Antinociceptive and Antioxidant Activities of the Dillenia indica Bark
M. Badrul Alam,
M. Saifur Rahman,
M. Maruf Khan,
Dillenia indica Linn. (Dilleniaceae) is widely used as food and reputed in the folk medicine of Bangladesh and India in particular, to relieve pain associated with gastrointestinal disturbance. Methanolic extract of the bark of Dillenia indica Linn. (MDI) was subjected to evaluate for its analgesic and antioxidant properties. The analgesic activity of MDI (200 and 400 mg kg-1 b.wt. p.o.) was determined for its central and peripheral pharmacological actions using hotplate as well as tail immersion method and acetic acid-induced writhing test with formalin induced pain in mice, respectively. To evaluate antioxidant potential of MDI, total antioxidant activity, scavenging of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical as well as total Reactive Oxygen Species (ROS) and assessment of reducing power were used. The extract, at the dose of 200 and 400 mg kg-1, produced a significant (p<0.05) increase in pain threshold in hotplate and tail immersion methods whereas significantly (p<0.05) reduced the writhing caused by acetic acid and the number of licks induced by formalin in a dose dependent manner. In DPPH and total ROS scavenging method, MDI showed good antioxidant potentiality with the IC50 value of 12.32±0.16 and 34.72±0.48 μg mL-1, respectively with a good reducing power. The findings of the study suggested that MDI has strong analgesic and antioxidant effects, conforming the traditional use of this plant for pain alleviation to its antioxidant potentiality.
Received: November 05, 2011;
Accepted: February 17, 2012;
Published: March 27, 2012
Pain transmission is a mechanism that involves a very complex interaction of
peripheral and central structures from the skin surface to the central cerebral
cortex (Susanna, 1999). According to the International
Association for the Study of Pain, pain has been defined as an unpleasant sensory
and emotional experience associated with actual or potential tissue damage or
described in terms of such damage (Merskey and Bogduk, 1994).
There are several types of pain, namely nociceptive, neurogenic,
neuropathic and psychogenic which are associated with
a stimulation of nociceptors, damage to neuronal tissue, dysfunction of a nerve
or psychological factors, respectively (Millan, 1999).
The direct and indirect action of chemical mediators, such as arachidonic acid
metabolites (prostaglandins and leukotrienes), peptides, serotonin, acetylcholine,
cytokines, nitric oxide, among others which can be produced or released following
tissue injury or by exogenous irritants (formalin, acetic acid), are responsible
for the multiplicity of events that occur during pain transmission, in both
the peripheral and central nervous systems (Belfrage et
al., 1995; Dray, 1997; Sawynok,
1998; Besson, 1999). Moreover, various free radicals
as well as reactive oxygen species (ROS) are also responsible for the induction
of short-term algesia (Chung, 2004) and triggers some
second messengers, are involved in sensitization of dorsal horn neurons that
plays a fundamentally important role in neuropathic pain (Ali
and Salter, 2001; Zhang et al., 2003). Oxidative
damage caused by the free radicals or ROS is considered to play a causative
role in aging and several stress related diseases including heart disease, inflammation,
diabetes, cognitive dysfunction, cancer and Alzheimers disease (Gulcin
et al., 2002). Undoubtedly, in vivo suppression of these reactive
species is important for the human body to eliminate the toxicity induced by
these reactive species. For several years, many researchers have been investigated
powerful and nontoxic antioxidants from natural sources, especially edible or
medicinal plants to prevent the above reactive species related disorders in
human as well as replace the synthetic compounds which are in use may have carcinogenic
activity and harmful to the lungs and liver (Rechner et
Plant extracts have been used for centuries, as popular remedies against several
health disorders. The study of plants that have been traditionally used as pain
killers should still be seen as a fruitful and logical research strategy in
the search for new analgesic drugs and pain mechanisms (Calixto
et al., 2000). Despite the severe adverse effects of morphine, steroidal
or non steroidal anti-inflammatory agents are used for the treatment of pain
clinically; naturally occurring agents with reduced side effects are required
to substitute this chemical therapeutics. The genus Dillenia has sixty
species, of which Dillenia indica Linn., belongs to the family Dilleniaceae,
is the most common edible species. Originated from Indonesia, this evergreen
tropical tree is now found from Bangladesh, India and Nepal to China. The common
names include Chulta (Bengali, Hindi), Bhavya (Sanskrit) and Elephant apple
(English). The leaf, bark and fruit of this plant are used as traditional medicine.
The juice of D. indica leaves, bark and fruits are mixed and given orally
(5-15 mL, two to five times daily) in the treatment of cancer and diarrhea (Sharma
et al., 2001). The fruit juice of this plant has anti-leukemic effect
(Kumar et al., 2009), cardiotonic effect, used as
cooling beverage in fever and also employed in cough mixture. The leaves and
bark are used as a laxative and astringent. Bruised bark is applied as a cataplasm
for patients with arthritis (Shome et al., 1980).
The solvent extracts of fruits and leaves of D. indica are reported to
have antioxidant activity (Abdille et al., 2005).
CNS depressant activities (Bhakuni et al., 1969)
and anti-inflammatory activity (Yeshwante et al.,
2009) in mice were found from the alcoholic extract of the leaves of D.
indica. Literature reviews indicated that no studies combining the analgesic
and antioxidant of the bark of Dillenia indica have so far been undertaken.
Taking this in view and as a part of our ongoing research (Alam
et al., 2011; Rahman et al., 2011)
on Bangladeshi medicinal plants, the present study aimed to evaluate the analgesic
activity of the bark extracts of Dillenia indica (MDI) along with their
in vitro antioxidant activity.
MATERIALS AND METHODS
Plant materials: The bark of the plant of Dillenia indica Linn. was collected from the botanical garden of Pharmacy department, Jahangirnagar University, Bangladesh during January 2009. The plant material was taxonomically identified by the National Herbarium of Bangladesh whose voucher specimen No. JU/32234 is maintained in our laboratory for future reference.
Chemicals: Ammonium molybdate, Folin-Chiocaltu phenol reagent, were purchased from E. Merck (Germany). 1,1-diphenyl-2-picryl-hydrazyl (DPPH), ascorbic acid, quercetin and potassium ferric cyanide and 2',7'-dichlorfluorescein-diacetate (DCFH-DA) were purchased from Sigma Chemical Company (St. Louis, MO, USA). Nalbuphine, Diclofenac-Na was collected from Square Pharmaceuticals Ltd., Bangladesh. All other chemicals and reagents were of analytical grade.
Preparation of plant extract: The plant material was shade-dried with occasional shifting and then powdered with a mechanical grinder, passing through sieve No. 40 and stored in a tight container. The dried powder material (1.5 kg) was refluxed with MeOH for 3 h. The total filtrate was concentrated to dryness, in vacuo at 40°C to render the MeOH extract (490 g).
In vivo analgesic activity
Animal: Albino mice (25-30 g) of both sexes were used for assessing biological activity. The animals were maintained under standard laboratory conditions and had free access to food and water ad libitum. The animals were allowed to acclimatize to the environment for 7 days prior to experimental session. The animals were divided into different groups, each consisting of five animals which were fasted overnight prior to the experiments. Experiments on animals were performed in accordance with guidelines of the Institutional Animal Ethics Committee, Atish Dipankar University of Science and Technology, Dhaka, Bangladesh. Animal treatment and maintenance for acute toxicity and analgesic effects were conducted in accordance with the Principle of Laboratory Animal Care (NIH publication No. 85-23, revised 1985) and the Animal Care and Use Guidelines of Atish Dipankar University of Science and Technology, Dhaka, Bangladesh.
Acute toxicity study: Acute oral toxicity assay was performed in healthy
nulliparous and non pregnant adult female albino Swiss mice (25-30 g) divided
into different groups. The test was performed using increasing oral dose of
the MDI in water (50, 100, 200, 500, 1000 mg kg-1 b.wt.), in 20 mL
kg-1 volume to different test groups. Normal group received water.
The mice were allowed to feed ad libitum, kept under regular observation
for 48 h, for any mortality or behavioral changes (Sanmugapriya
and Venkataraman, 2006).
Hot plate method: The animals were divided into four groups with five
mice in each group. Group I animals received vehicle (1% Tween 80 in water,
10 mL kg-1 b.wt.), animals of Group II received nalbuphine at 10
mg kg-1 b.wt. while animals of Group III and Group IV were treated
with 200 and 400 mg kg-1 b.wt. (p.o.) of the MDI. The animals were
placed on Eddys hot plate kept at a temperature of (55±0.5)°C.
A cut off period of 15 sec, was observed to avoid damage to the paw (Franzotti
et al., 2000). Reaction time was recorded when animals licked their
fore or hind paws or jumped prior to 0, 30, 60 and 90 min after oral administration
of the samples (Eddy and Leimback, 1953; Malairajan
et al., 2006; Toma, 2003).
Tail immersion test: The procedure is based on the observation that
morphine like drugs selectively prolongs the reaction time of the typical tail
withdrawal reflex in mice (Toma, 2003). The animals
were treated as discussed above. From 1-2 cm of the tail of mice was immersed
in warm water kept constant at 55°C. The reaction time was the time taken
by the mice to deflect their tails. The first reading was discarded and the
reaction time was recorded as a mean of the next three readings. A latency period
of 20 sec was defined as complete analgesia and the measurement was then stopped
to avoid injury to mice. The latent period of the tail-flick response was determined
before and 0, 30, 60 and 90 min after the administration of drugs.
Acetic acid-induced writhing test: The analgesic activity of the samples
was also studied using acetic acid-induced writhing model in mice. Test samples
and vehicle were administered orally 30 min before intraperitoneal administration
of 0.7% v/v acetic acid but Diclofenac-Na was administered intraperitoneally
15 min before injection of acetic acid. After an interval of 5 min, the mice
were observed for specific contraction of body referred to as writhing
for the next 10 min (Ahmed et al., 2004).
Formalin test: The antinociceptive activity of the drugs was determined
using the formalin test described by Dubuisson and Dennis
(1977). Control group received 5% formalin. Twenty microliter of 5% formalin
was injected into the dorsal surface of the right hind paw 60 min after administration
of MDI (200 and 400 mg kg-1, p.o.) and 30 min after administration
of diclofenac Na (10 mg kg-1, i.p.). The mice were observed for 30
min after the injection of formalin and the amount of time spent licking the
injected hind paw was recorded. The first 5 min post formalin injection is referred
to as the early phase and the period between 15 and 30 min as the late phase.
The total time spent licking or biting the injured paw (pain behavior) was measured
with a stop watch.
In vitro antioxidant activity
Determination of total antioxidant capacity: The antioxidant activity of
the MDI was evaluated by the phosphomolybdenum method according to the procedure
of Prieto et al. (1999).
Free radical scavenging activity measured by 1,1-diphenyl-2-picryl-hydrazyl
(DPPH): The free radical scavenging activity of MDI, based on the scavenging
activity of the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical, was
determined by the method described by Braca et al.
Measurement of the inhibition of the total ROS generation: Mice kidney
homogenates, prepared from the kidneys of freshly killed male Swiss albino mice,
weighing 30-39 g, were mixed with or without a suspension of extracts and then
incubated with 12.5 μM DCFH-DA, at 37°C for 30 min. Phosphate buffer
(50 mM, pH 7.4) was used. DCFH-DA is a stable compound which easily diffuses
into cells and is hydrolyzed by intracellular esterase to yield a reduced non-fluorescent
compound, DCFH which is trapped within the cells. The ROS produced by cells
oxidize the DCFH to the highly fluorescent 2',7'-dichlorodihydrofluorescein
(DCF). The fluorescence intensity of the oxidized DCF was monitored on a microplate
fluorescence spectrophotometer (Bio-Tek Instruments Inc., Winooski, VT), with
excitation and emission wavelengths of 460 and 530 nm, respectively (Label
and Bondy, 1990). IC50 value was calculated from the equation
of line obtained by plotting a graph of concentration versus % inhibition.
Reducing power activity: The reducing power of MDI was determined according
to the method previously described (Oyaizu, 1986). Extracts
at different concentrations in 1 mL of 10% DMSO were mixed with 2.5 mL of phosphate
buffer (0.2 M, pH 6.6) and 2.5 mL potassium ferricyanide [K3Fe (CN)6]
(1%) and then the mixture was incubated at 50°C for 30 min. Afterwards,
2.5 mL of trichloroacetic acid (10%) was added to the mixture which was then
centrifuged at 3000 rpm for 10 min. Finally, 2.5 mL of upper layer solution
was mixed with 2.5 mL distilled water and 0.5 mL FeCl3 (0.1%) and
the absorbance was measured at 700 nm. Increased absorbance of the reaction
mixture indicated increased reducing power.
Statistical analysis: All values were expressed as the Mean±SEM
of three replicate experiments. The analysis was performed by using SPSS statistical
package for WINDOWS (version 16.0; SPSS Inc, Chicago). Results related to the
reducing power activities were statistically analyzed by applying the Student
t-test and p<0.001 were considered to be statistically significant. All in
vivo data are subjected to ANOVA followed by Dunnetts test and p<0.05
were considered to be statistically significant.
In vivo analgesic activity
Hot plate method: Both doses of the extract produced a dose dependent increase in latency time when compared with the vehicle (Fig. 1). The result was found to be statistically significant (p<0.05-0.001).
||Effects of the MDI on latency to hotplate test. Values are
Mean±SEM (n = 5); *p<0.05, Dunnett test as compared to control.
Group I: Vehicle (1% Tween 80 in water), Group II: Nalbuphine 10 mg kg-1
b.wt., Group III and Group IV : 200 and 400 mg kg-1 b.wt. (p.o.)
of the crude extract of D. indica
|| Effects of the MDI on acetic acid-induced writhing in mice
|Values are Mean±SEM (n = 5), *p<0.05, Dunnett test
as compared to vehicle control. Group I: Vehicle (1% Tween 80 in water),
Group II: Diclofenac Na 10 mg kg-1 b.wt., Group III and Group
IV: 200 and 400 mg kg-1 b.wt. (p.o.) of the MDI
|| Effect of MDI in hindpaw licking in the formalin test in
|Values are Mean±SEM (n = 5), * p<0.05, Dunnett test
as compared to vehicle control. Group I: Vehicle (1% Tween 80 in water),
Group II: Diclofenac Na 10 mg kg-1 b.wt., Group III and Group
IV: 200 and 400 mg kg-1 b.wt. (p.o.) of the MDI
Tail immersion test: The tail withdrawal reflex time following administration of the MDI was found to increase with increasing dose of the sample. The result was statistically significant (p<0.05-0.001) and was comparable to the reference drug nalbuphine (Fig. 2).
Acetic acid-induced writhing test: Table 1 shows the effects of the extract of on acetic acid-induced writhing in mice. The oral administration of both doses of MDI significantly (p<0.001) inhibited writhing response induced by acetic acid in a dose dependent manner.
Formalin test: MDI (200 and 400 mg kg-1, p.o.) significantly
(p<0.001) suppressed the licking activity in either phase of the formalin-induced
pain in mice in a dose dependant manner (Table 2).
||Effects of the MDI on tail withdrawal reflex of mice induced
by tail immersion method. Values are Mean±SEM (n = 5); *p<0.05,
Dunnett test as compared to control. Group I: Vehicle (1% Tween 80 in water),
Group II: Nalbuphine 10 mg kg-1 b.wt., Group III and Group IV:
200 and 400 mg kg-1 b.wt. (p.o.) of the crude extract of D.
MDI, at the dose of 400 mg kg-1 b.wt., showed the more licking activity
against both phases of formalin-induced pain than that of the standard drug
In vitro antioxidant activity
Total antioxidant capacity: Total antioxidant capacity of D. indica is expressed as the number of equivalents of ascorbic acid. Total antioxidant capacity of MDI was found to be 213.9±0.69 mg g-1 m equivalent of ascorbic acid (Table 3).
DPPH radical scavenging activity: The percentage (%) scavenging of DPPH radical was found to be concentration dependent with the IC50 value of 12.32±0.16 μg mL-1, while IC50 value of standard ascorbic acid was found to be 12.76±0.11 μg mL-1 (Table 3).
Inhibition of total ROS generation: The percentage inhibition of ROS generation was illustrated in Table 3 and it is observed that scavenging of ROS by the extract is also concentration dependent with the IC50 value of 34.72±0.48 μg mL-1, while IC50 value of standard trolox was found to be 8.66±0.11 μg mL-1.
Reducing power ability: For the measurement of the reductive ability,
we investigated the Fe3+ to Fe2+ transformation in the
presence of MDI and compared with standards (galic acid, quercetin and ascorbic
acid) (Fig. 3).
||Reducing power of MDI, quercetin, ascorbic acid and galic
acid by spectrophotometric detection of Fe3+ to Fe2+
Like the antioxidant activity, the reducing power of MDI was found to be concentration
dependent and statistically significant (p<0.001).
The hotplate and tail immersion methods are commonly used for assessing central
antinociceptive response. Both methods are further distinguished by their tendency
to respond to the pain stimuli conducting through neuronal pathways as tail
immersion mediates a spinal reflex to nociceptive stimuli, while the hot plate
involves higher brain functions and is a supraspinally organized response (Chapman
et al., 1985). Narcotic analgesics inhibit both peripheral and central
mechanism of pain, while non steroidal anti-inflammatory drugs inhibit only
peripheral pain (Elisabetsky et al., 1995; Pal
et al., 1999). As noted, nalbuphine, the reference narcotic analgesic
drug (5 mg kg-1, p.o.) exhibited significant and paramount analgesic
effects in both the hot plate (supra spinal) as well as the tail immersion (spinal)
test; whereas, MDI (200 and 400 mg kg-1, p.o.) also produced a statistically
significant but lesser in degree antinociceptive response to that of nalbuphine
in both test suggesting that the plant extract may act as a narcotic analgesic.
However, the mechanism(s) behind the central analgesic response of MDI in both
tested methods is not completely understood and may need further investigation.
On the otherhand, acetic acid induced writhing response is a sensitive procedure
to evaluate peripherally acting analgesics and represents pain sensation by
triggering localized inflammatory response. Such pain stimulus leads to the
release of free arachidonic acid from the tissue phospholipid (Ahmed
et al., 2006). The response is thought to be mediated by peritoneal
mast cells (Ribeiro et al., 2000), acid sensing
ion channels (Voilley, 2004) and the prostaglandin pathways
(Hossain et al., 2006). The organic acid has
also been postulated to act indirectly by inducing the release of endogenous
mediators which stimulates the nociceptive neurons that are sensitive to NSAIDs
and narcotics (Adzu et al., 2003). It is well
known that non-steroidal anti-inflammatory and analgesic drugs mitigate the
inflammatory pain by inhibiting the formation of pain mediators at the peripheral
target sites where prostaglandins and bradykinin are proposed to play a significant
role in the pain process (Hirose et al., 1984).
In addition, it was suggested that non narcotic analgesics produce their action
by interfering with the local reaction to peritoneal irritation thereby reducing
the intensity of afferent nervous stimulation in the acetic acid induced writhing
test, a model of visceral pain (Vogel and Vogel, 1997).
Therefore, it is likely that MDI might have exerted its peripheral antinociceptive
action by interfering with the local reaction caused by the irritant or by inhibiting
the synthesis, release and/or antagonizing the action of pain mediators at the
target sites and this response in agreement with the previous studies with other
parts of D. indica, leaves (Bose et al., 2010).
The above findings clearly demonstrated that both central and peripheral mechanisms
are involved in the antinociceptive action of MDI. The analgesic activity of
MDI could also be linked to the mechanism of action either on central nervous
system or peripheral nervous system. Interestingly, compounds like flavonoids
(Kim et al., 2004a) and steroids, triterpenes
in part, have been shown to possess anti-inflammatory, analgesic activity and
the claim made by Pritam et al. (2011). Based
on the classes of compounds detected in MDI extract, several mechanisms of action
could be used to explain the observed activities of MDI extract.
The formalin model normally postulates the site and the mechanism of action
of the analgesic (Chau, 1989). This biphasic model is
represented by neurogenic (0-5 min) and inflammatory pain (15-30 min), respectively
(Hunskaar and Hole, 1987). Drugs that act primarily on
the central nervous system such as narcotics inhibit both as steroids and NSAIDs
suppress mainly the late phase (Adzu et al., 2003).
The suppression of neurogenic and inflammatory pains by the extract might imply
that it contains active analgesic principle that may be acting both centrally
and peripherally. This is an indication that the extract can be used to manage
acute as well as chronic pain. The mechanism by which formalin triggers C-fibers
activation remained unknown for a relatively long time. Recently, however, McNamara
et al. (2007) demonstrated that formalin activates primary afferent
neurons through a specific and direct on TRPA1, a member of the transient receptor
potential family of cation channels, expressed by a subset of C-fiber nociceptors
and this effect is accompanied by increased influx of Ca2+ ions.
TRPA1 cation channels at primary sensory terminals were also reported to mediate
noxious mechanical stimuli (Kerstein et al., 2009).
These experiments suggest that Ca2+ mobilization through TRPA1 cation
channels is concomitant with noxious chemicals and mechanical stimuli as they
produce their analgesic action. It is likely that the inhibitory effect of MDI
to pain response is due to inhibit the increase of the intracellular Ca2+
through TRPA1, presumably evoked by formalin. So, the bark extract of D.
indica may contain substances that affect the metabolism of Ca2+.
Literature survey revealed that tannins, triterpenoids and flavonoid are the
major phytoconstituents of D. indica (Abdille et
al., 2005; Parvin et al., 2009; Muhit
et al., 2010). Flovonoids, for example, have been found to suppress
the intracellular Ca2+ ion elevation in a dose dependent manner,
as well as the release of proinflammatory mediators such as TNFα (Kempuraj
et al., 2005).
To determine the efficacy of natural antioxidants either as pure compounds
or as plant extract, a great number of in vitro methods have been developed
in which antioxidant compounds act by several mechanisms. Determination of specific
antioxidant species might be less useful than the knowledge of the total antioxidant
capacity of a sample. The knowledge of total antioxidant activity can be useful
in the analysis of changes in plasma antioxidant activity related to oxidative
stress or the understanding of structureactivity relationships of pure
antioxidant species. Because of its simplicity and the cheap reagents it uses,
the phosphomolybdenum method is an alternative to the methods already available
for the evaluation of total antioxidant capacity. The phosphomolybdenum method
was based on the reduction of Mo(VI) to Mo(V) by the compounds having antioxidant
property and is successfully used to quantify vitamin E in seeds (Prieto
et al., 1999). DPPH• is a stable free radical that accepts
an electron or hydrogen radical to become a stable diamagnetic molecule (Nakayama,
1994) and is usually used as a substrate to evaluate the antioxidant activity
of a compound (Chang et al., 2002). Based on
the data obtained from this study, DPPH radical scavenging activity of MDI (IC50
12.32±0.16 μg mL-1) was similar to the standard ascorbic
acid (IC50 12.76±0.11 μg mL-1). These findings
agree with previous reports on scavenging of free radicals with other parts
of D. indica, leaves and fruits. (Abdille et al.,
2005; Parvin et al., 2009). Moreover, it
was revealed that MDI did show the proton donating ability and could serve as
free radical inhibitor or scavenger. A direct correlation between antioxidant
capacity and reducing power of certain plant extracts has been reported (Tanaka
et al., 1988). The reducing properties are generally associated with
the presence of reductones which have been shown to exert antioxidant action
by breaking the free radical chain by donating a hydrogen atom (Duh
et al., 1999). Because a substance may act as an antioxidant due
to its ability to reduce ROS by donating hydrogen atom (Jayaprakasha
et al., 2001; Khanam et al., 2004),
the ferric reducing property of plant extracts (Fig. 3) implies
that they are capable of donating hydrogen atom in a dose dependent manner.
Polyphenolic compounds, like flavonoids, tannins and phenolic acids, commonly
found in plants have been reported to have multiple biological effects, including
antioxidant activity (Kahkonen et al., 1999).
Phenolic compounds are understood to induce the cellular antioxidant system;
increase approximately 50% cellular glutathione concentration. Flavonoids are
important in the modulation of γ-glutamylcysteine synthase in both cellular
antioxidant defenses and detoxification of xenobiotics (Muchuweti
et al., 2007). A flavonoid dillenetin have been isolated from the
MDI (Muhit et al., 2010) and the total phenol
and flavonoid content was found to be the highest in methanolic extract (Abdille
et al., 2005) and this may be the cause for the highest antioxidant
activity in different model.
Flavonoids may increase the amount of endogenous serotonin or may interact
with 5-HT2A and 5-HT3 receptors which may be involved
in the mechanism of central analgesic activity (Annegowda
et al., 2010). Previous researchers reported the presence of several
therapeutically valued flavonoids from the D. indica (Arbianti
et al., 2007; Bose et al., 2010).
Moreover, MDI showed significant analgesic activity in the entire experimental
model which may be due to its high flavonoid content as well as free radical
scavenging activity, as these free radicals are involved during pain stimulation
and antioxidants showed reduction in such pain (Kim et
al., 2004b). It is well established that sensitization dorsal horn cells
in the spinal cord (central sensitization) plays a fundamentally important role
in neuropathic pain. Excessive ROS affects central sensitization and triggers
second messengers system that involved in sensitization of dorsal horn neurons
and also activates spinal glial cells which in turn play an important role in
chronic pain (Raghavendra et al., 2003). In addition,
recent studies suggest that the inflammatory tissue damage is due to the liberation
of reactive oxygen species form phagocytes invading the inflammation sites (Parke
and Sapota, 1996). There are also reports on the role of flavonoid, a powerful
antioxidant (Vinson et al., 1995; Brown
and Rice-Evans, 1998), in analgesic activity primarily by targeting prostaglandins
(Ramesh et al., 1998; Rajnarayana
et al., 2001). Moreover, flavonoids have the ability to inhibits
eicosanoid biosynthesis. Eicosanoids, such as prostaglandins are involved in
various immunological responses and are the end products of the cyclooxygenase
and lipoxygenase pathways (Jothimanivannan et al.,
2010). Tannins are also found to have a contribution in antinociceptive
activity (Ramprasath et al., 2006). Again the
plant extract demonstrated good antioxidant action in the tested models. So
it can be assumed that cyclooxygenase (COX) inhibitory activity along with antioxidant
activity may reduce the production of free arachidonic acid from phospholipid
or may inhibit the enzyme system responsible for the synthesis of prostaglandins
and ultimately relieve pain-sensation.
Based on the results of the present study, we conclude that the plant extract possesses strong analgesic and antioxidant potential. However, further studies are necessary to examine underlying mechanisms of analgesic and antioxidant effects and to isolate the active compound(s) responsible for these pharmacological activities.
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