Soaked Almonds Exhibit Vitamin E-dependent Memory Protective Effect in Rodent Models

Abstract

Background and Objective: It is believed in South-Asian traditions that a small dose of over-night soaked almonds taken in empty stomach have the potential to improve memory. This study aimed to investigate the comparative efficacy of whole and soaked almonds together with vitamin E estimation, given with or without food, for their memory protective effects in different animal models. Methodology: In study 1, different groups of mice (n = 9-10) were fed with three different doses (3, 6 and 12 g kg⁻¹) of almonds (whole, soaked and blanch-control) for 14 days. Memory protection was assessed using Morris water maze (MWM) in scopolamine-induced amnesia. Afterwards, mice were sacrificed and acetylcholinesterase (AChE) inhibition in hippocampus and frontal cortex was estimated. In study 2, High Fat Diet (HFD) was given to rats (n = 8) for 6 weeks for inducing memory impairment, together with different doses (1, 2 and 4 g kg⁻¹) of almonds (whole, soaked and blanch-control). Learning ability was tested through MWM performance in the last week. Besides, HPLC analysis was performed to see the effect of soaking on vitamin-E content of almonds. Results: Almond supplementation prevented scopolamine-induced amnesia in mice and improved learning ability in HFD-fed rats, respectively. Soaking led to an increase in vitamin-E content of almonds. Soaked almonds, consumed without food, protected memory and enhanced learning ability at a lower dose than the whole almonds in both models. With a dose-dependent trend, soaked almonds without food were found to be more effective in improving MWM performance and inhibiting AChE in hippocampus and frontal cortex. Conclusion: It is concluded that overnight soaking which enriches the vitamin-E content of almonds, effectively ameliorates memory impairments at low doses when consumed in empty stomach.

Full text

OPEN ACCESS International Journal of Pharmacology
ISSN 1811-7775
DOI: 10.3923/ijp.2017.448.456
Research Article
Soaked Almonds Exhibit Vitamin E-dependent Memory
Protective Effect in Rodent Models
1Jamshed Arslan, 2Touqeer Ahmed and 1,3Anwarul-Hassan Gilani
1Natural Product Research Unit, Department of Biological and Biomedical Sciences, The Aga Khan University Medical College,
74800 Karachi, Pakistan
2Neurobiology Laboratory, Atta-Ur-Rahman School of Applied Biosciences, National University of Science and Technology,
44000 Islamabad, Pakistan
3Pakistan Council for Science and Technology, G-5/2, Islamabad, Pakistan
Abstract
Background and Objective: It is believed in South-Asian traditions that a small dose of over-night soaked almonds taken in empty
stomach have the potential to improve memory. This study aimed to investigate the comparative efficacy of whole and soaked almonds
together with vitamin E estimation, given with or without food, for their memory protective effects in different animal models.
Methodology: In study 1, different groups of mice (n = 9-10) were fed with three different doses (3, 6 and 12 g kgG1) of almonds
(whole, soaked and blanch-control) for 14 days. Memory protection was assessed using Morris water maze (MWM) in scopolamine-
induced amnesia. Afterwards, mice were sacrificed and acetylcholinesterase (AChE) inhibition in hippocampus and frontal cortex was
estimated. In study 2, High Fat Diet (HFD) was given to rats (n = 8) for 6 weeks for inducing memory impairment, together with
different doses (1, 2 and 4 g kgG1) of almonds (whole, soaked and blanch-control). Learning ability was tested through MWM
performance in the last week. Besides, HPLC analysis was performed to see the effect of soaking on vitamin-E content of almonds.
Results: Almond supplementation prevented scopolamine-induced amnesia in mice and improved learning ability in HFD-fed rats,
respectively. Soaking led to an increase in vitamin-E content of almonds. Soaked almonds, consumed without food, protected memory
and enhanced learning ability at a lower dose than the whole almonds in both models. With a dose-dependent trend, soaked almonds
without food were found to be more effective in improving MWM performance and inhibiting AChE in hippocampus and frontal cortex.
Conclusion: It is concluded that overnight soaking which enriches the vitamin-E content of almonds, effectively ameliorates memory
impairments at low doses when consumed in empty stomach.
Key words: Acetylcholinesterase, vitamin-E, Morris water maze, mice, rats
Received: April 04, 2017 Accepted: May 16, 2017 Published: June 15, 2017
Citation: Jamshed Arslan, Touqeer Ahmed and Anwarul-Hassan Gilani, 2017. Soaked almonds exhibit vitamin E-dependent memory protective effect in
rodent models. Int. J. Pharmacol., 13: 448-456.
Corresponding Author: Anwarul-Hassan Gilani, Natural Product Research Unit, Department of Biological and Biomedical Sciences,
The Aga Khan University Medical College, 74800 Karachi, Pakistan Tel: +92-51-9204416 Fax: 9222172
Copyright: © 2017 Jamshed Arslan
et al
. This is an open access article distributed under the terms of the creative commons attribution License, which
permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.
Competing Interest: The authors have declared that no competing interest exists.
Data Availability: All relevant data are within the paper and its supporting information files.

Int. J. Pharmacol., 13 (5): 448-456, 2017
INTRODUCTION
Human life expectancy has increased. People now live
long enough to experience chronic metabolic and
neurological diseases which their aged bodies are not
sufficiently capable to deal with. Among the neurological
disorders, Alzheimers Disease (AD), the major cause of
dementia in elderly, appears to be a serious threat1. The AD is
a complex disorder, while, the efficacy of the available
treatments is limited2, with multiple side effects; hence,
natural products are warranted that could target multiple
pathways and have fewer side effects3-5.
Edible nuts are bestowed with these properties and they
appear to be important natural remedies for improving
cognitive performance, particularly in old age6. Among edible
nuts, folkloric recommendations have placed huge emphasis
on almonds (
Prunus dulcis
) for improving memory.
Specifically, the traditional recommendation in South Asian
culture to enhance memory is to consume a handful of
almonds (about 10 g) in empty stomach after overnight
soaking and peeling. As far as this mode of consumption
(soaking) is concerned, our laboratory has used it on human7
and animal studies8 for features related to cardiovascular
diseases, not memory. This process of soaking
per se
has been
shown to change the chemical composition of certain food
items9. However, data are missing regarding effects of soaking
on almonds and its memory-enhancing potential.
The process of soaking, breaks dormancy and the seed
prepares to germinate, which is facilitated by certain chemical
changes like catabolic conversion of complex carbohydrates
to simpler ones and enrichment of vitamin9. Almonds are
seeds with high vitamin-E content10. Research over the past
decade has particularized the memory protective potential of
vitamin-E, be it in Alzheimers disease, or other age-associated
memory impairments11 . The radical scavenging potential of
vitamin-E is particularly important in this context, since one of
the contributing molecular mechanisms of dementia includes
oxidative stress causing neurological dysfunction and
degradation. It is hypothesized that soaking increase
almonds vitamin-E content, thereby enhancing the memory
effects.
When testing potential treatment options in AD, the
significance of appropriate animal models to depict important
aspects of AD cannot be overstated. For example, in AD, there
is cholinergic hypo-function in the brain. Subsequently,
scopolamine-induced amnesia mice model is frequently used
to depict brains cholinergic decline that occurs in AD
patients12. To overcome cholinergic hypo-function, inhibition
of the acetylcholinesterase (AChE) enzyme, to reduce
acetylcholines breakdown, is among the predominant
mechanisms of most of the AD-therapies approved by FDA2.
A second animal model that can reflect AD-like learning
disability is the High Fat Diet (HFD) rat model5,13. Hence, both
of these models were used to assess the short term and
long-term effects of almonds on learning and memory.
Overall, it is aimed to extensively elaborate on the
memory-effects of different modes of almond consumption,
using two different animal models: Scopolamine-induced
amnesia model that involves 2 weeks of almond
consumption and HFD rat model involving 6 weeks of almond
consumption.
MATERIALS AND METHODS
Animals and diets: Mice, aged 4-6 months, with C57Bl/6
background and adults of Sprague-Dawley rats (180-200 g) of
either gender, housed in plastic cages with sawdust at The
Aga Khan University animal house (at 23-25EC) were used.
Animals were given tap water
ad libitum
. All the mice
received standard diet. All rats received HFD comprising
cholesterol-cholate-butter fat as described by Jamshed
et al
.5,
except the normal diet group, which received standard diet.
The experiments conducted were in accordance with the
guidelines for care and use of laboratory animals provided by
The National Research Council14. Protocol of this study was
approved by the Ethical Committee for Animal Care and Use,
The Aga Khan University, Karachi, Pakistan (Approval No.
008-Ani-BBS-13).
Chemicals: Physostigmine, scopolamine, electric eel
acetylcholinesterase, acetylthiocholine iodide 5, 5-dithiobis
(2-nitrobenzoic) acid and chemicals in HPLC analysis were
obtained from Sigma Chemical Company, St. Louis, MO,
USA. Drug solutions were made fresh on the day of
experiment.
Almond administration: The almonds used were imported
from California Sheld (Karmal), purchased from the local
market. Knowing how difference in size of animal affects the
metabolic rate, the formula recommended by FDA15 was
employed for dose translation of almonds from humans to
mice and rats. Smaller animals have higher metabolism and
therefore higher unit doses (per kilogram) compared to larger
animals and/or human. Correspondingly, human doses of 14,
28 and 56 g dayG1 (or rou ghly 14 , 28 a nd 56 almon ds da yG1)
were translated into equivalent mice doses of 3, 6 and
12 g kgG1, respectively, while 10, 20 and 40 g human doses
were converted into rat doses of 1, 2 and 4 g kgG1,
respectively.
449

Int. J. Pharmacol., 13 (5): 448-456, 2017
Almonds were given in any of the three forms: Whole,
soaked or blanched. Soaking was done by keeping almonds in
tap water overnight (10 h) and removing the skin in the
morning. To control the effects of skin removal
per
se
,
blanching was carried out by boiling almonds in tap
water (3 min) and removing the skin. In both soaked and
blanch-control almond groups, only the kernel was
administered after the skin-removal. The protocol for almond
administration was similar to that described in the earlier
study8. Briefly, weighed almonds were given without food in
which there was 60 min fasting for mice and 90 min for rats.
Once the almond was consumed, food was given back after
30 min. In the With food groups, this protocol (of fasting)
was not followed and almonds pieces were given
without food-withdrawal. The placebo group represented
diseased-control and received diet-flour 6 g kgG1 instead of
almonds.
Study design
Scopolamine-induced amnesia mice model: Two studies
were designed in which effects of short-term dosing of
almonds were observed. In study 1, multiple doses of whole
and soaked almonds were compared (Table 1). In study 2,
comparative effects of almond supplementation with food
and without food were observed (Table 2).
Protocol of Ahmed and Gilani16 was followed, in which
mice were injected with scopolamine (2 mg kgG1 i.p.),
Table 1: Design of study 1, comparing whole, soaked and blanched almonds in
scopolamine-induced amnesia mice model
Groups (n = 9-10) Interventions
1 Saline
2 Placebo
3 Physostigmine
4 Whole almonds (3 g kgG1)
5 Whole almonds (6 g kgG1)
6 Whole almonds (12 g kgG1)
7 Soaked almonds (3 g kgG1)
8 Soaked almonds (6 g kgG1)
9 Soaked almonds (12 g kgG1)
10 Blanched almonds (3 g kgG1)
11 Blanched almonds (6 g kgG1)
12 Blanched almonds (12 g kgG1)
Table 2: Design of study 2, comparing soaked almonds with and without food,
in scopolamine-induced amnesia mice model
Groups (n = 9-10) Interventions
1 Saline
2 Placebo
3 Physostigmine
4 Soaked almonds (3 g kgG1)
5 Soaked almonds (6 g kgG1)
6 Soaked almonds (12 g kgG1)
7 Soaked almonds (3 g kgG1) (with food)
8 Soaked almonds (6 g kgG1) (with food)
9 Soaked almonds (12 g kgG1) (with food)
except for the normal control group that received
saline injection instead of scopolamine. Physostigmine
(0.2 mg kgG1 i.p.) served as positive control. The flow of study
and behavioral tests are described in Table 3. After the
behavioral investigations, animals were sacrificed and brains
were isolated for AChE inhibitory assay in hippocampus and
frontal cortex.
High Fat Diet (HFD)-induced model of memory impairment
in rats:
Again, two studies (study 3 and 4) were designed, in
which effects of long-term dosing of almonds were observed.
In study 3, multiple doses of whole and soaked almonds were
compared (Table 4). In study 4, comparative effect of almond
administration with food and without food was observed
(Table 5). Protocol of Jamshed and Gilani8 was followed.
Briefly, normal group was fed with normal diet, whereas other
group was fed with a cholesterol-cholate-butter fat-containing
Table 3: Progression of behavioral studies on scopolamine-induced amnesia
mice model
Days Intervention
1-6 Only almonds
7 Almonds+open field
8 Almonds+acclimatization for Novel Object Recognition (NOR)
9 Almonds+NOR with scopolamine
10-13 Almonds+Morris Water Maze (MWM)
14 Almonds+MWM with scopolamine
Table 4: Design of study 3, comparing whole, soaked and blanched almonds
on high fat diet-induced rat model of memory impairment
Groups (n = 8) Interventions
1 Normal diet
2 High fat diet
3 Whole almonds (1 g kgG1)
4 Whole almonds (2 g kgG1)
5 Whole almonds (4 g kgG1)
6 Soaked almonds (1 g kgG1)
7 Soaked almonds (2 g kgG1)
8 Soaked almonds (4 g kgG1)
9 Blanched almonds (1 g kgG1)
10 Blanched almonds (2 g kgG1)
11 Blanched almonds (4 g kgG1)
Table 5: Design of study 4, comparing soaked almonds with and without food,
in high-fat diet-induced rat model of memory impairment
Groups (n = 8) Interventions
1 Normal diet
2 High fat diet
3 Soaked almonds (1 g kgG1)
4 Soaked almonds (2 g kgG1)
5 Soaked almonds (4 g kgG1)
6 Soaked almonds (1 g kgG1) (with food)
7 Soaked almonds (2 g kgG1) (with food)
8 Soaked almonds (4 g kgG1) (with food)
450

Int. J. Pharmacol., 13 (5): 448-456, 2017
** ** ** ** **
Sal Pla Phy 3612 3612 3612
Whole Soaked Blanched
Almonds (g kg )
G
1
Scopolamine (2 mg kg )
G
1
35
30
25
20
15
10
5
0
Whole almonds Soaked almonds Blanched almonds
Escape latency (sec)
HFD. Almonds were given for 6 weeks. During the memory
analysis through MWM, almond administration along with
HFD continued.
Behavioral paradigms
Open Field (OF) test: The OF test was performed both in mice
and rats for assessing locomotor performance. The parameters
observed were line crossing, rearing and grooming, as
described by Okoli
et al
.17.
Novel Object Recognition (NOR): The NOR was performed
only in mice for assessing memory, as described by
Dere
et al
.18. Briefly, 5 min acclimatization was carried out first,
in which mice were individually placed in a wooden box to get
familiarized. The following day, a 5 min acquisition trial with
two identical objects was conducted after scopolamine
administration (2 mg kgG1 i.p.). Following 3 h, a 4 min test trial
was conducted in which one object was replaced with a novel
object. The time of exploration of familiar (Tf) and novel object
(Tn) were recorded after analyzing the recordings. The
Discrimination Index (DI) or the difference between the time
spent exploring novel and familiar objects was calculated
as Eq. 1:
(1)
Tn-Tf
Tn+Tf
Morris Water Maze (MWM): It was performed for assessing
memory19 as described by Ahmed and Gilani16, in both mice
and rats. Briefly, for mice, after the completion of training
trials, on 5th day, the trained mice were injected with
scopolamine (2 mg kgG1 i.p.); then mice were allowed to swim
for 90 sec and the time spent to reach the platform was
measured. The rats, on the other hand, were allowed to swim
for 7 days: Two trials on first day and one trial in the remaining
6 days, as described by Jamshed
et al
.5.
Acetylcholinesterase (AChE) Activity: After behavioral
examinations, mice brains were isolated for
in vivo
AChE
inhibition testing20 in frontal cortex and hippocampus, as
described by Ahmed and Gilani16.
Vitamin-E ("-tocopherol) estimation: The "-tocopherol
content in whole, soaked and blanched almonds was
estimated by HPLC method21,22.
Statistical analysis: The software Graph PrismPad 4 was used
for statistical analysis and for plotting graphs. One-way ANOVA
followed by Dunnetts post-test was applied to calculate the
statistical significance. The p-values <0.05 were considered
significant and the values were represented as Mean±SEM.
RESULTS
Memory-restoring effect of soaked almonds on
scopolamine-induced amnesia in mice: The OF test showed
no locomotor dysfunction among mice from any group. The
NOR memory test showed similar effects (p>0.05) among
all the groups treated with almonds in study 1. Figure 1 shows
MWM in which, whole almonds showed amnesia-prevention
only at the highest dose tested (12 g kgG1; p<0.01), while
soaked almonds were efficacious even at a lower dose of
6 g kgG1 (p<0.01).
The AChE inhibition assay, overall, validated the findings
of MWM. Both in hippocampus (Fig. 2a) and frontal cortex
(Fig. 2b), soaked almonds at 6 and 12 g kgG1 markedly
inhibited AChE enzyme (p<0.01), similar to physostigmine.
Whole almonds significantly inhibited AChE (p<0.01) only at
the highest dose of 12 g kgG1 in frontal cortex but not in the
hippocampus. Blanched almonds had no effect on behavior
and/or enzyme inhibition (p>0.05) at both the tested doses.
Memory-restoring effect of soaked almonds on
HFD-induced memory impairment in rats: In study 3, using
HFD model, the OF test indicated no prior locomotor disability,
as indicated by the observation that rats from all the groups
performed in a similar manner (p>0.05).
Fig. 1: Effect of soaking on efficacy of almonds in preventing
scopolamine-induced amnesia in mice
Sal: Saline, Pla: Placebo, Phy: Physostigmine. Almond doses (3, 6 and
12 g kgG1) given for 2 weeks. One-way ANOVA followed by Dunnett's
test was app lie d. Al l va lue s ar e Me an±S EM, n = 9 -10 (**p<0.01)
451

Int. J. Pharmacol., 13 (5): 448-456, 2017
** **
Pla Phy 3 6 12 3 6 12 3 6 12
Whole Soaked Blanched
Almonds (g kg )
G
1
Scopolamine (2 mg kg )
G
1
0.30
0.25
0.20
0.15
0.10
0.05
0.00
**
**
(b)
** **
Pla Phy 3 6 12 3 6 12 3 6 12
Whole Soaked Blanched
Almonds (g kg )
G
1
Scopolamine (2 mg kg )
G
1
0.30
0.25
0.20
0.15
0.10
0.05
0.00
**
Protein (OD mg
G
1
)
(a)
Protein (OD mg
G
1
)
Whole almonds Soaked almonds Blanched almonds
ND HFD 1 2 4
Whole Soaked Blanched
Almonds (g kg )
G
1
High Fat Diet (HFD)
35
30
25
20
15
10
5
0
Whole almonds Soaked almonds Blanched almonds
*
Escape latency (sec)
(a)
124 124
***
*
(b)
*
*
**
ND HFD 124
Whole Soaked Blanched
Almonds (g kg )
G
1
High Fat Diet (HFD)
124 124
30
25
20
15
10
5
0
Escape latency (sec)
Fig. 2(a-b): Effect of soaking on almonds efficacy in inhibiting
acetylcholinesterase enzyme in scopolamine-
induced amnesic mice (a) Hippocampus and
(b) Frontal cortex
Pla: Placebo, Phy: Physostigmine. Almond doses (3, 6 and
12 g kgG1) given for 2 weeks. One-way ANOVA followed by
Dunnett's test was applied. All values are Mean±SEM, n = 5
(**p<0.01)
Further, it can be seen that in 6 weeks, HFD led to
noticeable learning disability (p<0.05) as shown by
significantly increased escape latency (Fig. 3). In the first
5 days of 6 week, there was no observable difference among
the performances of rats from all groups. At 6 day (Fig. 3a)
and 7 day (Fig. 3b), soaked almonds were effective in
preventing slowness of learning from HFD at low (2 g kgG1)
and larger (4 g kgG1) doses, while whole almonds were
effective (p<0.05) only at the highest tested dose of 4 g kgG1.
Effect of soaking on vitamin-E content of almonds: T he HP L C
analysis showed that vitamin-E ("-tocopherol) concentration
was 119.4 mg kgG1 for whole almonds, 259 mg kgG1 for
soaked almonds and 189 mg kgG1 for blanched almonds.
In other words, there was 117% increase in vitamin-E
Fig. 3(a-b): Effect of soaking on almonds efficacy to improve
learning ability in High Fat Diet (HFD)-fed rats on
Morris water maze (a) Day 6 and (b) Day 7
ND: Normal diet, Almond doses (1, 2 and 4 g kgG1) given for
6 weeks. One-way ANOVA followed by Dunnett's test was
applied. All values are Mean±SEM, n = 8 (*p<0.05 and **p<0.01)
content in soaked almonds and 58% increase in blanched
almonds, as compared to the whole almonds.
Effect of food on almonds efficacy in scopolamine-induced
amnesia in mice:
The OF test showed that all mice in study
group 2 were physically fit for further behavioral testing. The
NOR test showed no difference in performances of almond
treated groups (p>0.05).
Figure 4 shows that efficacy of almonds, particularly at
low dose (6 g kgG1 soaked) is markedly compromised in the
presence of food as can be seen from the higher escape
latency of groups consuming almonds with food compared to
Without food.
The
ex vivo
AChE is inline with these findings as the
memory improving effect is compromised by food
accompanying almonds. In both, hippocampus (Fig. 5a) and
frontal cortex (Fig. 5b) of these mice, profound inhibition of
452

Int. J. Pharmacol., 13 (5): 448-456, 2017
** ** ** **
Sal Pla Phy 3 g kg
G
1
Soaked almonds (g kg )
G
1
Scopolamine (2 mg kg )
G
1
40
30
20
10
0
E
scape latency (sec)
6 g kg
G
1
12 g kg
G
1
Without food With food
*
ND HFD 1 g kg
G
1
Soaked almonds (g kg )
G
1
High Fat Diet (HFD)
35
30
25
20
15
10
5
02 g kg
G
1
4 g kg
G
1
Without food Wit h foo d
*
(a)
Escap
e
lat
e
nc
y
(s
e
c)
*
**
ND HFD 1 g kg
G
1
Soaked almonds (g kg )
G
1
High Fat Diet (HFD)
30
25
20
15
10
5
02 g kg
G
1
4
**
(b)
Escap
e
lat
e
nc
y
(s
e
c)
*
g kg
G
1
** **
Pla Phy 3 g kg
G
1
Soaked almonds (g kg )
G
1
Scopolamine (2 mg kg )
G
1
0.30
0.25
0.20
0.15
0.10
0.05
0.00 6 g kg
G
1
12 g kg
G
1
Wit hout food Wit h food
*
**
(a)
** **
Pla Phy 3 g kg
G
1
Soaked almonds (g kg )
G
1
Scopolamine (2 mg kg )
G
1
0.30
0.25
0.20
0.15
0.10
0.05
0.00 6 g kg
G
1
12 g kg
G
1
**
(b)
Protein (OD mg
G
1
)Protein (OD mg )
G
1
Fig. 4: Effect of food on efficacy of soaked almonds to prevent
scopolamine-induced amnesia in mice
Sal: Saline, Pla: Placebo, Phy: Physostigmine. Almond doses (3 , 6 a nd
12 g kg G1) gi ve n fo r 2 w eek s. O ne- wa y AN OVA fol low ed by D unn ett 's
test was applied. All values are Mean±SEM, n = 9-10 (**p<0.01)
Fig. 5(a-b): Effect of food on efficacy of soaked almonds
to inhibit acetylcholinesterase enzyme in
scopolamine-induced amnesic mice (a)
Hippocampus and (b) Frontal cortex
Pla: Placebo, Phy: Physostigmine. Almond doses (3, 6 and
12 g kgG1) given for 2 weeks. One-way ANOVA followed by
Dunnett's test was applied. All values are Mean±SEM, n = 5
(**p<0.01)
Fig. 6(a-b): Effect of food on efficacy of soaked almonds to
improve learning ability in High Fat Diet (HFD)-fed
rats on Morris water maze (a) Day 6 and (b) Day 7
ND: Normal diet, Almond doses (1, 2 and 4 g kgG1) given or
6 weeks. One-way ANOVA followed by Dunnett's test was
applied. All values are Mean±SEM, n = 8 (*p<0.05 and **p<0.01)
AChE was observed by physostigmine as well as by all the
three doses of soaked almonds (3, 6 and 12 g kgG1) only when
given without food (p<0.01).
Effect of food on almonds efficacy in HFD-induced memory
impairment in rats: The OF test on rats of study group 4,
using the HFD model, indicated no locomotor abnormality in
any group. In the 6th week of treatment, MWM showed no
marked difference in the performances of rats from different
groups, in the first 5 days. On day 6 (Fig. 6a) and day 7 (Fig. 6b),
HFD induced some slowness in learning ability. The two high
doses of soaked almonds (2 and 4 g kgG1), only when given
without food, effectively prevented this impairment
(p<0.05) on day 6 and 7 (2 g kgG1 at p<0.05 4 g kgG1 at
p<0.01).
DISCUSSION
Almonds alleviate memory loss in two different animal
models was observed. Interestingly, overnight soaking
453

Int. J. Pharmacol., 13 (5): 448-456, 2017
enhanced the vitamin-E content of almonds improving their
efficacy compared to whole or blanched almonds. Even a low
dose of soaked almonds was effective but only when given
without accompanying food. While exploring the probable
underlying mechanisms, it was also elaborated inhibition of
AChE in hippocampus and frontal cortex by almonds.
This study was distinctive in a number of ways: Two
species of experimental animals was used to test hypotheses
of the present study, also tested the effect of soaking on
almonds content and showed vitamin-E enrichment to
escalate efficacy in terms of neuronal function and that food
can hinder the bio-effectiveness of almonds.
To the best of our knowledge, this is first study to explore
the memory improving effects of almonds in different animal
models. Although Kulkarni
et al
.23 have reported some
preliminary findings of memory improvement by almond
administration but there were some limitations of that study
that warranted further exploration. For example, the study
design was weak in the sense23 that used Elevated Plus Maze
(EPM) for memory evaluation, even though EPM is primarily
meant to assess anxiety-like behaviors24. Secondly, it was
found non-reproducibility of method of almond paste
administration mentioned therein. Interestingly, another
study25 described administration of almond suspension to the
scopolamine-induced amnesic rats but the fact that such an
oral administration would cause distress to the animal was
considered.
The study conducted by Kulkarni
et al
.23 and Batool
et al
.25
addressed only one mode of almond consumption in one
model. Since, there is no single animal model that could truly
represent AD because of its complexity26,27, this study
investigated effects of different doses of various modes of
almond consumption in two different animal models.
Batool
et al
.25
observed a reduced cholinergic functioning
with scopolamine and noticed that almonds not only reversed
that but also attenuated scopolamine-induced amnesia in rats.
Results of present study are in line with these findings but it
was observed AChE inhibition by whole almonds at the
highest dose tested (12 g kgG1) only in the frontal cortex
(Fig. 2). Moreover, Kulkarni
et al
.23 also reported reduction in
AChE levels by almonds but they observed AChE levels in
brain homogenates, rather than estimation in specific brain
components like hippocampus and frontal cortex.
Previously, soaked almonds were tested in a clinical trial
on coronary artery disease patients7 but the study did not
explore change in almond constituents due to soaking. This
was perhaps because the efficacy of soaked and un-soaked
almonds seemed to be similar on cardio-metabolic
parameters. It is now known that vitamin-E has almost
negligible beneficial effect on the cardiovascular system28.
Current study addresses the literature gap by measuring
one of key components in almonds, vitamin-E29. The
soaking-induced increase in vitamin E ("-tocopherol) in this
study is in line with previous reports where a similar increase
in various constituents has been reported for other food items
like cereal grains9. Soaking leads to seed germination, which
is evident by sprouting. This also results in conversion of
complex nutrients like carbohydrates to simpler forms and
degradation of anti-nutrients like phytates8. Since almonds are
known to possess the highest amount of vitamin-E among
nuts10, soaking was e xpected to increase the potential of
almonds against memory impairment, based on incr ease d
"-tocopherol level observed in this study. Interestingly,
vitamin-E has previously shown effectiveness against AD30, as
well as mild cognitive impairment31 , which explains enhanced
effect of soaked almonds in this study.
Oxidative stress, resulting from endogenous
overproduction of reactive oxygen species (ROS) is injurious to
all body cells but particularly to the post-mitotic neuronal and
glial cells. With increasing age, the cells competence to
combat oxidative threats is compromised. This signifies the
role of natural and dietary antioxidants like vitamin-E, which
come to rescue in the fight against progressively detrimental
ROS11.
It was observed that, it was the soaked almonds, not the
blanched almonds that led to improvement in memory. This
indicated that, it is not just the removal of almond-skin but
also the process of removing the skin that has positive
effects on the role of almonds in memory improvement.
Although blanched almonds also showed some increase in
"-tocopherol but this mode of almond consumption did not
lead to any improvement in memory, indicating the blanching
might bring some chemical changes in the almond that
prevent bioavailability of "-tocopherol (vitamin-E). This also
indicated that vitamin-E is just one of the components that
enhance soaked almonds memory-improving potential.
Future investigations into the effects of soaking and blanching
on the bioavailability of vitamin-E and other constituents are
warranted. The efficacy of soaked almonds that are
noteworthy at lower dose is dependent on its consumption in
empty stomach.
The duration of 60 and 90 min for mice and rats,
respectively, before administering almonds is likely to ensure
the stomach emptying to mimic the consumption style in
humans, as rodents, like mice and rats, eat relatively constantly
as opposed to human, who eat in intervals. Once the almond
was consumed, food was given back after 30 min to ensure
maximum absorption of almonds content in gastrointestinal
tract without probable hindrance from any food material. The
presence of food compromised the memory enhancing effects
454

Int. J. Pharmacol., 13 (5): 448-456, 2017
of almonds was observed. This was not unusual as several
studies on digestive models have shown that the
accompanying food matrix limits the bio-accessibility of
almond constituents from GI tract32. This indicated that there
is greater absorption of the almond constituents when
stomach is empty. These results are in line with the previous
study in which almonds when given in empty stomach
showed enhanced efficacy in cardiovascular parameters8.
It appears that fasting or empty stomach enhances
bioavailability of almond contents, which warrants further
investigations.
All animals were equivalent in terms of locomotor
abilities, as indicated by the OF tests, highlighting that almond
administration does not cause any locomotor disabilities
which could influence the results in NOR or MWM. In other
words, the results obtained in the memory testing were purely
based on memory performance and not on locomotor
competencies of animals.
The reason why almonds failed to show effect in NOR is
probably because almonds may require a minimum of 14 days
of administration to exhibit any memory improvement.
Second reason could be the fact that NOR paradigm has less
motivation than MWM18. In other words, MWM may be more
vigorous than NOR in terms of assessing memory. The reversal
of amnesia by the positive control, physostigmine can be
intriguing as it did not require long-term administration like
almonds do, which is not surprising as food or natural
products in their crude form usually takes more time in their
action compared to single chemical entity. However, further
studies are needed to explore the mechanisms involved.
CONCLUSION
This study provided evidence that overnight soaking
elevated the vitamin-E content of almonds thereby improving
its efficacy in memory impairment in rat and mice model of
dementia and memory impairment. Low doses of soaked
almonds, when given in empty stomach, showed greater
efficacy than other forms and modes of almond
administration. This is mediated possibly through AChE
inhibitory actions in the brain along with raised vitamin-E, a
well-known natural anti-oxidant, although additional
mechanisms cannot be ruled out. By extension, it can be
hoped that almonds can target dementia of AD through
multiple mechanisms, an aspect commonly seen in natural
products. This study is likely to offer safer remedy when
compared to established chemical drugs, which are
extraordinarily expensive and possess multiple side-effects,
yet with limited efficacy. Based on the findings in this study,
recommendation can be made to use almonds in empty
stomach after overnight soaking, thus allowing lower dose
resulting in cost-effectiveness, an important component when
life-long supplementation is warranted.
ACKNOWLEDGMENTS
The author would like to thank the Pakistan Academy
of Sciences/Higher Education Commission for funding this
research. The authors are also grateful to Ms. Sadaf Zahoor
(Department of Chemistry, University of Sargodha, Pakistan)
for estimating the "-tocopherol in this study, under guidance
of Dr. Farooq Anwar.
REFERENCES
1. Abbott, A., 2011. Dementia: A problem for our age. Nature,
475: S2-S4.
2. Rafii, M.S. and P.S. Aisen, 2015. Advances in Alzheimer's
disease drug development. BMC Med., Vol. 13.
10.1186/s12916-015-0297-4
3. Gilani, A.H. and Atta-ur-Rahman, 2005. Trends in
ethnopharmacology. J. Ethnopharmacol., 100: 43-49.
4. Ahmed, T. and A.H. Gilani, 2014. Therapeutic potential of
turmeric in Alzheimer's disease: Curcumin or curcuminoids?
Phytother. Res., 28: 517-525.
5. Jamshed, H., J. Arslan and A.H. Gilani, 2014. Cholesterol-
cholate-butterfat diet offers multi-organ dysfunction in rats.
Lipids Health Dis., Vol. 13. 10.1186/1476-511X-13-194
6. O'Brien, J., O. Okereke, E. Devore, B. Rosner, M. Breteler and
F. Grodstein, 2014. Long-term intake of nuts in relation
to cognitive function in older women. J. Nutr. Health Aging,
18: 496-502.
7. Jamshed, H., F.A.T. Sultan, R. Iqbal and A.H. Gilani, 2015.
Dietary almonds increase serum HDL cholesterol in coronary
artery disease patients in a randomized controlled trial. J.
Nutr., 145: 2287-2292.
8. Jamshed, H. and A.H. Gilani, 2015. Lower dose of almonds
exhibits vasculo-protective effect when given in empty
stomach. Int. J. Pharmacol., 11: 122-129.
9. Chavan, J.K., S.S. Kadam and L.R. Beuchat, 1989. Nutritional
improvement of cereals by sprouting. Crit. Rev. Food Sci.
Nutr., 28: 401-437.
10. Yada, S., K. Lapsley and G. Huang, 2011. A review of
composition studies of cultivated almonds: Macronutrients
and micronutrients. J. Food Compos. Anal., 24: 469-480.
11. Uttara, B., A.V. Singh, P. Zamboni and R.T. Mahajan, 2009.
Oxidative stress and neurodegenerative diseases: A review of
upstream and downstream antioxidant therapeutic options.
Curr. Neuropharmacol., 7: 65-74.
455

Int. J. Pharmacol., 13 (5): 448-456, 2017
12. Kwon, S.H., H.K. Lee, J.A. Kim, S.I. Hong and H.C. Kim
et al
.,
2010. Neuroprotective effects of chlorogenic acid on
scopolamine-induced amnesia via anti-acetylcholinesterase
and anti-oxidative activities in mice. Eur. J. Pharmacol.,
649: 210-217.
13. Kosari, S., E. Badoer, J.C. Nguyen, A.S. Killcross and
T.A. Jenkins, 2012. Effect of western and high fat diets on
memory and cholinergic measures in the rat. Behav. Brain
Res., 235: 98-103.
14. ILAR., 1996. Guide for the Care and Use of Laboratory Animals.
National Academies Press, Washington, DC.
15. Reagan-Shaw, S., M. Nihal and N. Ahmad, 2008. Dose
translation from animal to human studies revisited. FASEB J.,
22: 659-661.
16. Ahmed, T. and A.H. Gilani, 2009. Inhibitory effect of
curcuminoids on acetylcholinesterase activity and
attenuation of scopolamine-induced amnesia may explain
medicinal use of turmeric in Alzheimer's disease. Pharmacol.
Biochem. Behav., 91: 554-559.
17. Okoli, C.O., A.C. Ezike, O.C. Agwagah and P.A. Akah, 2010.
Anticonvulsant and anxiolytic evaluation of leaf extracts of
ocimum gratissimum
, a culinary herb. Pharmacogn. Res.,
2: 36-40.
18. Dere, E., J.P. Huston and M.A. De Souza Silva, 2007. The
pharmacology, neuroanatomy and neurogenetics of one-trial
object recognition in rodents. Neurosci. Biobehav. Rev.,
31: 673-704.
19. Morris, R., 1984. Developments of a water-maze procedure
for studying spatial learning in the rat. J. Neurosci. Methods,
11: 47-60.
20. Isoma, K., M. Ishikawa, M. Ohta, Y. Ogawa, H. Hasegawa,
T. Kohda and J. Kamei, 2002. Effects of T-82, a new quinoline
derivative, on cholinesterase activity and extracellular
acetylcholine concentration in rat brain. Jpn. J. Pharmacol.,
88: 206-212.
21. Anwar, F., R. Naseer, M.I. Bhanger, S. Ashraf, F.N. Talpur and
F.A. Aladedunye, 2008. Physico-chemical characteristics of
citrus seeds and seed oils from Pakistan. J. Am. Oil Chem. Soc.,
85: 321-330.
22. Manzoor, M., F. Anwar, T. Iqbal and M.I. Bhanger, 2007.
Physico-chemical characterization of
Moringa concanensis
seeds and seed oil. J. Am. Oil Chem. Soc., 84: 413-419.
23. Kulkarni, S.K., S.B. Kasture and S.A. Mengi, 2010. Efficacy study
of
Prunus amygdalus
(almond) nuts in scopolamine-induced
amnesia in rats. Indian J. Pharmacol., 42: 168-173.
24. Walf, A.A. and C.A. Frye, 2007. The use of the elevated plus
maze as an assay of anxiety-related behavior in rodents. Nat.
Protocols, 2: 322-328.
25. Batool, Z., S. Sadir, L. Liaquat, S. Tabassum and S. Madiha
et al
.,
2016. Repeated administration of almonds increases brain
acetylcholine levels and enhances memory function in
healthy rats while attenuates memory deficits in animal
model of amnesia. Brain Res. Bull., 120: 63-74.
26. Gotz, J. and L.M. Ittner, 2008. Animal models of Alzheimer's
disease and frontotemporal dementia. Nat. Rev. Neurosci.,
9: 532-544.
27. Gotz, J., J.R. Streffer, D. David, A. Schild and F. Hoerndli
et al
.,
2004. Transgenic animal models of Alzheimer's disease and
related disorders: Histopathology, behavior and therapy.
Mol. Psychiatry, 9: 664-683.
28. Asplund, A., 2002. Antioxidant vitamins in the prevention of
cardiovascular disease: A systematic review. J. Internal Med.,
21: 372-392.
29. Jambazian, P.R., E. Haddad, S. Rajaram, J. Tanzman and
J. Sabate, 2005. Almonds in the diet simultaneously improve
plasma "-tocopherol concentrations and reduce plasma
lipids. J. Am. Diet. Assoc., 105: 449-454.
30. Sano, M., C. Ernesto, R.G. Thomas, M.R. Klauber and
K. Schafer
et al
., 1997. A controlled trial of selegiline,
"-tocopherol, or both as treatment for Alzheimer`s disease.
The Alzheimer`s disease cooperative study. N. Engl. J. Med.,
336: 1216-1222.
31. Petersen, R.C., R.G. Thomas, M. Grundman, D. Bennett and
R. Doody
et al
., 2005. Vitamin E and donepezil for the
treatment of mild cognitive impairment. N. Engl. J. Med.,
352: 2379-2388.
32. Mandalari, G., M.M. Grundy, T. Grassby, M.L. Parker and
K.L. Cross
et al
., 2014. The effects of processing and
mastication on almond lipid bioaccessibility using novel
methods of
in vitro
digestion modelling and micro-structural
analysis. Br. J. Nutr., 112: 1521-1529.
456