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Journal of Medicinal Plants Research Vol. 6(21), pp. 3724-3731, 9 June, 2012
Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR11.743
ISSN 1996-0875 ©2012 Academic Journals
Full Length Research Paper
Phytochemical screening, antioxidant and analgesic
activities of Croton argyratus ethanolic extracts
N. I. Mohd Ali, H. V. Annegowda, S. M. Mansor, S. Ismail, S. Ramanathan and M. N. Mordi*
Centre for Drug Research, Universiti Sains Malaysia 11800, Penang, Malaysia.
Accepted 4 July, 2011
Series of experiments were conducted to screen phytochemical constituents, antioxidant and analgesic
activities of the ethanolic extracts of the plant Croton argyratus. The extracts obtained from leaves,
stem and root of the plant were evaluated for their antioxidant activity by means of 2,2- Diphenyl -1-
picrylhydrazyl (DPPH) radical scavenging activity, reducing power and total antioxidant capacity as well
as total phenolic and flavonoid contents was studied. To determine analgesic property of the
antioxidant rich extract, and formalin induced pain, hot plate and tail flick test were performed. The
leaves extract showed the highest value of antioxidant activity based on DPPH radical scavenging
activity, reducing power and total antioxidant capacity. The leaf extract also produced the highest total
phenolic and total flavonoid content and have a significant activity in late phase of the formalin induced
pain test at the dose of 200 mg/kg p.o. However, in the hot plate and tail flick tests, the extract did not
show any significant analgesic effects. The results suggested the potential use of C. argyratus plant
extracts as a natural source of antioxidant and may act peripherally to relieve pain.
Key words: DPPH, reducing power, total antioxidant capacity, total phenolic content, total flavonoid content,
peripheral analgesic.
INTRODUCTION
Croton argyratus is a small or medium-sized tree that can
grow up to 60 ft and can be found throughout Malaysia,
Burma (Myanmar) and Bali (Burkill, 1966). C. argyratus is
locally known as hamba raja, cheret budak, semelit
mayor or akar cheret budak. This plant is also recognized
as ‘Silver Croton’ as the undersides of the plant leaves
are silvery white or silvery brown. The decoction of the
leaves and stems are used by locals to cure purging and
to aid recovery from childbirth. The biological and
pharmacological aspects of Croton have been studied for
anti-inflammatory (Suarez et al., 2006), anticancer
(Sylvestre et al., 2006) and cytotoxicity (Morales et al.,
2005) activities. Horgen et al. (2001) has carried out
cytotoxicity test on human lung cancer line using the
methanol extract of the leaves/twigs, roots and stem bark
of C. argyratus. Results showed that the extracts of C.
argyratus displayed toxicity to cancer cells with an IC50
*Corresponding author. E-mail: mnizam@usm.my. Tel: (604)-
6533272. Fax: (604)-6568669.
values of <5.0 µg/ml. All extracts showed selectivity of
>10-fold against Lu-1 cell line compared with other cell
lines tested. In addition, C. argyratus also showed a good
antiplasmodial activity to Plasmodium falciparum
sensitive strain D10 (Noor et al., 2007).
Free radicals are chemically reactive species bearing
one or more unpaired electrons. They were found to play
a potent role in affecting human health. Radicals and
reactive oxygen species such as the superoxide anion
(O2•‾), hydroxyl radical (OH•) and peroxy radical (ROO•)
are known as mediators for degenerative and chronic
deteriorative including carcinogenesis, coronary heart
disease, inflammation, arthritis, diabetes and aging
(Ames et al., 1993; Heliovaara et al., 1994; Moskovitz et
al., 2001).
Antioxidants are essential substances that help to
protect the body from damage caused by free radicals
induced oxidative stress (Orhan et al., 2009). Synthetic
antioxidants such as butylated hydroxytoluene (BHT) and
butylated hydroxyanisole (BHA) have been shown to be
toxic and may cause mutagenic effect (Grice, 1986).
Currently, research interest to find natural antioxidants
has risen and the present work was carried out to explore
the in vitro antioxidant property of C. argyratus. An effort
has also been made to evaluate analgesic activity
possessed by C. argyratus leaves ethanolic extract
(CAE) as recent studies have shown that free radicals
are responsible for producing pain and inflammation (Gao
et al., 2007; Koblyakov, 2001).
MATERIALS AND METHODS
Chemicals
2,2-Diphenyl-1-picrylhydrazyl (DPPH), ferric chloride (FeCl3), Follin-
Ciocalteu reagent, aluminium chloride (AlCl3), aspirin,
formaldehyde, ammonium molybdate, ascorbic acid, gallic acid, and
(±)-catechin hydrate standards were obtained from Sigma-Aldrich
(St.Louis, MO, USA). Methanol, 95% ethanol, sulphuric acid
(H2S04), potassium ferricyanide [K3Fe(CN)6] and trichloroacetic acid
(TCA) were obtained from Merck (Germany). Sodium nitrite
(NaNO2), sodium carbonate (Na2CO3), sodium phosphate (Na3PO4)
and sodium hydroxide (NaOH) were purchased from Fluka (USA).
Morphine was obtained from Hospital Universiti Sains Malaysia
(Universiti Sains Malaysia, Kubang Kerian, Kelantan, Malaysia).
Plant material
C. argyratus was collected from Gunung Jerai, Kedah, Malaysia
and identified by Dr. Rahmad Zakaria, from the School of Biological
Sciences, Universiti Sains Malaysia (USM). A voucher specimen
(number 11196) was kept at School of Biological Sciences, USM.
The different parts of the plant were washed to remove the dirt and
dried in oven at 40°C. The dried leaves, stem and root were then
grinded to fine powder.
Preparation of extracts
Leaves, stem and root of C. argyratus (200 g each) were extracted
using Soxhlet apparatus at 50°C using 95% ethanol (2.5 L). The
extracts were then filtered, concentrated in a rotary evaporator and
followed by freeze drying to obtain dry powder of extracts. All
samples were kept in air tight glass container and stored at 4°C
until further analysis.
Animals
Analgesic experiments were conducted using male Swiss Albino
mice after protocol approval by the Animal Ethics Committee of
USM. The mice weighed between 25 to 30 g were obtained from
Animal House, USM. The animals were acclimatized to the lab
conditions for one week prior to the experiments. They were kept at
room temperature (37 under a light/dark cycle of 12 h and fed with
free access food and tap water ad libitum.
Preliminary phytochemical screening
Phytochemical screening of C. argyratus ethanolic extracts was
performed according to the method described by Sofowara (1982),
Harborne (1973), and Siddiqui and Ali (1997). This experiment was
carried out to detect the presence of amino acids, antraquinones,
flavonoids, saponin, steroid, terpenoids, cardiac glycoside, tannins,
alkaloids and reducing sugar.
Ali et al. 3725
Determination of total phenolic and flavonoid content
The total phenolic content of ethanolic extracts of different parts of
C. argyratus was measured using Folin-Ciocalteu method described
by Slinkard and Singleton (1977). To an aliquot of 0.4 ml sample,
2.0 ml of prediluted Folin-Ciocalteu reagent (1:10) was added. After
4 min, 1.6 ml of Na2CO3 solution (75 g/L) was added to the mixture
and mixed well. After 1 h, the absorbance of the resulting mixture
was measured at absorbance of 765 nm. Total phenolic content
was calculated from the calibration curve of gallic acid standard
solution and the values were expressed as mg gallic acid
equivalent (GAE)/g dry extract.
The total flavonoid content was determined according to the
method described by Sakanaka et al. (2005). In brief, 0.25 ml of
sample was mixed with 1.25 ml distilled water followed by 75 µl of
5% NaNO2. After 6 min, 150 µl of 10 % AlCl3 was added and the
mixture was allowed to stand for 5 min followed by addition of 0.5
ml of 1 M NaOH. Immediately, distilled water was added to the
mixture to make up the final volume of 2.5 ml. The absorbance of
this solution was determined at 510 nm. The total flavonoid content
was interpolated from the calibration curve of catechin solution and
the values were expressed in mg of catechin equivalents (CE)/g dry
extract.
Determination of antioxidant activity
DPPH radical scavenging activity
DPPH radical scavenging activity C. argyatus plant extracts as well
as ascorbic acid was determined using the method described by
Brand-Williams et al. (1995). Briefly, 77 µl of each extract and
ascorbic acid ranging from 0.01 to 1 mg/ml was transferred into 3
ml of 6 × 10-5 M methanolic DPPH solution. The samples were
incubated in the dark for 15 min at room temperature followed by
measuring the absorbance at 517 nm. The percent inhibition of
radical scavenging activity was calculated using the following
equation:
Ao - Ae
× 100
Ao
Where; Ao = absorbance of control, Ae = absorbance of extract
The IC50 values were calculated by linear regression of plots where
x-axis represented the concentration (mg/ml) and y-axis
represented the scavenging effect (% inhibition).
Reducing power
The reducing power of C. argyratus plant extracts, ascorbic acid
and gallic acid were carried out according to the method described
by Oyaizu (1986). To 0.1 ml of sample solution, 2.5 ml of 0.2 M
phosphate buffer (pH 6.6) and 2.5 ml of 1% K3Fe(CN)6 was added
followed by mixing and then incubated at 50°C for 20 min. After 20
min, 2.5 ml of 10 % TCA, 2.5 ml of distilled water and 0.5 ml of
0.1% FeCl3 were added. Then the absorbance of the final solution
was measured at 700 nm. A higher absorbance value of the
reaction mixture suggested a stronger reducing power activity.
Total antioxidant capacity
Total antioxidant capacity was determined using the method
described by Dasgupta and De (2004). To the 0.3 ml of ethanolic
3726 J. Med. Plants Res.
Table 1. Qualitative phytochemical screening of C. argyratus
plant extracts.
Constituent
Leaf
Stem
Root
Amino acids
-
-
-
Antraquinones
-
-
-
Flavonoids
+
+
+
Saponin
-
-
-
Steroid
+
++
+
Terpenoids
+++
+++
+++
Cardiac glycosides
+
+
-
Tannins
+
+
-
Alkaloids
-
-
-
Reducing sugar
+
+
-
+++ = abundance; ++ = moderately present; + = weakly present;
- = absent.
extracts of different parts of C. argyratus, respectively, 3 ml of
reagent solution (0.6 M H2SO4, 28 mM sodium phosphate and 4
mM ammonium molybdate) was added. The test tubes were
capped incubated at 95°C for 90 min. After the samples had cooled
to room temperature, absorbance of the mixture was measured at
695 nm. The antioxidant activity was expressed as milligram
ascorbic acid or gallic acid equivalent antioxidant capacity/g extract.
All the experiments were conducted in triplicates.
Analgesic activity
Hot plate
The hot plate test was performed using the method of Woolfe and
MacDonald (1944). Male Swiss Albino mice (n = 6) were
administered with CAE (50, 100 and 200 mg/kg), negative control
cosolvent (propylene glycol:tween 80:water = 4:1:4, v/v/v), and
positive control morphine sulphate (5 mg/kg s.c), respectively. After
30 min treatment (except 15 min for morphine), the mice were
placed on hot plate analgesia meter maintained at 55 ± 1°C. Time
taken for licking paw and jumping were recorded as response
latency. The animals were observed for every 15 min over 90 min
periods. The cut off time 45 s was chosen to prevent tissue
damage.
Tail flick
The experiment was carried out as described by D’Amour and
Smith (1941). Mice were administered orally with CAE (50, 100 and
200 mg/kg), cosolvent (propylene glycol: tween 80: water = 4:1:4,
v/v/v) as control and morphine sulphate (5 mg/kg, s.c) respectively.
The tail flick response of the mice was carried out by gently placing
the mice tail at the central position of the light beam and mice’s
response to the light by flicking or removing its tail was referred as
latency time (s). The cut off time of 10 s was maintained to prevent
tissue injury to the mice tail.
Formalin induced pain
The method used was similar to that described previously by
Hunskaar and Hole (1987) was followed to study formalin induced
pain Mice, which were treated with C. argyratus leaves extract (50,
100 and 200 mg/kg), cosolvent (propylene glycol: tween 80:
water = 4:1:4, v/v/v) and morphine sulphate (5 mg/kg. s.c)
respectively, 30 min before formalin injection (15 min for morphine).
After 30 min, 20 µl of 2.5% formalin in saline was injected to the
right hind paw of mice. The mice were placed in a glass cylinder
and the time spent (s) licking and biting the injected paw in the early
phase (0 to 5 min) and late phase (15 to 30 min) was recorded as
indicative of pain.
Statistical analysis
The results of antioxidant and analgesic activity were presented as
mean ± S.D from triplicate determination. Analysis of variance
(ANOVA) followed by Tukey’s test and Dunnet’s test were
performed to determine the significant difference between samples
(p < 0.05) using SPSS version 18 and Sigmaplot version 11.
RESULTS
Preliminary phytochemical screening
The result of qualitative phytochemical screening is
presented in Table 1. All extracts contained flavonoids,
terpenoids and steroids. Cardiac glycosides, tannins and
reducing sugar were detected in leaves and stem but
absent in root extract. However, amino acids,
antraquinones, saponin and alkaloids were completely
absent in all extracts.
Determination of total phenolic and total flavonoid
content
Table 2 shows the total phenolic and flavonoid contents
in different parts of C. argyratus. Total phenolic contents
was determined using the Follin-Ciocalteu reagent and
reported as gallic acid equivalents (GAE) by reference to
a standard curve (y = 8.470x + 0.285, r2 = 0.993).
Meanwhile, total flavonoid content was calculated in
comparison with standards of catechin equivalent (y =
3.093x + 0.0558, r2 = 0.995) and the result expressed as
in terms of catechin equivalent/g (CE/g) dry sample. The
results showed that leaves contained highest phenol
content followed by stem and root with the values of
40.62, 17.95 and 12.0 1mg GAE/g dry sample,
respectively. The highest flavonoid content was found in
leaves extract followed by stem and root with total
flavonoid content of 17.78, 14.29 and 7.08 mg CE/g dry
extract.
DPPH radical scavenging activity
As shown in Figure 1, the highest activity was observed
in the leaves extract followed by stem and root. The
scavenging abilities on DPPH radicals at 1 mg/ml of
extracts were leaves (62.54%), stem (30.48%) and root
(25.02%), respectively. Ascorbic acid (a standard
antioxidant) showed 96.04% inhibition of DPPH radical at
a concentration of 1 mg/ml. The IC50 value of C.
Ali et al. 3727
Table 2. Total phenolic and flavonoid content and IC50 values of C. argyratus plant extracts.
Extract
Total phenolic content
mg GAE/g extract
Total flavonoid content
mg CE/g extract
DPPH assay (IC50
mg/ml)
Leaves
40.62 ± 3.12
17.78 ± 0.34
1.45
Stem
17.95 ± 0.67
14.29 ± 0.13
ND
Root
12.01 ± 0.55
7.08 ± 0.73
ND
Ascorbic acid
-
-
0.09
Data shown as mean ± SD, n = 3. ND, could not be determined.
0.0 0.5 1.0 1.5
0
50
100
150
AA
leaves
stem
root
a
b
c
concentration (mg/mL)
% inhibition
Concentration (mg/ml)
Figure 1. Free radical scavenging activity of ethanolic extracts from different parts of C.
argyratus measured by DPPH assay. Results are mean ± SD (n = 3). a indicates
significant differences as compared to the control ascorbic acid at p < 0.05; b indicates
significant differences as compared to the control ascorbic acid at p < 0.01; c indicates
significant differences as compared to the control ascorbic acid at p < 0.01.
1.8
1.77
1.77
1.24
0.74
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
Ascorbic acid
Gallic acid
Leaves
Stem
Root
Absorbance at 700 nm
Figure 2. Reducing power of the extracts from C. argyratus plant. Ascorbic acid and
gallic acid were used as reference antioxidants. Values are mean ± SD (n = 3).
3728 J. Med. Plants Res.
Table 3. Total antioxidant capacities of different parts of C. argyratus.
Sample
Equivalent to ascorbic acid (mg)/g of extract
Equivalent to gallic acid (mg)/g of extract
Leaf
0.087 ± 0.0020c
0.14 ± 0.0030c
Stem
0.046 ± 0.0042b
0.074 ± 0.0066b
Root
0.032 ± 0.0013a
0.023 ± 0.0010a
Data expressed as mean ± SD, n = 3. Means with different superscript letters were significantly different at the level p < 0.05.
Table 4. Effect of C. argyratus leaves extract (CAE) and morphine on pain induced by hot plate test.
Treatment
Dose (mg/kg)
Latency period (min)
30
45
60
75
90
Control
0
15.24 ± 4.25
11.04 ± 3.12
14.68 ± 5.75
15.34 ± 6.83
17.3 ± 2.51
CAE
50
16.56 ± 6.57
19.95 ± 13.46
14.25 ± 3.50
15.19 ± 3.19
21.06 ± 10.60
CAE
100
16.03 ± 8.62
19.1 ± 7.10
23.17 ± 6.41
23.44 ± 5.31
20.96 ± 9.98
CAE
200
16.13 ± 9.28
16.03 ± 6.79
16.73 ± 5.64
18.64 ± 7.11
19.13 ± 10.33
Morphine
5
31.01 ± 7.61*
34.46 ± 7.55*
30.71 ± 11.10*
27.41 ± 10.26*
29.47 ± 11.08
Value expressed as mean ± SD (n = 6). *p < 0.05 when compared with control values.
Table 5. Effect of C. argyratus leaves extract (CAE) and morphine on pain induced by tail flick test.
Treatment
Dose (mg/kg)
Latency period (min)
30
45
60
75
90
Control
0
2.26 ± 0.54
1.77 ± 0.61
1.95 ± 0.29
2.08 ± 0.41
2.52 ± 1.12
CAE
50
1.85 ± 0.44
1.60 ± 0.31
2.24 ± 0.85
2.45 ± 1.46
2.53 ± 0.56
CAE
100
2.64 ± 0.23
2.28 ± 0.63
2.47 ± 0.81
3.36 ± 1.33
2.42 ± 1.11
CAE
200
3.42 ± 2.02
2.10 ± 0.71
1.73 ± 0.69
3.34 ± 1.53
1.54 ± 1.54
Morphine
5
9.71 ± 0.46*
10.00 ± 0.00*
10.00 ± 0.00*
10.00 ± 0.00*
9.62 ± 0.59*
Value expressed as mean ± SD (n = 6). *p < 0.05 when compared with control values.
argyratus leaves extract was 1.45 mg/ml and is higher
than IC50 for ascorbic acid, 0.09 mg/ml (Table 2).
Reducing power
Figure 2 shows the reducing capability of different parts
of C. argyratus. Leaves exhibited the stronger reducing
power (1.77) than stem (1.24) and root (0.74)
respectively, while ascorbic acid (1.80) had the strongest
reducing power among all the samples tested.
Total antioxidant capacity
Results of total antioxidant capacity method were present
in Table 3. Similar trend of antioxidant activity was also
found in this method as leaves extract of C. argyratus
had a higher capacity followed by stem and root.
Hot plate and tail flick tests
C. argyratus leaves extract did not show any significant
increase in the reaction time in both tests. In contrast,
morphine showed significant increase in reaction time (p
< 0.05) as compared with control group (Tables 4 and 5).
Formalin test
C. argyratus leaves extract did not shown any effect in
the early phase of the formalin test (0 to 5 min). However,
in the late phase (15 to 30 min), the extract at the higher
dose of 200 mg/kg showed significant reduction in the
licking and biting of paw. Even aspirin was also
significantly active (p<0.05) in the second phase however
morphine was active in both phases of formalin induced
pain (Figure 3).
DISCUSSION
Phenolic compounds are known to possess biological
effects such as antioxidants, anti-aging, cardioprotection
and anti-cancer activities (Han et al., 2007). In this study,
Ali et al. 3729
0
20
40
60
80
100
120
140
160
Control
50 mg/kg
100 mg/kg
200 mg/kg
Aspirin
Morphine
Paw licking (s)
Early Phase
Late Phase
*
*
*
*
Figure 3. Effect of C. argyratus leaves extract, morphine and aspirin on formalin test. Value expressed
as mean ± SD (n = 6).*p < 0.05 when compared with control values.
C. argyratus leaves contain higher phenols and
polyphenolic compounds than its stem and root. This is in
agreement with previous studies which reported that total
phenolic content in leaf was higher than other parts of the
plant for Cucumis melo, Petroselinum crispum,
Coriandrum sativum and Beta vulgaris (Ismail et al.,
2010; Pyo et al., 2004; Wong and Kitts, 2006). In this
study, leaves extract of C. argyratus also exhibited the
highest total flavonoid content, followed by stem and root
(Table 1). This is not surprising as flavonoids are the
most common and widely distributed group of plant
phenolic compounds which are very effective antioxidants
(Pokorny et al., 2001). Flavonoids are also responsible
for the free radical scavenging activity in plants (Das and
Pereira, 1990).
DPPH assay has been widely used for screening
antioxidant activity and is sensitive enough to detect the
active ingredients at low concentrations (Sánchez-
Moreno, 2002). The DPPH radical scavenging activity of
1 mg/ml ascorbic acid was the highest, followed by C.
argyratus leaves, stem and root respectively. The quality
of the antioxidants potency in the extracts was
determined by the IC50 value whereby, a low IC50 value
indicates strong antioxidant activity. The IC50 value of
leaves extract was determined at 1.45 mg/ml. On the
other hand, the IC50 value of stem and root extract could
not be determined due to the low percentage of inhibition
(<50 %) even at high concentration of 5 mg/ml. Even
though antioxidant activity of C. argyratus leaves extract
(IC50 = 1.45 mg/ml) was moderate compared to ascorbic
acid (IC 50 = 0.09 mg/ml), it is comparable to other fruits
which are considered to have good antioxidant activity
(orange IC50 = 5.40 ± 1.30 mg/ml, guava IC50 = 2.11 ±
0.63 mg/ml, star fruit IC50 = 3.80 ± 2.10 mg/ml) (Pin-Der-
Duh, 1998).
C. argyratus leaves extract showed the most reducing
power than stem and root. The reducing ability of a
compound is generally correlated with the presence of
reductones (Duan et al., 2007) which exert antioxidant
action by breaking the free radical chain via donating a
hydrogen atom (Shimada et al., 1992). In this assay,
Fe3+/ferricyanide complex was used as an indicator of
electron-donating activity. The presence of reductants
(antioxidants) in the sample caused the reduction of the
Fe3+/ferricyanide complex to its ferrous form. The Fe2+
can be monitored by measuring the formation of Perl’s
Prussian blue at 700 nm. The ability to reduce Fe3+ might
be due to hydrogen donation from phenolic compounds
(Prieto et al., 1999).
Total antioxidant capacity was estimated using
phosphomolybdenum method based on the reduction of
Mo (VI) to Mo (V) by the formation of a green
phosphate/Mo (V) complex at acidic pH (0.6 to 1.0) with
a maximum absorbance at 695 nm (Nagendra et al.,
2009). This assay is quantitative since the antioxidant
activity is expressed as the number of equivalents of
ascorbic acid and gallic acid. Leaves of C. argyratus had
the higher capacity than stem and root (Table 3). The
total antioxidant capacity of plant extract could be
attributed to their chemical composition and phenol
content. Free radicals have been implicated to be a
contributing factor in modulation of pain and tissue injury
(Khalil et al., 1999). Based on results, C. argyratus leaves
extract (CAE) gave the best antioxidant result and was
chosen for analgesic study. The analgesic effect of CAE
was examined using thermal models (hot plate and tail
flicks) and chemical model (formalin induced pain). Hot
plate and tail flicks methods are usually applied to
discover the involvement of central analgesic effect. The
hot plate test is thought to involve supraspinal reflex (Pini
et al., 1997); whereas, tail flick test involve spinal motor
reflex (D’amour and Smith, 1941). In this study, CAE did
3730 J. Med. Plants Res.
did not show any significant analgesic effect on pain
induced by both hot plate and tail flick tests in animals.
On the other hand, morphine (5 mg/kg), a centrally acting
analgesic produced significant effect on both tests. Based
on this observation, it can be suggested that CAE does
not act on the central analgesic mechanism.
The formalin injection causes an immediate and
intense increase in the spontaneous activity of afferent C
fibers and induces distinct quantifiable behavior indicate
of pain, for example, licking of the injected paw (Heapy et
al., 1987). The formalin test produces a distinct biphasic
nociceptive response that is neurogenic and inflammatory
phase. It has been reported that the early phase is
caused by a direct effect of formalin on nociceptors while
the late phase is a tonic response in which inflammatory
processes are involved and neurons in the dorsal horns
of the spinal cord are activated (Tjølsen et al., 1992).
CAE produced a significant reduction of licking activity in
the late phase at dose 200 mg/kg but did not give any
responses in the early phase. Centrally acting drugs such
as morphine (opiods) inhibited both phases whereas
peripherally acting drugs, aspirin (NSAIDs) inhibited the
late phase of formalin test (Santos et al., 1994; Shibata et
al., 1989). Thus, it can be deduced that CAE acts
peripherally and not centrally. Similar findings also have
been reported by using methanolic extract of
Cinnamomun iners and ethanolic extract of Curcuma
xanthorriza Roxb (Mustafa et al., 2010; Devaraj et al.,
2010).
Previous work with the root of C. argyratus identified
clerodane type diterpene, (-)-junceic acid (Norizan et al.,
2007) which has an anti-inflammatory effect (Bruno et al.,
1993). Although, (-)-junceic acid is a potential compound
to justify the analgesic effect of C. argyratus, this
compound was isolated from the root of C. argyratus and
not from leaves, the material used in this study. As
phytochemical screening revealed the presence of
flavonoids, terpenoids and steroids in C. argyratus, these
constituents might also be responsible for the antioxidant
and analgesic activities (Galati et al., 1994; Saeed et al.,
2010).
Based on the results, C. argyratus has a natural
antioxidant capacity as an alternative to synthetic
antioxidants. A bio-assay guided fractionation of this
extract is now in progress to identify the bioactive
substance(s) in the ethanol extract of C. argyratus as well
as the mechanisms of action involved in the effects
described in this work.
ACKNOWLEDGEMENTS
This project was funded by Research University Grant
from Universiti Sains Malaysia. N. I. Mohd Ali was
supported by Ministry of Higher Education, Malaysia and
Institute of Postgraduate Studies, Universiti Sains
Malaysia.
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