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Withanolide A offers neuroprotection, ameliorates stress resistance and prolongs the life expectancy of Caenorhabditis elegans


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Withanolide A (steroidal lactone) forms the major constituent of the most popular herbal drug in Ayurvedic medicine, Ashwagandha. It has been used since ancient times as an alternative medicine for the treatment of a variety of age related disorders. Here we provide multiple lines of evidence indicating that Withanolide A improves healthspan, delays age-associated physiological changes and also extends lifespan of C. elegans. We also report several neuroprotective benefits of this natural product, including its anti-amyloidogenic effects, alleviation of α-synuclein aggregation and neuroprotection through modulation of neural mediators like acetylcholine. We observed that Withanolide A mediates lifespan extension and promotes stress resistance via insulin/insulin-like growth factor signaling pathway. Such findings could be helpful to develop a therapeutic medicine from this natural product for the prevention or reversal of age-related ailments and to improve the survival of patients suffering from Alzheimer's or Parkinson's disease.
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Withanolide A offers neuroprotection, ameliorates stress resistance and
prolongs the life expectancy of Caenorhabditis elegans
Bashir Akhlaq Akhoon, Swapnil Pandey, Sudeep Tiwari, Rakesh Pandey
Microbial Technology and Nematology Department, CSIR Central Institute of Medicinal and Aromatic Plants, Lucknow 226015, India
abstractarticle info
Article history:
Received 9 September 2015
Received in revised form 15 February 2016
Accepted 2 March 2016
Available online 5 March 2016
Section Editor: Holly M. Brown-Borg
Withanolide A (steroidal lactone) forms the major constituent of the most popular herbal drug in Ayurvedic
medicine, Ashwagandha. It has been used since ancient times as an alternative medicine for the treatment of a
variety of age related disorders. Here we provide multiple lines of evidence indicating that Withanolide A im-
proves healthspan, delays age-associated physiological changes and also extends the lifespan of Caenorhabditis
elegans. We also report several neuroprotective benets of this natural product, including its anti-
amyloidogenic effects, alleviation of α-synuclein aggregation and neuroprotection through modulation of neural
mediators like acetylcholine. We observed that Withanolide A mediates lifespan extension and promotes stress
resistance via insulin/insulin-like growth factor signaling pathway. Such ndings could be helpful to develop a
therapeutic medicine from this natural product for the prevention or reversal of age-related ailments and to
improve the survival of patients suffering from Alzheimer's or Parkinson's disease.
© 2016 Elsevier Inc. All rights reserved.
Withanolide A
Insulin/IGF-1 pathway
Caenorhabditis elegans
1. Introduction
Aging is a challenge to every living organism and all human beings
have to encounter it. It has been considered as the main risk factor for
other most prevalent diseases, including several neurological disorders
(Niccoli and Partridge, 2012). In fact, numerous genetic pathways
such as the insulin/IGF-1 pathway that inuence aging also provides
neuroprotection and surprisingly such pathways are evolutionarily
conserved (Kenyon, 2005, 2010; de la Monte and Wands, 2005;
Bartke, 2008).
There is now growing evidence that both healthspan and/or lifespan
can be prolonged by genetic and/or dietary interventions (Chen et al.,
2013a; Lucanic et al., 2013; Argyropoulou et al., 2013) and phytochem-
icals seem promising in this endeavor. Phytochemicals are non-
nutritive components that occur naturally in plants and possess sub-
stantial biological activities. Fruits and vegetables contain thousands of
biologically active phytochemicals that are likely to interact in a number
of ways to prevent disease and promote health (Surh, 2003;
Argyropoulou et al., 2013). Withanolide A (WA), a steroidal lactone, is
the major constituent of Indian herbal drug Ashwagandha (root of
Withania somnifera)(Baitharu et al., 2014). WA has been reported to
have potential therapeutic value for several neurological disorders,
including Alzheimer's disease-associated amyloid pathology, regenera-
tion of neuritis, recovery of damaged synapses, axonal outgrowth etc.
(Zhao et al., 2002; Kuboyama et al., 2002, 2005; Baitharu et al., 2014;
Kurapati et al., 2013). Also, Ashwagandha has been found to have a
remarkable area of applications in Ayurvedic medicine for a variety of
age related ailments, including its use as an adaptogen to help the
body cope with daily stress (Winston and Maimes, 2007). In the
Ayurvedic literature, Ashwagandha is known as Avarada that means
rejuvenation or youthfulness. While WA has been reported by several
researchers to have neuromodulatory effects, there is no scienticvali-
dation for its use as an anti-aging agent to delay aging and or increase
Caenorhabditis elegans, a free living soil nematode has contributed
enormously to our understanding of several neurological disorders
and aging (Kenyon, 2005, 2010; Markaki and Tavernarakis, 2010; Li
and Le, 2013). It is an appealing model for aging neuroscience owing
to its short life-cycle, fully sequenced genome, 6080% human gene
counterparts, and amenability to classical and reverse genetics. Such
features of C. elegans make it a powerful platform for the discovery of
novel anti-aging and neuroprotective compounds. The C. elegans
model system has been successfully exploited by numerous investiga-
tors to discover compounds that impact neurobiology of aging
(Marvanova and Nichols, 2007; Petrascheck et al., 2007; Cho et al.,
2010; Argyropoulou et al., 2013; Lucanic et al., 2013; Fu et al., 2014).
The present study used this multifaceted animal model system
C. elegans to explore neuroprotective and anti-aging health benets of
WA and to shed some insights into its underlying mechanism.
Experimental Gerontology 78 (2016) 4756
Corresponding author at: Microbial T echnology and Nematology, CSIRCentral
Institute of Medicinal and Aromatic Plants (CSIRCIMAP), Lucknow 226015, India.
E-mail address: (R. Pandey).
0531-5565/© 2016 Elsevier Inc. All rights reserved.
Contents lists available at ScienceDirect
Experimental Gerontology
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2. Experimental procedures
2.1. Nematodes and pharmacological compound
The nematode strains used in this study were wild-type Bristol N2,
TK22: mev-1 (kn1), DA1116: eat-2 (ad1116), VC345: sgk-1 (ok538),
PS3551: hsf-1 (sy441), GR1307: daf-16 (mgDf50), EU1: skn-1 (zu67),
TJ356: zIs356 (daf-16::gfp +rol-6), CL2166: dvIs19 (gst-4p::GFP),
CF1553: muIs84 (sod-3p::GFP +rol-6). All the strains were procured
from the Caenorhabditis Genetics Center at the University of Minnesota.
Withanolide A (procured from Natural Remedies, India) was
dissolved in dimethyl sulfoxide (DMSO) to prepare 1 mM stock solution.
Worms were treated with various concentrations of WA viz., 2 μM, 5 μM,
25 μM, and 50 μM to examine the effect of WA on the lifespan of
C. elegans. We used 0.1% DMSO as a vehicle control in all our experiments.
2.2. Lifespan analysis
Lifespan analysis was performed at 20 °C on triplicate NGM plates
(50 worms/plate). Late L4 worms were transferred to NGM plates spot-
ted with Escherichia coli OP50 and containing 5 μM, 25 μM and 50 μM
WA concentrations spread uniformly over the OP50. 50 μM 5-Fluoro-
2deoxyuridine (FUdR) was also added to the NGM plates to inhibit
the progeny growth. Worms were transferred to new NGM plates
every 23 days and scored for survival using touch provoked
2.3. Measurement of body size and body movement
Worms were cultured at 20 °C similar to lifespan analysis, however
FudR was not added to the NGM plates. For body size measurement,
the pictures of 10 randomly selected worms were taken on days 2, 5
and 10 using Leica Application Suite V3 software (version 3.4.0). The
thrashing rate was determined as previously described (Vilchez et al.,
2012). In Brief, ten days 2, 5 and 10 worms were transferred to a drop
of M9 buffer, allowed to adapt for 30 s, and then the number of body
bends were recorded for 30 s.
2.4. Pharyngeal pumping and chemotaxis
For pumping assay, theage-synchronized worms werebred on NGM
plates and the movement of pharynx terminal bulb in worms (n = 10)
was recorded on days 2, 5 and 10 for 20-s intervals at room
Chemotaxis assay was performed using a 9 cm agar plate containing
1.5% agar, 1 mM CaCl
, 1 mM MgSO
NaCl salt solution (at pH 6.0) was added to the attractant spot and 10 μl
of double-distilled H
(Rabinowitch et al., 2014). Sodium azide (10 μl of 1 M) was added
10 min before the assay at the gradient peak to immobilize the worms
once they reach their targeted spot. Age-synchronized 50 day 5 adult
worms were washed in M9 buffer and placed in the center of agar
plates. Worms were allowed to move over the agar surface for a one-
and-a-half hours before the chemotaxis index (CI) was calculated as
CI = (A
) / 50, where A is the number of worms at the attractant
(NaCl) location and B is the number of worms on the control (H
O) side.
2.5. Fecundity assay
The fecundity assay was performed as described (Schulz et al.,
2007). Briey, 10 worms were cultured on NGM plates with or without
WA and allowed to lay eggs for 12 h. Parent worms were removed and
the progeny was allowed to develop for 48 h before quantication of
hatched nematodes.
2.6. Aβ-induced paralysis assay
For paralysis assay, we used a CL4176 strain of C. elegans.Thistrans-
genic strain has been widely used to examine Aβtoxicity. Worms were
raised at 15 °C and after 48 h the temperature was upshifted to 25 °C for
induction of transgenic expression of Aβ. Paralyzed worms were scored
using touch provoked movement. The experiment was repeated twice
with each group containing 3040 worms.
2.7. Relative quantication of acetylcholine and acetylcholinesterase levels
Age-synchronized worms were added to treatment plates and incu-
bated at 22 °C for 48 h. The worms were then washed thrice using M9
buffer to clear off any adhering bacteria and sonicated in 1 × reaction buff-
er for 3 min. Worm suspension was then centrifuged at 7000 g for 7 min
and subsequently, 100 μl of supernatant was assayed for acetylcholine
(ACh) and acetylcholinesterase (AChE) levels using the Amplex Red
ACh/AChE Assay Kit (Cat No. A12217; Invitrogen) according to the
manufacturer's protocol. Fluorescence measurements were performed
using 96 well plate uorimeter (BMG Polarstar Galaxy) at excitation
544 nm and emission at 590 nm and the relative uorescence was nor-
malized with respect to protein content.
2.8. Stress resistance assays
Resistance to thermal stress was determined with minor modica-
tions as previously described (Vilchez et al., 2012). As described for
lifespan analysis, worms were cultured at 20 °C on WA and control
NGM plates. Heat-shock was given to day 3 adults for three and a half
hours at 35 °C, followed by a daily examination of the survival rate
until all nematodes were dead.
For oxidative stress assay, nematodes were maintained on WA or con-
trol plates at 20 °C until their progeny production ceased. On the sixth day
(after L4) worms were transferred to fresh NGM plates containing 10 mM
paraquat and checked daily for viability (Zarse et al., 2012).
2.9. Quantication of ROS formation
Reactive Oxygen Species (ROS) levels were quantied with minor
modications as described earlier (Shukla et al., 2012). Approximately,
100 young (day 3) adults were used for the assay. Fluorescence spectral
measurements were captured by Cary Eclipse uorescence spectropho-
tometer (Agilent Technologies) at the excitation wavelength of 485 nm
and the emission wavelength of 535 nm.
2.10. α-Synuclein protein/lipofuscin aggregation assay and visualization of
GFP uorescence
NL5901 (an integrated α-synuclein: YFP fusion construct driven by
the unc-54 promoter) strain of C. elegans was used to analyze the effect
of 5 μM WA on the aggregation of Parkinson's disease associated protein
α-synuclein. Worms were raised like the lifespan assay and after 72 h of
WA treatment from late L4, twenty randomly selected adult worms
were mounted on 3% agarose pads and anesthetized by 2% sodium
azide for visualization of autouorescence. Images were captured with
auorescence microscope (Leica, DMI3000) using GFP lter (with exci-
tation at 365 nm and emission at 420 nm). Quantication of data was
performed by using ImageJ software. A similar protocol was followed
for Lipofuscin aggregation in N2 and other GFP uorescence measure-
ments carried out in this study.
2.11. Modeling of WA binding
The molecular docking approach wasusedtomodelWAintotheSGK-
1 binding site. For docking, the three-dimensional structure of WA was
retrieved from the PubChem database, one of the linked databases within
48 B.A. Akhoon et al. / Experimental Gerontology 78 (2016) 4756
the NCBI's Entrez information retrieval system. The WA structure was
submitted to the idTarget server (Wang et al., 2012)foritspossiblepro-
tein target predictions using the Z-score (b0) as a lter. Other in silico
methods employed are described in the Results and discussion section.
2.12. Real-time RT-PCR analysis
The total RNA was harvested using RNAzol reagent (Invitrogen,
USA). cDNA was synthesized using a cDNA synthesis kit (Invitrogen)
and quantitative real-time PCR was carried out using 7900HT Fast
Real-Time PCR System (Applied Biosystems) with SYBR green dye ac-
cording to the manufacturer's instructions. Relative gene expression
was normalized to act-1 and the data was analyzed using the ΔΔCt rel-
ative quantitation method (Livak et al., 2001).
2.13. Statistical analyses
All the statistical calculations relevant to lifespan and stress resistance
assays were performed using MedCalc software (Log-rank signicance
test). Other statistical analyses were carried out using ANOVA in
ASSISTAT 7.7 beta statistical assistance software. In all experiments, a
P-value less than 0.05 was considered statistically signicant.
3. Results and discussion
3.1. WA treatment signicantly enhanced both lifespan and healthspan of
C. elegans
To investigate the impact of WA on the lifespan of a multicellular eu-
karyote, we exposed adult C. elegans to WA at 5 μM, 25 μM, and 50 μM
concentrations. When treated with the above-mentioned concentrations
of WA, the worm showed a signicant extension of lifespan as compared
with the negative control group (untreated) (Table 1). We noticed that
WA caused a signicant extension (P0.0001) of mean and median
lifespan by 29% and 21% respectively at 5 μM concentration (Fig. 1A).
We examined the lipofuscin (aging biomarker) appearance of day 10
worms to check whether the WA treatment delayed the initiation of
aging or just prolonged the aged stage and found that the appearance
of lipofuscin was signicantly reduced by approximately 27.54%
(Pb0.01 **) in worms treated with WA (Fig. 1B), showing that worms
treated with WA initiated the aging process more slowly than the control
worms. The rate of motor activity declines with age and has been report-
ed to be a physiological parameter of animal healthiness (Hsu et al.,
2009). Therefore, we were interested to check the effect of the optimal
concentration of WA (5 μM) on the motor activity and learning ability
of N2 worms. We found that the rate of the motor activity decay was
also reduced in WA treated animals (Fig. 2A). Also, it was observed that
C. elegans pre-exposed to WA-treatment showed increased levels of che-
motaxis (Fig. 2B) which is reported to decline with age (Glenn et al.,
2004). These results show that WA extend both healthspan and lifespan
of worms. Increased longevity has been noticed to be associated with re-
ductions in fecundity and growth and several dietary supplements that
extend adult lifespan has been reported to signicantly affect the progeny
production and body size in worms (Harrington and Harley, 1988;
Houthoofd et al., 2002; Liao et al., 2011). Therefore, we monitored the re-
productive capacity of both treated and control worms. In our study, we
observed 9% less progeny of WA-treated worms (12 h survey) in compar-
ison to untreated ones (Fig. 2C), showing that WA also modulates the re-
productive ability of worms that is associated with lifespan as reported by
earlier studies. Consequently, we measured the body size (length and
width) of wild-type adult animals pre-treated with WA. We found no-
ticeable variation in the body size of WA-treated worms on the day 2,
day 5 and day 10 of adulthood as the worms were longer and wider
than untreated animals (Fig. 2D). These results signify that WA mediated
lifespan extension is associated with body size of worms. Since WA has
been reported to have several neuromodulatory effects (as stated in the
Introduction section), we were interested to check if WA shows any neu-
roprotective effect in C. elegans.
α-Synuclein aggregation, a major culprit behind parkinsonism
culminates in neuronal cell death in the substantia nigra of Parkinson's
disease (PD) patients (Stefanis, 2012). We checked the effect of WA on
proteopathies by employing a C. elegans strain (NL5901) exhibiting mus-
cular expression of α-synuclein tagged with Yellow uorescence Protein
(YFP). We observed substantial decrease (38%) in α-synuclein levels
(compared to control group) in NL5901 worms treated with 5 μMof
WA (Fig. 3A), implicating benecial effects of WA for PD patients.
Amyloid β(Aβ) is an obligate player behind Alzheimer's disease
(AD) and its excessive accumulation is toxic to the cells (Karran et al.,
2011; Villemagne et al., 2013). In order to detect the effect of 5 μM
WA on Aβaggregation, a paralysis assay was conducted using a trans-
genic CL4176 strain of C. elegans. The employed strain expresses muscu-
lar Aβupon temperature upshift. In comparison to control worms
(52.00 ± 2.30), a signicant decrease (Pb0.001) in the percentage of
paralyzed CL4176 worms (27.33 ± 1.76) was noticed after WA treat-
ment (Fig. 3B). Such results highlight the anti-amyloidogenic effects of
WA in C. elegans as large numbers of worms paralyzed indicates
increased toxicity of Aβ.
Impaired neurotransmission and cholinergic abnormalities/deple-
tion are one of the marked symptoms of AD patients (Perry et al.,
2000). Moreover, the degeneration of the cholinergic system is also a
contributing factor to the learning and memory decits observed in
AD patients or during normal aging. In order to determine the effect of
WA on ACh levels, the total ACh was estimated through the Amplex
Red assay kit. In comparison to the relative ACh levels in a control
group (1.00 ± 0.1), a signicant increase (Pb0.05) in gross ACh levels
(1.78 ± 0.01, P-value: 0.05) was observed in 5 μM WA treated group
(Fig. 3C). We were also interested to check the effect of WA on AChE
activity. We used an Amplex red AChE kit for this purposeand observed
that AChE activity as observed in control group (1.00 ± 0.01), was also
slightly increased after WA treatment (1.34 ± 0.09), however, the in-
crease was insignicant (Fig. 3D). A signicant increase in Ach levels
without alteration in Ache levels is of particular interest in the context
of the decits prevalent in AD. Our results showed the benecial effect
of WA in curtailing the cholinergic dysfunction through a possibly
enhanced synthesis of Ach, without affecting the gross Ache activity.
3.2. WA-mediated lifespan extension is independent of calorie restriction
Restricting dietary consumption of compounds without malnutri-
tion extends lifespan and attenuates age-related declines/diseases in
multiple species (Masoro, 2005), thereby suggesting a conserved un-
derlying mechanism from nematodes to humans (McCay et al., 1935;
Table 1
Lifespan analysis of wild-type worms cultured at 20 °C.
Strains Treatment (μM) Mean lifespan SE No. of worms (N) % change Pvalue Median lifespan Max. mean lifespan (±SE)
N2 Control 13.700 0.475 120 Pb0.0001 14 17.66 ± 0.13
2μM 14.293 0.561 117 4.32 Pb0.0001 14 19 ± 0.33
5μM 17.763 0.593 121 29.65 Pb0.0001 17 25.66 ± 0.17
25 μM 15.864 0.659 128 15.79 Pb0.0001 16 22 ± 0.15
50 μM 15.182 0.615 152 10.81 Pb0.0001 15 20.33 ± 0.26
49B.A. Akhoon et al. / Experimental Gerontology 78 (2016) 4756
Fontana et al., 2010). Since many external molecules extend lifespan by
dietary restriction phenomenon (Bass et al., 2007; Pallauf et al., 2013),
we asked whether WA acts as a dietary restriction mimetic. We tested
this hypothesis by prospecting the rate of pharyngeal pumping in
worms. We did not notice any signicant effect on the rate of pharyn-
geal pumping (Fig. 4A). We also found that the lifespan of eat-2
(ad1116) mutant animals, which exhibit the phenomenon of calorie re-
striction because of slower pumping (Lakowski and Hekimi, 1998), was
extended by WA treatment (Fig. 4B). Additionally, we examined the ef-
fects of WA treatment on the eat-2 gene expression of wild-type ani-
mals. WA treatment failed to affect the eat-2 expression of N2 animals.
These results suggest that calorie restriction per se is unlikely to be
the cause of the lifespan extension observed in WA-treated worms.
Therefore, to predict the possible targets of WA, we performed large
scale in silico protein screening using the idTarget. Our docking results
showed that WA recognizes human serine/threonine-protein kinase
SGK1 (PDB ID: 2R5T) with a good binding afnity (9.63 kcal/mol).
3.3. sgk-1 is required for lifespan extension by WA
The insulin/IGF-1 signaling (IIS) pathway has been implicated in lon-
gevity in various organisms and its reduction leads to lifespan extension
Fig. 1. WithanolideA (WA) extends C. eleganslifespan. (A) DoseresponseKaplanMeier survival curves of wild-type (N2) populations exposedto 0 μM (control),2, 5, 25 and 50 μMWAat
20 °C. Treatmentwith 5 μM, 25 μMand50μM WA signicantly (Pb0.0001) extendedthe mean lifespanof worms, whereastreatment with 2 μMWAdidnotcauseasignicant increasein
lifespan. The maximum mean and median lifespan extension by 29% and 21% respectively was seen in worms treated with 5 μM WA. (B) WA delays the progression of aging. Higher
accumulation of lipofuscin was seen in control worms compared with their respective 5 μM WA treated animals. Representative images show intestinal lipofuscin autouorescence of
untreated (Control) and WA-treated (5 μM) day 10 N2 animals (n = 20). Images were quantied using the ImageJ software. Scale bar, 30 μm.
Fig. 2. WA effects on the healthspan of C. elegans. (A) Effect of 5 μM WA on the motility of N2 worms evaluated as the mean numberof body bends in a 30-s period in 10 individual worms
on days 1, 3 and 5 adulthood. WAtreatment delays the age-relateddecline in body movement. Data represent the average number ofbends. (B) Salt chemotaxis in wild-type (day 5) N2
animals. Worms pretreated with WA showed improved levels of chemotaxis. (C) Average fertility of C. elegans treated with 5 μM WA. Worms when maintained on WA (72 h) showed
decrease in total progeny production (12 h survey). Plot is a representative of 2 independent experiments with a total of 10 nematodes per group. (D) Quantication of body size
measurements. The body length and width of each worm at days 2, 5 and 10 was measured. Values represent mean size of worms (n = 10). Body size of WA treated worms was
signicantly increased in comparison to control animals. Asterisks (** and *) indicate signicant changes at Pb0.01 and Pb0.05.
50 B.A. Akhoon et al. / Experimental Gerontology 78 (2016) 4756
(Tazearslan et al., 2011). The DAF-2 insulin receptor-like signaling path-
way in C. elegans also controls its lifespan by phosphorylating and
inhibiting the nuclear translocation of DAF-16/FoxO or SKN-1
accumulation in intestinal nuclei, where SGK-1 (C. elegans homolog
of SGK), functions in parallel to AKT-1 and AKT-2 to mediate this sig-
naling. SGK-1 contains a kinase domain which is 78% similar and 67%
identical to the human SGK kinase domain (Hertweck et al., 2004).
sgk-1 gain-of-function mutation enhances the lifespan in C. elegans
(Chen et al., 2013b). To analyze in vivo whether WA has any effect
on the sgk-1 of C. elegans, we assessed the impact of WA on the
lifespan of sgk-1 (ok538) null mutant. sgk-1 (ok538) harbors an
852 bp deletion that eliminates the seventh and most of the eighth
exon that forms the major part of the kinase domain, critical for
SGK-1 activity (Hertweck et al., 2004). Incubation with WA has no
inuence on the lifespan of sgk-1 mutant (Table 2; Fig. S1A), showing
that sgk-1 is required for the lifespan extension observed in WA-
treated worms.
Moreover, our bioinformatics analysis showed that SGK-1 kinase do-
main is conserved in many species, from C. elegans to Homo sapiens
(Fig. S2). We aligned the kinase domain of 2R5T (human SGK-1) with
the SGK-1 of C. elegans and found 62.8% identity and 79.1% similarity
among them at sequence level (Fig. 5A). Such results reveal that SGK-
1ofC. elegans shares a similar kinase domain organization as of
human and have an evolutionary and likely functional relationship. To
examine the structural differences, we modeled the SGK-1 of
C. elegans using the I-TASSER (Roy et al., 2010). The program uses
Fig. 3. WA shows multifunctional neuroprotective roles in C. elegans.(A)αsynuclein levels in NL5901 worms. A substantial decrease in alphasynuclein level was seen in WA treated
worms, compared to control. Fluorescence intensity was quantied by ImageJ software . (B) Graphical representation of Aβ-induced toxicity in CL4176 worms. The worms were
cultivated at 15 °C fo r 48 h. At the 48 h time point, the temperature was up-shifted to 25 °C. The worms were scored at 18 h after the initiation of upshift and the sc oring was
continued in 1 h increments until all worms were paralyzed. WA signicantly reduces the percentage of CL4176 worms paralyzed as a result of Aβtoxicity. (C, D) Effect of WA on
neurotransmitter acetylcholine and acetylcholinesterase in wild-type worms. WA elevates both ACh (C) and AChE (D) levels however the acetylcholinesterase increase was found
insignicant. ***Pb0.001, **Pb0.01, *Pb0.05, ns not signicant. Scale bar, 30 μm.
Fig. 4. WA-mediated lifespan extension is independent of calorie restriction in C. elegans. (A) Effect of WA on pharyngeal pumping. WA failed to affect the rate of pharyngeal pumping.
(B) Survival curves of eat-2 mutant animals (n = 100) either untreated or treated with 5 μM WA throughout their adult life. WA augments the lifespan of eat-2 mutants.
51B.A. Akhoon et al. / Experimental Gerontology 78 (2016) 4756
restraints from templates identied by multiple threading programs to
build full-length model using replica-exchange Monte-Carlo simula-
tions and reports a condence score (C-score) of the resultant model
to estimate the quality of the predicted models (Akhoon et al., 2014).
A C-score that ranges from 5 to 2 has been extensively tested in
large-scale benchmarking tests (Zhang and Skolnick, 2004; Zhang,
2008). In our case, we observed that C. elegans SGK-1 model was built
by I-TASSER using restraints from PDB templates viz., 2R5T, 4CRS,
3PFQ, 4L9I, 3A8X with a better C-score of 1.38. I-TASSER has another
scoring function, TM-score, to assess the structural similarity of model
and template protein structures. The score has been reported to over-
come the problem of RMSD which is sensitive to the local error. Irre-
spective of the protein length, a TM-score N0.5 indicates a model of
correct topology and a TM-score b0.17 means a random similarity.
The modeled protein (Fig. 5B) was found to have TM-score of 0.54 ±
0.15, indicating a better structural match of the C. elegans SGK-1 with
the templates. We also applied the ProSA-web service to check the over-
all model quality and to compare it with other experimentally deter-
mined protein structures of the same size. The z-score (a statistical
score for overall model quality) (Wiederstein and Sippl, 2007) of the
modeled SGK-1 protein was found to be 8.02, a value too close to
the experimentally resolved structures (Fig. 5C). Furthermore, overlap-
ping of the kinase domain of crystal structure 2R5T and the modeled
SGK-1 protein from C. elegans by CLICK (Nguyen et al., 2011) revealed
that these proteins were substantially similar in structure with a Cα
RMSD deviation of 1.64 A° (Fig. 5D). Such information was in favor as
signicant structural deviations could affect or modify the activity of
the protein (Srivastava et al., 2010). Many researchers have implement-
ed molecular docking approaches for the computational prediction of
receptor-ligand binding interactions/afnities (Pant et al., 2014;
Baloria et al., 2012; Akhoon et al., 2010, 2011). To get additional in-
sights, we modeled the docking of WA into the SGK-1 model through
an automated molecular-docking procedure using the web-based
SwissDock program (Grosdidier et al., 2011). Docking carried out in
SwissDock using the Accurateparameter in otherwise default parame-
ters, with no region of interest dened (blind docking) produced 30
clusters (docking poses) for WA. Analysis of all clusters showed a single
cluster (cluster no. 20) with the binding mode of WA similar to the ref-
erence structure (2R5T-WA complex modeled by idTarget) (Fig. 6),
showing that WA has preferred interaction (8.09 kcal/mol) towards
C. elegans SGK-1 and form 3 hydrogen bonds with LYS143, SER145,
PHE146 amino acid residues.
Fig. 5. Modeling of Serum/glucocorticoid-regulated kinase 1 in C. elegans. (A) A kinasedomain sequencealignment ofSGKs from human andC. elegans. The conserved (identical and similar
amino acid residues) are highlighted in black and gray colors. (B) 3D structure of the modeled kinase domain of SGK-1 protein in C. elegans. (C) Investigation of the modeled SGK-1
structure using the ProSA-web service. The z-score of this model is 8.02, a value too close to the experimentally resolvedstructures. (D) Structural overlay of the homology model of
SGK-1 protein of C. elegans (yellow) with the SGK of H. sapiens (cyan) (PDB ID: 2R5T).
Table 2
Lifespan of C. elegans mutants treated with Withanolide A.
Strains Treatment (μM) Mean lifespan (±SE) No. of worms (N) % change Pvalue Max. mean lifespan (±SE)
sgk-1 (ok538) Control 17.603 ± 0.500 126 22.23 ± 1.4
5μM 18.075 ± 0.455 119 2.68 Pb0.0001 23.66. ± 1.1
daf-16 (mgDf50) Control 11.098 ± 0.381 106 15.5 ± 1.3
5μM 10.849 ± 0.418 108 2.24 Pb0.0001 15 ± 1.1
skn-1 (zu67) Control 11.693 ± 0.326 114 16.5 ± 0.21
5μM 11.254 ± 0.306 113 3.75 P= 0.0001 16 ± 0.43
hsf-1 (sy441) Control 10.205 ± 0.256 105 15 ± 0.13
5μM 10.667 ± 0.305 111 4.5 Pb0.0001 15 ± 0.17
mev-1 (kn1) Control 9.151 ± 0.342 84 12.66 ± 0.26
5μM 10.571 ± 0.290 86 15.51 Pb0.0001 15.66 ± 0.53
52 B.A. Akhoon et al. / Experimental Gerontology 78 (2016) 4756
3.4. WA-mediated lifespan extension is DAF-16 dependent
DAF-16/FoxO, a major transcriptional output of IIS pathway, is a pri-
mary mediator of increased longevity and stress resistance in C. elegans.
The negative regulation of DAF-16 (responsible for increased longevity
and stress resistance) is controlled by DAF-2 via PI3K-AKT/SGK signal-
ing pathway. SGK-1 acts parallel to AKT-1 and promotes C. elegans lon-
gevity in a DAF-16 dependent manner (Chen et al., 2013b). We asked
whether WA treatment has any effect on the lifespan of DAF-16 and
to address this possibility, we assayed the lifespan of worms harboring
the daf-16 (mgDf50) mutation. WA did not extend the lifespan of daf-
16 animals (Table 2; Fig. S1B), indicating that WA promotes longevity
in an IIS-dependent manner.
3.5. WA induces sod-3 expression, but did not inuence the DAF-16/FoxO
subcellular localization
To further examine the idea that WA act on a component of the IIS
pathway, DAF-16, to inuence longevity, we examined the nuclear lo-
calization of DAF-16 using a GFP reporter strain (TJ356). However, we
did not nd nuclear-localized DAF-16::GFP fusions in the body of
worms after 72 h of treatment with WA. Since, it has been reported
that sgk-1 gain-of-function mutations did not inuence the DAF-16/
FoxO subcellular localization (Chen et al., 2013b) and had different ef-
fects on the expression of distinct DAF-16/FoxO target genes, therefore,
we monitored GFP uorescence in sod-3 transgenic strain CF1553 (SOD-
3GFP) of C. elegans following WA treatment (72 h). The level of sod-3:
GFP induction in WA-treated worms was signicantly higher (Pb0.01)
than control worms (Fig. 7A). Our results reveal that WA extends
lifespan, possibly through indirect regulation of a subset of DAF-16/
FoxO targets without affecting the subcellular localization of DAF-16.
3.6. WA activate SKN-1 and HSF-1 transcriptional activity
SKN-1, a C. elegans Nrf family transcription factor and a major com-
ponent of IIS, controls numerous biological processes including stress
resistance and longevity (Hsu et al., 2003). Earlier reports show that
SGK-1 may promote longevity by regulating other proteins that
functionally and/or physically interact with DAF-16/FoxO, such as
SKN-1 (Tullet et al., 2008)andHSF-1(Hsu et al., 2003). Heat shock
factor 1 (HSF-1) overexpression increases heat resistance and extends
lifespan in a DAF-16 dependent manner (Tullet et al., 2008). Additional-
ly, it has also been suggested that daf-16 and hsf-1 function in a common
pathway to regulate longevity (Morley and Morimoto, 2004). Based on
these reports, we tested the effect of WA on the lifespan of SKN-1 and
HSF-1 animals. We did not observe lifespan extension upon WA
treatment in skn- (zu67) (Fig. S1C) and hsf-1 (sy441)mutants
(Fig. S1D), suggesting that WA-mediated longevity is IIS pathway
dependent. Similar to the regulation of DAF-16 by insulin signaling,
the IIS kinases phosphorylate SKN-1 and reduces IIS, which leads to con-
stitutive SKN-1 accumulation in intestinal nuclei whereby its target
gene activation modulate worm longevity (Tullet et al., 2008; Choe
et al., 2009). SKN-1, expressed in the intestine and in the ASI neurons
of worms, mediates the phase 2 stress response (An and Blackwell,
2003). GST-4 is a phase II enzyme and is tightly regulated by the SKN-
1 transcription factor in response to oxidative stress (Choe et al., 2009;
Kell et al., 2007; Landis and Murphy, 2010). To gain insights into the
Fig. 6. Dockedconformationof WA with SGK of C. elegansan d H.sapiens. WA is indicatedin CPK, blue colored.The putative interacting residuesof C. elegans SGK-1forming hydrogen bonds
with WA are labeled and shown as sticks.
Fig. 7. WA up-regulates stress-responsive genes in C. elegans. Fluorescent photomicrographs of (A) SOD-3::GFP in CF1553 and (B) GST-4:GFP transgene in the CL2166 strains. GFP
expression in WA-treated worms is higher thanthat in control worms. Quantication of images was performed by ImageJ software. **Pb0.01. Scale bar, 30 μm.
53B.A. Akhoon et al. / Experimental Gerontology 78 (2016) 4756
effects of WA on gst-4 activity, we tested the expression of a gst-4p: GFP
transgene in the CL2166 (dvIs19 [pAF15] (gst-4::GFP::NLS)) transgenic
worm strain treated with WA. We observed increased uorescence in
the pharynx region of CL2166 worms (Fig. 7B). All these observations
show that WA acts through the IIS pathway.
3.7. WA increases stress resistance and lowers ROS generation
Besides longevity, these components (DAF-16, SKN-1, and SGK-1) of
IIS pathway also play a key role in the regulation of stress resistance in
C. elegans (Tatar et al., 2003; Murphy et al., 2003; Wolff et al., 2006;
Chen et al., 2013b). It has also been frequently demonstrated that
there is a strong correlation between lifespan extension and resistance
to multiple environmental stresses (Kenyon, 2010). Enhanced stress
tolerance is a common characteristic of long-lived mutants and over-
expression of some antioxidant enzyme genes also extends the lifespan
(Lithgow et al., 1995; Johnson et al., 2000; Kim et al., 2010; Zhang et al.,
2013). Therefore, to address this, we explored anti-stress effects of WA
in C. elegans.
To examine whether WA could have any effect on antioxidant stress
resistance, we subjected N2 worms to oxidative stress induced by para-
quat, an intracellular reactive oxygen species (ROS) generator. We no-
ticed that WA-treated nematodes were more resistant than control
worms, to oxidative stress induced by growing the worms in the pres-
ence of paraquat, and lived markedly longer than control worms
(Fig. 8A). Since increased antioxidant activity can result from lower re-
active oxygen species (ROS) generation, also ROS impinges on so many
cellular processes, including lifespan; we chose to further study wheth-
er WA treatment has any effect on ROS levels. We assessed total ROS of
WA-treated and control worms, the observation showed that the total
ROS levels were signicantly decreased (Pb0.01) in treated worms
compared to control (Fig. 8B). Moreover, we also checked the effect of
WA on mev-1 (kn1) mutant that has a defective electron transport in
complex II. The mev-1 mutant is short lived and is highly susceptible to oxida-
tive damage due to higher levels of ROS (Adachi et al., 1998). WA treatment
extended the lifespan of mev-1 mutants by 15% (Fig. 8C), conrming that the
antioxidant properties of WA are relevant in this context that explains the en-
hanced survival of WA-treated mev-1 mutants compared to control. Together,
these results indicate that it is the decreased level of ROS that consequently
lead to the prolonged survival of WA treated worms (Table 2).
It has been observed that many antioxidant compounds that
enhance oxidative stress resistance also confer thermotolerance. To ex-
plore whether WA has any effect on thermal resistance, we subjected
worms to a heat shock for three and a half hours at 35 °C. We observed
that treatment of adult C. elegans with WA lead to a high level of ther-
motolerance by 19% (Fig. 8D). Taken together, these results suggest
that WA reduces oxidative stress and render the worms resistant to
thermal stress.
3.8. Genetic requirements for WA-mediated lifespan extension in C. elegans
In order to verify the ndings further, we conducted gene expression
studies using quantitative real-time PCR after exposing the worms to
WA for 72 h. We found that WA enhances the expression of sgk-1 by
2.4 fold (Fig. 9). Our observations were in agreement with the recent
ndings whereby sgk-1 gain-of-function promotes longevity (Chen
et al., 2013b) and contrast with the earlier reported lifespan extension
induced by sgk-1 RNAi (Hertweck et al., 2004). Since sgk-1 is a molecular
component of the C. elegans IIS pathway, we examined the effect of WA
on daf-16, a key component of the IIS cascade. From our qRT-PCR results,
we found that WA treatment leads to the induction of the daf-16 and en-
hances its expression by 1.9 fold (Fig. 9). We measured the expression
Fig. 8. WA increases stress resistance of C. elegans. (A) Oxidative stress induced by paraquat. The experiment was done in the no-FUdR condition and worms were exposed to paraquat
(10 mM) after their cease of progeny production (sixth day after L4). WA (5 μM) increases antioxidant defense in C. elegans and prolongs survival of worms under oxidative stress
conditions. (B) Relative formation of reactive oxygen species (ROS) after 72 h of exposure to 5 μM WA. Less ROS was produced in WA pretreated worms as assessed with the total ROS
dye indicator DCF-DA, compared to control. (C) WA increases the % mean survival (15%) of mev-1 (kn1) mutants that are highly susceptible to oxidative damage due to higher levels
of ROS generation. (D) Thermo survival of nematodes pre-exposed (72 h after L4) to 5 μM WA. WA enhances C. elegans resistance to thermal stress.
54 B.A. Akhoon et al. / Experimental Gerontology 78 (2016) 4756
level of sod-3, a known DAF-16 target gene involved in stress responses.
Notably, our results data revealed that sod-3 expression levels were
almost 1.7 fold higher (Fig. 9) in WA-treated worms compared to con-
trol. To further ascertain whether DAF-16 is involved in WA-mediated
lifespan extension, the expression level of another direct target of
DAF-16 (fkh-9) was checked. We found higher fkh-9 expression levels
(2.3 fold) inWA-treated nematodes as compared to control worms. To-
gether, these ndings support the notion that WA-mediated lifespan
extension is DAF-16 dependent. Our qRT-PCR experiments showed
that WA also upregulates the mRNA expression levels of another impor-
tant component of IIS, skn-1 and its target gene gst-4 by 2.7 and 3.1 folds
respectively. Moreover, we also found signicant upregulation of ther-
mal shock response factors, hsf-1 (2 fold) and its target gene hsp-16.2
(1.6 fold) in WA-treated N2 worms (Fig. 9).
In summary, our ndings highlight the anti-aging and neuroprotec-
tive rolesof WA and demonstrate the involvement of IIS pathwayin WA
mediated lifespan extension in C. elegans. SGK1 activated by WA does
not affect DAF-16 translocation but the transcription of selective DAF-
16 targets. Further, WA enhances the transcriptional level of other im-
portant transcription factors suchas the antioxidant response transcrip-
tion factor SKN-1 and the heat-shock transcription factor HSF-1. Taken
together, these results suggest that SKN-1 and HSF-1 likely act in con-
cert with DAF-16 in WA-treated worms to increase neuron functionali-
ty, stress resistance, and lifespan. Our study demands more research on
this multifaceted natural product (WA) to gain a better understanding
of its therapeutic value for human beings.
Disclosure statement
The authors declare that they have no conict of interest.
We are grateful to the Director, CSIRCIMAP, Lucknow, India for his
kind support. BAA thanks Virendra Shukla, Shreesh Sammi and Laxmi
Rathor for their useful comments and assistance in the experimental
protocols. BAA also acknowledges useful discussions with Shailendra
K Gupta, Shishir K Gupta and Aakansha Pant. All the strains were obtain-
ed from the Caenorhabditis Genetic Center at the University of Minneso-
ta. BAA and ST were nancially supported by CSIR India (31/029(0251)/
2013-EMR-I and 31/34(157)/2013-EMR-I) through SRF grants.
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56 B.A. Akhoon et al. / Experimental Gerontology 78 (2016) 4756
... Thus, maximum increase in the lifespan was recorded at 100 µg/mL CPE dosage. It was also previously reported that the lifespan extension in C. elegans is dosagedependent [59][60][61], and a particular dosage will be more impactful than in comparison to other dosages. Our study is on par with the previous reports that a particular dose was effective in increment of maximum lifespan (100 µg/mL CPE), which was further used for comparative study with crude extract from the infected leaf sample. ...
... increase) and 200 µg/mL (10.4%) compared to untreated control animals ( Table 1, Figu 10). Thus, maximum increase in the lifespan was recorded at 100 µg/mL CPE dosage was also previously reported that the lifespan extension in C. elegans is dosage-depende [59][60][61], and a particular dosage will be more impactful than in comparison to oth dosages. Our study is on par with the previous reports that a particular dose was effect in increment of maximum lifespan (100 µg/mL CPE), which was further used comparative study with crude extract from the infected leaf sample. ...
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Citation: Soni, S.K.; Mishra, M.K.; Mishra, M.; Kumari, S.; Saxena, S.; Shukla, V.; Tiwari, S.; Shirke, P. Papaya Leaf Curl Virus (PaLCuV) Infection on Papaya (Carica papaya L.) Plants Alters Anatomical and Physiological Properties and Reduces Bioactive Components. Plants 2022,
... Thus, maximum increase in the lifespan was recorded at 100 µg/mL CPE dosage. It was also previously reported that the lifespan extension in C. elegans is dosagedependent [59][60][61], and a particular dosage will be more impactful than in comparison to other dosages. Our study is on par with the previous reports that a particular dose was effective in increment of maximum lifespan (100 µg/mL CPE), which was further used for comparative study with crude extract from the infected leaf sample. ...
... increase) and 200 µg/mL (10.4%) compared to untreated control animals ( Table 1, Figu 10). Thus, maximum increase in the lifespan was recorded at 100 µg/mL CPE dosage was also previously reported that the lifespan extension in C. elegans is dosage-depende [59][60][61], and a particular dosage will be more impactful than in comparison to oth dosages. Our study is on par with the previous reports that a particular dose was effect in increment of maximum lifespan (100 µg/mL CPE), which was further used comparative study with crude extract from the infected leaf sample. ...
Full-text available
Citation: Soni, S.K.; Mishra, M.K.; Mishra, M.; Kumari, S.; Saxena, S.; Shukla, V.; Tiwari, S.; Shirke, P. Papaya Leaf Curl Virus (PaLCuV) Infection on Papaya (Carica papaya L.) Plants Alters Anatomical and Physiological Properties and Reduces Bioactive Components. Plants 2022,
... KSM-66, the W. somnifera varietal employed in this study had withanolides as the major active ingredients. The use of withanolides for treatment of aspects of Parkinson's disease has been reported previously (Akhoon et al., 2016;Kuboyama et al., 2005;Vegh et al., 2021). Withanolide A was reported to induce regeneration of axons, dendrites, preand post-synapses in the neuron of rat Parkinson's disease model (Kuboyama et al., 2005). ...
... Withanolide A also showed proteopathies effects by decreasing α-synuclein levels 38% in of NL5901 Caenorhabditis elegans, a Parkinson's disease model, suggesting beneficial effects of withanolide A for PD patients (Akhoon et al., 2016). In addition, root extract of W. somnifera, containing approximately 12% withanolides, alone or in combination with a water-soluble formulation of coenzyme-Q10 was reported to enhance activation of pro-survival astroglia and inhibited pro-inflammatory microglia. ...
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Parkinson's disease is the most frequent neurodegenerative motor disorder. The clinical syndrome and pathology involve motor disturbance and the degeneration of dopaminergic neurons in the substantia nigra. Root extracts of Withania. somnifera, commonly called Ashwagandha, contain several major chemical constituents known as withanolides. Studies have shown that W. somnifera extracts exhibit numerous therapeutic effects including inflammation and oxidative stress reduction, memory and cognitive function improvement. This study aimed to evaluate the protective effects of KSM-66, W. somnifera root extract, on 6-hydroxydopamine (6-OHDA)-induced toxicity in the human neuroblastoma SH-SY5Y cell line, as well as the associated oxidative response protein expression and redox regulation activity focused on S-glutathionylation. SH-SY5Y cells were treated with 6-OHDA preceded or followed by treatment with the KSM-66 extract. Using KSM-66 concentrations ranging from 0.25 to 1 mg/ml before and after treatment of the cells with 6-OHDA has resulted in an increased viability of SH-SY5Y cells. Interestingly, the extract significantly increased glutathione peroxidase activity and thioltransferase activity upon pre- or post- 6-OHDA treatment. KSM-66 also modulated oxidative response proteins: peroxiredoxin-I, VGF and vimentin proteins upon 6-OHDA pre/post treatments. In addition, the extract controlled redox regulation via S-glutathionylation. Pre-treatment of SH-SY5Y cells with KSM-66 decreased protein-glutathionylation levels in the cells treated with 6-OHDA. The rescue of mitochondria with 0.5 mg/ml KSM-66 extract showed an increase in ATP levels. These findings suggest that W. somnifera root extract acts as a neuroprotectant, thereby introducing a potential agent for the treatment or prevention of neurodegenerative diseases.
... It has conserved genes that play similar functions in higher eukaryotes including humans. Therefore, C. elegans have been used to evaluate natural compounds for their potential anti-aging and stress resistance properties and in understanding their molecular mechanisms [12,13]. ...
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Pueraria lobata is a perennial legume, commonly used as a food source in China. The polysaccharides extracted from P. lobata have demonstrated various biological activities. However their anti-aging effects and the underline mechanisms are largely unknown. In this study, water-soluble polysaccharides (WSPS) from P. lobata were extracted and demonstrated antioxidant activity against DPPH radicals and hydroxyl radicals in vitro. Using nematode Caenorhabditis elegans as a model, we found that WSPS remarkably prolonged the survival, increased growth and locomotion under heat stress. To investigate the possible mechanism, the levels of reactive oxygen species (ROS) and lipid peroxidation product malondialdehyde (MDA) were determined. WSPS significantly decreased ROS and MDA levels which is consistent with increased activity of superoxide dismutase (SOD). Meanwhile, WSPS upregulated the expression of stress resistance genes sod-1, sod-5, hsf-1, hsp-12.6, hsp-16.2, skn-1 and gst-4. Together, these results suggest that the anti-aging activity of WSPS under heat stress was mediated most likely by activation of the target genes of heat-shock transcription factor (HSF)-1 and skinhead (SKN)-1, and thus inducing endogenous ROS scavenging response.
... However, α-Tocopherol, either in free form or encapsulated with SDNF (soluble dietary fiber-based nanofibers), may provide the longevity benefits via different routes [114]. Lifespan extension potentials have also been ascribed to additional high molecular weight isoprenoid-based compounds such as withanolide-A, specioside, ursolic acid, and glycyrrhetinic acid [60,[115][116][117]. Examples of these terpenoids with longevity-modulating effects are summarized in Table 2. ...
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In the forms of either herbs or functional foods, plants and their products have attracted medicinal, culinary, and nutraceutical applications due to their abundance in bioactive phytochemicals. Human beings and other animals have employed those bioactive phytochemicals to improve health quality based on their broad potentials as antioxidant, anti-microbial, anti-carcinogenic, anti-inflammatory, neuroprotective, and anti-aging effects, amongst others. For the past decade and half, efforts to discover bioactive phytochemicals both in pure and crude forms have been intensified using the Caenorhabditis elegans aging model, in which various metabolic pathways in humans are highly conserved. In this review, we summarized the aging and longevity pathways that are common to C. elegans and humans and collated some of the bioactive phytochemicals with health benefits and lifespan extending effects that have been studied in C. elegans. This simple animal model is not only a perfect system for discovering bioactive compounds but is also a research shortcut for elucidating the amelioration mechanisms of aging risk factors and associated diseases.
... Food & Function lifespan. 44,45 Furthermore, the activities of CAT and SOD increased markedly after FSPA pretreatment ( Fig. 3C and F). However, only a significant increase in CAT activity was observed in the PSPA treated group. ...
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Anthocyanins have anti-inflammatory, anticarcinogenic and antioxidant properties and anti-aging effects as well as potential application as pigments. The metabolism of anthocyanins in fermented food has attracted increasing attention. However, the effect of lactic acid bacteria (LAB) fermentation on its anti-aging activity remains mostly unknown. The current study aimed to investigate the compositions, antioxidant activities and anti-aging effect of fermented purple sweet potato anthocyanins (FSPA) on aging Caenorhabditis elegans compared to raw purple sweet potato anthocyanins (PSPA). Results showed that anthocyanins were degraded into more bioavailable phenolic acids by Weissella confusa fermentation. PSPA and FSPA can extend the lifespan of C. elegans by 26.7% and 37.5%, respectively, through improving the activity of antioxidant enzymes as well as decreasing MDA content, ROS levels and lipofuscin accumulation. Pretreatment of the worms with PSPA and FSPA induced their potential to resist to thermal tolerance and oxidative stress, and FSPA exerted a higher anti-stress effect than PSPA. Moreover, FSPA supplementation upregulated the mRNA expressions of genes daf-16, hsp-16.2, sir-2.1, skn-1 and sod-3 and downregulated the expression of daf-2 in the nematodes, whereas PSPA only induced the increase in the expressions of sir-2.1, skn-1 and sod-3. Overall, FSPA can improve stress resistance and extend the lifespan of C. elegans by both insulin/IGF-1 signaling pathway and dietary restriction pathway, providing a theoretical basis for the application of PSPA in fermented food as functional pigments.
Alzheimer’s disease (AD) is an age-dependent, progressive disorder affecting millions of people. Currently, the therapeutics for AD only treat the symptoms. Although they have been used to discover new products of interest for this disease, mammalian models used to investigate the molecular determinants of this disease are often prohibitively expensive, time-consuming and very complex. On the other hand, cell cultures lack the organism complexity involved in AD. Given the highly conserved neurological pathways between mammals and invertebrates, Caenorhabditis elegans has emerged as a powerful tool for the investigation of the pathophysiology of human AD. Numerous models of both Tau- and Aβ-induced toxicity, the two prime components observed to correlate with AD pathology and the ease of performing RNA interference for any gene in the C. elegans genome, allow for the identification of multiple therapeutic targets. The effects of many natural products in main AD hallmarks using these models suggest promising health-promoting effects. However, the way in which they exert such effects is not entirely clear. One of the reasons is that various possible therapeutic targets have not been evaluated in many studies. The present review aims to explore shared therapeutical targets and the potential of each of them for AD treatment or prevention.
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Alzheimer’s disease (AD) and dementia are disorders of the aging population and becoming major health care burden worldwide due to unavailability of complete therapy. AD is the most frequent cause of dementia among 60% to 80% patients and has effected 45 million people globally which is estimated to triple by 2050 (Alzheimer’s, 2015). AD is a progressive, neurodegenerative disorder, characterized by behavioral turbulence, cognitive dysfunctions, imperfection in routine life activities, thus putting a huge socioeconomic burden on the health care system ( Ahmad et al., 2015; Ali et al., 2017; Ayaz et al., 2017b). Among the pathophysiological hallmarks of the disease are the deficiency of vital neurotransmitter acetylcholine (ACh), deposition of amyloid plaques (Aβ), highly phosphorylated tau proteins, and imbalance in gluatamatergic system ( Ayaz et al., 2017a; Khalil et al., 2018 ; Ovais et al., 2018a). Only five drugs are linically approved for use, among which tacrine, galantamine, donepezil, and rivastigmine are cholinesterase inhibitors whereas the fifth one memantine is glutamatergic system modulator (Ayaz et al., 2015 ; Kamal et al., 2015). These drugs have limited efficacy and are associated with side effects like tacrine is hepatotoxic (Watkins et al., 1994). Currently, results from clinical trials performed in mild to moderate AD dementia have directed researchers to find more effective yet safe alternatives from natural sources (Yiannopoulou and Papageorgiou, 2013 ; Cummings et al., 2014 ; Ovais et al., 2018b).
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Plant secondary metabolites (PSMs) are vital for human health and constitute the skeletal framework of many pharmaceutical drugs. Indeed, more than 25% of the existing drugs belong to PSMs. One of the continuing challenges for drug discovery and pharmaceutical industries is gaining access to natural products, including medicinal plants. This bottleneck is heightened for endangered species prohibited for large sample collection, even if they show biological hits. While cultivating the pharmaceutically interesting plant species may be a solution, it is not always possible to grow the organism outside its natural habitat. Plants affected by abiotic stress present a potential alternative source for drug discovery. In order to overcome abiotic environmental stressors, plants may mount a defense response by producing a diversity of PSMs to avoid cells and tissue damage. Plants either synthesize new chemicals or increase the concentration (in most instances) of existing chemicals, including the prominent bioactive lead compounds morphine, camptothecin, catharanthine, epicatechin-3-gallate (EGCG), quercetin, resveratrol, and kaempferol. Most PSMs produced under various abiotic stress conditions are plant defense chemicals and are functionally anti-inflammatory and antioxidative. The major PSM groups are terpenoids, followed by alkaloids and phenolic compounds. We have searched the literature on plants affected by abiotic stress (primar-ily studied in the simulated growth conditions) and their PSMs (including pharmacological activities) from PubMed, Scopus, MEDLINE Ovid, Google Scholar, Databases, and journal websites. We used search keywords: "stress-affected plants," "plant secondary metabolites, "abiotic stress," "climatic influence," "pharmacological activities," "bioactive compounds," "drug discovery," and "medicinal plants" and retrieved published literature between 1973 to 2021. This review provides an overview of variation in bioactive phytochemical production in plants under various abiotic stress and their potential in the biodiscovery of therapeutic drugs. We excluded studies on the effects of biotic stress on PSMs.
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The rapid appearance of resistant malarial parasites after introduction of atovaquone (ATQ) drug has prompted the search for new drugs as even single point mutations in the active site of Cytochrome b protein can rapidly render ATQ ineffective. The presence of Y268 mutations in the Cytochrome b (Cyt b) protein is previously suggested to be responsible for the ATQ resistance in Plasmodium falciparum (P. falciparum). In this study, we examined the resistance mechanism against ATQ in P. falciparum through computational methods. Here, we reported a reliable protein model of Cyt bc1 complex containing Cyt b and the Iron-Sulphur Protein (ISP) of P. falciparum using composite modeling method by combining threading, ab initio modeling and atomic-level structure refinement approaches. The molecular dynamics simulations suggest that Y268S mutation causes ATQ resistance by reducing hydrophobic interactions between Cyt bc1 protein complex and ATQ. Moreover, the important histidine contact of ATQ with the ISP chain is also lost due to Y268S mutation. We noticed the induced mutation alters the arrangement of active site residues in a fashion that enforces ATQ to find its new stable binding site far away from the wild-type binding pocket. The MM-PBSA calculations also shows that the binding affinity of ATQ with Cyt bc1 complex is enough to hold it at this new site that ultimately leads to the ATQ resistance.
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Withania somnifera root extract has been used traditionally in ayurvedic system of medicine as a memory enhancer. Present study explores the ameliorative effect of withanolide A, a major component of withania root extract and its molecular mechanism against hypoxia induced memory impairment. Withanolide A was administered to male Sprague Dawley rats before a period of 21 days pre-exposure and during 07 days of exposure to a simulated altitude of 25,000 ft. Glutathione level and glutathione dependent free radicals scavenging enzyme system, ATP, NADPH level, γ-glutamylcysteinyl ligase (GCLC) activity and oxidative stress markers were assessed in the hippocampus. Expression of apoptotic marker caspase 3 in hippocampus was investigated by immunohistochemistry. Transcriptional alteration and expression of GCLC and Nuclear factor (erythroid-derived 2)-related factor 2 (Nrf2) were investigated by real time PCR and immunoblotting respectively. Exposure to hypobaric hypoxia decreased reduced glutathione (GSH) level and impaired reduced gluatathione dependent free radical scavenging system in hippocampus resulting in elevated oxidative stress. Supplementation of withanolide A during hypoxic exposure increased GSH level, augmented GSH dependent free radicals scavenging system and decreased the number of caspase and hoescht positive cells in hippocampus. While withanolide A reversed hypoxia mediated neurodegeneration, administration of buthionine sulfoximine along with withanolide A blunted its neuroprotective effects. Exogenous administration of corticosterone suppressed Nrf2 and GCLC expression whereas inhibition of corticosterone synthesis upregulated Nrf2 as well as GCLC. Thus present study infers that withanolide A reduces neurodegeneration by restoring hypoxia induced glutathione depletion in hippocampus. Further, Withanolide A increases glutathione biosynthesis in neuronal cells by upregulating GCLC level through Nrf2 pathway in a corticosterone dependenet manner.
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Neural circuits are functional ensembles of neurons that are selectively interconnected by chemical or electrical synapses. Here we describe a synthetic biology approach to the study of neural circuits, whereby new electrical synapses can be introduced in novel sites in the neuronal circuitry to reprogram behaviour. We added electrical synapses composed of the vertebrate gap junction protein Cx36 between Caenorhabditis elegans chemosensory neurons with opposite intrinsic responses to salt. Connecting these neurons by an ectopic electrical synapse led to a loss of lateral asymmetry and altered chemotaxis behaviour. In a second example, introducing Cx36 into an inhibitory chemical synapse between an olfactory receptor neuron and an interneuron changed the sign of the connection from negative to positive, and abolished the animal's behavioural response to benzaldehyde. These data demonstrate a synthetic strategy to rewire behavioural circuits by engineering synaptic connectivity in C. elegans.
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Parkinson's disease (PD) is the second most common degenerative disorder of the central nervous system that impairs motor skills and cognitive function. To date, the disease has no effective therapies. The identification of new drugs that provide benefit in arresting the decline seen in PD patients is the focus of much recent study. However, the lengthy time frame for the progression of neurodegeneration in PD increases both the time and cost of examining potential therapeutic compounds in mammalian models. An alternative is to first evaluate the efficacy of compounds in Caenorhabditis elegans models, which reduces examination time from months to days. n-Butylidenephthalide is the naturally-occurring component derived from the chloroform extract of Angelica sinensis. It has been shown to have anti-tumor and anti-inflammatory properties, but no reports have yet described the effects of n-butylidenephthalide on PD. The aim of this study was to assess the potential for n-butylidenephthalide to improve PD in C. elegans models. In the current study, we employed a pharmacological strain that expresses green fluorescent protein specifically in dopaminergic neurons (BZ555) and a transgenic strain that expresses human α-synuclein in muscle cells (OW13) to investigate the antiparkinsonian activities of n-butylidenephthalide. Our results demonstrate that in PD animal models, n-butylidenephthalide significantly attenuates dopaminergic neuron degeneration induced by 6-hydroxydopamine; reduces α-synuclein accumulation; recovers lipid content, food-sensing behavior, and dopamine levels; and prolongs life-span of 6-hydroxydopamine treatment, thus revealing its potential as a possible antiparkinsonian drug. n-Butylidenephthalide may exert its effects by blocking egl-1 expression to inhibit apoptosis pathways and by raising rpn-6 expression to enhance the activity of proteasomes. n-Butylidenephthalide may be one of the effective neuroprotective agents for PD.
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Inhibition of DAF-2 (insulin-like growth factor 1 [IGF-1] receptor) or RSKS-1 (S6K), key molecules in the insulin/IGF-1 signaling (IIS) and target of rapamycin (TOR) pathways, respectively, extend lifespan in Caenorhabditis elegans. However, it has not been clear how and in which tissues they interact with each other to modulate longevity. Here, we demonstrate that a combination of mutations in daf-2 and rsks-1 produces a nearly 5-fold increase in longevity that is much greater than the sum of single mutations. This synergistic lifespan extension requires positive feedback regulation of DAF-16 (FOXO) via the AMP-activated protein kinase (AMPK) complex. Furthermore, we identify germline as the key tissue for this synergistic longevity. Moreover, germline-specific inhibition of rsks-1 activates DAF-16 in the intestine. Together, our findings highlight the importance of the germline in the significantly increased longevity produced by daf-2 rsks-1, which has important implications for interactions between the two major conserved longevity pathways in more complex organisms.
Beta-caryophyllene (BCP) is a natural bicyclic sesquiterpene and is a FDA approved food additive, found as an active ingredient in essential oils of numerous edible plants. It possesses a wide range of biological activities including anti-oxidant, anti-inflammatory, anti-cancerous and local anesthetic actions. We used the well established Caenorhabditis elegans model system to elucidate the stress modulatory and lifespan prolonging action of BCP. The present study is the first time reporting of the lifespan extension and stress modulation potential of BCP in C. elegans. On evaluation, it was found that 50μM dose of BCP increased the lifespan of C. elegans over 22% (P≤0.0001) and significantly reduced intracellular free radical levels, maintaining cellular redox homeostasis. Moreover, the results suggest that BCP modulates feeding behaviour, pharyngeal pumping and body size effectively. Further, this compound also exhibited significant reduction in intestinal lipofuscin levels. In the present investigation, we have predicted possible biological molecular targets for BCP using molecular docking approaches and BCP was found to have interaction with SIR-2.1, SKN-1 and DAF-16. The prediction was further validated in vivo using mutants and transgenic strains unravelling underlying genetic mechanism. It was observed that BCP increased lifespan of mev-1 and daf-16 but failed to augment lifespan in eat-2, sir-2.1 and skn-1 mutants. Relative quantification of mRNA demonstrated that several genes regulating oxidative stress, xenobiotic detoxification and longevity were modulated by BCP treatment. The study unravels the involvement of multiple signalling pathways in BCP mediated lifespan extension.