Sideritis scardica extracts inhibit aggregation and toxicity of amyloid- β in Caenorhabditis elegans used as a model for Alzheimer’s disease

Abstract

Background
Beyond its traditional uses in the Balkan area, Sideritis scardica (known as Greek mountain tea, Lamiaceae) is currently extensively investigated for its pharmacological activity in the central nervous system. Antidepressant, psychostimulating, cognition-enhancing and neuroprotective properties have been described. In this study, we tested hydroalcoholic extracts of S. scardica for their potential to counteract amyloid-β toxicity and aggregation, which plays a crucial role in the pathogenesis of Alzheimer’s disease.

Methods
For this purpose, we have chosen the nematode Caenorhabditis elegans , which is used as a model organism for neurodegenerative diseases. The concentration of different polyphenols in extracts prepared from water, 20, 40, 50, and 70% ethanol was analysed by HPLC. Additionally, polar and unpolar fractions were prepared from the 40% ethanolic extract and phytochemically analysed.

Results
Essentially, the contents of all measured constituents increased with the lipophilicity of the extraction solvents. Treatment of transgenic C. elegans strains expressing amyloid-β with the extracts resulted in a reduced number of peptide aggregates in the head region of the worms and alleviated toxicity of amyloid-β, observable through the degree of paralysed animals. The mid-polar extracts (40 and 50% ethanol) turned out be the most active, decreasing the plaque number by 21% and delaying the amyloid-β-induced paralysis by up to 3.5 h. The more lipophilic extract fractions exhibited higher activity than the hydrophilic ones.

Discussion
Sideritis scardica extracts demonstrated pharmacological activity against characteristics of Alzheimer’s disease also in C. elegans , supporting current efforts to assess its potential for the treatment of cognitive decline. The active principle as well as the mode of action needs to be investigated in more detail.

Full text

Submitted 11 December 2017
Accepted 9 April 2018
Published 30 April 2018
Corresponding author
Felix Heiner, felixheiner@gmail.com
Academic editor
Elena González-Burgos
Additional Information and
Declarations can be found on
page 13
DOI 10.7717/peerj.4683
Copyright
2018 Heiner et al.
Distributed under
Creative Commons CC-BY 4.0
OPEN ACCESS
Sideritis scardica extracts inhibit
aggregation and toxicity of amyloid-β
in Caenorhabditis elegans used as a model
for Alzheimer’s disease
Felix Heiner1, Björn Feistel2and Michael Wink1
1Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Heidelberg, Germany
2Finzelberg GmbH & Co. KG, Andernach, Germany
ABSTRACT
Background. Beyond its traditional uses in the Balkan area, Sideritis scardica (known
as Greek mountain tea, Lamiaceae) is currently extensively investigated for its phar-
macological activity in the central nervous system. Antidepressant, psychostimulating,
cognition-enhancing and neuroprotective properties have been described. In this study,
we tested hydroalcoholic extracts of S. scardica for their potential to counteract amyloid-
βtoxicity and aggregation, which plays a crucial role in the pathogenesis of Alzheimer’s
disease.
Methods. For this purpose, we have chosen the nematode Caenorhabditis elegans, which
is used as a model organism for neurodegenerative diseases. The concentration of
different polyphenols in extracts prepared from water, 20, 40, 50, and 70% ethanol
was analysed by HPLC. Additionally, polar and unpolar fractions were prepared from
the 40% ethanolic extract and phytochemically analysed.
Results. Essentially, the contents of all measured constituents increased with the
lipophilicity of the extraction solvents. Treatment of transgenic C. elegans strains
expressing amyloid-βwith the extracts resulted in a reduced number of peptide
aggregates in the head region of the worms and alleviated toxicity of amyloid-β,
observable through the degree of paralysed animals. The mid-polar extracts (40 and
50% ethanol) turned out be the most active, decreasing the plaque number by 21% and
delaying the amyloid-β-induced paralysis by up to 3.5 h. The more lipophilic extract
fractions exhibited higher activity than the hydrophilic ones.
Discussion.Sideritis scardica extracts demonstrated pharmacological activity against
characteristics of Alzheimer’s disease also in C. elegans, supporting current efforts to
assess its potential for the treatment of cognitive decline. The active principle as well as
the mode of action needs to be investigated in more detail.
Subjects Cognitive Disorders, Drugs and Devices, Pharmacology
Keywords Sideritis scardica, Lamiaceae, Caenorhabditis elegans, Amyloid-β, Neurodegenerative
diseases, Neuroprotection, Alzheimer, Greek mountain tea
INTRODUCTION
Alzheimer’s disease (AD) is the most common type of dementia and also the most common
neurodegenerative disorder in general. As nowadays more people reach a high age than
How to cite this article Heiner et al. (2018), Sideritis scardica extracts inhibit aggregation and toxicity of amyloid-βin Caenorhabditis ele-
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in the past and a cure is still missing, AD is a rising concern for modern civilizations.
According to the World Alzheimer Report 2015, about 47 million people suffered from
dementia in 2015, and numbers may double every 20 years (Prince et al., 2015). A variety
of possible causes for AD are being discussed, of which amyloid-βpeptides (Aβ) still play
a key role in Alzheimer research and which could be targeted by drugs therapeutically or
preventively (Hardy & Selkoe, 2002). Aβis derived from the amyloid precursor protein
(APP) by cleavage through β- and γ-secretases (Selkoe, 1997). The monomers aggregate to
oligomers, to polymers and finally to senile plaques, which are abundant in the brain
of patients suffering from AD (Lansbury, 1999). The traditional formulation of the
amyloid hypothesis blamed those mature aggregates for neurodegeneration, but the
smaller oligomers were discovered to be the most neurotoxic Aβspecies (Lambert et al.,
1998;Walsh & Selkoe, 2004).
Sideritis scardica Griseb. (Lamiaceae) is a perennial shrub endemic to the Balkan
peninsula, with Bulgaria as its main habitat. Depending on the area, it is commonly
known as Greek mountain tea, Shepherd’s tea, Ironwort, Mursalski tea, Pirinski tea, or Caj
Mali. A broad range of traditional uses of S. scardica are known, including the treatment
of bronchitis, asthma, sore throat, the prevention of anemia, and the use as tonic or
poultice (Todorova & Trendafilova, 2014). Concerning the traditional use against cough
associated with common cold and gastrointestinal discomfort, a HMPC (2016) monograph
is available. The plant is rich in polyphenols, such as flavonoids, hydroxycinnamic
acid derivatives, and phenylethanoid glycosides (Evstatieva, 2002;Petreska et al., 2011).
Pharmacological activities like antimicrobial, gastroprotective and anti-inflammatory
activity are mostly accredited to this class of secondary metabolites (Tadic et al., 2007;Tadic
et al., 2012a;Tadic et al., 2012b). Recently the effects of S. scardica extracts on the central
nervous system were addressed in a number of studies. Hydroalcoholic extracts were able
to inhibit the reuptake of the monoamine neurotransmitters noradrenaline, dopamine
and serotonin in vitro (Feistel & Appel, 2013;Knörle, 2012). Furthermore, they showed
antidepressant and psychostimulating effects, as well as a modulation of AMPA-dependent
neurotransmission in rats (Dimpfel, 2013;Dimpfel, Schombert & Feistel, 2016a). In mice,
cognitive enhancement and Aβ-counteracting effects were observed (Hofrichter et al.,
2016). Also, clinical studies have already been performed. S. scardica extracts were able to
improve the mental performance of healthy subjects under stress conditions and of subjects
suffering from mild cognitive impairment (MCI), which is a precursor of AD (Behrendt et
al., 2016;Dimpfel, Schombert & Biller, 2016b). A double-blind, randomized, and placebo-
controlled clinical trial currently demonstrates a significant effect of a combination of
S. scardica and Bacopa monnieri extract (memoLoges R
) on the mental performance of
subjects suffering from MCI (Dimpfel et al., 2016c).
To further investigate the influence of hydroalcoholic S. scardica extracts on
neurodegenerative diseases and especially on Aβtoxicity and aggregation, we have chosen
Caenorhabditis elegans as a model organism (Link, 2006). In transgenic strains expressing
human Aβ(1–42), in vivo effects can be observed, that, unlike in vitro studies, also consider
bioavailability and other biological influences on a multicellular organism. In the present
study, we also tried to figure out the influence of extraction solvents on the content of
Heiner et al. (2018), PeerJ , DOI 10.7717/peerj.4683 2/16

polyphenolic compounds and pharmacological activity, if a dose-dependency exists, and
which extract fractions are potent in order to explore the active principle.
MATERIALS AND METHODS
Plant material
The drug Sideritidis scardicae herba from cultivation in Bulgaria was obtained from
Finzelberg GmbH & Co. KG, Andernach, Germany (Item 2232000; Batch 10018839).
Voucher specimens are deposited at the Department of Biology, Institute of Pharmacy and
Molecular Biotechnology, Heidelberg University, Germany (registration number P8562)
and at the Department of Pharmacognosy and Natural Products Chemistry, Faculty of
Pharmacy, University of Athens, Greece (Specimen-No. PAS101), where the plant material
was identified and specified. Five crude extracts with water, ethanol 20%, 40%, 50%,
and 70% (V/V) were prepared by exhaustive extraction with twofold moved maceration.
After filtering and uperisation (3 s at 120 ◦C) they were dried under vacuum. The 40%
ethanolic extract was additionally fractionated through liquid–liquid extraction (aqueous
and butanolic phase), reprecipitation in 70% ethanol (V/V) (supernatant and precipitate)
and solid–liquid separation with an Amberlite R
XAD7HP (Sigma-Aldrich, St. Louis, USA)
adsorber resin (aqueous and ethanolic phase). For the latter one, an aqueous solution of
the primary extract was applied onto the column and the compounds were eluted with
water and, subsequently, with increasing concentrations of ethanol. All test substances
were stored at 4 ◦C.
Phytochemical analysis
The extracts and fractions were analysed for total polyphenols with a Folin-Ciocalteu
UV method following chapter 2.8.14. of the European Pharmacopoeia (2017a) and for
specific polyphenolic compounds (flavonoids, acteoside, caffeoylquinic acids) with a HPLC
method. For this purpose, a Luna R
C18/2 column (Phenomenex, Torrance, CA, USA;
250 mm length, 4.6 mm inner diameter, 5 µm particle size) was used at a temperature of
40 ◦C in a Shimadzu LC10 HPLC system. 10 µL of about 5 mg/mL sample were injected. The
mobile phase was composed of water +0.1% H3PO4(H2O) and acetonitrile +0.1% H3PO4
(ACN) with the following gradient: From 95% H2O/5% ACN (0 min) to 50% H2O/50%
ACN in 41 min; 100% ACN from 45 to 50 min to 95% H2O/5% ACN until 52 min; 65
min in total. The compounds were detected by DAD at 330 nm and calculated through
scutellarin, chlorogenic acid, and acteoside (Phytolab, Vestenbergsgreuth, Germany) as
external standards (Fig. 1). Additionally, thin layer chromatography (TLC) was conducted
to highlight differences of the fractions. As the stationary phase, silica gel 60 F254 was
used. The plate was cleaned and activated with ethyl acetate/methanol 50:50 (V/V) and
dried at 105 ◦C for 30 min. 10 µL of preparations from 1 g S. scardica extract and 10 mL
ethanol 50% (10 min at 65 ◦C, filtered) were applied and separated within 15 cm using
dichloroethane/acetic acid/methanol/water 50:25:15:10 (V/V/V/V) as mobile phase. After
drying, anisaldehyde solution R (European Pharmacopoeia, 2017b) was sprayed on the
plate, which was dried again for 3 min at 120 ◦C.
Heiner et al. (2018), PeerJ , DOI 10.7717/peerj.4683 3/16

Figure 1 Exemplary chromatogram of 20% ethanolic S. scardica extract. Caffeoylquinic acids (cal-
culated as chlorogenic acid), acteoside, and flavonoids (calculated as scutellarin) could be quantified by
HPLC with UV detection at 330 nm.
Full-size DOI: 10.7717/peerj.4683/fig-1
C. elegans strains and culture conditions
Transgenic C. elegans strain CL2006 (genotype dvIs2 [pCL12(unc-54/human Abeta peptide
1–42 minigene) +pRF4]) constitutively expresses human Aβ(1–42) in its muscle cells. In
strain CL4176 (genotype smg-1(cc546) I; dvIs27 [myo-3p::A-Beta (1–42)::let-851 30UTR)
+rol-6(su1006)] X) the Aβexpression is temperature inducible through mutation of smg-1.
Strain CL802 (genotype smg-1(cc546) I; rol-6(su1006) II) also possesses the mutated smg-1
gene but is not able to express Aβ, representing a suitable control for CL4176. Additionally,
both strains used for paralysis assay contain a roller marker for visual discrimination of
phenotypes. All strains were obtained from the Caenorhabditis Genetics Center. The worms
were cultured on nematode growth medium (NGM) with E. coli OP50 as a food source at
20 ◦C (strain CL2006) or 16 ◦C (CL4176, CL802). To start with age-synchronized worms,
a hypochlorite treatment of gravid adults for 8 min, which isolates the eggs, was performed
before every assay (1% NaOCl, 0.5 M NaOH; Sigma-Aldrich, St. Louis, MI, USA).
Quantification of β-amyloid aggregates
Isolated eggs of strain CL2006 were incubated in S-medium containing about 109E. coli
OP50/mL for 48 h at 16 ◦C. The hatched worms were then transferred to NGM plates
containing the desired concentration of the test substances and E. coli OP50. 100 µg/mL
EGCG (Sigma-Aldrich, St. Louis, MI, USA) from green tea served as a positive control.
After 96 h of incubation at 16 ◦C the worms were fixed and Aβaggregates were stained
with 0.0125% thioflavin S (Sigma-Aldrich, St. Louis, MI, USA) in 50% ethanol as described
before (Fay et al., 1998). The Aβplaques in the head region of 20–25 worms per treatment
were counted using a Keyence BZ-9000 fluorescence microscope with a GFP filter
(excitation wavelength 480 nm, emission wavelength 510 nm).
Heiner et al. (2018), PeerJ , DOI 10.7717/peerj.4683 4/16

Paralysis assay (Aβtoxicity)
The assay was performed as described before (Dostal & Link, 2010). In brief, the treated
worms were kept at 16 ◦C for 48 h, then the temperature was upshifted to 25 ◦C to induce
the expression of Aβ. On the next day, scoring was conducted at least every 2 h for at least
12 h. The worms were counted as paralysed if they failed to respond to several touches with
a platinum wire.
Statistical analysis
All results are expressed as the mean ±S.E.M of at least three independently repeated
experiments. The median paralysis times (PT50) were obtained with a Kaplan–Meier
survival analysis. One-way ANOVA with Bonferroni post-hoc correction/independent two-
sample Student’s t-tests (equal variance) were carried out to analyze statistical differences
(as appropriate).
RESULTS
The phytochemical analysis revealed that basically the content of all tested plant compounds
increased with decreasing polarity of the extraction solvent (Table 1). Nevertheless,
compared to extraction solvents of stronger lipophilicity, the amount of total phenols in
the 40% ethanolic extract was surprisingly high which may be based on the lower drug-
extract ratio. From water to 70% ethanol the content of acteoside was enriched almost
13-fold in concentration. The more lipophilic fractions of the 40% ethanolic primary
extract (Liq-Liq BuOH, Reprecip. supernat, Resin EtOH) showed higher concentrations
of total polyphenols, acteoside and caffeoylquinic acids than the polar ones (Liq-Liq
H2O, Reprecip. precip., Resin H2O) and also compared to the primary extract (Fig. 2).
The butanolic fraction of the liquid–liquid extraction contained especially high yields
of the analyzed polyphenolic compounds. It contained concentrations of acteoside and
flavonoids that were approximately three times higher than the primary extract. Taking
the distribution of mass into consideration, acteoside especially seemed to selectively
accumulate in lipophilic solvents used for fractionation. The amount of flavanoids in the
fractions obtained from reprecipitation in 70% ethanol constituted the only case of a lower
concentration in the unpolar fraction (supernatant) compared to the polar one (precipitate)
and to the primary extract. However, the TLC consistently displays the generally higher
content of polyphenolic constituents in the lipophilic fractions (flavonoids Rf 0.3–0.5 and
tannins/hydroxycinnamic acids Rf 0.7–0.8) in comparison to those with stronger polarity
and to the original extract (Fig. 3).
The transgenic C. elegans strain CL2006 constitutively expresses human Aβ(1–42)
(Link, 1995). These peptides form aggregates, which were stained with thioflavin S for
quantification; Fig. 4 shows the visualized plaques in the head region of the worms (Fig.
4A). In a concentration of 600 µg/mL all S. scardica extracts significantly reduced the
number of Aβaggregates (Fig. 4B). The extract made of 20% ethanol clearly showed a
concentration-dependent activity (Fig. 4C), whereas the treatment with 50% ethanolic
extract seemed to lose effectiveness when the concentration was raised from 400 to
600 µg/mL. Worms that were treated with 400 µg/mL of lipophilic fractions prepared from
Heiner et al. (2018), PeerJ , DOI 10.7717/peerj.4683 5/16

Figure 2 Flowchart and phytochemical analysis of the fractions prepared from 40% ethanolic S.
scardica extract. Percentage distribution of mass is given for each pair of fractionation type in the
flowchart above. Occurring differences to 100% are loss of preparative handling.
Full-size DOI: 10.7717/peerj.4683/fig-2
Table 1 Phytochemical analysis of the S. scardica extracts prepared with different solvents. Total content of polyphenolic compounds was mea-
sured with an unspecific Folin-Ciocalteu method. Single groups of polyphenols were analysed by HPLC with UV detection.
Abbreviation H2O EtOH20 EtOH40 EtOH50 EtOH70
Extraction solvent Water 20% ethanol (V/V) 40% ethanol (V/V) 50% ethanol (V/V) 70% ethanol (V/V)
DER native 5.8:1 7.2:1 4.7:1 5.7:1 5.7:1
Polyphenols [%] 5.07 6.25 9.28 6.23 7.37
Flavonoids [%] 0.59 1.18 2.42 2.03 2.82
Acteoside [%] 0.12 0.41 1.41 0.94 1.54
Caffeoylquinic acids [%] 0.24 0.47 0.39 0.38 0.49
the 40% ethanolic extract showed similar plaque numbers to the actual primary extract
in the same concentration (Fig. 4D). In contrast, the hydrophilic fractions showed weak
or no significant activity (water phase of resin separation: 10.9 ±0.9% reduction). Taken
together, the lowest numbers of Aβplaques were seen in worms treated with 1,000 µg/mL
of the 20%, 400 and 600 µg/mL of the 40%, and 400 µg/mL of the 50% ethanolic extract
(20.3 ±1.4–22.4 ±0.4% reduction), which was slightly better than the positive control
EGCG (19.5 ±2.1%) studied previously in our laboratory (Abbas & Wink, 2010).
The temperature-inducible expression of human Aβ(1–42) makes worms of strain
CL4176 paralyse over time, which is an outcome of Aβtoxicity (Dostal & Link, 2010). The
Heiner et al. (2018), PeerJ , DOI 10.7717/peerj.4683 6/16

Figure 3 Thin layer chromatogram of polar constituents of 40% ethanolic S. scardica extract and its
fractions after spraying with anisaldehyde solution R. Allocation of the tracks: primary extract with 40%
ethanol (1); butanolic (2) and aqueous (3) phase of liquid–liquid extraction; supernatant (4) and precip-
itated fraction (5) of reprecipitation in 70% ethanol; ethanolic (6) and aqueous (7) phase of resin separa-
tion.
Full-size DOI: 10.7717/peerj.4683/fig-3
control strain CL802 does not express Aβ. The progression of this paralysis was traced
for at least 12 h (Fig. 5). The PT50, a median value describing the point in time when
exactly 50% of the worms were paralysed, was calculated to test for statistically significant
differences. Extracts and fractions were tested in two sets of experiments, which showed
slightly, but not significantly differing values of the negative control (0.5% ethanol) (Table
2). Nevertheless, all treatments were compared to the respective negative control of the
test series. All worms treated with 600 µg/mL of the different S. scardica extracts showed a
delay of the Aβ-induced paralysis similar to or better than the positive control 100 µg/mL
EGCG. The most active extract was the one prepared from 50% ethanol (more than 10%
delay), which was also acting in a concentration-dependent manner (Fig. 5B). 600 µg/mL
of the 40% ethanolic extract showed about 5% delay, but was tested in another series of
experiments, which makes a direct comparison inappropriate, especially as the percentage
delay of EGCG also differs from 6 to 3% in the two sets. Treatment with 400 µg/mL
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Figure 4 Effect of Sideritis scardica on Aβaggregation in C. elegans.(A) Fluorescence microscopic
image of the head region of a worm from strain CL2006. Arrowheads point out the β-amyloid plaques
that were stained with thioflavin S. (B, C, D) Reduction in number of the Aβaggregates. All extracts were
tested in a concentration of 600 µg/mL compared to 100 µg/mL EGCG as a positive control (B); the 20%
and 50% ethanolic extract were tested in additional concentrations to show dose-dependence (C). All frac-
tions were tested in a concentration of 400 µg/mL compared to the original 40% ethanolic extract in the
same concentration (D). Controls were treated with 0.5% ethanol. *p<0.05; ***p<0.001; concentrations
in µg/mL.
Full-size DOI: 10.7717/peerj.4683/fig-4
of the more lipophilic extract fractions attenuated the progression of the Aβ-induced
paralysis (about 6% delay each), whereas the polar fractions failed to increase the PT50
significantly (Table 2;Fig. 5C). Worms of the control strain (not expressing Aβ) that were
also treated with the extracts or fractions in the highest used concentration, did not exhibit
any paralysis.
DISCUSSION
All S. scardica extracts tested significantly reduced the number of Aβaggregates and
alleviated Aβtoxicity in transgenic C. elegans strains in a concentration of 600 µg/mL or
less. Taken together, the 40 and 50% ethanolic extracts were the most active, although
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Figure 5 Influence of S. scardica on Aβ-induced paralysis. (A, B) Paralysis curves from the first set of
experiments. Compared to the control (0.5% ethanol) CL4176 worms treated with 600 µg/mL extract pre-
pared from water, 20, 50, and 70% ethanol were paralysing slower and to a lesser extent (A). The control
strain, which is not expressing Aβ, did not show any paralysis. Amongst(continued on next page...)
Full-size DOI: 10.7717/peerj.4683/fig-5
Heiner et al. (2018), PeerJ , DOI 10.7717/peerj.4683 9/16

Figure 5 (... continued)
others, the 50% ethanolic extract showed a concentration-dependent activity (B). (C) Paralysis curves
from the second set of experiments. All worms treated with 400 µg/mL of the more lipophilic extracts
showed a delayed Aβ-induced paralysis, whereas 400 µg/mL of the polar ones failed to clearly shift the
curve to the right. Results of the fractions from liquid–liquid extraction as an example.
Table 2 Delay of Aβ-induced paralysis in C. elegans strain CL4176. PT50 are median values describing the point in time, when exactly 50% of the
worms are paralysed. Experiments were conducted in different test series with slightly differing values of the control; pvalues compared to the re-
spective control.
Series of
experiments
Treatment Concentration
[µg/mL]
PT50 ±S.E.M. [h] Significance
Set 1 Control (0.5% ethanol) 33.5 ±0.5
Set 1 EGCG 100 35.5 ±0.5 p<0.05
Set 1 H2O 600 35.5 ±0.5 p<0.05
600 35.0 ±0.6 p<0.05
800 35.5 ±0.5 p<0.05
Set 1 EtOH20
1,000 35.8 ±0.6 p<0.05
200 35.0 ±0.6
400 35.5 ±0.5 p<0.05
Set 1 EtOH50
600 37.0 ±0.0 p<0.001
Set 1 EtOH70 600 36.3 ±0.3 p<0.01
Set 2 Control (0.5% ethanol) 34.8 ±0.3
Set 2 EGCG 100 35.8 ±0.3 p<0.05
400 35.5 ±0.3
Set 2 EtOH40 600 36.5 ±0.3 p<0.01
Set 2 Liq-Liq BuOH 400 37.0 ±0.6 p<0.05
Set 2 Liq-Liq H2O 400 35.3 ±0.3
Set 2 Reprecip. supernat. 400 36.8 ±0.3 p<0.01
Set 2 Reprecip. precip. 400 35.5 ±0.3
Set 2 Resin EtOH 400 37.0 ±0.4 p<0.01
Set 2 Resin H2O 400 35.3 ±0.3
EtOH40 did not show a similar percentage delay of paralysis. But a direct comparison of the
values is difficult, as they were obtained from two different test series that were performed
about 1.5 years apart. In an in vivo system like C. elegans a lot of factors, including behavior,
can change to some extent. The worms of the second set basically started to paralyse some
hours later and showed slightly different paralysis progression (see Fig. 5). But obviously
data are consistent within the sets and none of the active substances tested in the same set
showed a significantly higher activity than EtOH40. Thus, compared to extracts prepared
from solvents of higher or lower polarity, the two mid-polar extracts showed the strongest
activity, although they did not contain the highest content of flavonoids, acteoside, and
caffeoylquinic acids as polyphenolic lead compounds. Reasons for this may be based on
pharmacodynamic synergisms of certain extract constituents, as plant extracts always
embody multicomponent mixtures. To elucidate this question, further studies must be
performed. Also, bioavailability of active compounds or even bioenhancing effects may
Heiner et al. (2018), PeerJ , DOI 10.7717/peerj.4683 10/16

play a role. Polyphenolics contained in S. scardica were reported to be bioavailable, as in a
clinical study 5% of polyphenols ingested with a cup of tea were found as metabolites in
urine samples via a HPLC-MS measurement (Petreska & Stefova, 2013). Drug absorption
is not very well investigated in C. elegans itself, but doubtless compounds have to show
similar properties as in vertebrates to be well absorbed from the intestine. According to
Zheng et al. (2013) the amount of drugs being absorbed is similar in C. elegans and mice.
Furthermore, the specifity of extraction solvents regarding the ratio of active to inactive
constituents could lead to higher activity of the mid-polar extracts.
The 20 and 50% ethanolic extracts were tested in different concentrations, showing a
dose-dependence in both assays. Only worms of strain CL2006 treated with 600 µg/mL
EtOH50 did not show a lower number of Aβplaques compared to 400 µg/mL. Beginning
toxic effects at this concentration are highly improbable, as all chosen treatments were
tested for their toxicity on C. elegans (data not shown). More likely, the extract is exhibiting
a U-shaped dose–response curve, that is more realistic in biological systems than linear
responses (Calabrese & Baldwin, 2001).
All the more lipophilic fractions of the primary 40% ethanolic extract showed a significant
reduction in number of Aβaggregates as well as a delayed Aβ-induced paralysis, with the
level of activity being similar to the primary extract or just slightly higher, which points out
that the lipophilicity of the extract constituents is perhaps not important alone, otherwise
the extract prepared from 70% ethanol would have also shown better results than the
mid-polar extracts. However, in most cases the polar fractions did not reveal significant
effects, but show trends. So, synergistic effects, maybe of polar and unpolar constituents,
are still worth being discussed and investigated. Considering the phytochemical profile of
the extract fractions, it is not completely clear if their Aβ-counteracting activities can be
attributed to their content of total polyphenols, or to a more specific class of compounds.
But as the precipitated fraction of the reprecipitation of EtOH40 in 70% ethanol that
contained more flavonoids than its lipophilic counterpart always showed lower activity,
this group of compounds may not play a central role. Contemplating the enrichment
of acteoside in the unpolar fractions, this phenylethanoid glycoside remains the most
promising compound for a potential causal correlation of content and activity.
The Aβ-counteracting activity of hydroalcoholic S. scardica extracts has already been
shown in mice (Hofrichter et al., 2016). Here the number of Aβdepositions, as well as the
level of soluble Aβ(1–42) was decreased, which is coherent with the results of the present
study. Hofrichter et al. (2016) also provided some evidence about the mode of action.
They found an intensified Aβclearance via enhancement of phagocytosis in microglia
and induction of ADAM10 expression, a crucial α-secretase, which cleaves Aβ(Esch et al.,
1990). An induction of ABC transporter could not be found. An influence of S. scardica
on secretases cannot be discussed using the results of the present study as the worms were
expressing Aβthrough a minigene, not by processing APP. Other possible mechanisms of
action against Aβtoxicity involve anti-inflammatory and antioxidant activities (Gilgun-
Sherki, Melamed & Offen, 2001;Heneka et al., 2015;Kadowaki et al., 2005;Shelat et al.,
2008). As S. scardica has already shown anti-inflammatory properties (Tadic et al., 2007),
the inhibition of neuroinflammation is a possible mechanism in vertebrates. But as the
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nematodes are lacking important structures and mediators which promote inflammation,
this is not applicable in the chosen model. Also, several antioxidant activities of Greek
mountain tea have been described in vitro (Todorova & Trendafilova, 2014), but no
antioxidant effects of the extracts or fractions, including the level of intracellular ROS
(reactive oxygen species) and defense against the pro-oxidant compound juglone, was
observed in C. elegans (data not shown). S. scardica is rich in polyphenols, which makes
a direct interaction with Aβpeptides highly probable, as this is described for many
polyphenolic compounds (Porat, Abramowitz & Gazit, 2006;Stefani & Rigacci, 2013).
Assembly of peptides can be inhibited by hydrogen or ionic bonds (hydroxyl groups of
polyphenols and amino groups of peptides), or by hydrophobic interactions. This direct
inhibition of Aβaggregation and oligomerisation is also described for EGCG, which was
used as the positive control (Abbas & Wink, 2010;Del Amo et al., 2012;Wang et al., 2010).
A reduced oligomerisation could likewise explain the alleviated Aβtoxicity.
CONCLUSIONS
In conclusion, it can be stated that hydroethanolic S. scardica extracts inhibit Aβaggregation
and toxicity in C. elegans with the mid-polar extracts being the most active. This
augments existing evidence and makes S. scardica highly interesting for the treatment
or prevention of neurodegenerative diseases like Alzheimer’s. Acteoside, a phenylethanoid
glycoside, represents a promising, potentially active substance in the extracts and fractions.
Nonetheless, further steps have to be taken to investigate the active principle of the extracts
and potential synergistic actions of its constituents. In addition, a detailed mechanism of
action cannot be stated at the moment; the hypothesized direct inhibition of Aβaggregation
needs further elucidation.
Abbreviations
AβAmyloid-β
ACN Acetonitrile
AD Alzheimer’s disease
APP amyloid precursor protein
DAD diode array detector
DER drug-extract ratio
EGCG (-)-epigallocatechin-3-gallate
EtOH40 40% ethanolic S. scardica extract
HMPC Committee on Herbal Medicinal Products
MCI Mild cognitive impairment
Rf Retardation factor
ACKNOWLEDGEMENTS
The C. elegans strains used in this study were provided by the CGC, which is funded by
NIH Office of Research Infrastructure Programs (P40 OD010440). Special thanks go to
Dr. Christopher Link, University of Colorado, for his great help concerning the strains he
and his group created.
Heiner et al. (2018), PeerJ , DOI 10.7717/peerj.4683 12/16

ADDITIONAL INFORMATION AND DECLARATIONS
Funding
The authors received no funding for this work.
Competing Interests
Björn Feistel is head of scientific affairs at Finzelberg GmbH & Co. KG. Michael Wink is
an Academic Editor for PeerJ.
Author Contributions
•Felix Heiner conceived and designed the experiments, performed the experiments,
analyzed the data, prepared figures and/or tables, approved the final draft.
•Björn Feistel conceived and designed the experiments, analyzed the data, contributed
reagents/materials/analysis tools, authored or reviewed drafts of the paper, approved the
final draft.
•Michael Wink analyzed the data, contributed reagents/materials/analysis tools, authored
or reviewed drafts of the paper, approved the final draft.
Supplemental Information
Supplemental information for this article can be found online at http://dx.doi.org/10.7717/
peerj.4683#supplemental-information.
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