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Research Article
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Sedative Activity of Methanolic Extract of Glochidion multiloculare (Rottler ex Willd) Voigt Leaves |
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M. Sekendar Ali,
M. Abdullah Al Mamun,
Mohammed Abu Sayeed,
Mohammad S. Rahman
and
Mohammad A. Rashid
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ABSTRACT
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Bangladesh is a good repository of medicinal plants. Traditional healers utilize them for treating many pathological states. Unfortunately, very few of them have been scientifically evaluated to know about the deep inside. The current study here is designed to evaluate the in vivo sedative activity of the leaves of Glochidion multiloculare (Rottler ex Willd) Voigt. With this purpose, the plant leaves were collected and powdered for extraction with methanol. Initially, the plant extract was subjected to brine shrimp lethality bioassay to monitor the presence of bioactive molecules. Later on, different neuropharmacological studies including hole cross, open field, thiopental-sodium induced sleeping time and Elevated-Plus Maze (EPM) tests were conducted to investigate sedative action. In the brine shrimp lethality bioassay, the LC50 value of the extract was found 37.19 μg mL-1, whereas the standard vincristine sulphate showed the LC50 10.50 μg mL-1. The moderate toxicity of the extract on brine shrimp indicated the existence of bioactive secondary metabolites in this extract. Besides, the extract decreased the locomotor activity of mice in hole cross, open field and EPM test indicating the CNS depression capability of the plant. Moreover, the extract was very much effective for prolonging the sleeping time (103 min) with quick onset of action (22 min) in comparison to the control group. The efficacy of the plant extract was found closer to the common sedative drug diazepam. Further investigations are required to explore the underlying mechanism of the sedative action and isolate bioactive principles.
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Received: March 15, 2013;
Accepted: April 27, 2013;
Published: November 26, 2013
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INTRODUCTION
Plants are the natural reservoir of numerous medicinal elements which globally
contribute a lot to the health management of a large number of populations globally.
The World Health Organization (WHO) acknowledges the herbal medicine in health
care due to their numerous benefits (Aschwanden, 2001).
In the recent trend, many countries are also promoting the standardization of
the plant materials for ensuring proper safety and efficacy of the drug (Huang
et al., 2008).
Bangladesh is located in the tropical zone and blessed with a lot of herbal
resources. Traditionally people of Bangladesh like to use plant derived drugs
as home remedy. Besides, a lot herbal preparations have been marketed for herbal
healing by many pharmaceutical companies. For exploring the multidimensional
effects of the plants of Bangladesh, proper scientific investigations are required.
People sometimes suffer a lot to have a comfortable sleep due to many pathological
or social factors. Sedatives are sometimes useful to provide relief. Plant sources
might be a very useful for displaying the sedative effects (Cardoso-Taketa
et al., 2008; Li et al., 2007; Ratnasooriya
et al., 2006; Rolland et al., 1991;
Schulz et al., 1998; Sharma
et al., 2012a, b). In this trend, we have
investigated the plant G. multiloculare.
G. multiloculare (Local name: Kudurpala, Family: Phyllanthaceae) indigenous
to south asian subcontinent. In Bangladesh it is found in the Hills of Chittagong,
Chittagong Hill Tracts, Comilla and Dinajpur. Fruits of this plant are used
to treat dysentery, diarrhoea, and cough. Previous phytochemical investigations
led to the isolation of glochidonol, glochidiol, glochidone, lupeol, daucosterol
and stigmasterol (Hasan et al., 2012).
The aim of the present study was to explore the biological potentiality of
this plant with special emphasis on sedative property.
MATERIALS AND METHODS
Plant materials: The fresh plant was collected from local area of Chittagong,
Bangladesh and identified by Dr. Sheikh Bokhtear Uddin, Department of Botany,
University of Chittagong.
Preparation of extract: The collected fresh plant leaves were washed
thoroughly with water and then air dried for a week at 35-40°C and pulverized
in electric grinder. The obtained powder was successively extracted in methanol
and filtered by Whatman filter paper. The filtrate so obtained was then concentrated
to dryness through the evaporation of solvent using rotary evaporator under
reduced pressure.
Animals: The study was conducted on Swiss albino mice purchased from
International Centre for Diarrheal Disease and Research, Bangladesh (ICDDR,
B). They were five to six weeks of age, weighing about 30-35 g, which were housed
in colony cages (six mice per cages) at an ambient temperature of 23±2°C
and relative humidity 50-60% with 12 h light and dark cycles having proper ventilation
in the room. The mice were fed normal diets purchased commercially from the
vendors and water ad libitum. The animals were allowed to acclimatize
to the laboratory environment for one week and then randomly divided into groups
for experiments.
Brine shrimp lethality bioassay: This assay was performed on brine shrimp
nauplii using method of Meyer et al., (1982).
In this experiment simple zoological organism (Artemia salina) was used
as a convenient monitor for the screening. The eggs of the brine shrimp were
collected from an aquarium shop of Chittagong, Bangladesh and hatched in artificial
seawater (3.8% NaCl solution) for 48 h to mature shrimp called nauplii. The
test sample of crude extract was prepared by dissolving them in DMSO (not more
than 0.01% v/v) plus sea water (3.8% NaCl in water) to attain concentrations
of 12.5, 25, 50, 100, 200 and 400 μg mL-1. A vial containing
DMSO diluted in seawater was used as a control. Standard vincristine sulphate
was used as positive control. Then matured shrimps were applied to each of all
experimental vials and control vial. After 24 h, the vials were inspected using
a magnifying glass and the number of survived nauplii in each vial was counted.
From the obtained data, the percent (%) of mortality of the brine shrimp nauplii
was calculated. The median lethal concentration, LC50 was then determined
using Probit analysis.
Hole cross test: This test was performed by the method described by
Takagi et al. (1971) for screening sedative activity
in mice. The animals were divided into three groups-control, positive control
and test. The test groups received methanolic extract of G. multiloculare
leaves at the doses of 200 mg kg-1 body weight (b.wt.) orally
whereas the control group received vehicle (1% Tween 80 in water) at dose of
10 mL kg-1 per oral (p.o). A steel partition was made in the middle
of a cage having a size of 30x20x14 cm. A hole of 3 cm diameter was made at
a height of 7.5 cm in the center of the cage. The total number of passages of
a mouse through the hole from one chamber to other was counted for a period
of 3 min on 0, 30, 60, 90 and 120 min after the oral treatment with test drugs.
In this test diazepam was used in the positive control group as reference standard
at the dose of 1 mg kg-1 intra peritoneal (i.p).
Open field test: The experiment was observed according to the methods
described by Gupta et al. (1971). The dose for
extract (200 mg kg-1 b.wt., p.o.) for vehicle (1% Tween 80 in water,
10 mL kg-1 p.o.) and for standard (Diazepam-1 mg kg-1,
p.o.) was maintained throughout the experiment. The floor of an open field of
half square meter was divided into a series of squares each alternatively colored
black and white. The wall of this apparatus was 40 cm height. During the study
period the total number of squares visited by the mice was counted for 3 min
on 0, 30, 60, 90 and 120 min.
Elevated plus maze test: This experiment was previously performed by
Lister (1987). The instrument used here consists of two
open arms (5x10 cm) and two closed arms (5x10xl5 cm) radiating from a platform
(5x5 cm) to give the apparatus a plus sign appearance. The apparatus was situated
40 cm above the floor in which the open arms edges were 0.5 cm in height to
keep the mice from falling and the closed-arms edges were 15 cm in height. Dark
opaque wood was used to make maze floor and walls. Sixty minutes after administration
of the test drugs; each animal was placed at the center of the maze facing one
of the enclosed arms. During the five min test period, the number of entry and
duration of staying into open arms was recorded. The entry into an arm was defined
as the point when the animal places all four paws onto the arm. The sound free
room and observations were made from an adjacent corner was conducted. The same
dose and route of hole cross test was used for this test.
Thiopental sodium induced sleeping time test: According to the experiment
of Ferrini et al. (1974), animals were randomly
divided into three groups consisting of five mice each. The test groups were
received methanolic extract of the leaves of G. multiloculare at dose
200 mg kg-1 (p.o.) body weight while the standard group was treated
with diazepam (1 mg kg-1, p.o.) and control group with vehicle (1%
Tween 80 in water, 10 mL kg-1 b. wt., p.o.). Twenty minutes later,
thiopental sodium (40 mg kg-1, i.p.) were administered to each mouse
to induce sleep. During the latent period (time between thiopental administrations
to loss of righting reflex) and duration of sleep i.e., time between the loss
and recovery of righting reflex the animals were observed.
Statistical analysis: All obtained data were expressed as mean±standard
deviation (n = 5) and were analyzed by one way ANOVA followed by using Dunnetts
test. The differences were considered significant at *p<0.05.
RESULTS
Brine shrimp lethality bioassay for bioactive compounds: The mortality
rate of brine shrimp napulii was increased with the increase with the concentration
of the sample. The LC50 value of the extract was 37.19 μg mL-1
where the LC50 of standard vincristine sulphate was 10.50 μg
mL-1. No mortality was found in the control group, using DMSO and
sea water.
Open field test: In this test, the total number of squares traveled
by the mice was suppressed significantly in the test group throughout the study
period (Table 1). The sedative activity obtained for extract
was statistically significant.
Hole cross test: The total numbers of hole crossed from one chamber
to another by mice were counted for control, standard and test group (Table
2). In the hole cross test, the extracts showed a decrease in locomotion
in the test animals during observation period as evident by the reduction in
number of hole crossed by the treated mice compared to the control group. The
result was comparable to the reference drug diazepam and was statistically significant
(*p<0.05).
Elevated plus maze (EPM) test: The methanolic extract of G. multiloculare
at the dose of 200 mg kg-1 body weight, showed the entries of
mice into the open arms and the time spent in the open arms 40.11 and 30.89%,
respectively. These values are significantly lower than those of control group.
Result of EPM test is presented in Table 3.
Table 1: |
CNS depressant activity of methanol extract of G. multiloculare
leaves on open field test in mice |
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All values are expressed as mean±standard deviation
(n = 5); One way Analysis of Variance (ANOVA) followed by Dunnetts
test. *p<0.05, significant compared to control |
Table 2: |
CNS depressant activity of methanolic extract of G. multiloculare
on hole cross test in mice |
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Values were expressed as mean±standard deviation (n
= 5); One way Analysis of Variance (ANOVA) followed by Dunnetts test.
*p<0.05, significant compared to control |
Table 3: |
CNS depressant activity of methanolic extract of leaves of
G. multiloculare on elevated plus maze test in mice |
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Values are expressed as mean±standard deviation (n
= 5); One way Analysis of Variance (ANOVA) followed by Dunnetts test.
*p<0.05, significant compared to control |
Table 4: |
CNS depressant activity of methanolic extract of G. multiloculare
on thiopental sodium induced sleeping time in mice |
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Values were expressed as mean±standard deviation (n
= 5); One way Analysis of Variance (ANOVA) followed by Dunnetts test.
*p<0.05, significant compared to control |
Thiopental sodium induced sleeping time test: In this test, the test
group was treated with the extract at 200 mg kg-1 showed significant
decrease in onset of sleep and increased duration of sleep. The extract significantly
showed the onset of sleep at 22 min with 103 min duration whereas the standard
diazepam displayed onset of sleep at 15 min with 145 min duration (Table
4).
DISCUSSION
Plants due to the presence of many secondary metabolites might show different
types of bioactivities. Usually bioactive compounds are toxic in higher doses.
So, the lethality originated from toxicity is considered as a marker of bioactive
compounds. For this purpose the brine shrimp lethality is utilized as bench
top bioassay to monitor the presence of bioactive metabolites in plant (Mclaughlin
et al., 1998). Our current study showed moderate toxicity in comparison
to the vincristine sulfate which confirms the presence of bioactive principles
in the G. multiloculare extract. It also justifies the use of this plant
for treating many pathological states as used by the traditional healers.
In the modern lifestyle, neurological disorders like seizure, anxiety, panic,
agitation and insomnia are very common. Various drugs like selective serotonin
reuptake inhibitors (SSRI), monoamine oxidase inhibitors (MAOIs), GABA analogues,
tricyclic anitdeprresant (TCAs) etc are used for counteracting them (Llorca
et al., 2002). These drugs are potent but also showed various side-effects.
Newer types of molecules are required to develop safer and effective drugs.
The γ-aminobutyric acid type A receptor (GABAAR) is an important
ionotropic receptor in neuropharmacological aspect. For this receptor, γ-aminobutyric
acid (GABA) is the major endogenous ligand, which causes hyperpolarization of
the neuron. So, the action potential is blocked and it results in the sedation
(Jiang et al., 2007).
The current neuropharmacological study with the methanolic extract of G.
multiloculare has been conducted. The plant extract demonstrated central
nervous system depressant activity as indicated by the decrease in locomotor
activity in mice in hole cross, open field and EPM test. The efficiency of the
plant extract was almost similar to that of common sedative drug diazepam.
Central Nervous System (CNS) stimulating drugs accelerate motor activities
such as locomotion, grooming and rearing behavior, while the CNS depressing
drugs inhibit those actions (Adebiyi et al., 2012;
Aderibigbe et al., 2010). Our present study
showed the significant level of inhibition of the spontaneous motor activities
in mice which ultimately indicates the effective induction of sedation by the
studied plant extract through CNS depression.
Previously it was reported that the CNS depressants prolong the drug induced
sleeping time (File and Wardill, 1975; Adebiyi
et al., 2012). The studied plant-extract increased the thiopental
sodium induced sleeping time compared to the normal saline treated group. This
ability to prolong the sleeping time demonstrates the capability of the G.
multiloculare to cause CNS depression.
However, plant extract usually contains numerous biosynthetic compounds. These
might show additive or synergistic action on single or multiple target sites
for displaying various types of bioactivities. Many plant extracts have already
been reported to act as ligands for GABAA receptor for showing the
sedative and hypnotic actions (Briskin, 2000; Jiang
et al., 2007). From the G. multiloculare some triterpenes
and steroids have been isolated, which might be responsible for acting as ligands
for GABAA receptor and displaying sedative action (Barua
et al., 2009).
CONCLUSION
The current investigation primarily confirmed the presence of bioactive molecules
in the plant extract as indicated by the brine shrimp toxicity assay. Among
the bioactive compounds, sedative components are available as proved by the
different assays for sedative action. However, further studies are needed to
understand the underlying mechanism of the observed activities and to isolate
the bioactive molecules from this plant.
ACKNOWLEDGMENT
We are grateful to the authority of International Islamic University Chittagong,
Bangladesh for providing adequate funds and other logistic supports to conduct
this research study.
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