-Daifotid,1, Nektarios Aligiannisc, Maria Halabalakic, Anthony Tsarbopoulosd,e,
Leandros A. Skaltsouniscand Despina Sanoudoua,b,
a4th Department of Internal Medicine, Clinical Genomics and Pharmacogenomics Unit, Medical School,
National and Kapodistrian University of Athens, Athens, Greece
bMolecular Biology Division, Center for Basic Research, Foundation for Biomedical Research of the Academy
of Athens, Athens, Greece
cDepartment of Pharmacognosy and Natural Product Chemistry, Faculty of Pharmacy, National and Kapodis-
trian University of Athens, Athens, Greece
dDepartment of Pharmacology, Medical School, National and Kapodistrian University of Athens, Athens, Greece15
eDepartment of Bioanalytical, GAIA Research Center, The Goulandris Natural History Museum, Kifissia, Greece16
Handling Associate Editor: Antonios Politis17
Accepted 9 May 2018
Background: Natural products are a significantly underutilized source of potential treatments against human disease.
Alzheimer’s disease (AD) is a prime example of conditions that could be amenable to such treatments as suggested by
recent findings.
Objective: Aiming to identify novel potentially therapeutic approaches against AD, we assessed the effects of Cichorium
spinosum and Sideritis scardica extracts, both distinct components of the Mediterranean diet.
Methods/Results: After the detailed characterization of the extracts’ composition using LC-HRMS methods, they were
evaluated on two AD neuronal cell culture models, namely the APP overexpressing SH-SY5Y-APP and the hyperphos-
phorylated tau expressing PC12-htau. Initially their effect on cell viability of SH-SY5Y and PC12 cells was examined, and
subsequently their downstream effects on APP and tau processing pathways were investigated in the SH-SY5Y-APP and
PC12-htau cells. We found that the S. scardica and C. spinosum extracts have similar effects on tau, as they both significantly
decrease total tau, the activation of the GSK3, ERK1 and/or ERK2 kinases of tau, as well as tau hyperphosphorylation. Fur-
thermore, both extracts appear to promote APP processing through the alpha, non-amyloidogenic pathway, albeit through
partly different mechanisms.
1This author passed away on March 17, 2016.
2These authors contributed equally to this work.
Correspondence to: Despina Sanoudou, PhD, FACMG, 4th
Department of Internal Medicine, Clinical Genomics and
Pharmacogenomics Unit, “Attikon” Hospital, Medical School,
National and Kapodistrian University of Athens, Rimini 1,
Chaidari 124-62, Greece. Tel.: +30 210 7462532; E-mail:
ISSN 1387-2877/18/$35.00 © 2018 – IOS Press and the authors. All rights reserved
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2I. Chalatsa et al. / Natural Products Against Alzheimer’s Disease
Conclusions: These findings suggest that C. spinosum and S. scardica could have a notable potential in the prevention and/or
treatment of AD, and merit further investigations at the in vivo level.
Keywords: Alzheimer’s disease, amyloid, amyloidosis, Cichorium spinosum, Mediterranean diet, mountain tea, neurode-
generative diseases, prevention, Sideritis scardica, tauopathies
Alzheimer’s disease (AD) is a progressive neu-
rodegenerative disease, the most common form of
diagnosed dementia (>44 million AD patients world-35
wide), and the 6th leading cause of death in the USA.36
By 2050, more than 100 million people are expected37
to be affected worldwide [1]. Currently, there are
no effective treatments, while the FDA approved
compounds exhibit short-term benefits and can have40
serious side effects [2, 3]. Natural products with neu-41
roprotective activities are believed to hold significant
promise in preventing or treating AD [4–8].43
It is anticipated that some of these products could44
demonstrate beneficial effects by preventing key AD
pathogenetic mechanisms namely the amyloidogenic46
and neurofibrillary tangles (NFTs) pathways [9]. The47
amyloid pathway is implicated in neuronal activity48
and differentiation, including synapse formation and49
transmission [10]. Under physiological conditions,50
the amyloid-protein precursor (APP) is processed51
by the alpha or beta proteolytic pathways. Through
the non-amyloidogenic alpha pathway, APP is53
cleaved by the alpha [11] and gamma secretases to
produce soluble APP alpha (sAPP) and APP-
C83 [12–14]. The active gamma secretases, PSEN1
and PSEN2, are the products of the respective
full-length protein cleavage and amino-/carboxy-58
terminal fragment (NTF/CTF) heterodimerization59
[15, 16], and are also encountered in the form of60
multiprotein complexes [17, 18]. Through the amy-
loidogenic beta pathway, APP is cleaved by beta62
secretase (BACE1) releasing sAPPto the extra-63
cellular space and leaving APP-C99 in the plasma
membrane. APP-C99 can be subsequently pro-
cessed by gamma secretase into amyloid-(A)of
different lengths (ranging from 37 to 46 amino acids)
and APP intracellular C-terminal domain (AICD)68
[12, 19]. Some species of Aare particularly toxic69
causing synaptic failure and neuronal death [20].70
The intracellular fibrils and NFTs formed dur-71
ing AD development consist primarily of the72
hyperphosphorylated and misfolded microtubule-
associated protein, tau. Normally located at the
axons, tau is important for neuronal differentiation
and development, maintenance of cellular morphol- 76
ogy and polarity, as well as axonal transport of 77
organelles, vesicles, or molecules [21]. Tau is sub- 78
jected to extensive post-translational modifications, 79
predominantly phosphorylation in >85 sites [22]. 80
The aberrant phosphorylation of tau in AD leads 81
to its dissociation from microtubules, microtubule 82
destabilization, loss of dendritic microtubules 83
and synapses, interruption of axonal transport, 84
plasma membrane degeneration, and eventually 85
neuronal loss [23, 24]. The hyperphosphorylated 86
tau molecules tend to self-assemble intracellu- 87
larly into filaments forming NFTs. Following the 88
death of tangle-bearing cells, tau filaments are 89
released in the extracellular space as neurotoxic 90
“ghost” tangles stimulating activation of microglial 91
cells and progressive neuronal degeneration 92
[2, 25, 26]. 93
The identification of natural products that could 94
disrupt the amyloid cascade or tau misprocessing and 95
prevent the accumulation of amyloid plaques or NFTs 96
is an area of intense research in the battle against AD. 97
Since dietary habits seem to significantly affect the 98
prevalence of cognitive impairment in a population 99
[27, 28], it has been proposed that the regular intake 100
of certain classes of compounds, such as antioxidants, 101
might act protectively against neuronal cell oxidation 102
and cognitive decline [29, 30]. Natural phenols, such 103
as those encountered in fresh fruits, green vegeta- 104
bles, red wine, and olive oil, are often regarded as 105
health-promoting options for maintaining cognitive 106
health [31–33]. In that sense, the model that combines 107
all of these nutritional elements, the Mediterranean 108
diet, should be thoroughly examined as a potent pre- 109
ventive tool in the battle against neurodegenerative 110
diseases. 111
The Mediterranean diet constitutes an exemplary 112
model diet with its benefits varying from low rates 113
of heart disease to higher survival rates [34, 35]. In 114
recent years, heightened attention has been drawn 115
to the link between the Mediterranean diet and 116
mental function in older adults [36]. In particular, 117
individuals adhering to a Mediterranean style diet 118
seem to have a reduced risk for developing AD [37] 119
and mild cognitive impairment (MCI), as well as 120
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for deterioration from MCI to AD [38]. Recently,121
a population study in rural Crete, demonstrated a
lower prevalence for dementia compared to global123
data [39]. Consequently, the study of the Cretan /124
Mediterranean diet, might serve as a rich source of125
new bioactive natural products.
Among the key components of the Mediterranean127
diet, the decoction of S. scardica leaves, commonly128
known as “mountain tea”, represents a daily habit129
and a traditional remedy. As such, the beneficial
properties of plants of the genus Sideritis have been131
extensively studied. A recent study demonstrated that132
S. scardica extracts inhibited the uptake of the neuro-
transmitters serotonin, noradrenaline, and dopamine,134
which are involved in multiple neurological disorders
[40], while other species of the Sideritis genus have136
been shown to exert antioxidant and anxiolytic-like137
properties [41], anticholinesterase activity [42, 43],
and to improve the spatial learning and memory in139
mice with A-induced amnesia [44]. Sideritis spp.140
extracts seem to reduce the amyloid plaque burden141
in transgenic mice, and significantly ameliorate their142
memory function [45], while a dietary supplement of143
S. scardica and selected B vitamins has been found144
to reduce stress-induced impairment of mental func-
tion in young adults [46]. S. scardica extract has also
been found to improve cognition and mental func-147
tion by modulating the AMPA receptor dependent
neurotransmission involved in synaptic plasticity and149
age-related cognitive decline [47].150
Another highly valued and extensively consumed
ingredient of the Mediterranean diet and especially152
the Cretan diet is the wild edible greens (ch´
of Crete, C. spinosum, an endemic Mediterranean
plant also known as “stamnagkathi” [48, 49], and it155
is thought to improve liver function [50]. Recently156
the C. intybus L. member of the Cichorium genus157
was suggested to improve amnesia and memory158
process impairment in rats [51]. Importantly, mul-159
tiple compounds present in C. spinosum extracts,
such as aesculetin, cichoric acid, chlorogenic acid,161
3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid,
4,5-dicaffeoylquinic acid, have been demonstrated to163
play a neuroprotective role [52–56]. Furthermore, the164
C.spinosum compound quercetin-3-O-glucuronide165
significantly reduces the generation of Apeptides166
and improves AD-type deficits in hippocampal for-167
mation basal synaptic transmission and long-term168
potentiation [57]. Furthermore, both S. scardica and
C. spinosum contain high levels of phenolic sub-
stances, such as phenolic acids and flavonoids, and
they have exhibited potent antioxidant activities in
in vitro models [58–61]. They therefore merit fur- 173
ther investigation for their potential in modulating 174
the molecular milieu of AD. 175
In this context, we assessed the effects of these two 176
plants’ natural extracts, distinct components of the 177
Mediterranean diet, in two established in vitro (cell 178
line) models of AD. We demonstrate that treatment 179
with “mountain tea” (S. scardica) or “stamnagkathi” 180
(C. spinosum) extracts it modulates multiple steps of 181
the APP misprocessing and tau hyperphosphoryla- 182
tion pathways, suggesting a potential preventive and 183
possibly therapeutic potential in AD. 184
Plant material extraction 186
For S. scardica, the dry plant material was 187
extracted with methanol using ultrasounds for 2 h, at 188
room temperature and the extract was concentrated 189
to dryness after filtration. For C. spinosum, the fresh 190
stems and leaves were boiled with distilled water in 191
a ratio of 1 kg plant material / 2 L of water and the 192
obtained decoction was filtered and lyophilized. 193
UHPLC-ESI(-)-HRMS analysis 194
Liquid chromatography analysis for S. scardica 195
was performed on an Accela®High-Speed LC Sys- 196
tem (Thermo Scientific) and for C. spinosum on an 197
Acquity®UPLC System (Waters). For both extracts, 198
detection was carried out on a LTQ-Orbitrap®199
XL hybrid mass spectrometer equipped with an 200
ESI source (Thermo Scientific), in negative mode. 201
Due to the different polarity of the extracts’ con- 202
stituents, two different gradient separation methods 203
were used. For S. scardica qualitative analyses, sep- 204
aration was achieved on a Fortis®C18 column 205
(100 mm ×2.1 mm, 1.7 m) using a gradient of water 206
containing 0.1% (v/v) formic acid (A) and acetonitrile 207
(B). Elution started with 95% A for 3 min and 208
decreased to 0% A in 21 min. These conditions 209
were maintained for 2 min before reverting to the 210
initial conditions for 7-min of re-equilibration. For 211
C. spinosum analysis, separation was achieved on 212
a Fortis®C18 column (150 mm ×2.1 mm, 1.7 m) 213
using the same solvent system. Elution started with 214
95% A and decreased to 5% in 23 min. These con- 215
ditions were maintained for 3 min before reverting 216
to initial conditions in 2 min, for a final 3-min 217
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re-equilibration. In both cases, the column was main-218
tained at 40C and the flow rate was set at 0.4 mL/min.219
10 L of water extracts at 200 g/mL were injected.220
MS data were acquired in negative-ion mode, in the221
full scan of 113–1000 m/z, with a resolution of 30000.222
Capillary temperature was set at 350C, whereas223
source voltage was 2.7 kV in ESI-. Tube lens and224
capillary voltage were tuned at –40 V and –10 V,225
respectively. Finally, nitrogen was used as sheath gas226
(40 arbitrary units) and auxiliary gas (10 arbitrary
units). For both extracts, a sample was prepared for228
analysis at a concentration of 0.2 mg/mL in a mixture229
of the mobile phase.
Cell culture and differentiation231
The human neuroblastoma SH-SY5Y and rat232
pheochromocytoma PC12 cell lines were used as well
established neuronal models that can differentiate
into neuron-like cells. Furthermore, SH-SY5Y-APP235
cells inducibly over-expressing APP695 (a kind236
gift of Dr S. Efthimiopoulos, Faculty of Biology,
National and Kapodistrian University of Athens,238
Greece) [62], and PC12-htau cells stably transfected239
with the human tau (htau; 3 R/0 N isoform) trans-240
gene and expressing hyperphosphorylated tau (a kind241
gift of Dr I. Sotiropoulos, Life and Health Sciences242
Research Institute (ICVS), School of Health Sci-
ences, University of Minho, Portugal)[63], were used244
as in vitro models of AD. All cells were cultured
in Dulbecco’s Modified Eagle’s Medium (DMEM)
without L-glutamine, maintained at 37C in a humid-247
ified 5% CO2environment. For SH-SY5Y cells,248
the medium was supplemented with 10% (vol/vol)249
heat-inactivated fetal bovine serum (FBS), 1% antibi-250
otic/antimycotic (10,000 units/mL of penicillin,251
10,000 g/mL of streptomycin, 25 g/mL of ampho-
tericin B) and 1% L-alanyl-L-glutamine. For SH-253
SY5Y-APP cells, the SH-SY5Y culture medium
contained an additional 100 g/mL of G418 (Gibco,255
Thermo Fisher Scientific Inc.). For PC12 cells, the
DMEM without L-glutamine was supplemented with257
5% (v/v) heat-inactivated horse serum (HS) and the258
cells were plated on collagen-treated flasks/plates259
[64]. For PC12-htau cells the culture medium con-260
tained an additional 100 g/ml of G418 (Gibco,261
Thermo Fisher Scientific Inc.). Differentiation of SH-262
SY5Y and SH-SY5Y-APP cells was achieved with
the addition of all-trans retinal (Sigma-Aldrich Co.)
to the culture media, to a final concentration of 105
M for 6 days. For the differentiation of PC12 and
PC12-htau cells, 0.75% FBS, 0.75% HS, 100 ng/ml 267
7 S nerve growth factor (NGF; Invitrogen), 1% antibi- 268
otic/antimycotic and 1% L-alanyl-L-glutamine were 269
added to the media for 7 days. 270
Natural products cell viability assays 271
Differentiated SH-SY5Y or PC12 cells were 272
exposed to a range of concentrations of either extract 273
(ranging from 0.04 g/ml to 400 g/ml for each natu- 274
ral extract) for 24 h or 72 h. The effect of the exposure 275
to each extract/concentration on cell viability was 276
evaluated with the Water Soluble Tetrazolium Salt 277
-1 assay (WST-1; Takara), a colorimetric test based 278
on the cleavage of WST-1 by mitochondrial dehy- 279
drogenase and the measurement of the absorbance 280
of the resulting formazan product at 450 nm in an 281
ELISA reader (Lucy 2; Anthos Labtec Instruments 282
GmbH). Since both extracts were diluted in DMSO, 283
matched concentrations of DMSO were used as con- 284
trol for each product concentration. All experiments 285
were performed at least three times. 286
Immunoblotting 287
Cells were lysed in lysis buffer (50 mM Tris, 288
pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% Triton) 289
supplemented with a mixture of protease inhibitors 290
(P8340; Sigma-Aldrich Co.), incubated on ice for 291
30 min and centrifuged at 13,000 rpm for 5 min. 292
For sAPPthe culture medium was collected and 293
condensed with centrifugal filter units (Amicon; 294
Millipore) at 4,000 rpm. The protein concentra- 295
tion was determined with the Bradford method 296
[65], using bovine serum albumin to generate a 297
standard curve. All samples were analyzed by SDS- 298
PAGE (Supplementary Table 1). GAPDH and actin 299
were used as loading controls in all cases, except 300
for sAPPwhere the volume of the initial cul- 301
ture media was used in sample normalization and 302
reciprocal sample volume loading. Proteins were 303
transferred to nitrocellulose membranes (Macherey- 304
Nagel GmbH & Co), which were then incubated with 305
primary antibodies (Supplementary Table 1). The 306
nitrocellulose membranes were subsequently washed 307
in 50 mM Tris-HCl, pH 7.5, 150mM NaCl, and 308
0.05% Tween 20 and incubated with a peroxidase- 309
conjugated anti-mouse (1:16,000 dilution; Sigma- 310
Aldrich Co.) or anti-rabbit (1:10,000 dilution; 311
BIO-RAD) secondary antibody. Protein signals were 312
detected using electrogenerated chemiluminescence 313
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(ECL) reagents according to the manufacturer’s pro-314
tocol (Thermo Fisher Scientific Inc.). The intensities
of the bands of interest from at least three dif-316
ferent experiments were quantified using Image J317
software (version 1.47v;
The measurements for all phosphorylated forms were
normalized to the total levels of the corresponding320
Statistical analysis322
Descriptive statistics were performed for all
experiments and data obtained are presented as324
mean ±standard deviation (SD). A pvalue 0.05325
was considered statistically significant. For the
immunoblotting assays, the integrated densities of
the bands obtained were analyzed by Student’s t-test.328
The ratios of protein isoforms to total protein were
assessed by chi-square.330
Plant extracts’ UHPLC-HRMS-ESI(-) profiles332
HRMS profiles were acquired using ESI, in
negative mode. Characterization of the detected
metabolites was achieved considering m/z val-
ues of the suggested elemental composition with336
a 5 ppm tolerance from the proposed theoretical337
mass, as well as RDB equivalent values (Fig. 1).338
A total of 40 secondary metabolites were iden-339
tified in the methanolic extract of S. scardica340
(Supplementary Table 2) and more than 30 com-341
pounds in the C. spinosum decoction (Supplementary
Table 3). The results of the analysis demonstrate
that the S. scardica extract is rich in phenylethanoic
glycosides, as well as flavonoids and their glyco-
sylated forms. The major components that were
detected under the class of phenylethanoic glyco-347
sides, were verbascoside, martynoside, echinacoside,348
lavandulofolioside, allysonoside, leucosceptoside,
forsythoside, samioside, as well as their iso-350
mers. Under the class of flavonoids, the main351
metabolites detected included scutellarein, isoscutel-
larein, hypolaetin, and apigenin, which were mainly
detected in their glycosylated forms, with or with-
out coumaroyl groups. The decoction of C. spinosum
appears to be rich in secondary metabolites belonging
to different chemical classes, including organic acids,357
such as citric, malic and cinnamic acid, flavonoid358
derivatives and sesquiterpene lactones. Caftaric, 359
cichoric and chlorogenic acid are the predominant 360
hydroxycinnamic acids in the extract, whereas in 361
the class of flavonoids, the principal compounds 362
were quercetin and luteolin glucuronides. Interest- 363
ingly, the extract appears to contain a small number 364
of sesquiterpene lactones such as the sulphonated 365
guianolide, 8-deacetylmatricarin-8-O-sulfate. 366
S. scardica and C. spinosum extracts do not 367
compromise viability of SH-SY5Y and PC12 cells 368
To evaluate the biological tolerance of neuron- 369
like cells for S. scardica and C. spinosum extracts, 370
we exposed them to a range of biologically rele- 371
vant concentrations for different time periods and 372
measured their effects on cell viability with WST- 373
1 assays [66–68]. In specific, differentiated wild type 374
SH-SY5Y and PC12 cells were exposed to con- 375
centrations ranging from 0.04 g/ml to 400 g/ml 376
for each natural extract (Figs. 2 and 3). Both S. 377
scardica and C. spinosum preserved intact the viabil- 378
ity of differentiated SH-SY5Y cells across all tested 379
concentrations and incubation times. These results 380
were confirmed by experiments in differentiated 381
PC12 cells. 382
Having determined that the specific S. scardica and 383
C. spinosum extracts do not compromise cell via- 384
bility, we proceeded to investigate their downstream 385
effects on APP processing and tau expression / phos- 386
phorylation in two AD neuronal cell culture models 387
(differentiated SH-SY5Y-APP and PC12-htau). 388
Effects on AβPP processing 389
Since APP processing is considered a central 390
pathogenetic mechanism for AD, we proceeded to 391
assess the effect of S. scardica and C. spinosum 392
on the expression of its key molecular players. Dif- 393
ferentiated SH-SY5Y-APP cells were treated with 394
the maximal non-toxic concentration, as determined 395
above, for 72 h (400 g/ml) and compared against 396
DMSO treatment. APP-C83 and sAPPwere used 397
as markers of the non-amyloidogenic alpha amyloid 398
pathway, while APP-C99 and -secretase (BACE1) 399
as markers of the amyloidogenic Apathway. The 400
levels of -secretases (PSEN1 and PSEN2), which 401
are implicated in both the alpha and beta amyloid 402
pathways, were also assessed (Fig. 4). 403
The C. spinosum extract significantly increased 404
APP-C99 by 73.3% and sAPPby 65.6%, thus 405
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Fig. 1. UHPLC-HRMS-ESI(-) profiles of: (A) S. scardica methanolic extract, and (B) C. spinosum decoction.
significantly altering the ratio of processed APP
peptides to total APP. Furthermore, it increased407
PSEN1-CTF by 69.7% and PSEN2-CTF by 166.4%,408
while it reduced BACE1 by 50.4% and PSEN1409
by 34.36%, overall affecting the ratio of processed410
PSEN1 and PSEN2 compared to their respective total411
protein levels.
The S. scardica extract significantly increased 413
cellular APP by 68.7%, PSEN1 by 146.7%, 414
PSEN1-CTF by 126.3% and PSEN2-CTF by 92.7%, 415
while decreasing BACE1 by 57.0% and the PSEN2 416
complexes by 36.1%. Overall it significantly affected 417
the ratio of processed APP peptides to total APP, 418
as well as those of PSEN1 and PSEN2. 419
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Fig. 2. Diagrammatic presentation of viability of differentiated SH-SY5Y cells at 24 and 72 h of treatment with different concentrations of:
(A) S. scardica extract, (B) C. spinosum extract (n=3, the concentration axis is log scaled).
Fig. 3. Diagrammatic presentation of viability of differentiated PC12 cells 24 and 72 h of treatment with different concentrations of: (A) S.
scardica extract, (B) C. spinosum extract (n=3, the concentration axis is log scaled).
Effects on tau phosphorylation420
Tau hyperphosphorylation is believed to be cru-
cial in AD pathogenesis by promoting the formation422
of NFTs and ultimately neural loss. Firstly, we con-423
firmed that tau is hyperphosphorylated at Thr231
and Ser199/Ser202 in PC12-htau cells compared425
to PC12 wt cells (Fig. 5). We then proceeded to
assess the effects of S. scardica and C. spinosum427
using differentiated PC12-htau cells expressing428
hyperphosphorylated human tau. The endpoints mea-
sured included tau phosphorylation (pThr231-tau and
pSer199/Ser202-tau), as well as expression and acti-
vation of the tau kinases GSK3and ERK1/2. It
should be noted that both GSK3and ERK1/2 were 433
activated in control PC12-htau cells, in agreement 434
with previous reports on AD (Fig. 6) [22, 69, 70]. 435
We found that treatment of differentiated PC12- 436
htau cells with the C. spinosum extract decreased total 437
tau (by 42%), the phosphorylation of tau (pThr231 438
by 77% and pSer199/Ser202 by 35%), ERK1 (by 439
38%), ERK2 (by 78%), pERK1 (by 55%), and 440
pERK2 (by 79%), while it increased the inactive 441
pSer9-GSK3(by 90%) compared to the DMSO 442
treatment. The S. scardica extract reduced total tau 443
(by 45%), the phosphorylation of tau (pThr231 by 444
75% and pSer199/Ser202 by 66%), ERK2 (by 35%) 445
and pERK2 (by 85%), and it increased the levels 446
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Fig. 4. Immunoblotting assessment of APP processing components after treatment of differentiated SH-SY5Y-APP cells with S. scardica
or C. spinosum extracts. A) Immunoblotting detection of cellular APP, APP-C99, APP-C83, sAPP, BACE1, PSEN1 complexes,
PSEN1, PSEN1-CTF, PSEN2 complexes, PSEN2, PSEN2-CTF, with GAPDH as internal control of protein expression. B) Diagrammatic
presentation of the percent change in expression of protein levels following treatment relative to DMSO control. Total APP protein levels
were based on the sum of the immunoblotting measurements of cellular APP, APP-C99, APP-C83, and sAPP. C) Diagrammatic
presentation of PSEN1 (top panel) and PSEN2 (bottom panel) processed forms to total protein levels. The size of the pie chart represents
the ratio of protein expression to total protein levels compared to DMSO control treatment of cells (*p< 0.05, t-test, n= 3).
Fig. 5. Immunoblotting assessment of tau hyperphosphorylation in PC12-htau cells. A) Immunoblotting detection of tau hyperphosphory-
lation at Thr231 and Ser199/Ser202 in PC12 and PC12-htau cells, with actin as internal control for protein expression. B) Diagrammatic
presentation of tau phosphorylation in PC12 and PC12-htau cells (*p< 0.05, t-test, n= 3).
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Fig. 6. Immunoblotting assessment of the tau phosphorylation pathway components after treatment of differentiated PC12-htau cells with
S. scardica or C. spinosum extracts. A) Immunoblotting detection of pThr231-tau, pSer199/Ser202-tau, total tau, pSer9-GSK3, GSK3,
pERK1/2, total ERK1/2, with actin as internal control of protein expression. B) Diagrammatic presentation of quantified protein expression
(*p< 0.05, t-test, n= 3).
of inactive pSer9-GSK3(by 150%) and ERK1 (by447
24%). Overall, C. spinosum and S. scardica have sim-
ilar effects on all investigated components of the tau449
pathway, with the exception of ERK1 (Fig. 6).
AD is characterized by the aggregation of amy-
loid plaques and the formation of NFTs. In search of
new therapeutic approaches against AD we screened
Sideritis scardica and Cichorium spinosum extracts,
as they are integral part of the Greek Mediterranean456
diet, rich sources of polyphenols (such as flavonoids457
and phenolic acids) and start to emerge as protective458
agents of memory and cognition [58, 59]. In order to459
determine the molecular effects of S. scardica and C.
spinosum we used two cell culture models of AD 461
and focused on key players of the APP and the 462
tau processing pathways, as they constitute promis- 463
ing targets against AD neurodegeneration and disease 464
progression [71–73]. 465
The first step in the evaluation of their therapeutic 466
potential requires the examination of their effects on 467
wild type cell viability. Towards this end, we used an 468
assay based on colorimetric tetrazolium salts cleav- 469
age to formazan by enzymes of metabolically active 470
cells [74]. Such assays are recommended because 471
they combine measurement of lethality, proliferation 472
and metabolism, and they are valuable for dose selec- 473
tion [75]. The assays were performed independently 474
for two types of differentiated neuron-like cells, origi- 475
nating from the well-established SH-SY5Y and PC12 476
cell lines. Both C. spinosum and S. scardica extracts 477
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10 I. Chalatsa et al. / Natural Products Against Alzheimer’s Disease
were very well tolerated at the cellular level, main-478
taining cell viability unaltered. The highest tested
biologically relevant and viability favorable doses480
(400 g/ml) were used for in depth studies at the481
APP and the tau processing pathway levels.482
Differentiated neuron-like SH-SY5Y-APP over-
expressing APP695, were used to dissect the effects484
of the C. spinosum extract on the key steps of485
the amyloidogenic and non-amyloidogenic APP486
processing pathways. Notably, we observed signif-
icantly increased levels of sAPP, the product488
of the non-amyloidogenic APP pathway. It has489
been shown that sAPPhas neurotrophic, neuro-
protective, and synaptotrophic properties, enhances491
neurite outgrowth, LTP, and memory retention and
is involved in the proliferation of neural precursor493
cells [76–82]. In addition, sAPPhas been found to494
act as a neuronal metallotransporter and metallochap-
erone, regulating metal homeostasis, an important496
process for Apeptides cleavage [77]. The increase497
in sAPPis likely mediated by the observed signif-498
icant increase in the active components, PSEN1-CTF499
and PSEN2-CTF, of the respective gamma secretases500
[83]. Meantime, the levels of PSEN1 were signif-501
icantly decreased. This could be explained by its
increased cleavage to PSEN1-CTF, a decrease in
protein expression or a combination of both. Incu-504
bation with C. spinosum also led to the reduction of
BACE1 levels, the beta secretase mediating Apro-506
duction which is reported increased in the brain of AD507
patients [84]. Consistently with our findings, cichoric
acid, a compound detected in our C. spinosum extract,509
has been shown to prevent memory impairment and
amyloidogenesis, at least in part, through the reg-
ulation of BACE1 levels [54]. Despite the reduced512
levels of BACE1, we observed increase of APP-513
C99. Although the mechanism behind this is unclear,514
it could potentially be due to accumulation of APP-515
C99 because of its reduced processing to A,asa516
consequence of the enhanced involvement of gamma
secretases in the non-amyloidogenic pathway, as518
described above. Overall, the C. spinosum extract
appears to promote APP processing preferentially520
through the alpha, non-amyloidogenic pathway.521
We proceeded to evaluate the effects of C.522
spinosum on tau hyperphosphorylation, using523
differentiated neuron-like PC12-htau cells which524
over-express hyperphosphorylated tau. Notably, a525
significant decrease of 42% was observed in total
tau levels. Tau proteins not only accumulate in the
brains of AD patients, but they are also emerging
as an informative predictor of a person’s cognitive
decline and potential response to treatment [85]. 530
Furthermore, the observed significant decrease in 531
phosphorylated pT231-tau and pSer199/Ser202-tau 532
by 77% and 35%, respectively, suggests a pro- 533
tective effect against phopsho-tau mediated AD 534
pathogenesis. 535
Since the phosphorylation of tau is primarily reg- 536
ulated by glycogen synthase kinase 3-beta (GSK3)537
and ERK1/2, we proceeded to assess both of these 538
pathways [22, 73, 86]. Treatment of the neuron-like 539
PC12-htau cells with C. spinosum led to signif- 540
icant increase of the inactive pSer9-GSK3, and 541
significant decrease of ERK1/2, as well as its 542
phosphorylated active forms pERK1 and pERK2. 543
Activation of GSK-3has been associated with the 544
formation of NFTs and the production of A[69], 545
while inhibition of GSK3through phosphoryla- 546
tion on Serine 9 (pSer9-GSK3) is neuroprotective 547
[22, 69, 70]. Activation of ERK1/2 increases both 548
tau phosphorylation and abnormal tau deposition in 549
AD, whereas inhibition of the ERK1/2 pathway pre- 550
vents tau-mediated cell death [87–90]. Consistently 551
with our observations, quercetin-3-O-glucuronide, 552
a compound detected in our C. spinosum extract, 553
has been shown to suppress the phosphorylation of 554
ERK1/2 and significantly reduced the generation of 555
Apeptides by primary neuron cultures [57, 91]. 556
Consequently, the C. spinosum extract, appears to 557
have a favorable impact on multiple steps of the tau 558
phosphorylation pathway, suggesting an overall neu- 559
roprotective effect. 560
The S. scardica extract also presented with sig- 561
nificant effects on the APP processing and tau 562
pathways. In specific, treatment of differentiated SH- 563
SY5Y-APP cells led to a significant decrease in 564
BACE1. This is in agreement with the reported 565
down-regulation of BACE1 by apigenin, a compound 566
detected in our S. scardica extract, which has been 567
shown to suppress amyloidogenesis and to amelio- 568
rate AD-associated learning and memory impairment 569
[44, 92]. BACE1 has been shown to regulate the lev- 570
els of full length APP [93]. Consistently with these 571
findings, we observed a significant increase in cellu- 572
lar APP. Furthermore, the expression of the PSEN1 573
complexes, PSEN1-CTF and PSEN2-CTF were sig- 574
nificantly increased, whereas the PSEN2 complexes’ 575
levels were decreased. These findings, in combination 576
with in vivo studies showing a significant reduc- 577
tion in buffer soluble A42 and decreased amyloid 578
plaque formation (number and size) post S. scardica 579
treatment [45], support the down-regulation of the 580
amyloidogenic pathway. 581
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I. Chalatsa et al. / Natural Products Against Alzheimer’s Disease 11
Importantly, S. scardica appears to also have a sig-582
nificant effect on tau related pathways, similar to this
of C. spinosum. In specific, treatment of PC12-htau584
cells resulted in a significant decrease of pThr231-585
tau, pSer199/Ser202-tau and pERK2, as well as the586
increase of the inactive pSer9-GSK3. These data
suggest that S. scardica could be a potential inhibitor588
of GSK3and ERK1/2 activation, and consequently589
of tau phosphorylation, all of which are directly590
implicated in AD pathogenesis. Indeed GSK3 is con-
sidered a promising therapeutic target for AD, and592
its inhibition by lithium reduced tau phosphoryla-593
tion in vivo and lowered the level of tau aggregates
[94, 95].595
Research on the potential of natural products in
preventing or treating specific diseases, including597
AD, is increasing exponentially [96]. Although there598
is substantial lack of scientific evidence or clinical
trials to support the use of natural products against600
AD, their market as nutritional supplements is boom-601
ing [96, 97]. Our findings demonstrate that the C.602
spinosum and S. scardica extracts cause significant603
molecular changes that could cumulatively reverse604
molecular processes associated with amyloidogene-605
sis and tau hyperphosphorylation. Furthermore, our
findings support and explain recent epidemiologi-
cal studies, suggesting the existence of a direct link608
between adherence to a Mediterranean style diet and
low risk for cognitive disease development [37–39].610
Recent data from the Hellenic Longitudinal Inves-611
tigation of Aging and Diet (HELIAD) suggest that
adherence to the Mediterranean diet has a neuropro-613
tective effect and is positively associated with better
cognitive performance and lower dementia rates in
Greek elders (prevalence of dementia in Greece is616
4.5%, the lowest in Europe) [98]. In conclusion,617
the Mediterranean style diet components Cichorium618
spinosum and Sideritis scardica extracts emerge as619
promising for the prevention and/or treatment of AD,620
through the inhibition of APP and tau misprocessing
pathways leading to AD.622
We are grateful to Professor S. Efthymiopoulos,624
Professor L. Stefanis, Dr. K. Vekrelis, Dr M. Xylouri,625
Dr N. Koulakiotis, Dr I. Dafnis, and M. Chatzis-626
This work has been supported by a “Large Scale628
Cooperative Project” (TreatAD, 09SYN-21-1003)
co-financed by the European Social Fund (ESF) and630
the General Secretariat for Research and Technology 631
in Greece. 632
Authors’ disclosures available online (https:// 633 634
The supplementary material is available in the 636
electronic version of this article: 637
10.3233/JAD-170862. 638
[1] Cummings J, Morstorf T, Zhong K (2014) Alzheimer’s dis- 640
ease drug-development pipeline: Few candidates, frequent 641
failures. Alzheimers Res Ther 6, 37. 642
[2] Giacobini E, Gold G (2013) Alzheimer disease therapy– 643
moving from amyloid-beta to tau. Nat Rev Neurol 9, 677- 644
686. 645
[3] Bond M, Rogers G, Peters J, Anderson R, Hoyle M, Min- 646
ers A, Moxham T, Davis S, Thokala P, Wailoo A, Jeffreys 647
M, Hyde C (2012) The effectiveness and cost-effectiveness 648
of donepezil, galantamine, rivastigmine and memantine for 649
the treatment of Alzheimer’s disease (review of Technol- 650
ogy Appraisal No. 111): A systematic review and economic 651
model. Health Technol Assess 16, 1-470. 652
[4] Essa M, Vijayan R, Castellano-Gonzalez G, Memon M, 653
Braidy N, Guillemin G (2012) Neuroprotective effect of 654
natural products against Alzheimer’s disease. Neurochem 655
Res 37, 1829-1842. 656
[5] Konrath E, Passos Cdos S, Klein LJ, Henriques A 657
(2013) Alkaloids as a source of potential anticholinesterase 658
inhibitors for the treatment of Alzheimer’s disease. J Pharm 659
Pharmacol 65, 1701-1725. 660
[6] Lakey-Beitia J, Berrocal R, Rao K, Durant A (2015) 661
Polyphenols as therapeutic molecules in Alzheimer’s dis- 662
ease through modulating amyloid pathways. Mol Neurobiol 663
51, 466-479. 664
[7] Mancuso C, Siciliano R, Barone E, Preziosi P (2012) Nat- 665
ural substances and Alzheimer’s disease: From preclinical 666
studies to evidence based medicine. Biochim Biophys Acta 667
1822, 616-624. 668
[8] Pinho B, Ferreres F, Valentao P, Andrade P (2013) Nature 669
as a source of metabolites with cholinesterase-inhibitory 670
activity: An approach to Alzheimer’s disease treatment. 671
J Pharm Pharmacol 65, 1681-1700. 672
[9] Dong S, Duan Y, Hu Y, Zhao Z (2012) Advances in 673
the pathogenesis of Alzheimer’s disease: A re-evaluation 674
of amyloid cascade hypothesis. Transl Neurodegener 675
1, 18. 676
[10] Bourdenx M, Koulakiotis N, Sanoudou D, Bezard E, Dehay 677
B, Tsarbopoulos A (2017) Protein aggregation and neu- 678
rodegeneration in prototypical neurodegenerative diseases: 679
Examples of amyloidopathies, tauopathies and synucle- 680
inopathies. Prog Neurobiol 155, 171-193. 681
[11] Haass C, Selkoe D (2007) Soluble protein oligomers in 682
neurodegeneration: Lessons from the Alzheimer’s amyloid 683
beta-peptide. Nat Rev Mol Cell Biol 8, 101-112. 684
[12] Haass C, Kaether C, Thinakaran G, Sisodia S (2012) Traf- 685
ficking and proteolytic processing of APP. Cold Spring Harb 686
Perspect Med 2, a006270. 687
Uncorrected Author Proof
12 I. Chalatsa et al. / Natural Products Against Alzheimer’s Disease
[13] WakabayashiT, De Strooper B (2008) Presenilins: Members
of the gamma-secretase quartets, but part-time soloists too.689
Physiology (Bethesda) 23, 194-204.690
[14] Zhang S, Zhang M, Cai F, Song W (2013) Biological func-691
tion of Presenilin and its role in AD pathogenesis. Transl692
Neurodegener 2, 15.
[15] Alves da Costa C, Mattson M, Ancolio K, Checler F694
(2003) The C-terminal fragment of presenilin 2 triggers695
p53-mediated staurosporine-induced apoptosis, a function696
independent of the presenilinase-derived N-terminal coun-
terpart. J Biol Chem 278, 12064-12069.698
[16] Laudon H, Mathews P, Karlstrom H, Bergman A, Farmery699
M, Nixon R, Winblad B, Gandy S, Lendahl U, Lundkvist J,700
Naslund J (2004) Co-expressed presenilin 1 NTF and CTF
form functional gamma-secretase complexes in cells devoid702
of full-length protein. J Neurochem 89, 44-53.703
[17] Garcia-Ayllon M, Campanari M, Brinkmalm G, Rabano704
A, Alom J, Saura C, Andreasen N, Blennow K, Saez-
Valero J (2013) CSF Presenilin-1 complexes are increased706
in Alzheimer’s disease. Acta Neuropathol Commun 1, 46.
[18] Hebert S, Godin C, Levesque G (2003) Oligomerization of708
human presenilin-1 fragments. FEBS Lett 550, 30-34.709
[19] Querfurth H, LaFerla F (2010) Alzheimer’s disease. N Engl710
J Med 362, 329-344.711
[20] Herrup K (2015) The case for rejecting the amyloid cascade
hypothesis. Nat Neurosci 18, 794-799.
[21] Spires-Jones T,Stoothoff W, de Calignon A, Jones P, Hyman
B (2009) Tau pathophysiology in neurodegeneration: A tan-
gled issue. Trends Neurosci 32, 150-159.
[22] Martin L, Latypova X, Wilson C, Magnaudeix A, Perrin M,717
Yardin C, Terro F (2013) Tau protein kinases: Involvement718
in Alzheimer’s disease. Ageing Res Rev 12, 289-309.719
[23] Berger Z, Roder H, Hanna A, Carlson A, Rangachari V,720
Yue M, Wszolek Z, Ashe K, Knight J, Dickson D, Andor-
fer C, Rosenberry T, Lewis J, Hutton M, Janus C (2007)722
Accumulation of pathological tau species and memory
loss in a conditional model of tauopathy. J Neurosci 27,
[24] Jaworski T, Kugler S, Van Leuven F (2010) Modeling726
of tau-mediated synaptic and neuronal degeneration in727
Alzheimer’s disease. Int J Alzheimers Dis 2010, 573138.
[25] Maccioni R, Farias G, Morales I, Navarrete L (2010) The
revitalized tau hypothesis on Alzheimer’sdisease. Arch Med730
Res 41, 226-231.
[26] Serrano-Pozo A, Frosch M, Masliah E, Hyman B (2011)
Neuropathological alterations in Alzheimer disease. Cold733
Spring Harb Perspect Med 1, a006189.734
[27] Solfrizzi V, Panza F, Capurso A (2003) The role of diet in
cognitive decline. J Neural Transm (Vienna) 110, 95-110.
[28] Solfrizzi V, Capurso C, D’Introno A, Colacicco AM, San-
tamato A, Ranieri M, Fiore P, Capurso A, Panza F (2008)738
Lifestyle-related factors in predementia and dementia syn-739
dromes. Expert Rev Neurother 8, 133-158.740
[29] Gomez-Pinilla F (2008) Brain foods: The effects of nutrients741
on brain function. Nat Rev Neurosci 9, 568-578.742
[30] Ishige K, Schuber D, Sagara Y (2001) Flavonoids protect
neuronal cells from oxidative stress by three distinct mech-
anisms. Free Rad Biol Med 30, 433-446.745
[31] Rigacci S, Stefani M (2015) Nutraceuticals and amyloid746
neurodegenerative diseases: A focus on natural phenols.747
Expert Rev Neurother 15, 41-52.748
[32] Ratnam DV, Ankola DD, Bhardwaj V, Sahana DK, Kumar
MN (2006) Role of antioxidants in prophylaxis and therapy:750
A pharmaceutical perspective. J Control Release 113, 189-751
[33] Porat Y, Abramowitz A, Gazit E (2006) Inhibition of amy- 753
loid fibril formation by polyphenols: Structural similarity 754
and aromatic interactions as a common inhibition mecha- 755
nism. Chem Biol Drug Des 67, 27-37. 756
[34] Trichopoulou A, Costacou T, Bamia C, Trichopoulos D 757
(2003) Adherence to a Mediterranean diet and survival in a 758
Greek population. N Engl J Med 348, 2599-2608. 759
[35] Estruch R, Ros E, Salas-Salvado J, Covas MI, Corella D, 760
Aros F, Gomez-Gracia E, Ruiz-Gutierrez V, Fiol M, Lapetra 761
J, Lamuela-Raventos RM, Serra-Majem L, Pinto X, Basora 762
J, Munoz MA, Sorli JV, Martinez JA, Martinez-Gonzalez 763
MA, Investigators PS (2013) Primary prevention of cardio- 764
vascular disease with a Mediterranean diet. N Engl J Med 765
368, 1279-1290. 766
[36] Feart C, Samieri C, Barberger-Gateau P (2010) Mediter- 767
ranean diet and cognitive function in older adults. Curr Opin 768
Clin Nutr Metab Care 13, 14-18. 769
[37] Scarmeas N, Stern Y, Tang MX, Mayeux R, Luchsinger JA 770
(2006) Mediterranean diet and risk for Alzheimer’s disease. 771
Ann Neurol 59, 912-921. 772
[38] Scarmeas N, Stern Y, Mayeux R, Manly JJ, Schupf N, 773
Luchsinger JA(2009) Mediterranean diet and mild cognitive 774
impairment. Arch Neurol 66, 216-225. 775
[39] Tsolaki M, Gkioka M, Verykouki E, Galoutzi N, Kavalou E, 776
Pattakou-Parasyri V (2017) Prevalenceof dementia, depres- 777
sion, and mild cognitive impairment in a rural area of the 778
island of Crete, Greece. Am J Alzheimers Dis Other Demen 779
32, 252-264. 780
[40] Knorle R (2012) Extracts of Sideritis scardica as triple 781
monoamine reuptake inhibitors. J Neural Transm (Vienna) 782
119, 1477-1482. 783
[41] Vasilopoulou C, Kontogianni V, Linardaki Z, Iatrou G, 784
Lamari F, Nerantzaki A, Gerothanassis I, Tzakos A, Mar- 785
garity M (2013) Phytochemical composition of “mountain 786
tea” from Sideritis clandestina subsp. clandestina and eval- 787
uation of its behavioral and oxidant/antioxidant effects on 788
adult mice. Eur J Nutr 52, 107-116. 789
[42] Ertas A, Ozturk M, Boga M, Topcu G (2009) Antioxidant 790
and anticholinesterase activity evaluation of ent-kaurane 791
diterpenoids from Sideritis arguta. J Nat Prod 72,792
500-502. 793
[43] Topcu G, Ertas A, Ozturk M, Dincel D, Kilic T, Halfon 794
B (2011) Ent-kaurane diterpenoids isolated from Sideritis 795
congesta. Phytochemistry Letters 4, 436-439. 796
[44] Liu R, Zhang T, Yang H, Lan X, Ying J, Du G (2011) 797
The flavonoid apigenin protects brain neurovascular cou- 798
pling against amyloid-beta(2)(5)(-)(3)(5)-induced toxicity 799
in mice. J Alzheimers Dis 24, 85-100. 800
[45] Hofrichter J, Krohn M, Schumacher T, Lange C, Feistel B, 801
WalbroelB, Pahnke J (2016) Sideritis spp. Extracts Enhance 802
Memory and Learning in Alzheimer’s beta-Amyloidosis 803
Mouse Models and Aged C57Bl/6 Mice. J Alzheimers Dis 804
53, 967-980. 805
[46] Behrendt I, Schneider I, Schuchardt JP, Bitterlich N, Hahn 806
A (2016) Effect of an herbal extract of sideritis scardica and 807
B-vitamins on cognitive performance under stress: A pilot 808
study. Int J Phytomed 8, 95-103. 809
[47] Dimpfel W, Schombert L, Feistel B (2016) Ex vivo char- 810
acterization of the action of sideritis extract using electrical 811
activity in the rat hippocampus slice preparation. Pharmacol 812
Pharm 7, 407-416. 813
[48] Petropoulos SA, Fernandes ˆ
A, Ntatsi G, Levizou E, Barros 814
L, Ferreira ICFR (2016) Nutritional profile and chemical 815
composition of Cichorium spinosum ecotypes. LWT 73, 95- 816
101. 817
Uncorrected Author Proof
I. Chalatsa et al. / Natural Products Against Alzheimer’s Disease 13
[49] Zeghichi S, Kallithraka S, Simopoulos AP (2003) Nutri-
tional composition of Molokhia (Corchorus olitorius) and819
Stamnagathi (Cichorium sponosum). World Rev Nutr Diet820
91, 1-21.821
[50] Hanlidou E, Karousou R, Kleftoyanni V, Kokkini S (2004)822
The herbal market of Thessaloniki (N Greece) and its rela-
tion to the ethnobotanical tradition. J Ethnopharmacol 91,824
[51] Mohajerani HH, N.; Salehi R (2015) Effect of hydroalco-826
holic extract of Cichorium intybus L. on passive avoidance
learning in male wistar rats. Int J Biol Pharm Allied Sci 4,828
[52] Heitman E, Ingram DK (2017) Cognitive and neuroprotec-830
tive effects of chlorogenic acid. Nutr Neurosci 20, 32-39.
[53] Kim JY, Lee HK, Hwang BY, Kim S, Yoo JK, Seong YH832
(2012) Neuroprotection of Ilex latifolia and caffeoylquinic833
acid derivatives against excitotoxic and hypoxic damage of834
cultured rat cortical neurons. Arch Pharm Res 35, 1115-
[54] Liu Q, Chen Y, Shen C, Xiao Y, Wang Y, Liu Z,
Liu X (2017) Chicoric acid supplementation prevents838
systemic inflammation-induced memory impairment and839
amyloidogenesis via inhibition of NF-kappaB. FASEB J 31,840
[55] Wang C, Pei A, Chen J, Yu H, Sun ML, Liu CF, Xu X
(2012) A natural coumarin derivativeesculetin offers neuro-
protection on cerebral ischemia/reperfusion injury in mice.
J Neurochem 121, 1007-1013.
[56] Wei M, Chen L, Liu J, Zhao J, Liu W, Feng F (2016) Protec-
tive effects of a Chotosan Fraction and its active components847
on beta-amyloid-induced neurotoxicity. Neurosci Lett 617,848
[57] Ho L, Ferruzzi MG, Janle EM, Wang J, Gong B, Chen850
TY, Lobo J, Cooper B, Wu QL, Talcott ST, Percival SS,
Simon JE, Pasinetti GM (2013) Identification of brain-852
targeted bioactive dietary quercetin-3-O-glucuronide as a
novel intervention for Alzheimer’s disease. FASEB J 27,
[58] Brieudes V, Angelis A, Vougogiannopoulou K, Pratsinis H,856
Kletsas D, Mitakou S, Halabalaki M, Skaltsounis L (2016)857
Phytochemical analysis and antioxidant potential of the
phytonutrient-rich decoction of Cichorium spinosum and
C. intybus. Planta Medica 82, 1070-1078.860
[59] Danesi F, Saha S, Kroon PA, Glibetic M, Konic-Ristic A,
D’Antuono LF, Bordoni A (2013) Bioactive-rich Sideritis
scardica tea (mountain tea) is as potent as Camellia sinensis863
tea at inducing cellular antioxidant defences and preventing864
oxidative stress. J Sci Food Agric 93, 3558-3564.
[60] Koleva II, Linssen JPH, van Beek TA, Evstatieva LN,
Kortenska V, Handjieva N (2003) Antioxidant activity
screening of extracts fromSideritis species (Labiatae) grown868
in Bulgaria. J Sci Food Agric 83, 809-819.869
[61] Petropoulos SA, Fernandes A, Barros L, Ferreira IC (2018)870
A comparison of the phenolic profile and antioxidant activ-871
ity of different Cichorium spinosum L. ecotypes. J Sci Food872
Agric 98, 183-189.
[62] Chatzistavraki M, Kyratzi E, Fotinopoulou A, Papazafiri
P, Efthimiopoulos S (2013) Downregulation of AbetaPP875
enhances both calcium content of endoplasmic reticulum876
and acidic stores and the dynamics of store operated calcium877
channel activity. J Alzheimers Dis 34, 407-415.878
[63] Sotiropoulos I, Catania C, Riedemann T, Fry J, Breen K,
Michaelidis T, Almeida O (2008) Glucocorticoids trigger880
Alzheimer disease-like pathobiochemistry in rat neuronal881
cells expressing human tau. J Neurochem 107, 385-397.882
[64] Fath T, Eidenmuller J, Brandt R (2002) Tau-mediated 883
cytotoxicity in a pseudohyperphosphorylation model of 884
Alzheimer’s disease. J Neurosci 22, 9733-9741. 885
[65] Bradford M (1976) A rapid and sensitive method for the 886
quantitation of microgram quantities of protein utilizing 887
the principle of protein-dye binding. Anal Biochem 72,888
248-254. 889
[66] Grossi C, Rigacci S, Ambrosini S, Ed Dami T, Luccarini I, 890
Traini C, Failli P, Berti A, Casamenti F, Stefani M (2013) 891
The polyphenol oleuropein aglycone protects TgCRND8 892
mice against Ass plaque pathology. PLoS One 8, e71702. 893
[67] Kostomoiri M, Fragkouli A, Sagnou M, Skaltsounis L, 894
Pelecanou M, Tsilibary E, Tzinia A (2013) Oleuropein, 895
an anti-oxidant polyphenol constituent of olive promotes 896
alpha-secretase cleavage of the amyloid precursor protein 897
(AbetaPP). Cell Mol Neurobiol 33, 147-154. 898
[68] Luccarini I, Ed Dami T, Grossi C, Rigacci S, Stefani 899
M, Casamenti F (2014) Oleuropein aglycone counteracts 900
Abeta42 toxicity in the rat brain. Neurosci Lett 558, 67-72. 901
[69] Forlenza O, Torres C, Talib L, de Paula V, Joaquim H, Diniz 902
B, Gattaz W (2011) Increased platelet GSK3B activity in 903
patients with mild cognitive impairment and Alzheimer’s 904
disease. J Psychiatr Res 45, 220-224. 905
[70] Medina M, WandosellF (2011) Deconstructing GSK-3: The 906
fine regulation of its activity. Int J Alzheimers Dis 2011,907
479249. 908
[71] Cui J, Wang X, Li X, Wang X, Zhang C, Li W, Zhang Y, Gu 909
H, Xie X, Nan F,Zhao J, Pei G (2015) Targeting the gamma- 910
/beta-secretase interaction reduces beta-amyloid generation 911
and ameliorates Alzheimer’s disease-related pathogenesis. 912
Cell Discov 1, 15021. 913
[72] Ben Halima S, Mishra S, Raja K, Willem M, Baici A, 914
Simons K, Brustle O, Koch P, Haass C, Caflisch A, 915
Rajendran L (2016) Specific inhibition of beta-secretase 916
processing of the Alzheimer disease amyloid precursor pro- 917
tein. Cell Rep 14, 2127-2141. 918
[73] Medina M, Avila J (2014) New perspectives on the role 919
of tau in Alzheimer’s disease. Implications for therapy. 920
Biochem Pharmacol 88, 540-547. 921
[74] Galanakis PA, Bazoti FN, Bergquist J, Markides K, Spy- 922
roulias GA, Tsarbopoulos A (2011) Study of the interaction 923
between the amyloid beta peptide (1-40) and antioxidant 924
compounds by nuclear magnetic resonance spectroscopy. 925
Biopolymers 96, 316-327. 926
[75] Huang R, Southall N, Cho MH, Xia M, Inglese J, Austin 927
CP (2008) Characterization of diversity in toxicity mecha- 928
nism using in vitro cytotoxicity assays in quantitative high 929
throughput screening. Chem Res Toxicol 21, 659-667. 930
[76] Caille I, Allinquant B, Dupont E, Bouillot C, Langer A, 931
Muller U, Prochiantz A (2004) Soluble form of amyloid 932
precursor protein regulates proliferation of progenitors in 933
the adult subventricular zone. Development 131, 2173-2181. 934
[77] Chasseigneaux S, Allinquant B (2012) Functions of Abeta, 935
sAPPalpha and sAPPbeta : Similarities and differences. 936
J Neurochem 120(Suppl 1), 99-108. 937
[78] Chow V, Mattson M, Wong P, Gleichmann M (2010) An 938
overview of APP processing enzymes and products. Neuro- 939
molecular Med 12, 1-12. 940
[79] Rohe M, Carlo A, Breyhan H, Sporbert A, Militz D, Schmidt 941
V, Wozny C, Harmeier A, Erdmann B, Bales K, Wolf 942
S, Kempermann G, Paul S, Schmitz D, Bayer T, Will- 943
now T, Andersen O (2008) Sortilin-related receptor with 944
A-type repeats (SORLA) affects the amyloid precursor 945
protein-dependent stimulation of ERK signaling and adult 946
neurogenesis. J Biol Chem 283, 14826-14834. 947
Uncorrected Author Proof
14 I. Chalatsa et al. / Natural Products Against Alzheimer’s Disease
[80] Thathiah A, De Strooper B (2011) The role of G protein-
coupled receptors in the pathology of Alzheimer’s disease.949
Nat Rev Neurosci 12, 73-87.950
[81] Muller U, Zheng H (2012) Physiological functions of951
APP family proteins. Cold Spring Harb Perspect Med 2,952
[82] Hunter S, Brayne C (2012) Relationships between the amy-954
loid precursor protein and its various proteolytic fragments955
and neuronal systems. Alzheimers Res Ther 4, 10.956
[83] Ahn K, Shelton C, Tian Y, Zhang X, Gilchrist M, Sisodia
S, Li Y (2010) Activation and intrinsic gamma-secretase958
activity of presenilin 1. ProcNatl Acad Sci U S A 107, 21435-959
[84] Rossner S, Sastre M, Bourne K, Lichtenthaler SF (2006)
Transcriptional and translational regulation of BACE1962
expression–implications for Alzheimer’sdisease. Prog Neu-963
robiol 79, 95-111.964
[85] Brier MR, Gordon B, Friedrichsen K, McCarthy J, Stern A,
Christensen J, Owen C, Aldea P, Su Y, Hassenstab J, Cairns966
NJ, Holtzman DM, Fagan AM, Morris JC, Benzinger TL,
Ances BM (2016) Tau and Abeta imaging, CSF measures,968
and cognition in Alzheimer’s disease. Sci Transl Med 8,969
[86] Kuret J, Chirita C, Congdon E, Kannanayakal T, Li G, Nec-971
ula M, Yin H, Zhong Q (2005) Pathwaysof tau fibrillization.
Biochim Biophys Acta 1739, 167-178.
[87] Ferrer I, Blanco R, Carmona M, Ribera R, Goutan E, Puig
B, Rey M, Cardozo A, Vi˜
nals F, Ribalta T (2001) Phospho-
rylated map kinase (ERK1, ERK2) expression is associated
with early tau deposition in neurones and glial cells, but not977
with increased nuclear DNA vulnerability and cell death,978
in Alzheimer disease, Pick’s disease, progressive supranu-979
clear palsy and corticobasal degeneration. Brain Pathol 11,980
[88] Amadoro G, Ciotti M, Costanzi M, Cestari V, Calissano982
P, Canu N (2006) NMDA receptor mediates tau-induced
neurotoxicity by calpain and ERK/MAPK activation. Proc
Natl Acad SciUSA103, 2892-2897.985
[89] Mazanetz M, Fischer P (2007) Untangling tau hyperphos-986
phorylation in drug design for neurodegenerative diseases.987
Nat Rev Drug Discov 6, 464-479.
[90] Pei J, Braak H, An W, Winblad B, Cowburn R, Iqbal K, 989
Grundke-Iqbal I (2002) Up-regulation of mitogen-activated 990
protein kinases ERK1/2 and MEK1/2 is associated with the 991
progression of neurofibrillary degeneration in Alzheimer’s 992
disease. Brain Res Mol Brain Res 109, 45-55. 993
[91] Yamazaki S, Miyoshi N, Kawabata K, Yasuda M, Shimoi K 994
(2014) Quercetin-3-O-glucuronide inhibits noradrenaline- 995
promoted invasion of MDA-MB-231 human breast cancer 996
cells by blocking beta(2)-adrenergic signaling. Arch 997
Biochem Biophys 557, 18-27. 998
[92] Zhao L, Wang JL, Liu R, Li XX, Li JF, Zhang L (2013) Neu- 999
roprotective, anti-amyloidogenic and neurotrophic effects 1000
of apigenin in an Alzheimer’s disease mouse model. 1001
Molecules 18, 9949-9965. 1002
[93] Nigam SM, Xu S, Ackermann F, Gregory JA, Lundkvist 1003
J, Lendahl U, Brodin L (2016) Endogenous APP accumu- 1004
lates in synapses after BACE1 inhibition. Neurosci Res 109,1005
9-15. 1006
[94] Mazanetz MP, Fischer PM (2007) Untangling tau hyper- 1007
phosphorylation in drug design for neurodegenerative 1008
diseases. Nat Rev Drug Discov 6, 464-479. 1009
[95] Noble W, Planel E, Zehr C, Olm V, Meyerson J, Suleman 1010
F, Gaynor K, Wang L, LaFrancois J, Feinstein B, Burns 1011
M, Krishnamurthy P, Wen Y, Bhat R, Lewis J, Dickson D, 1012
Duff K (2005) Inhibition of glycogen synthase kinase-3 by 1013
lithium correlates with reduced tauopathy and degeneration 1014
in vivo.Proc Natl Acad SciUSA102, 6990-6995. 1015
[96] Carrasco-Gallardo C, Farias G, Fuentes P, Crespo F, Mac- 1016
cioni R (2012) Can nutraceuticals prevent Alzheimer’s 1017
disease? Potential therapeutic role of a formulation con- 1018
taining shilajit and complex B vitamins. Arch Med Res 43,1019
699-704. 1020
[97] Mecocci P, Tinarelli C, Schulz R, Polidori M (2014) 1021
Nutraceuticals in cognitive impairment and Alzheimer’s 1022
disease. Front Pharmacol 5, 147. 1023
[98] Anastasiou CA, Yannakoulia M, Kosmidis MH, Dardio- 1024
tis E, Hadjigeorgiou GM, Sakka P, Arampatzi X, Bougea 1025
A, Labropoulos I, Scarmeas N (2017) Mediterranean diet 1026
and cognitive health: Initial results from the Hellenic Lon- 1027
gitudinal Investigation of Ageing and Diet. PLoS One 12,1028
e0182048. 1029
... chemical composition of the H. perforatum extract. A rapid and accurate method was developed, aiming to detect the major active compounds in the H. perforatum extract, as described elsewhere 30 . For the LC-HRMS, an Orbitrap high resolution mass analyzer was used in negative ionization mode. ...
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Due to increasing antibiotic resistance, the application of antimicrobial photodynamic therapy (aPDT) is gaining increasing popularity in dentistry. The aim of this study was to investigate the antimicrobial effects of aPDT using visible light (VIS) and water-filtered infrared-A (wIRA) in combination with a Hypericum perforatum extract on in situ oral biofilms. The chemical composition of H. perforatum extract was analyzed using ultra-high-performance liquid chromatography coupled with high resolution mass spectrometry (UPLC-HRMS). To obtain initial and mature oral biofilms in situ, intraoral devices with fixed bovine enamel slabs (BES) were carried by six healthy volunteers for two hours and three days, respectively. The ex situ exposure of biofilms to VIS + wIRA (200 mWcm−2) and H. perforatum (32 mg ml−1, non-rinsed or rinsed prior to aPDT after 2-min preincubation) lasted for five minutes. Biofilm treatment with 0.2% chlorhexidine gluconate solution (CHX) served as a positive control, while untreated biofilms served as a negative control. The colony-forming units (CFU) of the aPDT-treated biofilms were quantified, and the surviving microorganisms were identified using MALDI-TOF biochemical tests as well as 16 S rDNA-sequencing. We could show that the H. perforatum extract had significant photoactivation potential at a concentration of 32 mg ml−1. When aPDT was carried out in the presence of H. perforatum, all biofilms (100%) were completely eradicated (p = 0.0001). When H. perforatum was rinsed off prior to aPDT, more than 92% of the initial viable bacterial count and 13% of the mature oral biofilm were killed. Overall, the microbial composition in initial and mature biofilms was substantially altered after aPDT, inducing a shift in the synthesis of the microbial community. In conclusion, H. perforatum-mediated aPDT using VIS + wIRA interferes with oral biofilms, resulting in their elimination or the substantial alteration of microbial diversity and richness. The present results support the evaluation of H. perforatum-mediated aPDT for the adjunctive treatment of biofilm-associated oral diseases.
... Tau hyperphosphorylation is believed to be crucial in AD pathogenesis by promoting the formation of NFTs and ultimately neural loss. We have previously demonstrated that tau is hyperphosphorylated at Thr231 and Ser199/Ser202 in PC12htau cells compared to PC12 cells (Chalatsa et al., 2018). We therefore proceeded to assess the effects of trans-crocin 4 and trans-crocetin on this molecular mechanism using differentiated PC12-htau cells expressing hyperphosphorylated human tau. ...
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Crocus sativus L. natural compounds have been extensively used in traditional medicine for thousands of years. Recent research evidence is now emerging in support of its therapeutic potential for different pathologies including neurodegenerative diseases. Herein, the C. sativus L. natural compounds trans-crocin 4 and trans-crocetin were selected for in depth molecular characterization of their potentially protective effects against Alzheimer’s Disease (AD), utilizing two AD neuronal cell culture models (SH-SY5Y overexpressing APP and PC12 expressing hyperphosphorylated tau). Biologically relevant concentrations, ranging from 0.1 μM to 1 mM, applied for 24 h or 72 h, were well tolerated by differentiated wild type SH-SY5Y and PC12 cells. When tested on neuronally differentiated SH-SY5Y-APP both trans-crocin 4 and trans-crocetin had significant effects against amyloidogenic pathways. Trans-crocin 4 significantly decreased of β-secretase, a key enzyme of the amyloidogenic pathway, and APP-C99, while it decreased γ-secretases that generate toxic beta-amyloid peptides. Similarly, trans-crocetin treatment led to a reduction in β- and γ-secretases, as well as to accumulation of cellular AβPP. When tested on the neuronally differentiated PC12-htau cells, both compounds proved effective in suppressing the active forms of GSK3β and ERK1/2 kinases, as well as significantly reducing total tau and tau phosphorylation. Collectively, our data demonstrate a potent effect of trans-crocin 4 and trans-crocetin in suppressing key molecular pathways of AD pathogenesis, rendering them a promising tool in the prevention and potentially the treatment of AD.
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The aim of the study was to explain the effects of sesquiterpene lactones (SLs) from chicory (Cichorium intybus L.) root extracts as inhibitors of acetylcholinesterase (AChE) at the molecular level and to determine the inhibition of AChE activity by specific SLs (lactucin and lactucopicrin) and different chicory extracts. The obtained SLs-rich extracts were purified by the countercurrent partition chromatography (CPC) technique. AChE inhibitors were analyzed using two models: isothermal titration calorimetry (ITC) and docking simulation. The results of ITC analysis of the enzyme and the ligands’ complexation showed strong interactions of SLs as well as extracts from chicory with AChE. In a test of enzyme activity inhibition after introducing acetylcholine into the model system with SL, a stronger ability to inhibit the hydrolysis of the neurotransmitter was observed for lactucopicrin, which is one of the dominant SLs in chicory. The inhibition of enzyme activity was more efficient in the case of extracts, containing different enzyme ligands, exhibiting complementary patterns of binding the AChE active site. The study showed the high potential of using chicory to decrease the symptoms of Alzheimer’s disease.
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Background The Mediterranean dietary pattern has been associated with a decreased risk of many degenerative diseases and cognitive function in particular; however, relevant information from Mediterranean regions, where the prototype Mediterranean diet is typically adhered to, have been very limited. Additionally, predefined Mediterranean diet (MeDi) scores with use of a priori cut-offs have been used very rarely, limiting comparisons between different populations and thus external validity of the associations. Finally, associations between individual components of MeDi (i.e., food groups, macronutrients) and particular aspects of cognitive performance have rarely been explored. We evaluated the association of adherence to an a priori defined Mediterranean dietary pattern and its components with dementia and specific aspects of cognitive function in a representative population cohort in Greece. Methods Participants from the Hellenic Longitudinal Investigation of Ageing and Diet (HELIAD), an on-going population-based study, exploring potential associations between diet and cognitive performance in a representative sample from Greek regions, were included in this analysis. Diagnosis of dementia was made by a full clinical and neuropsychological evaluation, while cognitive performance was assessed according to five cognitive domains (memory, language, attention-speed, executive functioning, visuospatial perception) and a composite cognitive score. Adherence to MeDi was evaluated by an a priori score (range 0–55), derived from a detailed food frequency questionnaire. Results Among 1,865 individuals (mean age 73±6 years, 41% male), 90 were diagnosed with dementia and 223 with mild cognitive impairment. Each unit increase in the Mediterranean dietary score (MedDietScore) was associated with a 10% decrease in the odds for dementia. Adherence to the MeDi was also associated with better performance in memory, language, visuospatial perception and the composite cognitive score; the associations were strongest for memory. Fish consumption was negatively associated with dementia and cognitive performance positively associated with non-refined cereal consumption. Conclusions Our results suggest that adherence to the MeDi is associated with better cognitive performance and lower dementia rates in Greek elders. Thus, the MeDi in its a priori constructed prototype form may have cognitive benefits in traditional Mediterranean populations.
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The studies on the prevalence of dementia, depression, and mild cognitive impairment (MCI) in Greece are sparse and show major variations of prevalence depending on geographical areas, nutritional habits, and the way of living. The aim of this door-to-door study was to find the prevalence of dementia, depression, and MCI in a rural Greek population. Four hundred and forty-three individuals older than 61years following the application of specific criteria were diagnosed with: normal cognition, depression, MCI with and without depression, and dementia with and without depression. Four diagnostic methods were used, 2 of which included Mungas correction for age and education. After Mungas adjustment, the results were as follows-depression: 33.9%; MCI: 15.3%; MCI with depression: 8.6%; dementia: 2.0%; and dementia with depression: 7.2%. Dementia is less prevalent compared to global data and other Greek areas. Mild cognitive impairment is more prevalent than dementia. High percentages of depression may be related to low education.
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Chicoric acid (CA), a natural phenolic acid extracted from chicory and the echinacea (purple coneflower)plant (Echinacea purpurea), has been regarded as a nutraceutical that has powerful antioxidant and antiobesityactivities. We investigated the inhibitory effects of CA on systemic inflammation-induced neuroinflammation,amyloidogenesis, and cognitive impairment. C57BL/6J mice were treated with 0.05% CA in the drinking water for45 d. The mice were then treated by intraperitoneal injection of LPS (lipopolysaccharide). It was found that CAprevented LPS-induced memory impairment and neuronal loss through behavioral tests and histological exam-ination. Furthermore, amyloidogenesis in the CNS was detected. The results showed that CA prevented LPS-induced increases inb-amyloid (1-42 specific) (Ab1-42) accumulation, levels of amyloid precursor protein, andneuronalb-secretase 1 (BACE1), as well as the equilibrium cholinergic system in mouse brain. Moreover, CA down-regulated LPS-induced glial overactivation by inhibiting the MAPK and NF-kB pathway. Consequently, CA re-duced the levels of NF-kB transcriptionally regulated inflammatory mediators and cytokines such as iNOS,cyclooxygenase-2 (COX-2), IL-1b,andTNF-ain both mouse brain and BV2 microglial cells. These results demon-strated that CA alleviated memory impairment and amyloidogenesis triggered by LPS through suppressing NF-kBtranscriptional pathway, suggesting that CA might be a plausible therapeutic intervention for neuroinflammation-related diseases such as Alzheimer disease.
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A hydroethanolic extract (20% V/V) from Herba Sideritis scardica has been recognized to positively influence cognition. The present investigation aimed at the question if this extract would be able to modify intra-hippocampal communication after oral administration of 100 mg/kg daily for one week. The glutamatergic synapse between Schaffer Collaterals and pyramidal cells can be tested by electric stimulation using single pulses or theta burst stimulation. The resulting population spike is modulated by compounds acting at the central nervous system or other preparations directly or as ex vivo approach. In this case the effect of the special extract was tested in vitro the next day after repetitive in vitro administration. Conventional recording technique in the in vitro hippocampus slice revealed an increase of the population spike in the presence of single stimuli and theta burst stimuli resulting in increased long-term potentiation. This effect was tried to modulate by several glutamate receptor antagonists, among them compounds targeting at the ionic NMDA receptor (CGS19755), AMPA receptor (NBQX), Kainate receptor (UBP301) and targeting at three metabotropic glutamate receptors (mGluR I (YM298198), mGluRII ((RS)-APICA)) and mGluRIII (MSOP). Only NBQX was able to prevent the action of the Sideritis scardica extract. Since the AMPA receptor has been related to cognition in several reports in the literature, it is concluded from this result that the positive action of Sideritis scardica extract on brain function involves a modulation of AMPA receptor dependent neurotransmission.
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Nowadays, Alzheimer's disease is the most prevalent epiphenomenon of the aging population. Although soluble amyloid-β (Aβ) species (monomers, oligomers) are recognized triggers of the disease, no therapeutic approach is able to stop it. Herbal medicines are used to treat different diseases in many regions of the world. On the Balkan Peninsula, at the eastern Mediterranean Sea, and adjacent regions, Sideritis species are used as traditional medicine to prevent age-related problems in elderly. To evaluate this traditional knowledge in controlled experiments, we tested extracts of two commonly used Sideritis species, Sideritis euboea and Sideritis scardica, with regard to their effects on cognition in APP-transgenic and aged, non-transgenic C57Bl/6 mice. Additionally, histomorphological and biochemical changes associated with Aβ deposition and treatment were assessed. We found that daily oral treatment with Sideritis spp. extracts highly enhanced cognition in aged, non-transgenic as well as in APP-transgenic mice, an effect that was even more pronounced when extracts of both species were applied in combination. The treatment strongly reduced Aβ42 load in APP-transgenic mice, accompanied by increased phagocytic activity of microglia, and increased expression of the α-secretase ADAM10. Moreover, the treatment was able to fully rescue neuronal loss of APP-transgenic mice to normal levels as seen in non-transgenic controls. Having the traditional knowledge in mind, our results imply that treatment with Sideritis spp. extracts might be a potent, well-tolerated option for treating symptoms of cognitive impairment in elderly and with regard to Alzheimer's disease by affecting its most prominent hallmarks: Aβ pathology and cognitive decline.
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Alzheimer's disease (AD) is characterized by two molecular pathologies: cerebral b-Amyloidosis in the form of b-Amyloid (Ab) plaques and tauopathy in the form of neurofibrillary tangles, neuritic plaques, and neuropil threads. Until recently, only Ab could be studied in humans using positron emission tomography (PET) imaging owing to a lack of tau PET imaging agents. Clinical pathological studies have linked tau pathology closely to the onset and progression of cognitive symptoms in patients with AD. We report PET imaging of tau and Ab in a cohort of cognitively normal older adults and those with mild AD. Multivariate analyses identified unique disease-related stereotypical spatial patterns (topographies) for deposition of tau and Ab. These PET imaging tau and Ab topographies were spatially distinct but correlated with disease progression. Cerebrospinal fluid measures of tau, often used to stage preclinical AD, correlated with tau deposition in the temporal lobe. Tau deposition in the temporal lobe more closely tracked dementia status and was a better predictor of cognitive performance than Ab deposition in any region of the brain. These data support models of AD where tau pathology closely tracks changes in brain function that are responsible for the onset of early symptoms in AD.
Background: Wild greens are considered a rich source of phenolic compounds and antioxidants and an essential part of the so-called Mediterranean diet. In the present study, Cichorium spinosum L. ecotypes, cultivated or collected in situ from wild plants from the eastern Mediterranean were evaluated regarding their phenolic composition and antioxidant activity. Results: Significant differences were observed among the various studied ecotypes regarding their phenolic compounds content and profile, especially between wild and cultivated ecotypes, as well as the phenolic acids content between commercial products and cultivated plants. The antioxidant activity also varied among the various studied ecotypes and growing conditions, with commercial products having the highest antioxidant activity, whereas wild ecotypes showed lower antioxidant activity. Conclusions: In conclusion, C. spinosum leaves are a rich source of chicoric and 5-O-caffeoylquinic acid, while significant differences in total phenolic acids, flavonoids and phenolic compounds content, and antioxidant activity were observed among the studied ecotypes, as well as between the tested growing conditions. According to the results of the present study, further valorization of C. spinosum species has a great potential, since it could be used as a new alternative species in the food industry.
Chronic stress can impair cognitive functions including learning and memory. The current study investigated the reduction of (mental) stress and improvement of stress tolerance in 64 healthy men and women after six weeks intake of a dietary supplement containing an extract of Sideritis scardica and selected B-vitamins. Mental performance and visual attention were measured by Trail-Making Test (TMT) and Colour-Word-Test (CWT)before/after an acute stress stimulus (noise, CW-Interference). TMT improved upon product intake. The CWT reaction time accelerated upon product intake in situations of CW-Congruence (overall) (p=0.014), CW-conflict (overall) (p=0.024), CW-conflict (with noise) (p=0.001), CW-Congruence (without noise) (p=0.004) and CW-conflict (without noise) (p=0.017).CWT-changes upon product intake, differentiated for noise and CW-interference, showed (i) a bisection of CW-interference-related impairment of the reaction time in the presence of noise from 27 ms to 13.5 ms, (ii) a bisection of noise-related impairment of the reaction time in the presence of CW-conflict from 34 ms to 17 ms, (iii) an improvement of the impairment of the reaction time due to combined stress (noise plus CW-conflict) by 14.5 ms from 66 ms to 51.5 ms, (iv) despite of the improvement of the reaction time, no increase of the error rate. Safety blood parameters and the reporting of no adverse events argue for the product’s safety. These results may be relevant for persons solving cognitive tasks under conflict and/or noise (e.g. open-plan offices or car-driving) andsupport that the tested product alleviatesstress-induced impairment of executive functioning (working memory, cognitive flexibility, controlled behavioural inhibition).
Wild edible greens have been consumed as leafy vegetables throughout the centuries by many rural communities within the Mediterranean basin. In the present study, the nutritional profile and chemical composition of various Cichorium spinosum L. ecotypes was evaluated. For this purpose, ten ecotypes of C. spinosum collected in situ, grown in pots or purchased by retail supermarkets were examined. Nutritional value showed a great variation between the studied ecotypes for all the assessed parameters, whereas significant differences were observed between wild and cultivated ecotypes, as well as between conventionally cultivated and organic products. In terms of fatty acid composition, the conventionally grown ecotype had the highest nutritional value, as expressed by polyunsaturated fatty acids/unsaturated fatty acids (PUFA/SFA) and omega-6/omega-3 (n-6/n-3) fatty acids ratios. In conclusion, considering the differences between ecotypes, grown conditions and cultivation systems observed in this study, the selection of ecotypes with high nutritional value and their incorporation in commercial cultivation systems could allow for further exploitation of C. spinosum.
The Cretan diet, as the basis of the Mediterranean diet, has provided traditional remedies for the general well being of people through the long-established consumption of cooked wild greens and vegetables. The intake of the water decoctions of Cichorium spinosum and Cichorium intybus in the context of the daily dietary regime in Greece has been long associated with "liver detoxifying" properties. In the current study, we performed an in-depth investigation of the water decoctions traditionally prepared from C. spinosum and C. intybus through qualitative UHPLC-HRMS profiling and direct quantification of cichoric and caftaric acid as major antioxidant components of the decoction. In addition, we developed a one-step countercurrent chromatography method for the isolation of the two phenolic acids, along with a sulfoconjugate sesquiterpene lactone present only in the Cretan C. spinosum. All water decoctions were found not to be cytotoxic in human fibroblasts, whereas they all significantly reduced the intracellular reactive oxygen species, which is consistent with the major presence of strong antioxidant compounds such as cichoric acid. This work demonstrates that the intake of these decoctions in doses suggested by Greek traditional use is comparable to the ingestion of a phytomedical preparation of antioxidants. These results contribute to our current knowledge on the beneficial health effect of the Cretan diet. Georg Thieme Verlag KG Stuttgart · New York.