biomedicines

Article
Coconut (Cocos nucifera) Ethanolic Leaf Extract
Reduces Amyloid-β (1-42) Aggregation and Paralysis
Prevalence in Transgenic Caenorhabditis elegans
Independently of Free Radical Scavenging and
Acetylcholinesterase Inhibition
Rafael Vincent Manalo 1,† , Maries Ann Silvestre 2 , Aza Lea Anne Barbosa 2
and Paul Mark Medina 1,†, *
1 Department of Biochemistry and Molecular Biology, College of Medicine, University of the
Philippines Manila, Ermita, Manila 1000, Philippines; rmmanalo3@up.edu.ph
2 Juan R. Liwag Memorial High School, Gapan, Nueva Ecija 3105, Philippines;
mariesannrs@yahoo.com (M.A.S.); barbosa.azaleaanne@yahoo.com (A.L.A.B.)
* Correspondence: pmbmedina@post.upm.edu.ph; Tel.: +63-949-452-3202
† These authors contributed equally to this work.

Academic Editor: Wenbin Deng

Received: 16 January 2017; Accepted: 17 April 2017; Published: 21 April 2017

Abstract: Virgin coconut oil (VCO) has been the subject of several studies which have
aimed to alleviate Alzheimer’s disease (AD) pathology, focusing on in vitro antioxidant and
acetylcholinesterase (AChE) inhibitory activities. Here, we studied an underutilized and lesser-valued
part of the coconut tree, specifically the leaves, using in vitro and in vivo approaches. Coconut leaf
extract (CLE) was screened for antioxidant and AChE inhibitory properties in vitro and therapeutic
effects in two strains of transgenic Caenorhabditis elegans expressing amyloid-β1–42 (Aβ1-42 ) in muscle
cells. CLE demonstrated free radical scavenging activity with an EC50 that is 79-fold less compared
to ascorbic acid, and an AChE inhibitory activity that is 131-fold less compared to Rivastigmine.
Surprisingly, in spite of its low antioxidant activity and AChE inhibition, CLE reduced Aβ deposits
by 30.31% in CL2006 in a dose-independent manner, and reduced the percentage of paralyzed
nematodes at the lowest concentration of CLE (159.38 µg/mL), compared to dH2 O/vehicle (control).
Phytochemical analysis detected glycosides, anthocyanins, and hydrolyzable tannins in CLE, some
of which are known to be anti-amyloidogenic. Taken together, these findings suggest that CLE
metabolites alternatively decrease AB1–42 aggregation and paralysis prevalence independently of free
radical scavenging and AChE inhibition, and this warrants further investigation on the bioactive
compounds of CLE.

Keywords: Alzheimer’s disease (AD); sporadic inclusion body myositis (sIBM); coconut leaf extract;
Cocos nucifera; Caenorhabditis elegans

1. Introduction
Alzheimer’s disease (AD) afflicts more than 26 million people globally, and is said to be the most
common neurodegenerative disease worldwide. It is mainly idiopathic, with late-onset affecting more
than 90% of cases [1]. It is classified, across its pathological hallmarks, as a tauopathy-characterized by
hyperphosphorylated, filamentous tau aggregates prior to microtubule collapse—a major requisite for
the formation of neurofibrillary tangles [2,3]. The presence of amyloid-β (Aβ) plaques are thought to
constitute the main biomarkers for AD. The combined criteria from CERAD, Braak NFT, and Thal,

Biomedicines 2017, 5, 17; doi:10.3390/biomedicines5020017 www.mdpi.com/journal/biomedicines

Biomedicines 2017, 5, 17 2 of 13

for instance, maintain the definition of AD as a procession from a complex of clinically pathological
diagnoses of Aβ aggregates, neurofibrillary tangles, and cognitive dysfunction; however, recent case
series showing AD-diagnosed patients lacking Aβ deposits challenge the generalizability of the criteria,
as well as the role of protein aggregates in the development and progression of AD [4]. Nonetheless,
the combined presence of tangles and plaques, which are associated with progressive dementia and
neurodegeneration, is the most widely accepted view [5].
Interestingly, the presence of Aβ and tau pathologies in AD are said to associate with inflammatory
responses, the latter paving the way to the development of the disease in question. The soluble tau
oligomers, which are hypothesized to bring about more drastic adverse events related to tangle
formation, were shown to co-localize with inflammation-associated astrocytes, microglia, and related
cytokines [3]. In addition, a recent study by Laurent et al. showed that T-cells from the hippocampus
mediate the inflammation process and promote cognitive decline [6]. What is alarming is the fact
that tau proteins propagate their pathological assemblies in a prion-like manner [7], which tends to
aggravate the degree of inflammation and progression of AD.
Remarkably, the symptoms and general features of AD coincide with that of sporadic inclusion
body myositis (sIBM)—a form of skeletal muscle disease. As with AD, sIBM is by consensus a
combination of muscular degeneration and inflammation [8], often characterized by slow-onset
atrophy, lethargy, and dysphagia, with hallmark biomarkers of filamentous inclusions and intracellular
Aβ deposits in muscle cells [9]. As with AD, sIBM is late-onset, with the highest prevalence occurring
in older age groups, especially beginning at the age of 50 [8,10]. Thus, it is seen that both degenerative
diseases contribute to lessening the quality of life of the elderly, aggravated by the lack of a known cure.
There have been many researches on VCO and its potential to salvage neurons from
amyloid-induced degeneration, reduce inflammation, and provide ketone bodies for therapeutic
effects and increased cognitive function [11–14]. However, it is proposed that the anti-amyloidogenic
and anti-aggregatory properties are to be found in the phenolic compounds of the plant [15,16],
which motivated the present study. Here, we demonstrate the antioxidant and acetylcholinesterase
(AChE) inhibitory properties of coconut leaf extract (CLE) using 2,2-diphenyl-1-picryl-hydrazyl (DPPH)
scavenging and AChE inhibition assays. We then demonstrated that CLE reduced Aβ aggregation
and paralysis in vivo. Phytochemical analysis then revealed that glycosides, anthocyanins, and
hydrolyzable tannins were present in the extract, warranting further investigation on these bioactive
compounds in CLE.

2. Materials and Methods

2.1. Leaf Harvesting and Crude Ethanolic Extraction of Cocos nucifera Leaves
Leaves were harvested from mature coconut (Cocos nucifera) trees in ecologically acceptable
proportions at San Nicolas, Gapas City of the Nueva Ecija province, Philippines. For confirmation and
the correct classification of plant identity, samples were sent to the botany division of The National
Museum at Ermita, Manila, Philippines, and to the Industrial Technology Development Institute (ITDI)
of the Department of Science and Technology (Taguig, Philippines) for standardized crude ethanolic
extraction. For phytochemical analysis, the same outsource implemented the procedure, as requested.

2.2. Caenorhabditis elegans Strains

All strains were obtained from and provided by the Caenorhabditis Genetics Center (CGC) of
the University of Minnesota, which is funded by the NIH Office of Research Infrastructure Programs
(P40 OD010440). For the present study, the following strains were used:
N2—wild type
C. elegans var. Bristol
CL4176—expresses Aβ1–42
dvIs27 [myo-3p::A-β(1-42)::let-851 30 UTR]
Biomedicines 2017, 5, 17 3 of 13

CL2006—expresses Aβ1–42 ; temperature-sensitive

dvIs2 [pCL12(unc-54/human Aβ peptide 1-42 minigene) + pRF4]

CL2006 strains were maintained at 20 ◦ C, while CL4176 strains were kept at 16 ◦ C, and both were
periodically transferred to OP50-incubated nematode growth media (NGM) plates. For phenotypic
activation in CL4176, heat-sensitive nematodes were continually exposed at 25 ◦ C post-treatment.
All plates were prepared according to the protocol of Steirnagle [17].

2.3. Antioxidant and Acetylcholinesterase Inhibitory Activities of Crude CLE Extracts

Leaf extracts were tested for their antioxidant properties and capability of inhibiting AChE via
the DPPH and AChE inhibition assays, respectively. For the DPPH scavenging assay, five treatments
of CLE with progressing concentrations (in µg/mL) and one control were prepared, as follows. For the
positive control, ascorbic acid at the same concentrations (in µg/mL) as the CLEs were tested. On the
other hand, methanol was used as the negative control. Absorbance was then taken after 30 min at
517 nm and dose-response curves for both CLE and ascorbic acid were graphed, from which the EC50
value was directly obtained. The EC50 is the effective concentration where a half-maximal effect is
observed. The AChE inhibition assay was similarly performed, consisting of 11 treatment groups.
The positive control used in this assay was Rivastigmine, which is a known AChE inhibitory drug
and is currently used as a treatment for patients with AD [18]. Meanwhile, the protocol for the AChE
inhibitory assay was adopted from Ellman et al. [19]. Enzyme activity was obtained at 25-second
intervals for 31 readings at 420 nm, using the Thermoscientific Multiskan. Both assays were done
in triplicate.

2.4. Nematode Toxicity Assay

To eliminate the confounding factor of toxicity in the procedure, nematodes – wild-type (WT) and
transgenic (TG) – were exposed to increasing concentrations of CLE (12.75, 127.5, 1275 µg/mL). Further,
nematodes were exposed to sterile dH2 O as the negative control. All treatment groups were exposed
to intervention for 24 h, adopted from Qiao et al. [20], after which the percent mortality was obtained
using a stereoscope from the Department of Biochemistry and Molecular Biology, College of Medicine,
University of the Philippines Manila (Manila, Philippines). The toxicity assay aforementioned was
done in triplicate.

2.5. Amyloid-β Aggregation and Paralysis Tests

To test the protective effect of CLE against AD and sIBM in vivo, two C. elegans strains (CL2006
and CL4176) were exposed to varying concentrations of CLE. In all procedures, sterile dH2 O acted as
the negative control. For CL2006, nematodes were divided into groups and exposed to treatment at
20 ◦ C for five days. Nematodes were then sampled via worm-picking and placed on 2% agarose pads
with two drops of ~80% glycerol, before being placed on a microscope slide. At this point, some of
the worms may perish, so the glycerol may be lowered to ~50% or be viewed within five minutes or
less to prevent confounding degradation. Aβ deposits were viewed under bright-field microscopy
to improve protein deposit scoring. For CL4176, treatment was administered prior to heat activation.
Nematodes were divided into groups and exposed at different concentrations of CLE at 16 ◦ C for 36 h.
The temperature was then raised to 25 ◦ C for two hours, and the proportion of paralyzed nematodes
was obtained every 12 for 36 h. Likewise, the aggregation assay was employed using similar conditions
to the paralysis assay (Table 1). For both strains, the assays were done in triplicate.

2.6. Statistical Analyses

The effects of intervention and control were compared via analysis of variance (ANOVA) and IC50
and EC50 were determined using GraphPad Prism 6.0 (GraphPad Software, Inc., La Jolla, CA, USA).
For all of the statistical tests, a p-value of p < 0.05 was accepted as statistically significant. To further
Biomedicines 2017, 5, 17 4 of 13

test the validity of the results, post-hoc two-tailed t-tests (in least significant difference) were computed
where applicable.

TableBiomedicines
1. Coconut 2017, 5, 17 extract (CLE) treatment groups for Aβ
leaf 4 of 13
1–42 aggregation and paralysis assays.

further test the validity of the results, post-hoc two-tailed t-tests (in least significant difference) were
Treatment Group CLE Concentration (µg/mL) C. elegans Strain Temperature
computed where applicable.
Aβ1–42 Aggregation Assay
Table
1 (−) treatment groups for Aβ1–42 aggregation and paralysis assays.
1. Coconut leaf extract (CLE)
2 159.38
Treatment Group CLE Concentration (μg/mL) C. elegans Strain Temperature
3 318.75
CL2006
Aβ1–42 Aggregation Assay 20 ◦ C
4 637.5
5 1 (−)
1275
6 2 159.38
6375
3 318.75
CL2006 20 °C
4 637.5 Aβ1–42 Paralysis Assay
1 5 (1275
−)
2 6 6375
159.38
3 318.75 Aβ1–42 Paralysis Assay Pre-treatment period: 10–16 ◦ C
CL4176
4 1 637.5
(−) Post-treatment period: 24 ◦ C
5 2 1275
159.38
6 3 6375
318.75 Pre-treatment period: 10–16 °C
CL4176
4 637.5 Post-treatment period: 24 °C
5 1275
3. Results 6 6375

3.1. Coconut Leaf Extract (CLE) Neutralizes DPPH Radicals in a Dose-Dependent Manner
3. Results

In the3.1.
study conducted,
Coconut Leaf Extract leaves from Cocos
(CLE) Neutralizes DPPH nucifera
Radicals were first testedManner
in a Dose-Dependent in terms of their antioxidant
property and AChE In the study conducted, leaves from Cocos nucifera were first tested in terms of theirpurposes.
inhibitory activity, before being tested in vivo, for practical antioxidant Here, we
checked whether
property and AChE inhibitory activity, before being tested in vivo, for practical purposes. Here, we at 517 nm.
the CLE would neutralize the DPPH radical, thus lowering its absorbance
We found checked
that at whether
the λmax theofCLE wouldCLE
DPPH, neutralize the DPPHDPPH
neutralizes radical,with
thus lowering
an EC50itsofabsorbance
18.11 µg/mL at 517 (Table 2),
nm. We found that at the λmax of DPPH, CLE neutralizes DPPH with an EC50 of 18.11 μg/mL (Table
less than one-fifth of the concentration of the 4th treatment group (Figure 1A). This implies that at a
2), less than one-fifth of the concentration of the 4th treatment group (Figure 1A). This implies that at
relatively low concentration,
a relatively CLE elicits
low concentration, CLEanelicits
antioxidant effect.effect.
an antioxidant Further, we found
Further, we found thatthat
thethe
antioxidant
property ofantioxidant
CLE in vitro wasofdose-dependent,
property in that higherinconcentrations
CLE in vitro was dose-dependent, of CLE led
that higher concentrations to greater
of CLE led losses
to greater losses
of DPPH absorbance atof517
DPPH
nmabsorbance at 517Apparently,
(Figure 1A). nm (Figure 1A). Apparently,
there was no there was no difference
difference in
in the antioxidant
the antioxidant activity between 100–1000 μg/mL, as confirmed by one-way analysis of variance
activity between 100–1000 µg/mL, as confirmed by one-way analysis of variance (ANOVA). However,
(ANOVA). However, the antioxidant activity at these concentrations might have already been
the antioxidant activity
saturated. In terms atofthese
its ECconcentrations might have already been saturated. In terms of its
50, the antioxidant activity of CLE was about 79-fold less—implying a

EC50 , the antioxidant activity

possible contribution from ofantioxidant
CLE wasactivity
aboutagainst
79-fold
Aβless—implying
that is less manifoldathan
possible
if it werecontribution
ascorbic from
antioxidant acid.
activity against Aβ that is less manifold than if it were ascorbic acid.

2,2-diphenyl-1-picryl-hydrazyl
Figure 1. Figure 1. 2,2-diphenyl-1-picryl-hydrazyl (DPPH) scavenging
(DPPH) scavenging and and acetylcholinesterase
acetylcholinesterase (AChE) (AChE)
inhibition by ethanolic coconut (Cocos nucifera) leaf extract in vitro. The CLE was tested
inhibition by ethanolic coconut (Cocos nucifera) leaf extract in vitro. The CLE was tested at varying at varying
concentrations in vitro for antioxidant activity and the ability to inhibit standard AChE. (A) DPPH
concentrations in vitro for antioxidant activity and the ability to inhibit standard AChE. (A) DPPH
scavenging effect of CLE compared with the control (ascorbic acid). At 100 μg/mL, CLE neutralized
scavenging effect of CLE compared with the control (ascorbic acid). At 100 µg/mL, CLE neutralized
DPPH radicals by 93.85% (n = 3). *** p < 0.0001 when compared with the control at the same
concentration using a two-tailed t-test. p < 0.05 was used to compare treatment groups 4 to 6
(100–1000 µg/mL) using one-way ANOVA; (B) Dose-response curves comparing the AChE inhibitory
activity of CLE and positive control (Rivastigmine) in vitro.
Biomedicines 2017, 5, 17 5 of 13

Table 2. Scavenged DPPH free radicals and inhibited AChE at various concentrations of CLE.

Treatment Group Concentration (µg/mL) EC50 /IC50 (µg/mL) Positive Control

DPPH Scavenging Assay
CLE Control
1 0.1
2 1
Ascorbic acid
3 10 18.11 0.23
4 100
5 300
6 1000
AChE Inhibition Assay
1 0.01
2 0.1
3 1
4 5
5 10
6 50 3218.56 24.52 Rivastigmine
7 100
8 500
9 1000
10 3000
11 3500
EC50 /IC50 : the concentration at which a half-maximal response is observed, and depends on context.

3.2. CLE Inhibits AChE Less Effectively than Does Rivastigmine In Vitro
We then checked whether CLE inhibits AChE, which is postulated to confer protection from
complications in AD and is therefore a desired effect. The results showed that when CLE is
homogenized with AChE and incubated at 25 ◦ C for 15 min, it inhibits the activity on acetylcholine
metabolism with an IC50 of 3218.56 µg/mL. Using the standard of treatment (Rivastigmine), the IC50
value was found to be 24.52 µg/mL (Figure 1B). This implies the presence of an inhibitory activity
only at manifold higher concentrations, as compared with vehicle (Table 2).

3.3. CLE is Non-Lethal to Wild-type and Transgenic C. elegans CL2006 and CL4176
To eliminate the possibility that CLE has intrinsically deleterious effects in vivo, we exposed
strains N2, CL2006, and CL4176 at concentrations progressing to three orders of magnitude (12.75, 127.5,
1275 µg/mL). Observations post-treatment showed a 100% survival rate for all nematodes, indicating
that at these concentrations, any lethal effects that are observed are solely due to Aβ1–42 expression.

3.4. CLE Significantly Reduces Aβ Aggregate Deposits in Transgenic C. elegans Strains CL2006 and CL4176
We next determined whether CLE would elicit protective effects in vivo. Varying concentrations of
CLE were administered to groups of transgenic C. elegans after a maturation period of three days; then,
measurements were successively taken post-treatment, according to established protocol. For testing
the therapeutic effects of CLE, a C. elegans strain population continuously expressing Aβ aggregates in
the muscle cell walls (CL2006) was used.
This tested the efficacy of CLEs against plaque deposition after the formation of Aβ proteins.
In vivo, CLE protected against Aβ toxicity by reducing the deposits of visible aggregates in the cell
walls of CL2006 (Figure 2A–D). Compared to vehicle, a mean reduction of 30.31% in Aβ deposits
was observed. Two-tailed t-tests confirmed this observation as highly statistically significant at
p < 0.0001 (Figure 2E). Further, there were no significant differences observed on the deposition of Aβ
aggregates, regardless of the CLE concentration administered. This implies that the efficacy of CLE is
somewhat dose-independent.
Biomedicines 2017, 5, 17 6 of 13

of CL2006 (Figure 2A–D). Compared to vehicle, a mean reduction of 30.31% in Aβ deposits was
observed. Two-tailed t-tests confirmed this observation as highly statistically significant at p < 0.0001
(Figure 2E). Further, there were no significant differences observed on the deposition of Aβ
Biomedicines
aggregates, 2017, 5, 17
regardless of the CLE concentration administered. This implies that the efficacy of6CLE
of 13

is somewhat dose-independent.

Figure
Figure 2.2. CLE significantly reduces amyloid-β plaque deposits in transgenic C. C. elegans
elegans strains
strains CL2006,
CL2006,
independently
independently of of concentration. Nematodes were
concentration. Nematodes were transferred
transferred toto OP50-incubated
OP50-incubated NGM NGM plates
plates and
and
allowed to mature for three days at 20 ◦ C, after which they were fed with either vehicle or CLE at
allowed to mature for three days at 20 °C, after which they were fed with either vehicle or CLE at
varying concentrations ad
varying concentrations ad libitum. The amyloid-β
libitum. The amyloid-β deposits
deposits in
in CL2006
CL2006 were
were then
then counted
counted five
five days
days
post-treatment, and were pooled in triplicate. Dark portions in the cell wall of C.
post-treatment, and were pooled in triplicate. Dark portions in the cell wall of C. elegans indicateelegans indicate
the
the protein
protein deposits.
deposits. CL2006
CL2006 strains
strains werewere
fed fed
withwith either
either (A) (A) vehicle
vehicle or (C)
or (C) CLE.CLE. Detailed
Detailed images
images of
of the
the body walls of nematodes fed with (B) vehicle or (D) CLE are shown. White
body walls of nematodes fed with (B) vehicle or (D) CLE are shown. White circles identify the circles identify the
aggregate
aggregate deposits;
deposits; (E)
(E) Aβ
Aβ deposits
deposits were
were counted
counted per
per treatment
treatment group
group atat varying
varying concentrations
concentrations of of
CLE (in µg/mL). *** p
CLE (in μg/mL). *** p < 0.0001 when compared with vehicle using two-tailed t-test. p <
< 0.0001 when compared with vehicle using two-tailed t-test. p 0.05 was
< 0.05 was used
used
to
to test
test for
for significance
significance between
between treatment
treatment groups
groups using
using one-way
one-way ANOVA.
ANOVA.

3.5. Paralysis of CL4176 C. elegans Strains Was Partially Relieved

Relieved by
by Exposure
Exposure to
to CLE
CLE Prior
Prior to
to Heat
Heat
Activation of Aβ Expression
Activation of Aβ Expression
The
The next
next question
question to to ask
ask was
was whether
whether CLEsCLEs elicited
elicited beneficial
beneficial effects
effects when
when administered
administered at at an
an
earlier time point. Therefore, a strain only expressing Aβ proteins upon exposure
earlier time point. Therefore, a strain only expressing Aβ proteins upon exposure to a temperature of to a temperature
of ◦ C and above proved the utility. To this end, CL4176 C. elegans strains were exposed to CLE
25 25
°C and above proved the utility. To this end, CL4176 C. elegans strains were exposed to CLE for 36
for 36 h, before
h, before allowingallowing Aβ production.
Aβ production. This is This
in is in stark
stark contrast
contrast withwith CL2006,
CL2006, primarily
primarily because
because the
the prophylactic effect of CLE was now being tested. In all of triplicates
prophylactic effect of CLE was now being tested. In all of triplicates examined, nematodes assigned examined, nematodes
assigned
to treatmentto treatment
groups withgroups CLEwith CLE exposure
exposure had aprevalence
had a lower lower prevalence of paralysis
of paralysis at the at thehour
12th 12th post-
hour
post-induction,
induction, which then led to paralysis no better than or worse than the control, except at the
which then led to paralysis no better than or worse than the control, except at the
concentration of 159.38 µg/mL
concentration of 159.38 μg/mL (Figure 3E).(Figure 3E).
To
To assess
assess thethe link
link between
between aggregation
aggregation and and paralysis,
paralysis, the the CL4176
CL4176 strain
strain was
was used
used for for the
the
aggregation assay at the concentration with the lowest apparent effect
aggregation assay at the concentration with the lowest apparent effect in CL4176 (1275 μg/mL) at the in CL4176 (1275 µg/mL)
at thehour,
36th 36th with
hour, conditions
with conditionssimilarsimilar
to theto the paralysis
paralysis assay.assay.
As theAsresults
the results
showed, showed, aggregates
aggregates were
were lessened by up to 54.55% compared to vehicle at 12 h post-induction.
lessened by up to 54.55% compared to vehicle at 12 h post-induction. To this end, it is likely To this end, it is likely that
that
paralysis
paralysis reduction
reduction was was drastic
drastic atat this
this time
time point
point due
due toto aa high
high reduction
reduction in in Aβ deposits—however,
Aβ deposits—however,
the
the lack
lack of of aa trend
trend between
between paralysis
paralysis andand AβAβ aggregation
aggregation at at the
the 24th
24th and
and 36th
36th hours
hours suggests
suggests that that
CLE
CLE can only delay paralysis, and that Aβ aggregation is not the only determinant of paralysis
can only delay paralysis, and that Aβ aggregation is not the only determinant of paralysis in in
CL4176, since the concentration that worked was the lowest (159.38 µg/mL),
CL4176, since the concentration that worked was the lowest (159.38 μg/mL), which should also have which should also have
the
the lowest
lowest antioxidant
antioxidant and and AChE
AChE inhibitory
inhibitory activities
activities of
of all
all the
the CLECLE treatment
treatment groups.
groups. TheThe observed
observed
proportion of nematodes relieved of motor deficits was statistically confirmed
proportion of nematodes relieved of motor deficits was statistically confirmed with a value with a value of p <of0.05.
p<
0.05. There was a certain difficulty in distinguishing the proportion of relieved nematodes using the
CL2006 strains,
There was asocertain
the paralysis assay
difficulty was only donethe
in distinguishing on proportion
CL4176. This of difficulty is in fact warranted,
relieved nematodes using the
due to the
CL2006 inherent
strains, so theresistance
paralysisofassay
CL2006wastoonly
paralysis—indeed,
done on CL4176.the Thisvariability
difficultyofisparalysis between
in fact warranted,
nematodes was found to be unpredictable, with some nematodes never becoming paralyzed [21].
Hence, the need to use CL4176 for paralysis assays related to Aβ expression maintains its essentiality.
Biomedicines 2017, 5, 17 7 of 13

due to the inherent resistance of CL2006 to paralysis—indeed, the variability of paralysis between
nematodes was found to be unpredictable, with some nematodes never becoming paralyzed [21].
Biomedicines 2017, 5, 17 7 of 13
Hence, the need to use CL4176 for paralysis assays related to Aβ expression maintains its essentiality.

Figure3.3.CLE
Figure CLEreduces
reducesparalysis
paralysisprevalence
prevalencein inC.
C.elegans
elegansstrain
strainCL4176.
CL4176.Nematodes
Nematodeswere wereexposed
exposedtoto
(A)
(A)vehicle
vehicleor
or(C)
(C)CLE
CLEininconcentrations
concentrationssimilar
similartotothe
theAβ
Aβaggregation
aggregationassay
assayininvivo.
vivo.AβAβexpression
expression
was ◦ C after 36 h post-treatment of CLE or vehicle at 16 ◦ C for 36 h. Detailed images
wasinduced
inducedatat 25
25 °C after 36 h post-treatment of CLE or vehicle at 16 °C for 36 h. Detailed images of
of thebody
the bodywalls
wallsofofnematodes
nematodesfedfedwith
with(B)
(B)vehicle
vehicle or
or (D)
(D) CLE
CLE are
are shown.
shown. White
White circles
circles identify
identifythe
the
aggregate
aggregate deposits; (E) The proportion of paralyzed nematodes were gathered at 2, 12, 24,and
deposits; (E) The proportion of paralyzed nematodes were gathered at 2, 12, 24, and3636hh
post-induction.
post-induction.To Todetermine
determinethethelink
linkof
ofaggregation
aggregationin inparalysis,
paralysis,the
theaggregation
aggregationassay
assaywas
wasdone
doneinin
CL4176 using the paralysis assay conditions.
CL4176 using the paralysis assay conditions.

3.6.
3.6.Phytochemicals
PhytochemicalsPresent
Presentin
inCLE
CLEPossibly
PossiblyInteract
Interactwith
withAβ
Aβ1–42 Peptide
1–42 Peptide

In
Inthe
thephytochemical
phytochemicalanalysis,
analysis,screening
screeningof ofthe
thecompounds
compoundsresulted
resultedininthe
thepresence
presenceof ofglycoside
glycoside
compounds,
compounds, flavonoids (particularly anthocyanins), and hydrolyzable tannins (Table 3). Due
flavonoids (particularly anthocyanins), and hydrolyzable tannins (Table 3). Dueto tothe
the
presence of hydrolyzable tannins in the CLE, it was postulated that the metabolites
presence of hydrolyzable tannins in the CLE, it was postulated that the metabolites of such tannins of such tannins
could
couldalso
also contribute
contribute to to the
the efficacy of the
efficacy of the leaf
leaf extract.
extract. Urolithin
UrolithinA, A,aaderivative
derivativeofofellagitannin
ellagitanninthat
thatis
isknown
knowntotoinduce
inducemitophagy
mitophagyand andincrease
increasethe thelifespan
lifespan of C. elegans
of C. elegans [22–25];
[22–25]; gallic
gallic acid, a part of
acid, a part of
hydrolyzable tannins with known antimicrobial, antioxidant, and neuroprotective
hydrolyzable tannins with known antimicrobial, antioxidant, and neuroprotective effects [26–28]; effects [26–28];
and
and pyrogallol,
pyrogallol, aa derivative
derivative ofof gallic
gallic acid
acid whose
whose moiety
moiety isis associated
associated with β-secretase (BACE1)
with β-secretase (BACE1)
inhibition
inhibition[29]
[29]and andtogether
togetherwith
withgallic
gallicacid
acidisisknown
knownto tobe
beanti-amyloidogenic
anti-amyloidogenic[30], [30],could
couldbe bepossible
possible
contributors to the protective effect of CLE in vivo. It was noted that cardiac glycosides
contributors to the protective effect of CLE in vivo. It was noted that cardiac glycosides may may accumulate
in the central in
accumulate nervous systemnervous
the central [31], warranting investigation
system [31], on itsinvestigation
warranting alternative yet
onpromising effect on
its alternative yet
Aβ 1–42 aggregation.
promising effect on Aβ1–42 aggregation.
Table 3. Phytochemical analysis of crude ethanolic CLE.
Table 3. Phytochemical analysis of crude ethanolic CLE.

Test
TestParameter
Parameter Method
Method Result
Result
Alkaloids Mayer/Meyer test Alkaloids absent
Alkaloids Mayer/Meyer test Alkaloids absent
Anthraquinones Bornträger test Anthraquinones absent
Anthraquinones Bornträger test Anthraquinones absent
Cardenolides and bufadienolides Keller-Kiliani test Glycoside compounds present
Cardenolides and bufadienolides
Flavonoids
Keller-Kiliani test
Bate-Smith and Metcalf test
Glycoside compounds present
Anthocyanins present
Flavonoids
Tannins and polyphenolic compounds
Bate-Smith and Metcalf
Ferric chloride test
test Anthocyanins present
Hydrolyzable tannins present
Tannins and polyphenolic
Saponins
compounds Ferric chloride
Froth test
test Hydrolyzable tannins present
Saponins absent
Saponins Froth test Saponins absent

4. Discussion
4. Discussion
4.1. C. elegans as a Model for Alzheimer’s Disease (AD) and Sporadic Inclusion Body Myositis (sIBM)
4.1. C. elegans as a Model for Alzheimer’s Disease (AD) and Sporadic Inclusion Body Myositis (sIBM)
In the study conducted, Aβ aggregation and its toxicity in the form of paralysis were treated
in vivo with ethanolic extracts from coconut (Cocos nucifera) leaves. The transgenic Caenorhabditis elegans
strains used served as biological models of AD and sIBM—the former in terms of Aβ overexpression
Biomedicines 2017, 5, 17 8 of 13

and aggregation, and the latter in terms of expression in the muscle cell walls. Further, the two
strains modelled the effects of administration at two different time points-before the induction of Aβ
expression and after it was expressed. While higher forms of animal models would generate greater
external validity, the use of transgenic nematodes for this study holds several advantages. For one,
human orthologs of disease genes such as tau, relevant neuronal cells, ion channels, and transporters
are conserved in C. elegans, which are important in neurodegenerative disease studies.
To this end, nematodes have been utilized in pathway analysis and drug screening for diseases
such as AD, Parkinson’s disease (PD), and Huntington’s (HD) [32–34]. Further, the conservation
of 12 over 17 signalling cascades, a short generation time, and a life cycle of two to three weeks, as
well as the ease of observation provided by its transparent body lining, provide efficient tools for
scoring protein aggregation [35]. While it is arguable that aggregation in muscle walls does not entirely
represent the multiple factors associated with AD, expression in muscle cells grants a larger picture
of protein formation, and is useful for studies specifically targeting insoluble Aβ deposits, since it
is easier to visualize than if it were in neuronal cells and is actually the tissue of choice for studies
involving such proteins [36,37].

4.2. Reduction of Aβ1–42 Aggregation by CLE Is Independent of Free Radical Scavenging and AChE Inhibition
In the DPPH assay, the EC50 value for radical scavenging was found to be 18.11 µg/mL, and as the
concentration increased further, the antioxidant activity remained the same, as confirmed by ANOVA.
However, this antioxidant activity may or may not play a part in the amelioration of insoluble Aβ
deposits, due to the fact that CLE has a ~79-fold greater EC50 as compared to ascorbic acid (Table 2).
In the AChE inhibition assay, CLE showed even less effective results. In preventing the activity
of standard AChE, the IC50 of CLE was found to be more manifold than that of Rivastigmine
(3218.56 µg/mL versus 24.52 µg/mL), implying a dose-dependent activity whose efficacy is observed
at much higher concentrations. The critical results were manifested in the Aβ aggregation assay in
CL2006. In all treatment groups (159.38.5 to 6375 µg/mL), a steady decrease in protein deposition
of 30.31% was observed. Strikingly, this opposed the dose-dependent activity of CLE against AChE,
since at every concentration from 160 µg/mL, the efficacy of the extract was consistently maintained.
Were AChE inhibition to contribute significantly, the beneficial effect should be observed between
treatments 4 and 5 (1275 and 6375 µg/mL)—however, this was not the case. Hence, CLE acted against
Aβ deposits through a pathway that involved more than just AChE inhibition.
Further, the results opposed, in some way, the antioxidant activity of CLE against DPPH. CL2006
displayed a 30.31% Aβ reduction from 187.5 µg/mL of CLE, which was also dose-independent and
was statistically the same throughout all the concentrations. While this concentration is about nine
times that of the EC50 value in vitro, we argue that: (1) the composition of CLE as it acts in vivo
would likely change due to metabolism by the C. elegans machinery; and (2) it is expected that at
high concentrations, vitamin C, as well as other antioxidants, should become pro-oxidant in vitro and
in vivo [22–24]—therefore, at least at treatments 3 to 5 (with concentrations 35 to 350 times higher
than EC50 in vitro), Aβ was expected to increase. It is worth noting that previous in vivo studies
demonstrated the therapeutic effects of vitamin C at moderate doses [38,39], which at larger doses
of about 600 mg/kg, resulted in increased neurodegeneration and neuroinflammation in AD rat
models [40]. These findings reflect the duality of vitamin C in terms of its oxidizing ability [41,42],
which may promote Aβ deposition by reacting with metal ions similar to how copper (Cu2+ ) induces
Aβ-mediated H2 O2 generation upon binding to His residues and undergoing redox reactions [43].
Since CLE is non-inferior to ascorbic acid in terms of its antioxidant activity at high concentrations, a
similar trend to previous studies should be reflected in the treatment groups, which were 9–350 times
higher than the DPPH EC50 of CLE. Strikingly, this was not the case, and Aβ deposition neither
increased nor decreased significantly at higher CLE concentrations in vivo. To this end, we hypothesize
that CLE is also acting in a way that opposes the free radical scavenging duality at high concentrations.
Biomedicines 2017, 5, 17 9 of 13

increased nor decreased significantly at higher CLE concentrations in vivo. To this end, we
hypothesize that CLE is also acting in a way that opposes the free radical scavenging duality at high
Biomedicines 2017, 5, 17 9 of 13
concentrations.
We clarify that the alternative anti-aggregatory effect of CLE might be dose-dependent, but is
beingWe masked
clarifyby thethe
that increasing pro-oxidant
alternative activity of
anti-aggregatory antioxidants
effect at higher
of CLE might concentrations ofbut
be dose-dependent, CLE is
(Figure
being 4). This
masked byhypothesis
the increasingis not unlikely, activity
pro-oxidant since recent studies have
of antioxidants also shown
at higher neuroprotective
concentrations of CLE
effects that
(Figure are hypothesis
4). This either independent of antioxidant
is not unlikely, activity
since recent studiesorhave
address a moreneuroprotective
also shown complex interplay of
effects
factors
that are that constitute
either independentan antioxidant,
of antioxidantsuch as lipophilicity
activity or addressand molecular
a more complexweight [44,45].
interplay These
of factors
remarkable
that results
constitute point out the
an antioxidant, possible
such mode of action
as lipophilicity of CLE metabolites
and molecular apart from
weight [44,45]. Theseconventional
remarkable
criteria,point
results suchout asthe
interactions
possible modewithofAβ-related proteins
action of CLE like the
metabolites peroxisome
apart proliferator-activated
from conventional criteria, such
receptor
as gamma
interactions (PPARγ)
with and pro-apoptotic
Aβ-related proteins like theproteins—respectively known to be upregulated
peroxisome proliferator-activated receptor gamma and
downregulated
(PPARγ) by natural proteins—respectively
and pro-apoptotic products such as epigallocatechin gallate (EGCG)
known to be upregulated in green tea [29].
and downregulated by
Further,products
natural CLE may instead
such be interactinggallate
as epigallocatechin with known
(EGCG)proteins
in greenaffecting Aβ deposition
tea [29]. Further, CLE may in instead
C. elegans,
be
such as DAF-2
interacting and FOXO
with known proteins[46,47], all Aβ
affecting of deposition
which warrant furthersuch
in C. elegans, investigation
as DAF-2 and on FOXO
the possible
[46,47],
mechanisms
all on CLE further
of which warrant metabolites.
investigation on the possible mechanisms on CLE metabolites.

Figure 4.
Figure 4. Model
Model forfor
Aβ Aβ
aggregation and paralysis
aggregation reduction
and paralysis by CLEbyin CLE
reduction transgenic C. elegans.C.CLE
in transgenic acts
elegans.
by inhibiting
CLE reactive oxygen
acts by inhibiting reactive species
oxygen (ROS)
speciesand
(ROS)AChEand activities in C. elegans,
AChE activities albeit albeit
in C. elegans, at highat
concentrations—both
high concentrations—both of which would
of which wouldotherwise worsen
otherwise worsen Aβ-induced
Aβ-inducedpathology.
pathology.However,
However, at at high
concentrations, a pro-oxidant effect is expected due to antioxidant
antioxidant excess,
excess, resulting
resulting to to CLE-induced
CLE-induced
ROS production (broken arrow). At concentrations 9 to 350 times times higher
higher than
than thethe EC
EC5050 of CLE, the

effect on
effect on aggregation
aggregationwaswasdose-independent,
dose-independent,asasshown
shownininFigure
Figure2.2.Therefore,
Therefore, it it
is is possible
possible that
that CLE
CLE is
is acting
acting directly
directly through
through compounds
compounds that
that areare anti-aggregatory,
anti-aggregatory, which
which masks
masks thethe pro-oxidant
pro-oxidant activity
activity of
of antioxidants
antioxidants in CLE
in CLE at higher
at higher extract
extract concentrations
concentrations (red
(red “?”).
“?”).

We hypothesize,
We hypothesize, therefore,
therefore, that
that bioactive
bioactive compounds
compounds in in CLE
CLE directly
directly act
act totoinhibit
inhibitAβAβ1–42
1–42
aggregation (Figure
aggregation (Figure 4),
4),and
andififthese
theseanti-aggregatory
anti-aggregatory compounds
compounds in associated CLECLE
in associated are isolated and
are isolated
separated
and fromfrom
separated those thatthat
those confer protection
confer from
protection fromoxidative stress,
oxidative stress,a amuch
muchgreater
greatereffect
effect might be
might be
observed in
observed in vivo.
vivo.

4.3. Reduction
4.3. Reduction of
of Aβ-Induced
Aβ-Induced Paralysis
Paralysis by
by CLE
CLE Is
Is Independent of Free
Independent of Free Radical
Radical Scavenging
Scavenging and
and AChE
Inhibition
AChE Inhibition
In the
the paralysis
paralysisassay,
assay,CL4176
CL4176nematodes
nematodes displayed
displayed a higher
a higher incidence of paralysis
incidence 2 h post-
of paralysis 2 h
induction in treatment
post-induction groupsgroups
in treatment with CLE.withTheCLE. hypothesis that CLE
The hypothesis is deleterious
that in terms of
CLE is deleterious in paralysis
terms of
was testedwas
paralysis by tested
comparing the proportion
by comparing of nematodes
the proportion paralyzed 12
of nematodes h post-induction.
paralyzed The results
12 h post-induction.
showed
The thatshowed
results this was notthis
that thewascase.
not Inthe
thecase.
assays In performed, CL4176 nematodes
the assays performed, were relieved
CL4176 nematodes wereof
paralysisofcompared
relieved paralysis to the control
compared at the
to the lowest
control concentration
at the of CLE (159.38
lowest concentration of CLE μg/mL).
(159.38Since this
µg/mL).
concentration
Since also exhibits
this concentration alsothe lowestthe
exhibits antioxidant and AChEand
lowest antioxidant inhibitory activities, activities,
AChE inhibitory the effect observed
the effect
against paralysis
observed implies an
against paralysis actionanindependent
implies of antioxidant
action independent activityactivity
of antioxidant and AChEand inhibition. From
AChE inhibition.
here, ithere,
From mayitbemayinferred that the
be inferred factors
that determining
the factors the incidence
determining of paralysis
the incidence are different
of paralysis from those
are different from
that aggravate
those its progression.
that aggravate For instance,
its progression. it is known
For instance, that thethat
it is known lifespan of C. elegans
the lifespan of C. is short is
elegans (two to
short
(two to three weeks), more so with transgenic strains, and that as the nematodes age, the capacity to
maintain proteostasis decreases, aggravating the accumulation of insoluble proteins that exist even
Biomedicines 2017, 5, 17 10 of 13

in physiologic conditions [48,49]. Therefore, it is rational to hypothesize that the extracts might also
be increasing the lifespan of the paralyzed nematodes. Should this be the case, then the succeeding
cross-sectional observations would tend to obtain a higher proportion of paralyzed nematodes in
the CLE treatment groups, because more nematodes survive long enough to be counted and these
nematodes, due to an older age, develop a more profound form of paralysis. These results may have
implications in the age and concentration at which CLE administration would elicit its maximum
protective effect against AD and sIBM. Further, it is also possible that CLE has bioactive compounds
which are cytotoxic but non-lethal to the nematodes at very high concentrations, but was not readily
evident in the aggregation and nematode toxicity assays because of the suspected anti-aggregatory
bioactive compound.
We further compared the effects of CLE exposure in CL2006 and CL4176, and found that the
concentration of CLE does not matter in CL2006; rather, at any concentration, the nematodes benefited
in terms of the reduction in Aβ deposits. With CL4176, this was not the case. This is implicative of
two possibilities: (1) CLE contains a bioactive compound that is exclusively effective against Aβ1–42
aggregation; and (2) more factors other than Aβ aggregation, ROS production, and AChE action on
ACh may exist in the progression of paralysis in C. elegans. We recommend using a greater sample size
than n = 30 to assess whether the paralysis trend improves, as well as using higher animal models,
such as mice.

4.4. Anthocyanins, Tannins and Glycosides Are Candidate Compounds Against Aβ1–42 Induced Pathology
Phytochemical screening of CLE showed the presence of anthocyanins, hydrolyzable tannins, and
glycosides. It is important to note that the metabolism of CLE by C. elegans occurred throughout the
study; hence, the isolation of a single compound might be more complex. For instance, secondary
metabolites such as urolithin A, which has been shown to extend the lifespan of C. elegans and
improve muscle strength [22–25], or gallic acid and its derivative pyrogallol that are known as
anti-amyloidogenic compounds [30], are all known secondary metabolites of the phytochemicals
detected and may in part explain the observed differences in the assays performed. Indeed, the
metabolism of phytochemicals in vivo may play a role in the differential effects of CLE in vitro and
in vivo.

5. Conclusions
AD and sIBM are neurodegenerative and inflammatory muscle diseases, respectively, but are
related to each other due to known risk factors related to proteinopathies. One such factor is the
aggregation of Aβ, which allows a “two birds with one stone” approach to drug or extract screening
for cell-distinct diseases. The results of our study show that CLE has an antioxidant activity that is
inferior by 79-fold to ascorbic acid, and an AChE inhibitory activity 131-fold less than Rivastigmine,
a known AChE inhibitory drug being prescribed to AD patients. However, stark contrasts between
the antioxidant and AChE inhibitory activities in vitro, and the protective effect of CLE against Aβ
aggregation and paralysis in vivo, suggest that CLE may act against AD and sIBM in a way that is
independent of free radical scavenging and acetylcholinesterase inhibition. However, the protective
effects of CLE only delay the progression of paralysis and cannot fully salvage the nematodes from
deleterious motor deficits. This warrants further investigations on the time-dependence of CLE
administration and biofunctional activities apart from AChE inhibition and free radical scavenging.
Lastly, the presence of anthocyanins, hydrolyzable tannins, and glycosides direct future researchers to
a guided screening of compounds related to these in an effort to treat Aβ1–42 -induced pathology.

Acknowledgments: All strains were obtained from and provided by the Caenorhabditis Genetics Center
(CGC) of the University of Minnesota, which is funded by the NIH Office of Research Infrastructure Programs
(P40 OD010440). We wish to thank the Department of Education of the Republic of the Philippines for the funding
to do this research. Mary Lorraine Lorido is acknowledged for her valuable contribution in assisting Aza Lea and
Maries Ann. Further, their colleagues at Juan R. Liwag Memorial High School are also acknowledged. No funds
for covering the cost of open access publishing have been given.
Biomedicines 2017, 5, 17 11 of 13

Author Contributions: Paul Mark Medina conceived and designed the experiments, discussed the results,
and designed the figures with Rafael Vincent Manalo, and was the overall supervisor of the experiments;
Rafael Vincent Manalo wrote the paper, managed the C. elegans strains, did additional experiments with
Paul Mark Medina for manuscript revisions, and contributed ideas in discussing the results; Maries Ann Silvestre
and Aza Lea Anne Barbosa performed the experiments; In addition, all authors contributed to the analysis of
the paper.
Conflicts of Interest: The authors declare no conflict of interest. The funding sponsors had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the
decision to publish the results.

References
1. El Gaamouch, F.; Jing, P.; Xia, J.; Cai, D. Alzheimer’s Disease Risk Genes and Lipid Regulators.
J. Alzheimer’s Dis. 2016, 53, 15–29. [CrossRef] [PubMed]
2. Guo, J.L.; Narasimhan, S.; Changolkar, L.; He, Z.; Stieber, A.; Zhang, B.; Gathagan, R.J.; Iba, M.; McBride, J.D.;
Trojanowski, J.Q.; et al. Unique pathological tau conformers from Alzheimer’s brains transmit tau pathology
in nontransgenic mice. J. Exp. Med. 2016, 213, 1–20. [CrossRef] [PubMed]
3. Nilson, A.N.; English, K.C.; Gerson, J.E.; Whittle, T.B.; Crain, C.N.; Xue, J.; Sengupta, U.;
Castillo-Carranza, D.L.; Zhang, W.; Gupta, P.; et al. Tau Oligomers Associate with Inflammation in the Brain
and Retina of Tauopathy Mice and in Neurodegenerative Diseases. J. Alzheimer’s Dis. 2016, 55, 1083–1099.
[CrossRef] [PubMed]
4. Crary, J. Primary age-related tauopathy and the amyloid cascade hypothesis: The exception that proves the
rule. J. Neurol. Neuromed. 2016, 1, 53–57.
5. Hardy, J.; Selkoe, D.J. The amyloid hypothesis of Alzheimer’s disease: Progress and problems on the road to
therapeutics. Science 2002, 297, 353–356. [CrossRef] [PubMed]
6. Laurent, C.; Dorotheé, G.; Hunot, S.; Martin, E.; Monnet, Y.; Duchamp, M.; Dong, Y.; Légeron, F.P.;
Leboucher, A.; Burnouf, S.; et al. Hippocampal T cell infiltration promotes neuroinflammation and cognitive
decline in a mouse model of tauopathy. Brain 2016, 140, 1–17. [CrossRef] [PubMed]
7. Tardivel, M.; Bégard, S.; Bousset, L.; Dujardin, S.; Coens, A.; Melki, R.; Buée, L.; Colin, M. Tunneling nanotube
(TNT)-mediated neuron-to neuron transfer of pathological Tau protein assemblies. Acta Neuropathol. Commun.
2016, 4, 1–14. [CrossRef] [PubMed]
8. Karpati, G.; O’Ferrall, E. Sporadic inclusion body myositis: Pathogenic considerations. Ann. Neurol. 2009, 65,
7–11. [CrossRef] [PubMed]
9. Dalakas, M.C. Sporadic inclusion body myositis—Diagnosis, pathogenesis and therapeutic strategies.
Nat. Clin. Pract. Neurol. 2006, 2, 437–447. [CrossRef] [PubMed]
10. Oh, T.H.; Brumfield, K.A.; Kasperbauer, J.L.; Basford, J.R. Dysphagia in inclusion body myositis: Clinical
features, management, and clinical outcome. Am. J. Phys. Med. Rehabil. 2008, 87, 883–889. [CrossRef]
[PubMed]
11. Hu, Y.; de la Rubia Orti, J.E.; Selvi, S.; Sancho Castillo, S.; Rochina, M.J.; Manresa Ramon, R.;
Montoya-Castilla, I. Coconut Oil: Non-Alternative Drug Treatment Against Alzheimer’s Disease. Nutr. Hosp.
2015, 32, 2822–2827.
12. Nafar, F.; Mearow, K.M. Coconut oil attenuates the effects of amyloid-β on cortical neurons in vitro.
J. Alzheimer’s Dis. 2014, 39, 233–237.
13. Nonaka, Y.; Takagi, T.; Inai, M.; Nishimura, S.; Urashima, S.; Honda, K.; Aoyama, T.; Terada, S. Lauric Acid
Stimulates Ketone Body Production in the KT-5 Astrocyte Cell Line. J. Oleo Sci. 2016, 65, 693–699. [CrossRef]
[PubMed]
14. Dedea, L. Can coconut oil replace caprylidene for Alzheimer disease? J. Am. Acad. Phys. Assist. 2012, 25, 19.
[CrossRef]
15. Fernando, W.M.; Martins, I.J.; Goozee, K.G.; Brennan, C.S.; Jayasena, V.; Martins, R.N. The role of dietary
coconut for the prevention and treatment of Alzheimer’s disease: Potential mechanisms of action. Br. J. Nutr.
2015, 114, 1–14. [CrossRef] [PubMed]
16. Chan, E.W.C.; Lim, Y.Y.; Omar, M. Antioxidant and antibacterial activity of leaves of Etlingera species
(Zingiberaceae) in Peninsular Malaysia. Food Chem. 2007, 104, 1586–1593. [CrossRef]
Biomedicines 2017, 5, 17 12 of 13

17. Stiernagle, T. Maintenance of C. elegans. Available online: http://www.wormbook.org/chapters/www_

strainmaintain/strainmaintain.html (accessed on 20 December 2016).
18. Lefèvre, G.; Callegari, F.; Gstegier, S.; Xiong, Y. Effects of Renal Impairment on Steady-State Plasma
Concentrations of Rivastigmine: A Population Pharmacokinetic Analysis of Capsule and Patch Formulations
in Patients with Alzheimer’s Disease. Drugs Aging 2016, 33, 725–736. [CrossRef] [PubMed]
19. George, L.; Ellman, K.; Courtney, D.; Andres, V., Jr.; Featherstone, R. A new and rapid colorimetric
determination of acetylcholinesterase activity. Biochem. Pharmacol. 1961, 7, 90–95.
20. Qiao, Y.; Zhao, Y.; Wu, Q.; Sun, L.; Ruan, Q.; Chen, Y.; Wang, M.; Duan, J.; Wang, D. Full toxicity assessment
of Genkwa Flos and the underlying mechanism in nematode Caenorhabditis elegans. PLoS ONE 2014, 9, e91825.
[CrossRef] [PubMed]
21. Lublin, A.L.; Link, C.D. Alzheimer’s Disease Drug Discovery: In vivo screening using C. elegans as a model
for β-amyloid peptide-induced toxicity. Drug Discov. Today Technol. 2013, 10, e115–e119. [CrossRef] [PubMed]
22. Ryu, D.; Mouchiroud, L.; Andreux, P.A.; Katsyuba, E.; Moullan, N.; Nicolet-Dit-Felix, A.A.; Williams, E.G.;
Jha, P.; Lo Sasso, G.; Huzard, D.; et al. Urolithin A induces mitophagy and prolongs lifespan in C. elegans
and increases muscle function in rodents. Nat. Med. 2016, 22, 879–888. [PubMed]
23. Valero, T. Mitochondrial biogenesis: Pharmacological approaches. Curr. Pharm. Des. 2014, 20, 5507–5509.
[CrossRef] [PubMed]
24. Tomas-Barberan, F.A.; Gonzales-Sarrias, A.; Garcia-Villalba, R.; Nunez-Sanchez, M.A.; Selma, M.V.;
Garcia-Conesa, M.T.; Espin, J.C. Urolithins, the rescue of ‘old’ metabolites to understand a ‘new’ concept:
Metabotypes as a nexus between phenolic metabolism, microbiota dysbiosis and host health status. Mol. Nutr.
Food Res. 2016, 61, 1500901. [CrossRef] [PubMed]
25. Seeram, N.P.; Aronson, W.J.; Zhang, Y.; Henning, S.M.; Moro, A.; Lee, R.P.; Sartippour, M.; Harris, D.M.;
Rettig, M.; Suchard, M.A.; et al. Pomegrenate ellagitannin-derived metabolites inhibit prostate cancer growth
and localize to the mouse prostate gland. J. Agric. Food Chem. 2007, 55, 7732–7737. [CrossRef] [PubMed]
26. Mansouri, M.T.; Farbood, Y.; Sameri, M.J.; Sarkaki, A.; Naghizadeh, B.; Rafeirad, M. Neuroprotective effects
of oral gallic acid against oxidative stress induced by 6-hydroxydopamine in rats. Food Chem. 2013, 138,
1028–1033. [CrossRef] [PubMed]
27. Zhang, Z.X.; Li, Y.B.; Zhao, R.P. Epigallocatechin Gallate Attenuates β-Amyloid Generation and Oxidative
Stress Involvement of PPARγ in N2a/APP695 Cells. Neurochem. Res. 2017, 42, 468–480. [CrossRef] [PubMed]
28. Gul, F.; Khan, K.M.; Adhikari, A.; Zafar, S.; Akram, M.; Khan, H.; Saeed, M. Antimicrobial and antioxidant
activities of a new metabolite from Quercus incana. Nat. Prod. Res. 2016, 21, 1–19. [CrossRef] [PubMed]
29. Jeon, S.Y.; Bae, K.; Seong, Y.H.; Song, K.S. Green tea catechins as a BACE1 (β-secretase) inhibitor. Bioorg. Med.
Chem. Lett. 2003, 13, 3905–3908. [CrossRef] [PubMed]
30. Chan, S.; Kantham, S.; Rao, V.M.; Palanivelu, M.K.; Pham, H.L.; Shaw, P.N.; McGeary, R.P.; Ross, B.P.
Metal chelation radical scavenging and inhibition of Aβ42 fibrillation by food constituents in relation to
Alzheimer’s disease. Food Chem. 2016, 199, 185–194. [CrossRef] [PubMed]
31. Smith, T. Pharmakokinetics, bioavailability and serum levels of cardiac glycosides. J. Am. Coll. Cardiol. 1985,
5, 43A–50A. [CrossRef]
32. Alexander, A.; Marfil, V.; Li, C. Use of Caenorhabditis elegans as a model to study Alzheimer’s disease and
other neurodegenerative diseases. Front. Genet. 2014, 5, 1–21. [CrossRef] [PubMed]
33. Diomede, L.; Rigacci, S.; Romeo, M.; Stefani, M.; Salmona, M. Oleuropein Aglycone Protects Transgenic C.
elegans Strains Expressing Aβ42 by Reducing Plaque Load and Motor Deficit. PLoS ONE 2013, 8, e58893.
[CrossRef] [PubMed]
34. Chen, X.; Barclay, J.; Burgoyne, R.; Morgan, A. Using C. elegans to discover therapeutic compounds for
ageing-associated neurodegenerative diseases. Chem. Cent. J. 2015, 9, 1–20. [CrossRef] [PubMed]
35. Markaki, M.; Tavernarakis, N. Modeling human diseases in Caenorhabditis elegans. Biotechnol. J. 2010, 5,
1261–1276. [CrossRef] [PubMed]
36. Link, C.D. Expression of human β-amyloid peptide in transgenic Caenorhabditis elegans. Proc. Natl. Acad.
Sci. USA 1995, 92, 9368–9372. [CrossRef] [PubMed]
37. David, D.; Ollikainen, N.; Trinidad, J.; Cary, M.; Burlingame, A.; Kenyon, C. Widespread Protein Aggregation
as an Inherent Part of Aging in C. elegans. PLoS Biol. 2010, 8, e1000450. [CrossRef] [PubMed]
38. Heo, J.H.; Hyon-Lee; Lee, K.M. The Possible Role of Antioxidant Vitamin C in Alzheimer’s Disease Treatment
and Prevention. Am. J. Alzheimer’s Dis. 2013, 28, 120–125. [CrossRef] [PubMed]
Biomedicines 2017, 5, 17 13 of 13

39. Sarkar, S.; Mukherjee, A.; Swarnakar, S.; Das, N. Nanocapsulated Ascorbic Acid Combating Cerebral
Ischemia Reperfusion-Induced Oxidative Injury in Rat Brain. Curr. Alzheimer Res. 2016, 13, 1363–1373.
[CrossRef] [PubMed]
40. Sil, S.; Ghosh, T.; Gupta, P.; Ghosh, R.; Kabir, S.N.; Roy, A. Dual Role of Vitamin C on the Neuroinflammation
Mediated Neurodegeneration and Memory Impairments in Colchicine Induced Rat Model of Alzheimer’s
Disease. J. Mol. Neurosci. 2016, 60, 421–435. [CrossRef] [PubMed]
41. Rietjens, I.; Boersma, M.; de Haan, L.; Spenkelink, B.; Awad, H.; Cnubben, N.; van Zanden, J.;
van der Woude, H.; Alink, G.; Koeman, J. The pro-oxidant chemistry of the natural antioxidants vitamin C,
vitamin E, carotenoids and flavonoids. Environ. Toxicol. Pharmacol. 2002, 11, 321–333. [CrossRef]
42. Putchala, M.C.; Ramani, P.; Sherlin, H.J.; Premkumar, P.; Natesan, A. Ascorbic acid and its pro-oxidant
activity as a therapy for tumours of oral cavity—A systematic review. Arch. Oral. Biol. 2013, 58, 563–574.
[CrossRef] [PubMed]
43. Ginotra, Y.P.; Ramteke, S.N.; Walke, G.R.; Rapole, S.; Kulkarni, P.P. Histidine availability is decisive in
ROS-mediated cytotoxicity of copper complexes of Aβ1-16 peptide. Free Radic. Res. 2016, 50, 405–413.
[CrossRef] [PubMed]
44. Chaudhari, K.; Wong, J.M.; Vann, P.H.; Sumien, N. Exercise, but not antioxidants, reversed the
ApoE4-associated motor impairments in adult GFAP-ApoE mice. Behav. Brain Res. 2016, 305, 37–45.
[CrossRef] [PubMed]
45. Ohlow, M.J.; Sohre, S.; Granold, M.; Schreckenberger, M.; Moosmann, B. Why Have Clinical Trials of
Antioxidants to Prevent Neurodegeneration Failed?—A Cellular Investigation of Novel Phenothiazine-Type
Antioxidants Reveals Competing Objectives for Pharmaceutical Neuroprotection. Pharm. Res. 2016.
[CrossRef] [PubMed]
46. Kim, D.-K.; Kim, T.H.; Lee, S.-J. Mechanisms of aging-related proteinopathies in Caenorhabditis elegans.
Exp. Mol. Med. 2016, 48, e263. [CrossRef] [PubMed]
47. Ahmad, W.; Ebert, P.R. Metformin Attenuates Aβ Pathology Mediated Through Levamisole Sensitive
Nicotinic Acetylcholine Receptors in a C. elegans Model of Alzheimer’s Disease. Mol. Neurobiol. 2016, 1–13.
[CrossRef] [PubMed]
48. Kikis, E. The struggle by Caenorhabditis elegans to maintain proteostasis during aging and disease. Biol. Direct
2016, 11, 58. [CrossRef] [PubMed]
49. Cuanalo-Contreras, K.; Park, W.; Murkherjee, A.; Millán-Pérez, P.; Soto, C. Delaying aging in Caenorhabditis
elegans with protein aggregation inhibitors. Biochem. Biophys. Res. Commun. 2017, 482, 62–67. [CrossRef]
[PubMed]
50. Yang, J.; Liu, X.; Zhang, X.; Jin, Q.; Li, J. Phenolic Profiles, Antioxidant Activities, and Neuroprotective
Properties of Mulberry (Morus atropurpurea Roxb.) Fruit Extracts from Different Ripening Stages. J. Food Sci.
2016, 81, C2439–C2446. [CrossRef] [PubMed]
51. Di Giovanni, S.; Eleuteri, S.; Paleologou, K.E.; Yin, G.; Zweckstetter, M.; Carrupt, P.A.; Lashuel, H.A.
Entacapone and tolcapone, two catechol O-methyltransferase inhibitors, block fibril formation of α-synuclein
and β-amyloid and protect against amyloid-induced toxicity. J. Biol. Chem. 2010, 285, 14941–14954. [CrossRef]
[PubMed]
52. Du, X.T.; Wang, L.; Wang, Y.J.; Andreasen, M.; Zhan, D.W.; Feng, Y.; Li, M.; Zhao, M.; Otzen, D.; Xue, D.; et al.
Aβ1-16 can aggregate and induce the production of reactive oxygen species, nitric oxide, and inflammatory
cytokines. J. Alzheimer’s Dis. 2011, 27, 401–413.

© 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).