Elucidating the Effect of hesperidin on Behavioral and
biochemical markers of liver and kidney function in Sprague Dawley Rats
Pandian PaneerSelvam1,2, Usha Kumari1,
Dharma Raj Tanimale1,2, Mohamed Asem1,2,
Sherly Deborah George3, Subramani Parasuraman4
1Unit of Physiology,
Faculty of Medicine, AIMST University, Kedah, Malaysia.
2Clinical Skills
Centre, Faculty of Medicine, AIMST University, Kedah, Malaysia.
3Department
of Physiology, Faculty of Medicine, Manipal University College Malaysia, Melaka,
Malaysia.
4Department
of Pharmacology, Faculty of Pharmacy, AIMST University, Kedah, Malaysia.
*Corresponding Author E-mail: dr.pandianselvam@gmail.com
ABSTRACT:
Background: Hesperidin is a naturally occurring Bioflavonoid found
in citrus fruits. It has anti-inflammatory, antioxidant, antihypertensive, antimicrobial,
anti-carcinogenic and vasodilator activities. Hesperidin’s effect on normal behavioral
is not clear. Hence the present study is aimed to elucidate the effects of hesperidin
on behavioral and biochemical markers of liver and kidney function in Sprague Dawley
(SD) rats. Method: Healthy, adult, male SD rats were used for the study.
Animals were divided into 5 groups (n = 6) viz., Group I: Control, Group
II: Vitamin C(200mg/kg), Group III: Hesperidin (25mg/kg), Group IV: Hesperidin (50mg/kg)
and GroupV: Hesperidin (100mg/kg). The standard and test drugs were suspended in
0.5% w/v carboxymethyl cellulose and administered once daily through oral gavage
for 28 consecutive days. Throughout the study changes in behavioral functions (locomotor
activity, muscular strength, learning and memory) and body weight were monitored
at regular intervals. Blood samples were collected from all the experimental rats
and used for biochemical parameters analysis. Results: Vitamin C and hesperidin
did not show any significant alterations in locomotion, grip strength, muscular
strength and spatial memory when compared with normal control. Both vitamin C and
hesperidin improved the normal anxiety behavior without affecting regular body weight
gain and biochemical markers of liver and kidney function. Conclusion: The
experimental rats administered with hesperidin at the dose levels of 25, 50and100
mg/kg did not show any changes in psychomotor behavior and significantly improved
their alertness without affecting the biochemical markers of liver and kidney function.
KEYWORDS: Actophotometer, Citrus fruits, Forced swim test, Rotarod,
Wire grip strength.
INTRODUCTION:
Medicinal
plants are commonly used as ingredients to prevent or treat diseases. The early
documented records stating the usage of herbal medicine for both prevention and
treatment date back to around 5,000 years in India and China.1
Plantextract
and its phytoconstituents are commonly used for treating various diseases and disorders.
The extracts of the plants and their phytoconstituents are proven for their biological
activities.2 Flavonoids are one of the promising plant secondary metabolites
of over 6000 different components that can be found almost in all plants which are
also in-charge for the pigmentation of the fruits, is a ubiquitous group of polyphenolic
substances.3 Epidemiological and animal studies have shown that citrus
flavonoids influence various activities including antioxidant, anti-carcinogenic,
anti-inflammatory activities and neuroprotective effects.4
Hesperidin
is a naturally occurring bioflavonoid found in immature sour oranges, Citrus
unshiu, Ponderosa lemon and C. mitis. Hesperidin hasanaglycon
(hesperitinormethyleriodictyol) combined with rutinose[6-O-(-l-Rhamnopyranosyl)-D-glucopyranose]
and/or [6-O-(-l-Rhamnosyl)-D-glucose], as a disaccharide component, in its structure
and this makes hesperidin to be considered as -7-rutinoside of hesperetin.5
The bitter compound which is in the Citrusaurantium (bitterorange) is an
isomer of hesperidin which is known as neohesperidin.4
Hesperidin has antimicrobial, antioxidant,
antidiabetic, antihyperlipidemic, anticancer and antipsychotics and its effect on
the normal physiological condition is not clear. Hence the present study is planned
to investigate the dose-depended psychomotor behavior and biochemical changes in
rodents.
MATERIALS AND METHODS:
Animals:
Healthy, adult, male Sprague Dawley (SD) rats
weighing between 140–160 g was obtained from AIMST University’s Central Animal house.
The rats were housed in large, spacious polyacrylic cages at an ambient room with
a 12-hr-light/12-hr-dark cycle. The rats were fed with a normal rodent pellet diet
and water ad libitum. The study was carried out with prior ethical approval
(AUAEC/FOM/2020/06) from AIMST University Human and Animal Ethics Committee, and
the study was conducted according to the Animal Research Review Panel guidelines.
Effect of hesperidinon behavioral and biochemical
markers of liver and kidney function:
A total of 30 rats were used for the study.
The rats were divided into 5 groups (6 animals in each group) [n = 6/group] as follows:
Group I: Control
Group II: Vitamin C (200mg/kg)
Group III: Hesperidin (25mg/kg)
Group IV: Hesperidin (50mg/kg)
Group V: Hesperidin (100mg/kg)
The dose of vitamin C and hesperidin is selected
from the literature.6,7Vitamin C is a known antioxidant. Neurodegenerative
diseases typically involve high levels of oxidative stress and the antioxidant exhibited
potential therapeutic roles against ischemic stroke, Alzheimer's disease, Parkinson's
disease, and Huntington's disease.8In the present study, effect of hesperidin
is compared with antioxidant. Both vitamin C and hesperidin had been suspended in
0.5% w/v carboxymethyl cellulose (CMC) and administered via oral gavage once
daily for 28 consecutive days. The body weight of the rats was monitored at regular
intervals. The effect of hesperidin on behavior (locomotor activity, grip strength,
muscular strength, anxiolytic behavior, immunization time) was studied on pre-study
day, day 7, 14, 21 and 28 of the experiment. At the end of the study, the blood
sample was collected from the experimental rats through retro-orbital plexus puncture.
The serum was separated from the blood sample and was used for biochemical analysis.
Body weight analysis:
Body weight variations were monitored throughout
the study for all the experimental animals at regular intervals.
Behavioral analysis:
Effect of hesperidin on rodent locomotion,
grip strength, muscular strength, anxiolytic behavior, and immunization time were
studied using rodent activity cage (actophotometer), hanging wire grip test, rotarod
test, elevated plus maze test and forced swim test, respectively.
Locomotor activity:
The locomotor activity of the animal was monitored
using actophotometer. Individual rat activity was monitored for 10 minutes at room
temperature. Each rat was given the respective drug and its activity score was evaluated
and recorded after the interval of 15 min.9
Hanging wire grip test:
The hanging wire grip test was performed to
evaluate the strength of the neuromuscular and the coordination of movement of an
experimental animal. Two heavy-based retort stands were placed at a width of 55
cm, and a thread which is 1 mm thick was tied in between these two poles at 35cm
of height. During the test, the thread was tightly attached to the frame to avoid
vibration or unwanted displacement of the thread. The animal was placed in the center
of the wire, and the latency to falling time was observed. A covering of the bedding
material was set down to avoid trauma and injury to the animal when it falls.10
Muscular activities:
The muscle coordination test was done using
the rotarod apparatus. The speed of the rod rotation was adjusted to 4 rpm/min at
first and then was increased up to 20 rpm/min. Pre-training was done to the rats
using the rotarod for the duration of 1–3 h prior to the testing. The training was
incorporated by placing the rat on the rotating rod and measuring the latency to
fall, up to a maximum of 120 s.11
Anxiolytic behavior effect:
Anxiolytic behavior was studied using rodent
elevated plus maze. The elevated plus maze is made of two enclosed and open arms
with 50 × 10 (L × W) dimensions and heights of 40 cm enclosed arms. The two enclosed
and open arms were arranged opposite to each other, and the maze was elevated to
60 cm in height. Preceding the experiment, on the 7thand 14thday
of the experiment, the rat was placed in the center of the maze, facing one of the
enclosed arms and was monitored for the duration of 10 min. During the observation,
the number of entries to each arm and the time spent in each arm were recorded.
The elevated plus maze experiment was conducted inan environment which is sound
free.12
Forced swim test:
The forced swim test is used to evaluate “depressive-like”
states and behavioral despair. The training was done for 3 consecutive days, with
four consecutive trials/day for each of the experimental rats with a time interval
of 30 min.13 The water tank contained a round pool, filled with tap water
(23°C–26°C), to the depth of 0.3–0.4 m. The rodent was then put into the tank upon
which the time between the rodent stopped swimming and start sinking was recorded.
The rodent was removed immediately as soon as it started to sink. The procedure
was repeated for all the rodents. The water tank was then cleaned frequently to
avoid microbial contaminations.14
Biochemical parameters analysis:
At the end of the study, about 1 mL of blood
sample was collected through the retro-orbital plexus and the serum was separated
by centrifuging at 3000 RPM for 20 min.15 Then, the separated serum
sample was used for the estimation of alanine aminotransferase [ALT], aminotransferase
[AST], alkaline phosphatase [ALP], creatinine and urea using the Reflotron Plus
biochemical analyzer (Roche Diagnostics, Germany) with the help of commercially
available Reflotron strips.
Statistical analysis:
All the data was expressed as mean ± standard
error of the mean (SEM), and the statistical significance between the groups was
tested using one-way analysis of variance followed by Tukey’s post-hoc test.
P < 0.05 was considered statistically significant.
RESULTS:
Hesperidin (25, 50 and 100 mg/kg) and vitamin
C administered animals did not show any significant changes in the bodyweight throughout
the study when compared with the control animals (Figure – 1).
In behavioral analysis, hesperidin and vitamin
C did not cause any significant changes in locomotor activity, grip strength, muscular
strength and immobilization time when compared with the control (Figure – 2 - 5).
The anxiolytic effect of hesperidin was studied using an elevated plus maze. In
this, hesperidin 50 and 100 mg/kg showed significant reductions in time spent in
closed arms and increased in time spent in open arms from day 21 and day 14 onwards,
respectively when compared with the control. Vitamin C significantly reduced time
spent in closed arms and increased the time spent in open arms from day 14 onwards
compared with the control group (P<0.001). The results indicated that both hesperidin
(50 and 100 mg/kg) and vitamin C administered animals had improved the normal anxiety
behavior (Figure – 6 & 7).
In biochemical parameters analysis, both hesperidin
and vitamin C did not cause any significant changes in AST, ALT, ALP, bilirubin,
urea, and creatinine when compared with the control (Table - 1).
Figure
1: Effect of hesperidinon body weight of rats. All the values are expressed as mean± SEM (n =6).
Figure
- 2: Effect of hesperidinon locomotor
activity of rats. All the values are expressed
as mean ± SEM (n = 6).
Figure
- 3: Effect of hesperidinon grip strength
of rats. All the values are expressed as
mean± SEM (n =6).
Figure
- 4: Effect of hesperidinon muscle
coordination of rats. All the values are
expressed as mean ± SEM (n =6).
Figure
- 5: Effect of hesperidinon immobilization
time of rats. All the values are expressed
as mean± SEM (n =6).
Figure – 6: Effect of
hesperidin on anxiety (Time spend in elevated plus maze arms) of rats. Values are expressed as mean ± SEM(n=6).bP<0.01andcP<0.001compare
with control (One-way ANOVA followed by Tukey post-hoc test)
Figure
– 7: Effect of hesperidin on anxiety (Number of entries in elevated plus maze arms)
of rats. All the values are expressed as
mean± SEM (n =6).
DISCUSSION:
In the present study, effect of hesperidin
on the behavioral function and biochemical parameters were evaluated. The animals
administered with hesperidin 25, 50 and 100 mg/kg did not show any significant changes
in locomotor activity, grip strength, muscular strength, and biochemical parameters.
Also, hesperidin improved the anxiolytic behavior in rats.
Throughout the study, hesperidin did not affect
the body weight of the animals. Bodyweight is considered as a simple and sensitive
indicator of adverse effects after exposure to asubstance.16In the experimental
animals, hesperidin does not exhibit any negative effect on the growth, weight loss
or weight gain of the animals.17 Thenmozhi et al., also studied
the effect of hesperidin on body weight at the dose levels of 100 mg/kg body weight
in rats and observed no significant changes in regular body weight. Hesperidin also
prevented the metal ion-induced reduction in the body weight of experimental animals.18
In the present study, hesperidin did not affect
the locomotor activity. Kumar et al., studied the effect of hesperidin (50
mg/ kg) on locomotor activity and observed no significant changes in locomotor activity.19Wire
grip strength test has been done and it includes the functional observational battery
(FOB) to study the neurobehavioral toxicity and the changes that occur in grip strength
have been explained as evidence for motor neurotoxicity in experimental rats.20
Throughout the study, hesperidin did not show any changes in grip strength behavior.
Motor coordination is one of the complex behavioral
domains which could show muscle strength, balance, and patterned gait, as well as
the competence of the sensory system functions. Problems in motor performance can
disconcert the behavioral pattern of learning and memory, exploration, motivation,
and nociception.21 In the present study, hesperidin did not show any
significant changes in muscular strength. Kumar et al., studied the effect
of hesperidin on muscular function in rats and did not observe any significant changes
in the grip strength when compared with vehicle administered group.19
Hesperidin also didn’t affect the immobilization time in the forced swim test. A
forced swim test is used to monitor depressive-like behavior and assumes that immobility
reflects a measure of behavioral despair.13Javed et al., evaluated
the effect of hesperidin (100 and 200mg/kg) on the impairment of memory in an intracerebroventricular
streptozotocin-induced model of memory impairment (using Morri’swatermaze test)
in mice and observed memory enhancement effect.22 Hesperidin also has
a beneficial effect on neuroinflammation, neurobehavioral, oxidative stress and
lipid alteration in intra cerebroventricular streptozotocin-induced cognitive impairment
in mice.22,23
Hesperidin (50 and 100 mg/kg) and vitamin C
reduced time spend in closed arms and increased time spend in open arms when compared
with the control. The results indicated that both hesperidin (50 and 100 mg/kg)
and vitamin C administered animals had improved their normal anxiety behavior. The
elevated plusma zetest which is widely used for rodent models of anxiety in research
to investigate the neurochemical basis of anxiety and the psychological of the rats
[24]. Fu et al., reported the antidepressant effects of hesperidin. In chronic
unpredictable mild stress-induced mice, hesperidin (100, 200 mg/kg) significantly
relieved depressive-like behaviors[25]. Hesperidin (100 mg/kg) also, improved continuous
and interval swimming in rats.26
Hesperidin (25, 50 and 100 mg/kg) and vitamin
C did not show any significant changes in biochemical parameters. In four weeks
of continuous and interval swimming study, hesperidin lowered total cholesterol
and triglycerides and increased high-density lipoprotein cholesterol.26Hesperidin
is known for its antioxidant effect and this effect may contribute to its pharmacological
actions.27Hesperidin is predominantly metabolized to hesperedin-7-O-β-D-glucuronide
and hesperedin-3’-O-β-D-glucuronide. Both these metabolites have transactivation
activity towards peroxisome proliferator-activated receptor gamma and regulates
glucose and lipid homeostasis in human.28 Hesperidin also reduces inflammatory mediators including
cyclooxygenase and oxide synthase and prevents/ reduces oxidative stress-induced
cellular damage and improves the well-being.29,30
CONCLUSION:
The experimental rats administered with hesperidin
at the dose levels of 25, 50 and100 mg/kg did not show any changes in psychomotor
behavior such as locomotor activity, muscular strength, and immobilization time
and significantly improved their alertness when compared with control. Hesperidin
also did not show any significant changes in biochemical parameters at the dose
levels of 25, 50 and100 mg/kg. Thus, this study results suggest that hesperidin
can improve well-being (alertness) without producing any alterations in psychomotor
behavior and biochemical markers of liver and kidney function.
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