Protective Effect of Myricetin Microemulsion against Psychological Stress in Rat Model
Govindaraj Sakthivel, Prabhakaran Prajisha, Manoharan Deva Karunya, Rajan Ravindran*
Department of Physiology, Dr. A.L.M Post Graduate Institute of Basic Medical Sciences, University of Madras, India
*Corresponding author: Dr. Rajan Ravindran, Assistant Professor and Head i/c, Department of Physiology, Dr. ALM PG IBMS, University of Madras, Taramani, Chennai-600 113, India.Tel:+919444145990; Email: ravindran.unom@gmail.com
Received Date: 03 July, 2017;
Accepted Date: 09 August, 2017; Published Date: 19 August, 2017
Citation: Sakthivej G, Prajisha P, Karunya MD, Ravindran R (2017) Protective Effect of Myricetin Microemulsion against Psychological Stress in Rat Model. J Psychiatry Cogn Behav: JPCB-122. DOI: 10.29011/2574-7762.000022
1. Abstract
Undoubtedly stress is an integral part of human life. Stressful experiences disturb the normal biological homeostasis, which has a deleterious effect on normal physiological and psychological function. In the past two decades, there was not much progression in the development of drugs, effective drug delivery or therapy for Central Nervous System (CNS)related problems. Myricetin is a flavonoid, which is present in many dietary products and it has biologically diverse actions such as anticancer, antihypertensive, antioxidant etc.,nevertheless, it is unable to deliver to the brain due to its polar soluble nature. Hence, we developed microemulsion formulation of myricetin (MYR-ME) to enhance its bioavailability into the brain. An animal model of psychological stress was developed by using restrainer (6h/day for 21 days of restraint stress) and treatment with MYR-ME (10mg/kg BW) was administrated for 21 days orally. The experimental animals were randomly divided into four groups, Group I - Control, Group II - Vehicle, Group III - Restraint stress exposed animals and Group IV - Stress with MYR-ME treated animals.At the end stress procedure and stress with treatment, the next day animals were tested cognitive function and anxiety behaviors. The animals were exposed to chronic stress showed deleterious effects on cognitive function, learning and memory,as well as anxiety-related behavior. However, treatment with MYR-ME improved performance in the cognitive function and anxiety-related behavior, indicating that myricetin in the form of the microemulsion can enhance bioavailability of myricetin in the brain which could be demised stress-induced alterations and ultimately acts as a good nootropic and anxiolytic compound.
2.
Keywords: Behavior; Chronic
stress; Cognition; Learning and Memory; MYR-ME
1. Introduction
When homeostasis is threatened by stressful experience, the body undergoes myriad changes. The brain is the central target to stress, where hippocampus, amygdala and prefrontal cortex are the primary areas that undergo stress-induced structural remodeling which can ultimately alter the physiological and behavioral response. The stress can acts on the brain via multiple interacting pathways mediatedby glucocorticoids, excitatory amino acids, and ROS(reactive oxygen species) production. The result of this stressorcan lead to structural remodeling of neuronal architecture and thus, affectcognitive dysfunction, behavioralalteration, learning and memory impairment[1] and other neurological problems.
The stressful responseshas been regulated by two major hormonal system Hypothalamic-Pituitary-Adrenal (HPA) axis and Sympathetic Adrenal Medullary (SAM) axis.Further,elevated circulating glucocorticoidsactivates the release of excitatory amino acid glutamate, resulting in increased cellular Ca2+ concentration, the excess level of Ca2+is toxic to the cell and causes structural and functional changes and also shrinkage of thehippocampal neuron[2] and leads tomany pathological conditions such as cognitive decline, neurodegenerative diseases and neuronal death[3].
The earlier studies reported stress can induce behavioral alteration, depression, cognitive decline and may risk for many other neuropsychiatric and neurodegenerative diseases.Hence, it is important to develop ananimal model of human diseases for understanding pathophysiology and developing new therapeutic strategy against stress induced cognitive dysfunction and anxiety behavior. In the present study, spatial learning and memory was tested by eight arm radial maze [4], novel object recognition test [5].
Elevated plus maze activityconsidered as a valid animal behavioral study to detect anxiety by using natural stimuli like fear of a novel open space and fear of balancing on a relatively narrow, raised platform [6].Open field behavior is another method to test theexploratory, locomotor and anxiety or increased fear condition in rodents[7]. The light/dark test is based on the innate aversion of rodents to brightly illuminate areas and on the spontaneous exploratory behavior of rodents in responses to mild stressors that is novel environment and light[8].
There is a lack of effective drug delivery or therapeutic treatment for Central Nervous System (CNS) related diseases and disorders. There are many potentially active polar soluble drugs are unable to access to the brain. The biggest obstacle for achieving effective therapeutic treatment for the CNS related problem is the presence of Blood-Brain Barrier (BBB). There are many studies on neuroprotective and radical scavenging activity of polyphenols due to theantioxidantcapacities. Accumulating studies shows that polyphenols markedly reduce the risk of dementia [9]and improves the cognitive performances[10].
Myricetin is the drug in the present study,among many other flavonoids it hasmorea number of replaceable numbers of ahydrogen atom in its chemical structure[11]. Thehydrogen atom isresponsible for the free radical scavenging activity of the compound and other health promising activity of myricetin.Myricetin is present in many plant-derived foodstuffs, notably in grapes, berries, onions, walnuts, herbs, wines, vegetables, fruits and medicinal plants and is classified as a flavonoid with strong antioxidant effects [12]. In recent years, it has been reported that myricetin exhibits a variety of beneficial effects on hypoglycemic [13], antioxidant [14], anticancer effect, neuroprotective activity, anti-inflammatory, and antiviral actions in humans [15], it received great attention because of its varied biological activities. Myricetin is a partial water-solublecompound, which limits its curative effect of the drug due to the presence of BBB[16]. Myricetin has the biologically diverse actions though, it is inaccessible to the braindue to polar soluble nature[17]. Hence, we need an alternative effective method for drug delivery techniques or drug formulation methods to reachpolar soluble compounds into thebrain. Hence, we have developed MYR-ME delivery system to enhance bioavailability polar soluble drugs into the brain, this was achieved by technique described in this article.
The microemulsion can be prepared by oil-in-water (o/w) or of water-in-oil (w/o), microemulsion producing a transparent product that has a particle size of 10 to 100 nm and does not have the tendency to coalesce. The particle size determines the rate of drug release as well absorption. It is composed of the oil phase, surfactant, co-surfactant and an aqueous phase at appropriate ratios [18]. The earlier studies have reported that myricetin in the oil are phase protected from the microemulsion and thus avoiding the gastrointestinal and liver metabolism, thereby, it could increase the drug bioavailability by the microemulsion delivery system, microemulsion increase the bioavailability of drug (16.05 fold) when compared to a myricetin suspension by the oral administration [16].
Based on the
earlier literatures we developed a hypothesis that, myricetin in the form of
microemulsion may enhance the drug delivery to the brain and thereby it can
protect or prevent psychological stress induced cognitive dysfunction and
anxiety behavior the Wistar albino rats.
2. Materials and Methods
2.1. Chemicals and Agents
Myricetin was
purchased from Sigma-Aldrich and all other chemicals used are of an analytical
grade, purchased from Sisco Research Laboratories
(SRL), India.
2.2. Animals and Maintenance
Adult male
Wistar Albino rats weighing 200-220g were randomly divided into four groups
used for the study. All animal procedures were approved by the institutional
animal ethical committee and CPCSEA (IAEC No: 01/26/2014). Animals were housed
in a group of three rats per cage and animals were maintained at controlled room temperature 23 ± 2ºC with
12:12 hour light: dark cycle and allowed to free access to food and water. The
animals were fed ad libitum with a standard rat pellet diet and drinking water.
2.3. Experimental Design
Animals were
divided into four groups, each group consists ofsixanimals:Group
I- Non-stress control animals, Group II- Non stress control animal with vehicle
administrated, Group III- Restraint stressexposed
and Group IV- restraint stress with MYR-MEtreated group.
2.4. Psychological Stress Model
According to thepreviously described method, psychological
stress was developed by restraint the animals for 6 hours daily for twenty-one
days[19]. The restrainers were designed
by wire mesh, containsthesteel base, and steel mesh, a padlock and latch will
help to secure the rat in the restrainer.
Wire mesh is designed tightly to fit and restrict the movement of theanimal, without any discomfort, not restricting
breathing, interfering with thermoregulation,
or causing pain. Animals were subjected to chronic restraint stress 6h/day
for21days; the time period was randomly changed in ordered to avoid the
habituation. Each group contains six animals.
The
microemulsion was introduced by Hoar and Schulman,MYR-ME(O/W) was prepared by
the previously described method by Wang, et al.[16]. The
microemulsion was prepared by dissolving Tween 80 and Tween 20 in 1:2
ratio (surfactant) in ethanol (co-surfactant), then mixed well with oleic acid
(oil phase),subsequently theappropriate
amount of myricetin was added into the mixture. The formulations were formed
spontaneously at room temperature when the appropriate amount of phosphate
buffer(pH p6.5) was added by gently vibrating. MYR-ME (10mg/kg BW) dosage as
described in the previous study [17] was administrated orally for 21
days,before an hour of stress exposure.
2.6. Behavior Analysis
2.6.1. Open Field Test
Open Field Test (OFT), is used to measure the exploratory and
anxiety-related behavior in the novel place. The test was performed accordingly
by apreviously described method with slight modification [20], the apparatus made up of (40 cm × 40 cm × 49 cm) black arena with
wooden floor consists of equally divided (5 × 5) square. The apparatus is
illuminated with white light in the center. Rats were placed in the center or one of the four corners of the open field and
allowed to explore the apparatus for 5 minutes. After the 5-minute test, rats
were returned to their home cages.The test provides a unique opportunity to
assess three independent behavioral dimensions relating to the locomotor activity, exploration, and emotional activity, by placing the animal in the
corner of brightly lighted a large rectangular box. Thetested area was cleaned after every entry of an animal with 70%
alcohol. Emotional status of the animal and locomotor activity of the rat was
assessed by the parameters like peripheral ambulation, central ambulation,
rearing, grooming, immobilization, and defecation.
2.6.2. Elevated Plus Maze (EPM)
The
Elevated Plus Maze (EPM) test is used to assess anxiety-related behavior in a
rodent model. The elevated plus maze was made of wooden Perspex, with two
opposite open arms and two opposite closed arms of the same size, the entire
apparatus was elevated 50 cm above the floor. The apparatus was situated in a
darkened room, illuminated by a single 60 W white light bulb located
approximately 100cm above from the center of the maze. Rats were placed in the
central square of the maze, facing one of the open arms. Each animal tested to
elevated plus maze test only once, rats were randomly removed from their home
cages and tested for 5 min in an elevated plus maze to ensure anxiety levels.
The number of entries into open, closed arms and time spent in each arm were
scored for the first 5 min. The tested area was cleaned with 70% alcohol prior
to the introduction of each animal. Animals falling off the maze were
eliminated from the analysis. The parameters include the number of open, closed
arm entries and the number of head dips
(dipping the head below the
open arm of the EPM, with all four paws on an open arm) (Walf & Cheryl, 2007).
2.6.3. Light and Dark box test (Place preference task)
The light/dark
box also was used to assess the anxiety-like
behavior of rodents. The box was divided into two compartments, 18 × 15
× 15 inches(long, wide and high) light compartment with open at the top and 12
× 15 × 15 inches (long, wide and high) the dark compartment that was fully
enclosed. The divider between the two compartments and contained a 3 × 4 inch (wide, high) opening at floor level. This
allows the animal entries between compartments. At the beginning of testing,
each animal was placed in the center of the light compartment. Behavior
subsequently was videotaped for 5 min. Behaviors were scored by an observer who
was blind to the treatment conditions. The measures scored were (1) initial
latency to enter the dark compartment, (2) time spent in the bright area and
(3) time spent in a dark compartment [22].
2.6.4. Radial arm maze (RAM)
Spatial
learning and memory were tested by using an eight-arm radial maze apparatus as
described before with slight modification in the experimental procedure [23].The next day after the end of 21
days of restraint stress animals and restraint stress treated with MYR-ME
animals were tested in the eight-arm radial maze for testing memory after
stress and treatment. The eight-arm radial maze made of steel material, had an
octagonal central platform, 33.5 cm wide, around which were arranged 60 cm long
by 12 cm wide arms. The whole apparatus was elevated 40 cm from the floor in a
sound proof chamber. Before the training session,
the animals were 80% food deprived, during behavioral training the sucrose
pellet is kept as its reward so that they
could become habituated to the apparatus.
Initially, animals were allowed to freely explore the maze for 2 days with all
arms baited with sucrose pellets for 10 min. By the third day of training on
the spatial task, only four arms (fixed for that animal) were always baited and
food rewards placed at the end of the arms. Two training session were performed
for eight days between 09:30-11:00am and
4:30-6:00 pm every day. Each individual rat had its own set of four rewarded
arms. The room contained several visual reference cues on the wall. Each trial
began with the placement of the animal on central platform facing arm number
one and ended when the rat had visited the four baited. The following
parameters were measured on ninth day based on the Olton’s definition, i) Number of reference memory errors(i.e. each
entry into a non-baited arms), ii) Number of working memory errors(i.e.
re-entries into already visited baited arms were noted) and iii) Time is taken
to visit all the baited arms.
2.6.5. Spontaneous alteration T-maze
Spontaneous
alteration T-maze wasused to assess
cognitive ability and memory retention ability of rodents. The test is based on
the left and right arm discrimination, in this test,
therathas to discriminate either left or right arm of the T-maze to get
the food reward. The T-maze consisted of a start box (12x12 cm), stem (35x12
cm), two arms (35x12 cm) and each arm had a goal area of (15x12 cm) and the
side walls were off 40 cm height. The
first day animals were habituated to apparatus and allow to explore in
theT-maze for 10 minutes in both the arms were baited with sucrose pellets.
Followed by ten trials with inter-trial intervals of 2 minutes, rats were
trained to reach either right or left arm and when they reached to the goal
area pellets were provided. The rats were trained till reach 80%corrected
response and noted a number of days taken for reaching 80%. Followed
the80%corrected response, the rats were tested for memory retention after two
days as described before [24].
2.6.6. Novel object recognition test
The
novel object recognition test is used to analyze recognition memory. Behavioral
activity and neuronal activity were recorded first in the open field(40 cm × 40 cm × 49 cm), during the
initial exposure and subsequent familiarization to two identical objects with
same size and different shapes were used for the experiment. During habituation, animals were placed in the open
field apparatus for 10 mins and the next day the identical object was placed
and the rats were allowed to explore on objects for 5 mins after two hours of
interval one old object is replaced with novel object and animals were allowed
to explore for 5 mins. The objects and box were wiped by 70% ethanol after each
trial to remove the odor of the previous rat.
The positions of the objects were similar during training and test. Percentage
of time spent was calculated as TNovel/(TNovel × TFamiliar)
×100 where TNovel is time spent with
anovel object and TFamiliar is time spent with a familiar
object.The experiment involved a three session, one habituation to open field
apparatus and session two is for familiarization to identical objects. During
the third session, the first test of novelty was performed as described before [25].
2.7. Statistical analysis
Data were analyzed by using (SPSS 20)
version software and presented in the form of the bar diagram and expressed as
mean ± SEM. One-way ANOVA, followed by the
multiple comparisons by Tukey post hoc
multiple comparisonswasperformed
eight-arm radial maze, T-maze,novel
object recognition, open fieldbehavior,elevated
plus maze, and light and dark test.
p<0.05 was considered as significant. The “a” represent control group, “b”
represents a vehicle group,“c”
represent the stress group and “d”
represent stress with MYR-ME treated group.
3. RESULTS
3.1.1. Number of central square entries
The
stress group animals demonstrated anxiety related behavior as tested by number
of central squire entries which were significantly reduced (3.67 ± 0.42, 0.003), whereas those animals were treated
with MYR-ME the entries were recorded at the same frequency as the non-stressed
group (7.83 ± 0.60, p = 0.004). The parameters
studied in the open field behavior are summarized inTable 1.
3.1.2. Number of peripheral square entries
The
number of peripheral square entries were significantlyincreased
(53.16 ± 4.35, p = 0.004) in the restraint
stress exposed group. Whereas, the number of peripheral square entries were
significantly decreased (29.33 ± 3.14, p =
0.001) in stress with MYR-ME treated group, as similar to that of control group.
3.1.3. Number of grooming
Therestraint
stress exposed group showed increased number of grooming (14.66 ± 1.20, p = 0.005), this was significantly reduced
(7.83 ± 0.60, p = 0.001) in restraint stress
with MYR-ME treated, almost similar pattern of grooming was observed as
non-stressed group.
3.1.4. Number of rearing
There was markedly increased rearing(16.16 ± 1.57, p = 0.001)was observed in restraint stress exposed group, while this was reduced in MYR-ME treated group (8.66 ± 0.96, p = 0.003), this was restored almost as like that of control group.
3.1.5. Number of fecal pellets
The
stress exposed group showed increased number of fecal pellets (4.33 ± 0.42, p = 0.001) whereas, MYR-ME treated group
showed reduced number of fecal pellets at similar frequency as that of
non-stressed group (2 ± 0.36, p = 0,003).
3.2. Elevated plus maze (EPM)
3.2.1. Number of entries in open arm
The
one-way ANOVA followed by the Tukey posthoc
test revealed stress exposed group animals were showed high anxiety levels as
assessed by of open arms entries, which
were decreased in stress group (1.67 ± 0.33, P =
0.001), whereasMYR-ME treated group showed significantly increased as similar
that of non-stressed animals (5.33 ± 0.67, p =
0.006).The results of parameters studied were shown in Table 2.
3.2.2. Number of entries in closed arm
The increased
number of closed arm entries (8.33 ± 1.75, p =
0.001) were observed in restraint stress exposed group, whereas it was
significantlyreduced in MYR-ME treated group, as close to that of non-stressed
group (5 ± 0.89, p = 0.001).
3.2.3. Time spent in open arm
Stress
group animals showed less time spent in the open arm (29.67 ± 2.18, p = 0.005), this was significantlyincreased in
MYR-ME administrated group,assimilartothat of non-stressed animals (95.8 ± 1.92, p 0. 006).
3.2.4. Time spends in closed arm
The time spent in the closed arm was higher in stress exposed group (269 ± 3.74, p = 0.005), whereas it was significantlydecreased (197.83 ± 1.79, p = 0.004) in restraint stress with MYR-ME treated group.
3.2.5. Number of head dips
The
restraint stress exposed group showed significantly less (10.33 ± 0.67, p = 0.005) head dips whereas, it was almost
restored in MYR-ME treated group, as like that of control animals(24.5 ± 1.17, p = 0.001).
3.3. Light and dark test(Place-preference task)
3.3.1. Initial latency to enter into dark area
The
anxiety related behavior also tested by evaluating initial latency to enter
into dark area, restraint stress exposed group has taken less time (15 ± 1.80, p = 0.003) to enter into dark area, whereas
those who treated with MYR-ME animals showed significantly increased time was taken (29.16 ±
1.57, p = 0.001) to enter the dark area, as that of non-stressed animals, this isshown in Figure 1A.
3.3.2. Time spent in bright area
The
decreased time spent in bright area was observed in restraint stress exposed
group (29.16 ± 2.49, p= 0.001), while this was
significantly increased in MYR-ME treated group, the time spent in bright area
was as similar to that of non-stressed animals (87.16 ±
5.21, p = 0.02),the results are described in Figure 1B.
3.3.3. Time spent in dark area
The
stress exposed group animals showed increased time spent (233.5 ± 2.83 sec, p = 0.01) in dark area,however this was
significantly decreased when treated with MYR-ME, as same that of non-stressed
animals (176 ± 5.4sec, p = 0.001). Time spent in
the dark area of all groups is shown in Figure 1C.
3.4. Spatial memory assessment in eight-arm radial maze
3.4.1. Working memory error
The
stress exposed animals showed deficits in the spatial memory, this was tested
by assessing working memory error,the resulats
aresummarized in Figure 2A. Tukey posthoc test revealed that the working memory
error was significantly increased (1.67 ± 0.12,
p = 0.02) in restraint stress induced group, whereas MYR-ME treated group showed
significantly decreased (0.5 ± 0.12, p = 0.006),
the similar trend was recorded as like non-stressed animals.
. Reference memory error
The number of reference memory error was
more in restraint stress exposed (3.83 ± .30, p
= .003) group, whereas MYR-ME treated group showed asignificantlyless number of reference memory error (1.83 ± .30, p = .004), as same kindthat of non-stressed
animals, the results summarized in Figure 2B.
5.4.3. Time is taken to visit the entire baited arm
The
restraint stress exposed animalswere showed significantly increased (153. 16 ± 2.77, p = .003) time taken to visit all baited arms.
However, MYR-ME treated group showed significantly less time taken (47.17 ± 3.26, p = .009) to visit
all baited arms, alike non-stressed animals, the results are described in Figure 2C.
5.4.4. Novel object recognition test
The
restraint stress exposed group showed significantlyless
exploration to novel object (33.71 ±
3.43, p = 0.01), whereas MYR-ME treated group showed significantly increased novel
exploration (74.58 ± 2.68, p = 0.003), this was restored
almost as that of control animals. The results are shown in Figure 3.
5.4.5. Spontaneous alterationT-maze
Spontaneous
alteration T-maze was used toassessthecognitive
ability of rodents. The parameters
obtained arethe number of days taken to
attain 80% corrected response, spontaneous alteration scoring, theaverage time taken to each entry, memory retention scoring andtheaverage time taken for memory retention
scoring. The results described in Table 3.
5.4.6. Number of days taken for 80%correct choice
Restraint
stress exposed group showed impaired cognitive function, this was tested by
number of days taken to attain 80% correct choice (3.166 ± 0.30, p = .001), whereas,those who treated with MYR-ME
group showed similar days was observed as non-stressed animals(1.33 ± 0.21, p = 0.001).
5.4.7. Spontaneous alteration scoring
Spontaneous
alteration scoring was less (81.67 ± 1.67, p =
0.683) in restraint stress exposed group animals,although, this was restored in
MYR-ME treated group,as like non-stressed animals(85 ±
2.16).
5.4.8. Average time is taken to each entry
Restraint
stress exposed animals demonstrated impaired decision-making ability was tested
by average time to visit each arm entry (24.51 ±
1.99 sec, p = 0.01), however animals those were treated with MYR-ME the average
time taken wasnoted at the same frequency as the non-stressed group (14.73 ± 1.53sec, p = 0.001).
5.4.9. Memory retention scoring
Impaired memory retention ability was observed in restraint stress exposed (61.67± 3.07, p = 0.004), while MYR-ME treated group showed significantly (p<0.05) increased memory retention scoring as that of non-stressed animals (81.66 ± 3.07, p = 0.002).
5.4.10. Average time is taken in memory retention scoring
Average time taken for each entry during memory retention scoring significantly was more in rrestraint stress exposed group (44.11 ± 2.25sec, p = 0.004), whereas MYR-ME administrated group animals were taken significantly less time, as similar result observed in non-stressed animals (13.26 ± 051sec, p = 0.006).
6. Discussion
Stress has become an integral part of human life, stressful experiencedisturbs the imbalance in the body’s physiological function by elevated secretions of stress hormones, which damages the brain and it leads to impairment in cognitive function, learning and memory and also alters the behavior[26].
Restraint stress causes a wide range of anxiety behavior which leads to an alteration inlearning and memory [27]. In the present study, twenty-one days of chronic restraint stress exposed animals showed increased anxiety levelin the open field test, which is characterized by increasedperipheral squares entries and increase grooming and rearing.However, MYR-ME treated group showed significantly reduced peripheral squares entries, grooming, rearing and increased central square entries. The similar result was found in chronic restraint stress exposed group [19].Further, it was confirmed by elevated plus maze test, restraint stress exposed animals showed less open arm entries, more closed arm entries and also the time spent was more in the closed when compared to open arm, theanexiety level was significantly decreased in MYR-ME treated group. Our result is consistent with earlier findings, six days of restraint stress significantly increased anxiety behavior in the elevated plus maze behavior this was improved by treatemet with ocium santum and camellia sinensis.Further, Mohan et al, [28] reported that myricetin treated group significantly decreased anxiety behavior by altering 5-HT levels.
In addition, the restraint stress group animal spent more time in the dark areawhereas, less time was spent in the bright area in the light and dark test, this was reduced in MYR-ME treated group,the anxiety-likebehaviors as the time spent in the lighter area was increased and latency for entering the dark area was significantly reduced in MYR-ME treated group.
From thepresentstudy,it is observed thatMYR-ME effectively protects fromdeteriorating effects of in the rat model. MYR-ME treated animals showed significantly increased spatial learning and memory.It reduces working memory error, reference memory error and timetaken to visit the baited arms compare to restraint stress exposed animals. The present study supports the previous study that 21 days of restraint stress can impair spatial learning [29].
Chronic restraint stress exposed animals showed impaired object recognition memory by changing the dendritic morphology of limbic areas of the rat brain, such as hippocampus, amygdaloid complex, and prefrontal cortex [30], this alteration increases anxiety and impair both memory and spatial learning [31].
Restraintstress exposed group showed an average 3 days taken to attain 80% corrected response in the spontaneous alteration T-maze. In the 1st day, time taken for each entry to the baited arm was increased when compared to that of control, after two days ofT-maze training stress group showed average memory retention scoring 60%and increased time has taken to reach the baited arm (i.e. average time taken for each entry) was more in the restraint group, this indicates the impaired decision-making ability in the restraint stress groups. WhereasMYR-MEtreated group showed increased memory retention ability and less time taken for visiting arms, this indicates that MYR-ME treatment improved or protect the deleterious effect caused by restraint stress, the reason would be increased bioavailability, controlled drug delivery and longer half-life (t1/2) of MYR-ME when compare to myricetin [32].Twenty-one days of restraint stress exposure leads to adeficit in both acquisition and retentions in the T-maze [5].Chronic stress impairs the maintenance of novel short-term memory, i.e. working memory, which is the term applied to the aspect of memory responsible for the recall of information immediatelyafter it has been presented. These results may support that chronic psychosocial stress exaggerates the acquisition of spatial information/object recognition [33]. This memoryimpairment was reduced by MYR-ME treatment.
Myricetin in the form of microemulsion showed (16.05) fold increased bioavailability, increased half-life and controlled delivery system thesewould be reason for the increased activity of myricetin as anxiolytic and nootropic compound [16]. Mohan et al. have reported that myricetin acted as an anxiolytic compound [28], this was further confirmed by the present study MYR-ME was acted as anxiolytic as a compound. Myricetin as such or in the form of microemulsion it acts as antioxidants and it provides the protection against oxidative stress[34-36], this would be a possible protective mechanism of action of myricetin against restraint stress.
The
characteristic features of the study are, microemulsion formulation is one of
the suitable alternative drug delivery methods for polar soluble or partially
polar soluble compounds to enhance the drug delivery to the brain. The present
study extensively validated the anxiety related behavior by appropriate and
three different behavioral techniques and spontaneous alteration T - maze test
to assess cognitive functions such as decision making, memory retention ability.
However, the study also has limitations, the study doesn’t includesbiochemical,
neurochemical and also molecular mechanism behind the ameliorative effect
MYR-ME. Hence, it is necessary to carry out neurotransmitter estimation,
genomic and proteomic expressions to identify the clear mechanism behind the
ameliorative effect of MYR-ME, this will be carried out in future research.
7. Conclusion
Restraint stress havedeleterious
effect on cognitive function and spatial memory, and anxiety-related behavior. The compound of our interest, myricetin in the oil
phase protected from the microemulsion and avoiding the gastrointestinal and
liver metabolism thereby it could increase
the drug bioavailability and controlled drug delivery by microemulsion delivery system. So, thereby
myricetin enter the hepaticenteral
circulation and extend drug release, results in longer
residence time in rats.Restraint stressexposed animalswere
showeddecreased memory retention ability,
decision-making capacity,increased memory
retention ability, spatial learning and increased anxiety relatedbehaviour,
this was effectively protected by oral administration of (10 mg/kg) MYR-ME.The
microemulsion form of the myricetinmayhave protective effect against restraint
stress-induced cognitive dysfunction, spatial memory impairment, and other behavioral
alterations.
Declarations of interest
The authors declare no conflict of interest.
Acknowledgement
Thefinancial assistance from University of Madras and ICMR -SRF fellowships are
greatly acknowledged.
Figure 1:Light and dark box test, initial latency to enter into a dark area (1A), time spent in bright areas (1B) and time spent in a dark area (1C). The data were expressed as mean ± SEM. “a” represents compared to control, “b” represents compared to Vehicle, “c” compared to stress group and “d” represents compared to stress with myricetin microemulsion treated and the value p<0.05 are considered as significant.
Figure 2:Eight-arm radial maze, working memory error (2A), reference memory (2B) and time was taken (2C). The data were expressed as mean ± SEM. “a” represents compared to control, “b” represents compared to Vehicle, “c” compared to stress group and “d” represents compared to stress with myricetin microemulsion treated and the value p<0.05 are considered as significant.
Figure
3:Novel
object recognition test-Novel object
recognition test, the data were expressed as mean ±
SEM. The data were expressed as mean ± SEM. “a”
represents compared to control, “b” represents compared to Vehicle, “c”
compared to stress group and “d” represents compared to stress with myricetin
microemulsion treated and the value p<0.05 are considered as significant.
Parameters
|
Control |
Vehicle |
Stress |
Stress + MYR-ME |
Central squares entries (Count) |
8.33 ± 0.61 |
8.3 ± 0.42 |
3.67 ± 0.42a,b,d |
7.83 ± 0.60c |
Peripheral squares entries (Count) |
33.5 ± 3.27 |
33.83 ± 4.11 |
53.16 ± 4.35a,b,d |
29.33 ± 3.14c |
Number of grooming (Count) |
9.33 ± 0.42 |
9.00 ± 0.73 |
14.66 ± 1.20a,b,d |
7.83 ± 0.60c |
Number of rearing (Count) |
8.00 ± 0.73 |
7.83 ± 1.01 |
16.16 ± 1.57a,b,d |
8.66 ± 0.96c |
Number of faecal pellets (Count) |
1.83 ± 0.30 |
2.16 ± 0.47 |
4.33 ± 0.42a,b,d |
2.00 ± 0.36c |
Table 1: Open field behavior-Describes parameters studied in the open field behavior. The data were expressed as mean ± SEM. *a compared to control, *b compared to vehicle, *c compared to stress group and *d has compared to stress with myricetin microemulsion treated and the value p<0.05 are considered as significant.
Parameters
|
Control |
Vehicle |
Stress |
Stress + MYR-ME |
Open arm entries (Count) |
7.00 ± 0.51 |
6.67 ± 0.49 |
1.67 ± 0.33a,b,d |
5.33 ± 0.67c |
Closed arm entries (Count) |
4.33 ± 1.21 |
4.16 ± 0.75 |
8.33 ± 1.75a,b,d |
5.00 ± 0.89c |
Time spent in open arm (Count) |
128.33 ± 4.64 |
119.0 ± 4.87 |
29.67 ± 2.18a,b,d |
95.83 ± 1.92c |
Time spent in closed arm (Count) |
171.5 ± 4.50 |
177.16 ± 3.63 |
269.0 ± 3.74a,b,d |
197.83 ± 1.79c |
Head dipping (Count) |
24.5 ± 1.78 |
24.5 ± 1.87 |
10.33 ± 0.67a,b,d |
24.5 ± 1.17c |
Table 2: Elevated plus maze-Describes parameters studied in the elevated plus maze. The data were expressed as mean ± SEM. *a compared to control, *b compared to vehicle, *c compared to stress group and *d has compared to stress with myricetin micro emulsion treated and the value p<0.05 are considered as significant.
Parameters
|
Control |
Vehicle |
Stress |
Stress + MYR-ME |
Days taken to attain 80% (Days) |
1.5 ± 0.22 |
1.66 ± 0.21 |
3.1 ± 0.30a,b,d |
1.33 ± 0.21c |
Spontaneous alteration scoring (%) |
85.00 ± 2.23 |
85.00 ± 2.36 |
81.67 ± 1.67 |
85.00 ± 2.16 |
Average time taken to each entry (sec) |
15.93 ± 1.36 |
15.9 ± 1.23 |
24.51 ± 1.99a,b,d |
9.73 ± 0.53a,c |
Memory retention scoring (%) |
81.67 ± 1.67 |
80.00 ± 2.58 |
61.67 ± 3.07a,b,d |
81.67 ± 3.07c |
Average time taken for memory retention scoring (sec) |
15.70 ± 0.89 |
15.00 ± 1.32 |
44.11 ± 2.25a,b,d |
13.26 ± 0.517 |
Table 3: Spontaneous alteration T-maze test-Describes parameters studied in the spontaneous alteration T-maze. The data were expressed as mean ± SEM. *a compared to control, *b compared to vehicle, *c compared to stress group and *d has compared to stress with myricetin microemulsion treated and the value p<0.05 are considered as significant.
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