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Sideritis scardica extracts inhibit aggregation and toxicity of amyloid-β in Caenorhabditis elegans used as a model for Alzheimer's disease
Note that a Preprint of this article also exists, first published December 22, 2017.
Felix Heiner1, Björn Feistel2, Michael Wink1
1Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Heidelberg, Germany
2Finzelberg GmbH & Co. KG, Andernach, Germany
Elena González-Burgos
Cognitive Disorders, Drugs and Devices, Pharmacology
Sideritis scardica, Lamiaceae, Caenorhabditis elegans, Amyloid-β, Neurodegenerative diseases, Neuroprotection, Alzheimer, Greek mountain tea
© 2018 Heiner et al.
Heiner F, Feistel B, Wink M. 2018. Sideritis scardica extracts inhibit aggregation and toxicity of amyloid-β in Caenorhabditis elegans used as a model for Alzheimer's disease. PeerJ 6:e4683 https://doi.org/10.7717/peerj.4683
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.
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.
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.
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.
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 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-β peptides (Aβ) still play a key role in Alzheimer research and which could be targeted by drugs therapeutically or preventively (Hardy & Selkoe, 2002). Aβ is 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 Aβ species (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®) 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 Aβ toxicity 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 polyphenolic compounds and pharmacological activity, if a dose-dependency exists, and which extract fractions are potent in order to explore the active principle.
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® 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® 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.
Figure 1: Exemplary chromatogram of 20% ethanolic S. scardica extract.
Caffeoylquinic acids (calculated as chlorogenic acid), acteoside, and flavonoids (calculated as scutellarin) could be quantified by HPLC with UV detection at 330 nm.
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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 3′UTR) + rol-6(su1006)] X) the Aβ expression 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 109 E. 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 Aβ aggregates were stained with 0.0125% thioflavin S (Sigma-Aldrich, St. Louis, MI, USA) in 50% ethanol as described before (Fay et al., 1998). The Aβ plaques 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).
Paralysis assay (Aβ toxicity)
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.
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).
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).
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.
Phytochemical analysis of the S. scardica extracts prepared with different solvents.
Total content of polyphenolic compounds was measured 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
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 precipitated fraction (5) of reprecipitation in 70% ethanol; ethanolic (6) and aqueous (7) phase of resin separation.
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 Aβ aggregates (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 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 Aβ plaques 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).
Figure 4: Effect of Sideritis scardica on Aβ aggregation 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 Aβ aggregates. 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 fractions 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.
The temperature-inducible expression of human Aβ (1–42) makes worms of strain CL4176 paralyse over time, which is an outcome of Aβ toxicity (Dostal & Link, 2010). The 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 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.
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 prepared 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. Amongstothers, 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.
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; p values compared to the respective control.
Series of experiments
Concentration [µg/mL]
PT50 ± S.E.M. [h]
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
Set 1 EtOH20 600 35.0 ± 0.6 p < 0.05
800 35.5 ± 0.5 p < 0.05
1,000 35.8 ± 0.6 p < 0.05
Set 1 EtOH50 200 35.0 ± 0.6
600 37.0 ± 0.0 p < 0.001
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
All S. scardica extracts tested significantly reduced the number of Aβ aggregates and alleviated Aβ toxicity 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 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 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 Aβ plaques 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 Aβ aggregates 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 Aβ depositions, 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 Aβ clearance 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 Aβ through a minigene, not by processing APP. Other possible mechanisms of action against Aβ toxicity 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 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 Aβ peptides 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 Aβ aggregation 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 Aβ toxicity.
In conclusion, it can be stated that hydroethanolic S. scardica extracts inhibit Aβ aggregation 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 Aβ aggregation needs further elucidation.
Figure 2 as Powerpoint
DOI: 10.7717/peerj.4683/supp-1
Growth control of worms treated with the substances
The worms were treated with the highest used concentrations of the extracts and fractions. A reduced body length would point to toxic effects to the worms or antimicrobial properties against the food source of the worms (E. coli). All concentrations were deemed suitable
Raw data of A beta plaques
Single values (mean plaque number of 20–25 worms) of up to 4 independently performed experiments. Results: means, SEM, statistics
Raw data of paralysis assay
First sheet: Single values (median values obtained from Kaplan-Meier analysis) of 4 independently performed experiments. Results: means, SEM, statistics
Second sheet: % of worms (test strain CL4176 expressing A beta) not paralysed for every time point. Data used for creating the graphs
Third sheet: % of worms (control strain CL802 not expressing A beta) not paralysed for every time point. Worms were treated with the highest concentration of the respective extract/ fraction. As worms were not paralysing, the delayed paralysis of CL4176 can be attributed to A beta-counteracting effects of the test substances.
Hagers monograph of Sideritis scardica
Hagers monograph of Sideritidis scardicae herba
Aβ
Acetonitrile
diode array detector
drug-extract ratio
(-)-epigallocatechin-3-gallate
EtOH40
40% ethanolic S. scardica extract
Committee on Herbal Medicinal Products
Retardation factor
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.
Björn Feistel is head of scientific affairs at Finzelberg GmbH & Co. KG. Michael Wink is an Academic Editor for PeerJ.
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.
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Kohelet Yeshiva High School was originally founded in 1997 as Stern Hebrew High School by Jewish communal leader and industrialist Harry Stern z"l. Under the direction of founding Head of School, Rabbi Moshe Simkovich, the school began operations in 2000 in the basement of the JCC Klein Branch with a charter class of 15 students. In 2003, the school acquired its own campus in the Rhawnhurst section of Northeast Philadelphia, where enrollment grew to 93 students with limited options to expand.
As it was completing its first decade, the Kohelet Foundation presented the school with the bold and
generous opportunity to relocate to the fully renovated, former Akiba Hebrew Academy four-acre campus in Merion Station, Pennsylvania. This historic structure was originally constructed in 1912 with a classroom building and gymnasium added in the 1970s. Coinciding with its move and the Kohelet Foundation's transformative gift, the school's name was changed to Kohelet Yeshiva High School on the Martha & Harry Stern Campus, beginning with the 2010-11 school year.
As the cornerstone of its extensive renovation, a soaring Beit Midrash addition was added to the campus in June 2011. This exquisite space provides the school with a fitting home for daily davening, use as a Judaic study hall, and as a center for community events.
A new and exciting era arrived as Kohelet Yeshiva High School merged with Yeshiva Lab School in August of 2016 to form Kohelet Yeshiva, a seamless K-12 highway of innovative Jewish education. Yeshiva Lab School was an initiative started by the Kohelet Foundation in the fall of 2015, whose purpose was to provide an educationally progressive Modern Orthodox elementary school option in the Lower Merion area, while advancing the field of Jewish education nationally. To do so, Yeshiva Lab School set out to design a program driven by the question "what do we know about how children learn?" rather than "what do we know about how schools have always operated?" In answering that question, Yeshiva Lab School crafted 10 Principles of Learning which drive every element of its innovative, student centered, program.
In the fall of 2017, Kohelet Yeshiva opened its final division - a Middle School to bridge the Lab School and High School. The Middle School program employs the same Principles of Learning as the Lab School and is powered by the Altitude Learning platform, positioning Kohelet Yeshiva Middle School as a nationally recognized pioneer in the area of personalized learning and competency based academic, social, and emotional growth.
In September 2019, we opened the doors to our new K-8 building, a 30,000 sq. ft. state-of-the-art learning space that has been designed based on what we know about how children learn. Kohelet Yeshiva's unique reputation for innovation, inspiration, rigor, and warmth has brought in students from across the greater Philadelphia/Delaware Valley region and well beyond. Enrollment is ever expanding, with more than 260 students currently in grades K-4 and 6-12. As of June 2019, Kohelet Yeshiva has graduated 16 classes, comprising 388 distinguished alumni who have gone on to top-ranked universities in the United States and to the finest Yeshivot and Midrashot in Israel.

redpajama_set_name: RedPajamaCommonCrawl


Business owners can find a custom sign company in just about every town or city in the United States. Custom sign companies provide business owners with a wide variety of signage products that range from simple banners, real estate signs and magnetic car signs to more complicated outdoor advertising products. Basically your local custom sign company is a jack of all trades.
So what's the difference between local custom sign companies and Signtronix?
Product Specialization: The first difference between Signtronix and other sign companies is that we focus on a very narrow range of sign products. As a sign manufacturer, we produce specific product lines for small to medium-sized businesses that include both indoor and outdoor sign products.
Marketing Driven: All of the sign products that Signtronix produces are created with one purpose in mind: Generating more customers for your business. Often you will find that when you order a sign from a local custom sign company they will ask you," what would you like on your sign?" At Signtronix are marketing professionals will guide you toward creating an image that promotes your brand and creates instant top of the mind awareness as to the products and services you offer. This generates instant additional foot traffic coming into your business location.
Quality Control: Because of our production techniques we have the ability to produce a signed product with much higher quality control. Each step of the sign creation process is overseen by a team of specialists that are experts at their craft. The finalized product that is delivered to our customers carries the UL (Underwriter Laboratories) listing and all of our outdoor sign products are designed to Dade County wind load standards to ensure that all of our products can withstand hurricane force winds. Every Signtronix sign face panel comes with a 10 year unconditional breakage warranty.
Design Customization: While the frame and components of our signs are mass-produced to the highest standards, the face panel is uniquely a custom design specifically for your business. Our sign faces start with high impact polycarbonate. The signed face is vacuum formed to create a multi-dimensional surface, providing for a higher range of visibility. Next our professional artists use airbrushing and graphic application techniques that bring your custom sign to life. We can expertly reproduce any logos, graphics and even photographs and incorporate them into your sign design. This creates the most eye-catching sign you can find on your street!
The Bottom Line: We combine the benefit of mass production which is manufacturing a higher-quality product at a lower price with the ability to deliver a custom sign designed specifically for your business. This is a process that cannot be matched by local custom sign companies. Use our Contact form to find a local Signtronix representative.
Discover the difference for yourself please browse our sign gallery or visit us on Facebook where we post a new sign that we have created everyday of the week.
Do you manufacture your face panels in house, or are they out-sourced? Sometimes the customizations require a professional plastics company to get it just right. Interested to see how you process is with that.
Hello Dennis. We do manufacture our panels in house.


This impressive coastal home has gorgeous views out across the Atlantic, a choice of beach and selection of amenities within walking distance, and provides a perfect base for an escape to the seaside!
Assemble your mates for a celebration by the beach at this eclectic retreat with startling views of the ocean. Strong statement design makes this beachside property a stylish getaway and a lovely place to laze away the holiday. You're close to endless stretches of sand battered by excellent waves that roll in off the Atlantic Ocean so for surfer groups this property is a must.
Artfully decorated in vibrant, block colours that characterise the designer's playful and clever stylistic approach, you'll immediately find yourself engulfed by that holiday feeling. Gather together all sixteen guests on the sofas around a crackling fire for evening's chatting, sharing nibbles and playing games or head out into the fresh air of the terrace charring up a BBQ.
In a sought-after holiday town like Bude, location is essential, and this property is located at the top of the sandy Crooklets Beach. As the sun rises, take the dogs to run along the shore, splashing through waves and digging in the sand or go for a refreshing morning swim in the tidal pool. Ride the surf or dive along the ocean floor at one of the Bude beaches or take the South West Coast Path 4 miles down to Widemouth. If you decide to escape the beaches for a day the steep cobbled streets of Clovelly is 14 miles and wild Bodmin Moor is 28 miles drive.
Great location close to the beach and town amenities. The size and layout of the property lends itself to being used by large groups or families, offering privacy for individual guests.
This property is personally managed by Belinda.


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Management of Pharmaceutical Household Waste – Report By OECD
National, | Aug 3, 2021
Organisation for Economic Co-operation and Development (OECD) published a document on the management of pharmaceutical household waste. The document describes global practices and regulations on medication take-back programs. Click here to read more.
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Nearly every adult in the U.S. has witnessed bullying in school. This violent behavior is equally prevalent in public and private schools, as well as in rural, urban, and suburban areas. In the past, bullying was seen as something children should contend with on their own. Victims of bullying were told to defend themselves and to get tough. However, bullying is now recognized as a preventable source of wide-ranging problems that can extend well into adulthood.
The reported rates of bullying have decreased dramatically over the past three decades. However, one in five children on average still report being bullied in one way or another. The decline in bullying at school is likely the result of improving strategies to combat it, such as increasing school security personnel and nearly ubiquitous teacher training, but parents have a role to play as well. As a parent, you can implement strategies of your own to help prevent your child from becoming a victim of bullying.
Verbal or written bullying strikes kids on an emotional level. These assaults may include name calling, threatening violence, teasing, or making unwanted sexual comments.
Humans are social by nature, so the withdrawal of social interaction hits each of us hard. This may take the form of exclusion of an individual or spreading rumors about that person.
When bullying becomes physical, evidence may be obvious. Bruises and cuts from hitting, punching, or kicking should signal an immediate intervention.
Regardless of what type of bullying your child has witnessed or experienced, the results can be devastating. Bullied kids regularly report it affecting their friendships, self-esteem, school work, and physical health. Any one of those effects can have repercussions that may last well into adulthood.
Bullying's consequences affect the victim and the aggressor, who often becomes the victim of future bullying. These effects often involve depression, anxiety, substance abuse, lower performance in school, and future incidence of sexual and physical violence. Bystanders may also experience negative effects, whether or not they encourage the behavior by laughing or taunting.
As a parent, the best thing you can do to prevent your child from being bullied is to instill in them a sense of self-confidence and of empathy. Kids who do not tolerate bullying are notoriously difficult to bully. Ending bullying will require a team effort, so each of us should be prepared to take at least the following steps for our children.
Incidents of bullying tend to decrease as children progress through school. Bullying tends to occur much less frequently in high school than middle school. When kids understand what bullying is and can recognize it, they are better equipped to do something about it. Rather than confronting bullies, kids can talk to a trusted adult, who may offer guidance and encouragement. They can also show kindness to a kid who is bullied or help them tell an adult. Instill the desire to find sensible alternatives to violence at an early age.
Bullying is often the result of a mob-like group mentality. Groups of bullies will single out and pounce on individuals perceived as being loners. Encourage your child to maintain healthy friendships with peers from an early age. Statistics show that more than half of all bullying situations stop entirely when a friend or bystander steps in on behalf of the person being bullied. Children should learn to step up for bullied kids and to expect their friends to do the same.
Whether out of embarrassment or reluctance to tattle, many kids avoid telling adults about bullying. In that case, the only thing a concerned parent can do is be aware of the telltale signs of bullying. Look for unexplained injuries, missing items, faking illness to avoid school, falling grades, or difficulty sleeping. Bullied kids may suddenly lose friends and become disinterested in social activities. Any one of these signs in isolation may not warrant much concern, but when they occur together a problem may be present.
The labels "bully" and "victim of bullying" are slowly dropping out of favor because their use can have unintended consequences. They may work well as a description of what is happening, but labels can stick in unwanted ways. They can prescribe a behavior for a child, disregarding that child's ability to change the situation. Instead of generalizing, refer to each incident of bullying as isolated, and the participants as the child or children who bullied or were bullied. This way, it is easier to move on from the incident.
In Utah, school-based programs that focus on violence prevention include anti-bullying education. These types of programs have been shown to reduce bullying incidents by as much as 25 percent. If you are concerned about bullying at your child's school, reach out to his or her teachers or principal. Educators in Utah have resources available to implement a youth-violence prevention strategy, and they should be made aware of any potential violence problem in their school. As a parent, you should expect that your concerns will be promptly addressed.
When seen as distinct from bullying that occurs at school, cyberbullying is becoming more prevalent every year. Nearly a quarter of all middle school students reported experiencing bullying online in the 2015-2016 school year, the most recent year for which statistics are available. That is nearly double the amount from only a decade prior. Kids should learn that online communication is the same as real-world communication. They should neither say nor accept anything that would not be acceptable in school or at home.
Students regularly report that the advice adults give them either fails to resolve the bullying or makes it worse. Telling children to "fight back" often has unintended consequences. Some of the other usual pieces of advice – telling the bully how it feels, ignoring the bully, etc. – have been shown to produce the most negative results. Rather, students consistently say that adults who listen to them and check on their well-being have the most positive effects. Kids should be encouraged to say, "Stop!", to use humor to deflect the behavior, or to simply walk away.
Bully-proofing a child does not necessarily mean changing anything about that child. Rather, it should be a concerted effort on the part of parents and teachers to foster a sense of well-being in all kids. Many kids are bullied because of something that makes them different from others, but it is our differences that make us unique. Often these differences are not anything a child can or should change, such as a disability or race. Instead of expecting a bullied child to correct the issue, parents should seek a multi-faceted solution involving the whole community.
Would you let your child take on 40,000 dollars in debt?