Analgesic and Antioxidant Properties of Ethanolic Extract of Terminalia catappa L. Leaves
The aim of the present study was to investigate the analgesic and antioxidant activities of ethanolic extract of Terminalia catappa (TCSE) leaves obtained by soxhlet extraction. The analgesic effects of TCSE extract was studied by formalin induced pain, hot plate and tail flick tests where as antioxidant activity was evaluated by ABTS radical scavenging and metal chelating assays. In formalin test, 80 mg kg-1 (p.o.) dose of TCSE extract inhibited both the phases (p<0.05) of animals nociception, but TCSE extracts (40 mg kg-1, p.o.) inhibited only late phase. TCSE extract (80 mg kg-1, p.o.) showed a significant (p<0.05) increase in the reaction time in hot plate test at the time interval of 60, 90 and 120 min. In contrast, both the doses of TCSE extracts did not show any analgesic effect in tail flick test, but morphine (5 mg kg-1, s.c.) shown significantly (p<0.05) higher analgesic effect at all time intervals. TCSE extracts showed moderate ABTS free radical scavenging activity compared to standard gallic acid and higher activity compared to BHT (88.07, 96.35 and 68.76% of inhibition, respectively) but shown less ability to chelate ferrous ion. It was concluded from our studies that TCSE extracts have analgesic and anti-inflammatory properties as well as better radical scavenging ability.
Received: July 27, 2010;
Accepted: September 15, 2010;
Published: October 04, 2010
Terminalia catappa L. (T. catappa L.) is a Combretaceous plant
which grows commonly in tropical and subtropical countries. The leaf, bark and
fruit of this plant have long been used in folk medicine for antidiarrheic,
antipyretic and haemostatic purposes in India, Phillippines, Malaysia and Indonesia
(Lin, 1992). T. catappa leaf has been reported
to possess antioxidative, hepatoprotective, antidiabetic, anti-inflammatory
and anti-HIV reverse transcriptase activity (Chyau et
al., 2002; Tang et al., 2004; Nagappa
et al., 2003; Middleton et al., 2000;
Fan et al., 2004; Tan et
al., 1991). Leaves of T. catappa contains a number of hydrolysable
tannins such as punicalagin, punicalin, chebulagic acid, corialgin, geranin,
terflavins A and B, tergallagin, gratin B (Tanaka et
al., 1986), flavonoids like, isovitexin, vitexin, isoorientin, rutin
(Lin et al., 2000) and triterpenoids such ursolic
acid and 2α, 3β, 23-trihydroxyurs-12-en-28 oic acid (Fan
et al., 2004).
The undesired effects of the analgesic compounds available in the market provided
an opportunity for new and better analgesic compounds (Katzung,
2007). Bioactive compounds derived from plants have been utilised since
from the earlier time for the various purposes including the treatment of pain.
Moreover, numerous plant species were found to possess analgesic and anti-inflammatory
activities (Gill et al., 2010; Jothimanivannan
et al., 2010; Mills and Bone, 2000). The plants
belonging to the family Combretaceae comprise of about 200 species of
Terminalia, which are most widely used for medicinal purposes (McGaw
et al., 2001).
The presence of wide number of bioactive compounds with variety of biological
activities in T. catappa attracted us to select this plant for our analgesic
study. Earlier researchers have reported the analgesic activity of aqueous juice
prepared by maceration from the tender leaves of T. catappa to support
its folklore use (Ratnasooriya et al., 2002).
The amount bioactive constituents present in the plant extract depend on the
method of extraction and type of solvent employed which directly influence the
biological activity of the plant extracts (Hayouni et
al., 2007). In this regard, our main objective was to evaluate the analgesic
activity of T. catappa leaf ethanolic extract obtained by soxhlet extraction.
Free radicals are involved during pain stimulation and antioxidants shown to
reduce such pain (Kim et al., 2004; Das
and Maulik, 1994). Hence, an attempt also been made to evaluate the antioxidant
activity of T. catappa leaf extract.
MATERIALS AND METHODS
Plant material: Fresh leaves of Terminalia catappa were collected
from the campus of Universiti Sains Malaysia (USM), Penang, Malaysia in November
2008. The plant was authentified by a botanist and the voucher specimen (No.
11048) was deposited in Herbarium, School of Biological Sciences, USM. Collected
leaves were then washed with running water to remove the dirt and adherent then
dried and grinds to get powder of 40 mesh size.
Preparation of extract: Hundred gram of ground sample was refluxed with 1000 mL of ethanol (99.5%) at 70°C for 48 h using soxhlet apparatus. The liquid extract was separated from solid residue by vacuum filtration through Whatman filter paper (No. 1), filtered ethanolic was then concentrated to small volume using rotary evaporator (Buchi Rotavapor R-215, Switzerland) followed by drying in freeze dryer (LABCONCO, Free Zone 6 Liter, USA) to get constant mass and kept in air tight container at 4°C until further analysis.
Chemicals: Potassium persulfate, 2,2-azinobis (ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), gallic acid, butylated hydroxytoluene (BHT), aspirin, ferrozine, formaldehyde and ethylenediamine tetraacetic acids (EDTA) were purchased from Sigma Aldrich, Germany. Morphine was obtained from Hospital USM, Kelantan, Malaysia. All solvents used were of analytical grade and purchased from Fisher Scientific (M) Sdn. Bhd, Malaysia.
Animals: Male Sprague Dawley rats (150-200 g) and Swiss albino mice (20-25 g) were obtained from animal house, USM. These animals were acclimatised to the laboratory one week before the experimental procedure. All animals had free access to water and food pellets and were fasted overnight prior to use. The experimental procedures were approved by Animal Ethics Committee, USM.
Formalin-induced pain: Modified method described by Santos
and Calixto (1997) was used in this study. The pain was induced by injecting
0.05 mL of 2.5% formaldehyde (40%) in saline in the subplantar region of rat
hind paw. Preliminary oral LD50 dose of TCSE in mice was 800 mg kg-1
hence rats (six in a group) were given with one-tenth LD50 dose of
TCSE extract (40 and 80 mg kg-1, p.o.), morphine (5 mg kg-1,
s.c.), aspirin (100 mg kg-1, p.o.) and co-solvent. The time spent
in licking and biting the injected paw was considered as indicative of nociceptive
behaviour was recorded. Two successive phases are involved following the injection
of formalin, first phase (0 to 5 min) representing neurogenic pain and second
phase (15 to 30 min) representing tonic and inflammatory pain response (Hunskaar
and Hole, 1987). The percentage inhibition for each rat and each group was
obtained as follows:
Percentage inhibition = [(Ao A1/Ao)x100]
where, Ao is the reading of the co-solvent and A1 is
the reading of samples.
Hot plate: The hot plate test to measure the latency response was performed
according to the method described by Woolfe and MacDonald
(1994), with slight modification (Santos et al.,
1998). Rats were placed on the heated surface of a hot plate analgesia meter
(IITC Life Science Series 8, Model PE34, Victory Blvd, Woodland Hills, CA 91367,
USA) maintained at 55±0.2°C. The time taken for licking, shaking
of forepaws or jumping was recorded as the index of response latency. Animals
were tested before administration of samples in order to obtain the baseline
latency and an automatic cut-off time of 45 sec was used in order to prevent
rat paw tissue damage. Rats (six in a group) were given with TCSE extract (40
and 80 mg kg-1, p.o.), morphine (5 mg kg-1, s.c.), aspirin
(100 mg kg-1, p.o.) and co-solvent (propylene glycol: Tween 80: water
= 4:1:4, v/v/v) served as control. The response latency was measured 30 min
before and 30, 60, 90 and 120 min after the administration of samples.
Tail flick: The method described by D'Amour and
Smith (1941) was used to measure response latency in the tail flick test.
A radiant heat analgesiometer (IITC Life Science Series 8, Model 33T, Victory
Blvd, Woodland Hills, CA 91367, USA) was used. Rats responded to a focused beam
of light by flicking their tail. An automatic cut-off time of 10 s was used
to avoid tissue damage. Animals were tested before administration of samples
in order to obtain the baseline latency. Rats (six in a group) were given with
TCSE extract (40 and 80 mg kg-1, p.o.), morphine (5 mg kg-1,
s.c.), aspirin (100 mg kg-1, p.o.) and co-solvent. The latency for
flicking the tail was determined 30 min before and 30, 60, 90 and 120 min after
the administration of samples and quantified by an average of three measurements.
BTS radical scavenging assay: ABTS radical scavenging activity of TCSE
was determined according to the previously described procedure (Re
et al., 1999). Fresh ABTS radical was prepared by adding 5 mL of
a 4.9 mM potassium persulfate solution to 5 mL of a 14 mM ABTS solution and
this solution was kept for 16 h in dark. This solution was diluted with methanol
to yield an absorbance of 0.700±0.02 at 734 nm and the same solution
was used for the antioxidant assay. The final reaction mixture (1 mL) of standard
and extract comprised 950 μL of ABTS solution and 50 μL of the extract
at variable concentration (0.5-10 μg mL-1). This reaction mixture
was vortex for 10 sec, after 6 min absorbance was recorded at 734 nm and compared
with the control ABTS solution.
ABTS radical scavenging activity of different extracts was calculated using
the following formula:
ABTS radical scavenging activity (%) = [(Ao
where, Ao is the absorbance of the control and A1 is
the absorbance of samples.
Metal chelating activity: The chelating ability of ferrous ions by TCSE
extract was evaluated by the method described by Dinis et
al. (1994). To the 2.5 mL of TCSE extract (0.250-2 mg mL-1)
and EDTA served as the positive control (0.0025-0.04 mg mL-1), 0.05
mL of FeCl2• 4H2O (2 mM) was added. The reaction
was initiated by the addition of 0.2 mL of ferrozine solution (5 mM) then the
mixture was vortexed and kept for 10 min at room temperature. Absorbance of
the resulting solution was then measured at 562 nm against the blank prepared
in the same way using FeCl2 and methanol. Sample without extract
served as the negative control. All tests were done in triplicate and the percentage
inhibition was calculated using the formula used for the ABTS assay.
Statistical analysis: The results of antioxidant and analgesic activity were expressed as means±SD and means±SEM respectively. Statistical analysis of variance was performed with one way ANOVA, followed by Tukeys HSD (Honestly Significant Difference) using SPSS 17 (SPSS Inc., Wacker Drive, Chicago, USA). p<0.05 were consider to be statistically significant when compared to control.
Formalin induced pain: The effect of TCSE extract, aspirin and morphine
in early phase (0-5 min, neurogenic pain) and late phase (15-30 min, inflammatory
pain) of formalin test are shown in Table 1. TCSE extract
significantly (p<0.05) blocked the neurogenic pain only at the dose of 80
mg kg-1 and at the same dose it inhibited the inflammatory pain (46.9%,
p<0.05) better than neurogenic pain (16.39%, p<0.05). TCSE extracts shown
significant (p<0.05) activity during the late phase. Similarly, morphine
produced significant inhibition (p<0.05) of both early and late phase. In
contrast, aspirin showed significant inhibition (p<0.05) only during the
Hot plate: The result of hot plate test was shown in Table
2. The analgesic effect of TCSE extract was significantly (p<0.05) more
with the dose of 80 mg kg-1 at the time interval of 60, 90 and 120
min whereas 40 mg kg-1 found to be not effective at all the time
|| Effect of TCSE extract, morphine and aspirin on formalin-induced
|Each value represents mean±SEM in seconds for six rats
treated with TCSE extracts or reference drugs. *p<0.05, significant from
|| Effect of TCSE extract and morphine on pain induced by hotplate
|Each value represents mean±SEM in seconds for six rats
treated with TCSE extracts or reference drugs. *p<0.05, significant from
|| Effect of TCSE extract and morphine on pain using tail flick
|Each value represents Mean±SEM in seconds for six rats
treated with TCSE extracts or reference drugs. *p<0.05, significant from
Whereas, morphine (5 mg kg-1) considerably (p<0.05) increased
the reaction time to the nociceptive response in comparison with the control,
TCSE extract and aspirin at all the tested time intervals. It was also found
that the antinociceptive activity of morphine was three and two and half times
more than that of 80 mg kg-1 TCSE extract at 30 and 60 min, respectively
and was found to be almost equal at 90 min time interval.
Tail flick: Results of tail flick test (Table 3) revealed that TCSE extracts did not show any analgesic effect in this test at the different intervals of time tested, whereas morphine showed significantly (p<0.05) higher analgesic activity at all the tested time interval. It was also found that the activity of morphine was significantly higher at 30 and 60 min and was found to decrease at 90 and 120 min.
||ABTS radical scavenging ability of TCSE and reference standards.
Each value is expressed mean±SD (n = 3)
||Chelating ability of TCSE and EDTA. Each value is expressed
mean±SD (n = 3)
Antioxidant activity: The free radical scavenging activity of TCSE extract was investigated using ABTS assay. TCSE extract inhibited the ABTS radicals in a concentration-dependent manner (Fig. 1). Scavenging activity of TCSE extract was marked (p<0.05) than the synthetic antioxidant BHT in all the concentration but was less than gallic acid. TCSE extract at the concentration of 10 μg mL-1 showed similar activity as that of gallic acid 4 μg mL-1 concentrations. TCSE extract was found to possess weak chelating ability in comparison with the positive control EDTA (Fig. 2). Chelating ability of TCSE extract at the concentration of 2 mg mL-1 was similar to that of EDTA at the concentration of 0.005 mg mL-1.
In this study, we evaluated the potential antinociceptive activity of TCSE extract using chemical model (formalin induced pain) and thermal models (hot plate and tail flick) of nociception in rats. The antinociceptive models used were such that both centrally and peripherally mediated effects were investigated. Antioxidant models used were to evaluate the radical scavenging and metal chelating ability of TCSE extract.
Formalin method was normally employed to elucidate the mechanism of pain and
analgesia which consists of early phase (0-5 min) because of the stimulation
of nociceptive receptors in the paw and the late phase probably reflects the
inflammation process (Tjolsen et al., 1992; Coderre
and Melzack, 1992). Centrally acting drugs inhibit both phases of formalin
induced pain, where as drugs like aspirin and which acts peripherally inhibit
only the late phase (Chan et al., 1995). Oral
administration of TCSE extract (80 mg kg-1) inhibited the early phase
of formalin test but the percentage of inhibition was four times less than that
of morphine, which shows that central mechanism may be involved in the analgesic
activity of TCSE extract. The second phase of formalin test is related to a
peripheral inflammatory process. Both the doses (40 and 80 mg kg-1)
significantly (p<0.05) inhibited the late phase. Hence, it can be deduced
that peripheral mechanism might also be involved in the anti-inflammatory effect.
This result was in agreement with the findings of Fan et
al. (2004) in which they isolated triterpenic acids which are responsible
for anti-inflammatory activity from T. catappa leaf. But our results
were contrary to the previous findings of Ratnasooriya et
al. (2002) in which administration of macerated aqueous juice of T.
catappa inhibited only the early phase not the late phase. The reason may
be the extraction method and the solvent we employed might have extracted out
the bioactive compounds responsible for the anti-inflammatory activity.
Hot plate test is normally employed to find out the involvement of central
analgesic activity (Paulino et al., 2003). In
the present study, TCSE extract showed analgesic activity at higher dose (80
mg kg-1) thus, the extract may act via central mechanism. Morphine
(5 mg kg-1) a centrally acting analgesic produced significant (p<0.05)
analgesic activity than TCSE extract (80 mg kg-1). Thus, TCSE extract
may have lower potency than morphine. Present findings were in agreement with
the previous findings of Ratnasooriya et al. (2002)
in which oral administration of macerated aqueous juice of T catappa
increased the antinociceptive reaction time.
Tail flick test is a very well known test used for discriminating morphine
like analgesics from non opiate analgesics (Vogel, 2002).
Both the doses of TCSE extracts (40 and 80 mg kg-1) were unable to
produce analgesic effect. We suspect it might be because of the dose of the
TCSE extract used was too low to induce the antinociception in this test. Present
results were in agreement with the previous findings of Ratnasooriya
et al. (2002) in which oral administration of macerated aqueous juice
of T. catappa did not exhibit analgesic effect in tail flick test.
Since, the TCSE extract inhibited both the early and late phase of formalin test and analgesic activity in hotplate test, both central and peripheral mechanism may be involved which implies that it has not only antinociceptive but also anti-inflammatory activity.
Production of oxyradicals as well as nitric oxide plays an important role in
various models of inflammation (Cuzzocrea et al.,
1998; Moncada et al., 1991). TCSE extract
was found to possess good radical scavenging activity and may partly contribute
for the anti-inflammatory activity of the extract. Certain flavonoids like quercetin
were found to inhibit cyclooxygenase, 5-lipooxygenase pathways and even phospholipidase
A2 responsible for the antiinflammatory response (Williams
et al., 1995). A study by Filho et al.
(1996) reported on the antinociceptive effect of quercetin through a central
mechanism. Flavonoids may increase the amount of endogenous serotonin or may
interact with 5-HT2A and 5-HT3 receptors (Lee
et al., 2005) which may be involved in the mechanism of central analgesic
activity (Colpaert et al., 2002). Previous researchers
reported the presence of several therapeutically valued flavonoids from the
leaves of T. catappa (Lin et al., 2000).
Even though TCSE extract was found to possess good radical scavenging activity,
it had weak metal chelating ability in comparison with the reference drug EDTA.
Based on the results of present study, we conclude that TCSE extract possess antinociceptive activity at higher dose and anti-inflammatory activities at lower dose. This activity may be related to the presence of phytochemicals such as flavonoids, tannins and triterpenes reported in leaf extract. This study also justifies the influence of extracting solvent and the method of extraction with respect to the biological activity of the plant extract. Taking into account its potent bioactivity, the plant is an ideal candidate for progress into advanced studies.
Further studies are currently underway to isolate and characterise the bioactive compounds and their antinociceptive mechanisms.
This project was funded by USM Research University Grant and Ministry of Science, Technology and Innovation, Malaysia (MOSTI). H.V. Annegowda gratefully acknowledges Institute of Postgraduate Studies of USM, Malaysia for granting USM Fellowship.
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