Avicenna Journal of Phytomedicine

Received: Oct 1, 2012; Accepted: Nov 27, 2012

Vol. 3, No. 2, Spring 2013, 112-125

Original Research Paper

Antioxidant, analgesic and anti-inflammatory activities of the methanolic

extract of Piper betle leaves

Badrul Alam1, Fahima Akter1, Nahida Parvin1, Rashna Sharmin Pia1, Sharmin Akter1, Jesmin
Chowdhury1, Kazi Sifath-E-Jahan1, Ekramul Haque1,2*

Abstract
Objective: The present study was designed to evaluate the antioxidant, analgesic, and anti-
inflammatory activities of the methanolic extract of Piper betle leaves (MPBL).
Materials and Methods: MPBL was evaluated for anti-inflammatory activity using carrageenan-
induced hind paw edema model. Analgesic activity of MPBL was evaluated by hot plate, writhing,
and formalin tests. Total phenolic and flavonoids content, total antioxidant activity, scavenging of 1,1-
diphenyl-2-picrylhydrazyl (DPPH) radical, peroxynitrate (ONOO) as well as inhibition of total ROS
generation, and assessment of reducing power were used to evaluate antioxidant potential of MPBL.
Results: The extract of MPBL, at the dose of 100 and 200 mg/kg, produced a significant (p<0.05)
increase in pain threshold in hot plate method 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. The
same ranges of doses of MPBL caused significant (p<0.05) inhibition of carrageenan-induced paw
edema after 4 h in a dose-dependent manner. In DPPH, ONOO-, and total ROS scavenging method,
MPBL showed good antioxidant potentiality with the IC50 value of 16.33±1.02, 25.16±0.61 , and
41.72±0.48 µg/ml, respectively with a significant (p<0.05) good reducing power.
Conclusion: The findings of the study suggested that MPBL has strong analgesic, anti-inflammatory,
and antioxidant effects, conforming the traditional use of this plant for inflammatory pain alleviation
to its antioxidant potentiality.

Keywords: Analgesic, Antioxidant, Anti-inflammatory, Piper betle

1- Department of Pharmacy, Atish Dipankar University of Science & Technology, Dhaka, Bangladesh
2- Department of Pharmacy, University of Rajshahi, Rajshahi, Bangladesh
*Corresponding author: Tel: +8801711952286
E-mail: [email protected]

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 Antioxidant, analgesic and anti-inflammatory activities of Piper betle

Introduction medicinal properties (Oladosu et al., 2011)

Free radicals cause depletion of immune so that, the study of plants that have been
system antioxidants (Ouattara et al., 2011), traditionally used as pain killers should still
change in gene expression and induce be seen as a fruitful and logical research
abnormal proteins, and contribute to more strategy in the search for new analgesic
than one hundred disorders in humans drugs and pain mechanisms (Calixto et al.,
including atherosclerosis, arthritis, 2000).
ischemia, and reperfusion injury of many Betelvine Piper betle (P. betle) belongs
tissues, central nervous system injury, to the family of Piperaceae, popularly
gastritis, cancer, and AIDS (Hela and regarded as a medicinal plant in the South
Abdullah, 2010). Moreover, various free East Asia region. Experimentally, leaves of
radicals are also responsible for the P. betle are shown to possess antimicrobial
induction of short term algesia (Chung, (Agarwal et al., 2012), gastroprotective
2004) as well as play an important role in (Majumdar et al., 2003), wound healing
the pathogenesis of inflammation (Winrow (Santhanam and Nagarajan, 1990),
et al., 1993). Inflammation is the response hepatoprotective (Saravanan et al., 2002),
to injury of cells and body tissues through antioxidant (Choudhary and Kale, 2002;
different factors such as infections, Santhakumari et al., 2003), anti-fertility on
chemicals, thermal, and mechanical injuries male rats (Ratnasooriya and Premakumara,
(Oyedapo et al., 2008). Various 1997), and antimotility effects on washed
endogenous mediators such as histamine, human spermatozoa (Ratnasooriya et al.,
serotonin, bradykinin, prostaglandins, etc. 1990). The chief constituent of the leaves is
are most abundant in inflammatory cells a volatile oil which contains phenols, betel-
and among them prostaglandins are phenol, chavibetol and chavicol, cadinene,
ubiquitous substances that indicate and and hydroxychavicol, which have been
modulate cell and tissue responses involved ascribed to possess anti-oxidant and anti-
in inflammation (Hossain et al., 2011). carcinogenic activities (Bhide et al., 1991;
These mediators, even in small quantities, Garg and Jain, 1992; Singh et al., 2009).
can elicit pain response. Pain results in The tribal population and aborigines of
dropped muscular activities, associated Bangladesh chew the leaves as a narcotic
with various free radicals as well as which causes swooning and profuse
reactive oxygen species (ROS) that trigger sweating and also helps to give warmth to
some second messengers and are involved the body during winter. The present study
in sensitization of dorsal horn neurons that was carried out to evaluate the antioxidant,
plays a fundamentally important role in analgesic, and anti-inflammatory activities
neuropathic pain (Ali and Salter, 2001; of crude extract of P. betle leaves in
Zhang et al., 2003). Having various and different experimental model.
severe adverse effects such as gastric
lesions for NSAIDs, adverse cardiovascular
thrombotic effects for selective Materials and Methods
cyclooxygenase-2 inhibitors (Chowdhury et Plant materials
al., 2009), and tolerance and dependence The leaves of the P. betle Linn were
induced by opiates, use of these drugs as collected from the adjoining area of
anti-inflammatory and analgesic agents Jahangirnagar University Campus,
have not been successful in all of the cases. Bangladesh, during February 2011. The
Therefore, new anti-inflammatory and plant material was taxonomically identified
analgesic drugs lacking those effects are by the National Herbarium of Bangladesh
being searched all over the world as and voucher specimen no. JU/33334 is
alternatives. Medicinal plant have great maintained in our laboratory for future
value to phytochemists because of their reference.

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 Badrul Alam et al.

Chemicals equivalents (GAE) and the flavonoid

Ammonium molybdate, Folin-chiocaltu contents of the extract was expressed in
phenol reagent, and carrageenan were terms of quercetin equivalent (the standard
purchased from E. Merck (Germany). 1,1- curve equation: y=0.009x-0.036).
diphenyl-2-picryl-hydrazyl (DPPH),
ascorbic acid, quercetin potassium ferric Determination of total antioxidant capacity
cyanide, 2',7'-dichlorfluorescein-diacetate The antioxidant activity of the MPBL
(DCFH-DA), 5,5’-dithiobis [2-nitrobenzoic was evaluated by the phosphomolybdenum
acid] (DTNB), L-penicillamine (L-2- method according to the procedure of
amino-3-mercapto-3-methylbutanoic acid), Prieto et al., (1999). The assay is based on
and diethylene triamine pentaacetic acid the reduction of Mo(VI)–Mo(V) by the
(DTPA) were purchased from Sigma Co. extract and subsequent formation of a green
(St. Louis, MO, USA). 6-Hydroxy-2,5,7,8- phosphate/Mo(V) complex at acid pH.
tetramethylchroman-2-carboxylic acid Extract (0.3 ml) was combined with 3 ml of
(Trolox) was purchased from Aldrich reagent solution (0.6 M sulfuric acid, 28
Chemical Co. (Milwaukee, WI, USA) The mM sodium phosphate, and 4 mM
high quality 2’,7’-dichlorofluorescein ammonium molybdate). The tubes
diacetate (DCFH-DA), dihydrorhoclamine containing the reaction solution were
123 (DHR 123), and ONOO- were incubated at 95 °C for 90 min. Then, the
purchased from Molecular Probes (Eugene, absorbance of the solution was measured at
Oregon, USA) and Cayman (Ann Arbor, 695 nm using a spectrophotometer
MI, USA), respectively. Nalbuphine, (Shimadzu, UV-150-02) against blank after
indomethacin, and diclofenac-Na were cooling to room temperature. Methanol (0.3
collected from Square Pharmaceuticals ml) is used as the blank experiment. The
Ltd., Bangladesh. All other chemicals and antioxidant activity is expressed as the
reagents were of analytical grade. number of equivalents of ascorbic acid
using the following formula:
Preparation of plant extract C = (c×V)/m, where, C: total antioxidant
The plant material was shade-dried with activity, mg/g plant extract, in ascorbic
occasional shifting and then powdered with acid; c: the concentration of ascorbic acid
a mechanical grinder, passing through sieve established from the calibration curve,
#40, and stored in a tight container. The mg/ml; V: the volume of extract, ml; m: the
dried powder material (1.5 kg) was weight of pure plant extract, g.
refluxed with MeOH for three hours. The
total filtrate was concentrated to dryness, in Free radical scavenging activity measured
vacuo at 40 ° C to render the MeOH extract by DPPH
(410 g). The free radical scavenging activity of
MPBL, based on the scavenging activity of
In vitro antioxidant activity the stable 1,1-diphenyl-2- picrylhydrazyl
The amount of phenolic compounds and (DPPH) free radical, was determined by the
flavonoids method described by Braca et al. (2001).
The total phenolic and flavonoid content MPBL (0.1 ml) was added to 3 ml of a
of methanolic extract was determined using 0.004% MeOH solution of DPPH.
Folin-ciocalteu reagent (Yu et al., 2002) Absorbance at 517 nm was determined
and aluminium chloride colorimetric after 30 min, and the percentage inhibition
method (Chang et al., 2002), respectively. activity was calculated from [(A0–
The content of total phenolics in MPBL A1)/A0]×100, where A0 is the absorbance of
was calculated from regression equation of the control and A1 is the absorbance of the
the calibration curve (y=0.013x+0.127, r2= extract/ standard. IC50 value was calculated
0.988) and is expressed as galic acid from the equation of line obtained by

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 Antioxidant, analgesic and anti-inflammatory activities of Piper betle

plotting a graph of concentration (µg/ml) DCFH-DA, at 37 °C for 30 min. Phosphate

versus % inhibition. buffer (50 mM, pH 7.4) was used. DCFH-
DA is a stable compound, which easily
Measurement of the ONOO- scavenging diffuses into cells, and is hydrolyzed by
activity intracellular esterase to yield a reduced
The ONOO- scavenging activity was non-fluorescent compound, DCFH, which
measured by monitoring the oxidation of is trapped within the cells. The ROS
DHR 123, by modifying the method of produced by cells oxidize the DCFH to the
Kooy et al. (1994). The DHR 123 (5 mM), highly fluorescent 2',7'-
in dimethylformamide, was purged with dichlorodihydrofluorescein (DCF). The
nitrogen, stored at -80 °C and used as a fluorescence intensity of the oxidized DCF
stock solution. This solution was then was monitored on a microplate
placed on ice and kept from exposure to fluorescence spectrophotometer (Bio-Tek
light, before the study. The buffer used Instruments Inc., Winooski, VT), with
consisted of 90 mM sodium chloride, 50 excitation and emission wavelengths of 460
mM sodium phosphate, 5 mM potassium and 530 nm, respectively (Label and
chloride at pH 7.4, and 100 mM Bondy, 1990). IC50 value was calculated
diethylenetriaminopentaacetic acid from the equation of line obtained by
(DTPA), each of which were prepared with plotting a graph of concentration versus %
high quality deionized water, and purged inhibition.
with nitrogen. The final concentration of
the DHR 123 was 5 µM. The background Reducing power activity
and final fluorescent intensities were The reducing power of MPBL was
measured 5 minutes after treatment, both determined according to the method
with and without the addition of authentic previously described (Oyaizu, 1986).
ONOO-. The DHR 123 was oxidized Extracts at different concentrations in 1 ml
rapidly by authentic ONOO-, and its final of 10% DMSO were mixed with 2.5 ml of
fluorescent intensity remained unchanged phosphatebuffer (0.2 M, pH 6.6) and 2.5 ml
over time. The fluorescent intensity of the potassium ferricyanide [K3Fe (CN)6] (1%),
oxidized DHR 123 was measured using a and then the mixture was incubated at 50 °C
microplate fluorescence reader, FL 500 for 30 min. Afterwards, 2.5 ml of
(Bio-Tek Instruments Inc.), with excitation trichloroacetic acid (10%) was added to the
and emission wavelengths of 480 and 530 mixture, which was then centrifuged at
nm, respectively. The results were 3000 rpm for 10 min. Finally, 2.5 ml of
expressed as the mean±standard error (n=3) upper layer solution was mixed with 2.5 ml
of the final fluorescence intensity minus the distilled water and 0.5 ml FeCl3 (0.1%),
background fluorescence. The effects were and the absorbance was measured at 700
expressed as the percentage of inhibition of nm. Increased absorbance of the reaction
the DHR 123 oxidation. IC50 was mixture indicated increased reducing
calculated from the equation of line power.
obtained by plotting a graph of
concentration (µg/ml) versus % inhibition. In vivo analgesic activity
Animal
Measurement of the inhibition of the total Swiss albino mice (25-30g) and Wistar
ROS generation rats (175-250 g) of both sexes were used
Mice kidney homogenates, prepared for assessing biological activity. The
from the kidneys of freshly killed male animals were maintained under standard
Swiss albino mice, weighing 30-39 g, were laboratory conditions and had free access to
mixed with or without a suspension of food and water ad libitum. The animals
extracts, and then incubated with 12.5 µM were allowed to acclimatize to the

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 Badrul Alam et al.

environment for 7 days prior to 90 min after oral administration of the

experimental session. The animals were samples.
divided into different groups, each
consisting of five animals which were Acetic acid-induced writhing test
fasted overnight prior to the experiments. The analgesic activity of the samples
Experiments on animals were performed in was also studied using acetic acid-induced
accordance with guidelines of the writhing model in mice. Test samples and
Institutional Animal Ethics Committee, vehicle were administered orally 30 min
Atish Dipankar University of Science & before intraperitoneal administration of
Technology, Dhaka, Bangladesh. Animal 0.7% v/v acetic acid but diclofenac-Na was
treatment and maintenance for acute administered intraperitonially 15 min
toxicity and analgesic effects were before injection of acetic acid. After an
conducted in accordance with the Principle interval of 5 min, the mice were observed
of Laboratory Animal Care (NIH for specific contraction of body referred to
publication No. 85-23, revised 1985) and as ‘writhing’ for the next 10 min (Ahmed et
the Animal Care and Use Guidelines of al., 2004).
Atish Dipankar University of Science &
Technology, Dhaka, Bangladesh. Formalin test
The antinociceptive activity of the drugs
Acute toxicity study was determined using the formalin test
Acute oral toxicity assay was performed described by Dubuission and Dennis
in healthy nulliparous and nonpregnant (1977). Control group received 20 µl of 5%
adult female albino Swiss mice (25-30 g) formalin via injection into the dorsal
divided into different groups. The test was surface of the right hind paw 60 min after
performed using increasing oral dose of the administration of MPBL (200 and 400
MPBL in water (50, 100, 200, 500, and mg/kg, p.o.) and 30 min after
1000 mg/kg body weight) in 20 ml/kg administration of diclofenac Na (10 mg/kg,
volume to different test groups. Normal i.p.). The mice were observed for 30 min
group received water. The mice were after the injection of formalin and the
allowed to feed ad libitum, kept under amount of time spent licking the injected
regular observation for 48 h for any hind paw was recorded. The first 5 min
mortality or behavioral changes post-formalin injection is referred to as the
(Sanmugapriya and Venkataraman, 2006). early phase and the period between 15 and
30 min as the late phase. The total time
Hot plate method spent licking or biting the injured paw (pain
The animals were divided into four behavior) was measured with a stop watch.
groups with five mice in each group. Group
I animals received vehicle (1% Tween 80 Anti-inflammatory activity
in water, 10 ml/kg body weight), animals of Carrageenan-induced paw edema test in
Group II received nalbuphine at 10 mg/kg rats
body weight while animals of Group III Wistar rats (175-250 g) of both sexes
and Group IV were treated with 100 and were divided into four groups of five
200 mg/kg body weight (p.o.) of the animals each. The test groups received 100
MPBL. The animals were placed on Eddy’s and 200 mg/kg body weight (p.o.) of the
hot plate kept at a temperature of (55±0.5) extract. The reference group received
°C. A cut-off period of 15 second, was indomethacin (10 mg/kg body weight, p.o.)
observed to avoid damage to the paw while the control group received 3 ml/kg
(Franzotti et al., 2000). Reaction time was body weight normal saline. After 1 h, 0.1
recorded when animals licked their fore or ml of 1% carrageenan suspension in normal
hind paws, or jumped prior to 0, 30, 60, and saline was injected into the subplanatar

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 Antioxidant, analgesic and anti-inflammatory activities of Piper betle

tissue of the right hind paw (Winter et al., Table 1 represents the content of both
1962). The paw volume was measured at 1, groups in MPBL extract. The content of
2, 3, and 4 h after carrageenan injection total phenolics in the extract of P. betle was
using a micrometer screw gauge. The determined using the Folin-ciocalteu assay,
percentage inhibition of the inflammation calculated from regression equation of the
was calculated from the formula: calibration curve (y=0.013x+0.127, r2=
% inhibition = (1-Dt/Do)×100, 0.988) and is expressed as galic acid
where, Do was the average inflammation equivalents (GAE) and the flavonoid
(hind paw edema) of the control group of contents of the extract was expressed in
rats at a given time and Dt was the average terms of quercetin equivalent (the standard
inflammation of the drug treated (i.e., curve equation: y=0.009x-0.036).
extract or reference indomethacin) rats at
the same time (Winter et al., 1962).
Total antioxidant capacity
Total antioxidant capacity of MPBL is
Statistical analysis
All values were expressed as the expressed as the number of equivalents of
mean±SEM of three replicate experiments. ascorbic acid (Table 1). Total antioxidant
The analysis was performed using SPSS capacity of MPBL was found to be 81.72±
statistical package for WINDOWS (version 0.48 mg/gm equivalent of ascorbic acid.
16.0; SPSS Inc, Chicago). Results related
to the reducing power activities were DPPH radical scavenging activity
statistically analyzed by applying the The percentage (%) scavenging of
Student t-test and p<0.001 were considered DPPH radical was found to be
to be statistically significant. All in vivo concentration-dependent with the IC50
data are subjected to ANOVA followed by value of 16.33±0.16 µg/ml, while IC50
Dunnett’s test and p<0.05 were considered value of standard ascorbic acid was found
to be statistically significant to be 12.10±0.02 µg/ml (Table 2).

Peroxynitrite (ONOO-) scavenging activity

Results The ONOO- scavenging activity was
Acute toxicity studies measured by monitoring the oxidation of
The acute toxicity studies mainly aim at DHR 123. The MeOH extract of MPBL
establishing the therapeutic index, i.e., the exhibited significant ONOO- scavenging
ratio between the pharmacologically effects in a dose-dependent manner, with
effective dose and the lethal dose on the IC50 values of 25.16±0.61µg/ml, whereas,
same strain and species. MPBL was safe up penicillamine, a well-known ONOO-
to a dose of 1000 mg/kg (p.o.) body weight. scavenger, with IC50 value of 10.20±0.32
Behavior of the animals was closely µg/ml. (Table 2).
observed for the first 3 h then at an interval
of every 4 h during the next 48 h. The Inhibition of total ROS generation
extract did not cause mortality in mice and The percentage inhibition of ROS
rats during 48 h observation but little generation was illustrated in Table 2 and it
behavioral changes, locomotor ataxia, is observed that scavenging of ROS by the
diarrhea, and weight loss were observed. extract is also concentration-dependent
Food and water intake had no significant with the IC50 value of 41.72±0.48 µg/ml,
difference among the group studied. while IC50 value of standard trolox was
found to be 12.32±0.11 µg/ml.
In vitro antioxidant activity
Total phenolic and flavonoid contents Reducing power ability

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 Badrul Alam et al.

For the measurement of the reductive Similar to the antioxidant activity, the
ability, we investigated the Fe3+ to Fe2+ reducing power of MPBL was found to be
transformation in the presence of MPBL concentration-dependent and statistically
and compared with standards (Galic acid, significant (p<0.001).
quercetin and ascorbic acid) (Figure 1).

Table 1. Yield, total amount of plant phenolic compounds, flavonoids, and total antioxidant capacity of
methanolic extract of Piper betle.

a b c
Total phenols mg/g plant Total flavonoids mg/g plant Total antioxidant capacity
Sample Yield (%)
extract (in GAE) extract (in QA) mg/g plant extract (in ASC)

MPBL 39.92 136.33 ± 1.02* 52.16 ± 0.61* 81.72 ± 0.48*

The GAE, QA, and ASC values are expressed as Means±SEM of triplicate experiments. aGalic acid equivalents
(GAE, mg/g of each extract) for the total phenolic content, bQuercetin equivalent (QA, mg/g of each extract) for
the total flavonoid content, cAscorbic acid equivalent (ASC, mg/g of each extract) for the total antioxidant
capacity.

Table 2. Scavenging/inhibitory effects of the Piper betle extract against DPPH, ONOO-, and Total ROS
generation.

DPPH ONOO- ROS

Sample
IC50(µg/ml) IC50(µg/ml) IC50(µg/ml)

MPBL 16.33±1.02* 25.16±0.61* 41.72±0.48*

Ascorbic acid 12.10±0.02

L-penicillamine 10.20±0.32

Trolox 12.32±0.19

IC50 values are mean±SEM (n=3)*; p < 0.001 by student t-test for values between the sample and the control.

1000.00 Reducing power assessment of MPBL vs Starndards

750.00
% Reducing power

500.00

250.00

0.00
0 50 100 150 200 250

Concentration (microgram/ml)

Quercetin Ascorbic acid Galic acid MPBL

Figure 1. Values are mean±SEM, Reducing power of MPBL, quercetin, ascorbic acid, and galic acid by
spectrophotometric detection of Fe3+ to Fe2+ transformation.

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 Antioxidant, analgesic and anti-inflammatory activities of Piper betle

In vivo analgesic activity formalin-induced pain in mice in a dose-

Hot plate method dependant manner (Table 4). MPBL, at the
Result of hot plate test is shown in dose of 200 mg/kg body weight, showed
Figure 2. Both doses of the extract almost similar licking activity against both
produced a dose-dependent increase in phases of formalin-induced pain than that
latency time when compared with the of the standard drug diclofenac Na.
vehicle. The result was found to be
statistically significant (p<0.05). Anti-inflammatory activity
Carrageenan-induced paw edema test
Acetic acid-induced writhing test Figure 3 shows the results of the anti-
Table 3 shows the effects of the extract edematous effects of orally administered
of on acetic acid-induced writhing in mice. methanolic extract of P. betle on
The oral administration of both doses of carrageenan-induced paw edema in rats.
MPBL significantly (p<0.05) inhibited MPBL showed statistically significant
writhing response induced by acetic acid in (p<0.05) dose-dependent anti-inflammatory
a dose-dependent manner. activity. MPBL showed remarkable anti-
inflammatory effects at 200 mg/kg dose
Formalin test (66.66% inhibition), whereas standard
MPBL (100 and 200 mg/kg, p.o.) indomethacin showed 72.72% of inhibition
significantly (P<0.05) suppressed the of paw edema.
licking activity in either phase of the

Figure 2. Effects of the MPBL on latency to hot plate test. Values are mean±SEM, (n=5); *p<0.05 as compared
with vehicle control (one-way ANOVA followed by Dunnett’s test). Group I animals received vehicle (1%
Tween 80 in water), Group II received nalbuphine 10 mg/kg body weight, Group III and Group IV were treated
with 100 and 200 mg/kg body weight (p.o.) of the crude extract of P. betle, respectively.

Table 3. Effects of the MPBL on acetic acid-induced writhing in mice.

Groups Dose (mg/kg) No. of writhing % inhibition

Group I Vehicle 34.40

Group II 10 10.60 69.19*
Group III 100 19.80 42.44*
Group IV 200 12.27 64.53*

Values are mean±SEM, (n=5); *p<0.05 as compared with vehicle control (one-way ANOVA followed by
Dunnett’s test). Group I animals received vehicle (1% Tween 80 in water), Group II received diclofenac Na 10
mg/kg body weight, Group III and Group IV were treated with 100 and 200 mg/kg body weight (p.o.) of the
MPBL.

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 Badrul Alam et al.

Table 4. Effect of MPBL in hindpaw licking in the formalin test in mice.

Groups Dose (mg/kg) Early phase (Sec) % protection Late phase (Sec) % protection

Group-I Vehicle 34.16 ± 1.38 - 47.0 ± 1.03 -

Group-II 10 16.83 ± 0.90* 50.73 19.83 ± 0.70* 57.80
Group-III 100 27.5 ± 0.76* 19.51 21.5 ± 0.95* 54.25
Group-IV 200 18.00 ± 0.65* 47.31 20.67 ± 1.46* 56.02

Values are mean±SEM, (n=5); *p<0.05 as compared to vehicle control (one-way ANOVA followed by
Dunnett’s test). Group I animals received vehicle (1% Tween 80 in water), Group II received diclofenac Na 10
mg/kg body weight, Group III and Group IV were treated with 100 and 200 mg/kg body weight (p.o.) of the
MPBL, respectively.

Figure 3. Effects of the MPBL on carrageenan-induced paw edema test. Values are mean±SEM, (n=5); *p<0.05
as compared to vehicle control (one-way ANOVA followed by Dunnett’s test). Group I animals received
vehicle (1% Tween 80 in water), Group II received indomethacin10 mg/kg body weight, Group III and Group
IV were treated with 100 and 200 mg/kg body weight (p.o.) of the crude extract of P. betle, respectively.

Discussion relationships of pure antioxidant species.

The upshots of oxidative stress are The phosphomolybdenum method was
serious and sometimes manifested by based on the reduction of Mo(VI) to Mo(V)
increased activities of enzymes involved in by the compounds having antioxidant
oxygen detoxification (Gupta et al., 2005). property and is successfully used to
Therefore, the identification of new quantify vitamin E in seeds (Prieto et al.,
antioxidant may reduce the risk of various 1999).
chronic diseases involved in free radicals. DPPH is a stable free radical that
To determine the efficacy of natural accepts an electron or hydrogen radical to
antioxidants either as pure compounds or as become a stable diamagnetic molecule
plant extract, a great number of in vitro (Lompo et al., 2007) and is usually used as
methods have been developed in which a substrate to evaluate the antioxidant
antioxidant compounds act by several activity of a compound (Braca et al., 2001).
mechanisms. The knowledge of total Based on the data obtained from this study,
antioxidant activity can be useful in the DPPH radical scavenging activity of MPBL
analysis of changes in plasma antioxidant (IC50 16.33±0.16 µg/ml) was similar to the
activity related to oxidative stress, or the standard ascorbic acid (IC50 12.10±0.02
understanding of structure–activity µg/ml). Moreover, it was revealed that

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 Antioxidant, analgesic and anti-inflammatory activities of Piper betle

MPBL did show the proton donating ability antinociceptive response to that of
and could serve as free radical inhibitor or nalbuphine suggesting that the plant extract
scavenger. A direct correlation between may act as a narcotic analgesic.
antioxidant capacity and reducing power of On the other hand, acetic acid-induced
certain plant extracts has been reported writhing response is a sensitive procedure
(Nakayama et al., 1993). to evaluate peripherally acting analgesics
The reducing properties are generally and represents pain sensation by triggering
associated with the presence of reductones, localized inflammatory response. Such pain
which have been shown to exert antioxidant stimulus leads to the release of free
action by breaking the free radical chain by arachidonic acid from the tissue
donating a hydrogen atom (Tanaka et al., phospholipid (Ribeiro et al., 2000). The
1988). Because a substance may act as an response is thought to be mediated by
antioxidant due to its ability to reduce ROS peritoneal mast cells (Voilley, 2004), acid
by donating hydrogen atom (Jayprakash et sensing ion channels (Hossain et al., 2006),
al., 2001), the ferric reducing property of and the prostaglandin pathways (Adzu et
plant extracts (Figure 1) implies that they al., 2003). The organic acid has also been
are capable of donating hydrogen atom in a postulated to act indirectly by inducing the
dose-dependent manner. Polyphenolic release of endogenous mediators, which
compounds, such as flavonoids, tannins, stimulates the nociceptive neurons that are
and phenolic acids, which are commonly sensitive to NSAIDs and narcotics (Alam et
found in plants, have been reported to have al., 2012). It is well known that non-
multiple biological effects, including steroidal anti-inflammatory and analgesic
antioxidant activity (Khanam et al., 2004). drugs mitigate the inflammatory pain by
Phenolic compounds are understood to inhibiting the formation of pain mediators
induce the cellular antioxidant system and at the peripheral target sites where
increase approximately 50% cellular prostaglandins and bradykinin are proposed
glutathione concentration. P. betle leaves to play a significant role in the pain process
are rich in phenol, polyphenol, and tannin (Kim et al., 2004). Therefore, it is likely
(Kahkonen et al., 1999) and may be that MPBL might have exerted its
responsible for causing the paramount peripheral antinociceptive action by
antioxidant effect which is supported by interfering with the local reaction caused by
previous studies (Arambewela et al., 2005; the irritant or by inhibiting the synthesis,
Choudhary and Kale, 2002; Santhakumari release, and/or antagonizing the action of
et al., 2003; Dasgupta and De, 2004.). pain mediators at the target sites. The above
The hot plate method is commonly used findings clearly demonstrated that both
for assessing central antinociceptive central and peripheral mechanisms are
response involving higher brain functions involved in the antinociceptive action of
and is a supraspinally organized response MPBL. Interestingly, compounds such as
(Chapman et al., 1985; Elisabetsky et al., flavonoids, steroids, and triterpenes in part,
1995). Narcotic analgesics inhibit both have been shown to possess anti-
peripheral and central mechanism of pain, inflammatory and analgesic activity as the
while nonsteroidal anti-inflammatory drugs claim made by Pritam et al. (2011). Based
inhibit only peripheral pain (Pal et al., on the classes of compounds detected in
1999; Ahmed et al., 2006). As noted, MPBL extract, several mechanisms of
nalbuphine, the reference narcotic analgesic action could be used to explain the
drug (5 mg/kg, p.o.) exhibited significant observed activities of MPBL extract.
and paramount analgesic effects in the hot The formalin model normally postulates
plate (supra spinal) test, whereas, MPBL the site and the mechanism of action of the
(100 and 200 mg/kg, p.o.) produced a analgesic. This biphasic model is
statistically significant but lesser in degree represented by neurogenic (0-5 min) and

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 Badrul Alam et al.

inflammatory pain (15-30 min), macrophages (Antonio and Brito, 1998;

respectively (Hunskaar and Hole, 1987). Gupta et al., 2006; Sawadogo et al., 2006).
Drugs that act primarily on the central Since the extract significantly inhibited
nervous system such as narcotics inhibit paw edema induced by carrageenan in the
both as steroids and NSAIDs suppress second phase, this finding suggests a
mainly the late phase (Alam et al., 2012). possible inhibition of cyclooxygenase
The suppression of neurogenic and synthesis by the extract and this effect is
inflammatory pains by the extract might similar to that produced by non-steroidal
imply that it contains active analgesic anti-inflammatory drugs such as
principle that may be acting both centrally indomethacin, whose mechanism of action
and peripherally. This is an indication that is inhibition of the cyclooxygenase enzyme.
the extract can be used to manage acute as Flavonoids and saponins are well known
well as chronic pain. The mechanism by for their ability to inhibit pain perception as
which formalin triggers C-fibers activation well as anti-inflammatory properties due to
remained unknown for a relatively long their inhibitory effects on enzymes
time. Recently, however, McNamara et al. involved in the production of the chemical
(2007) demonstrated that formalin activates mediator of inflammation (Pin et al., 2010).
primary afferent neurons through a specific This hypothesis is strongly supported by
and direct effect on TRPA1, a member of the previous study, which has shown that P.
the transient receptor potential family of betle possess anti-inflammatory activity
cation channels, expressed by a subset of due to the presence of high flavonoid
C-fiber nociceptors and this effect is content (Koblyakov, 2001; Vaghasiya et
accompanied by increased influx of Ca2+ al., 2007).
ions. TRPA1 cation channels at primary In addition, the release of ROS and
sensory terminals were also reported to excessive nitric oxide (NO) due to the
mediate noxious mechanical stimuli activation of neutrophils during tissue
(Kerstein et al., 2009). These experiments damage and inflammation is responsible for
suggest that Ca2+ mobilization through a variety of disease (Bhandare et al., 2010).
TRPA1 cation channels is concomitant Recent findings (Srivastava et al., 2000;
with noxious chemicals and mechanical Viana et al., 2003) suggest that polyphenols
stimuli as they produce their analgesic are potent inhibitors of NO synthase
action. It is likely that the inhibitory effect activity and NO production. As MPBL
of MPBL to pain response is due to showed significant free radical as well as
inhibiting the increase of the intracellular ONOO- scavenging activity, this can be
Ca2+ through TRPA1, presumably evoked responsible for the reduction of
by formalin. Therefore, MPBL may contain inflammation in the carrageenan-induced
substances that affect the metabolism of paw edema in rats. The results of the
Ca2+. experiments suggest that P. betle may be
Carrageenan-induced edema has been used as an alternative or supplementary
commonly used as an experimental animal herbal remedy for the treatment of pain and
model for acute inflammation and is inflammatory disease. Because of its
believed to be biphasic. The early phase (1- analgesic and anti-inflammatory effects, P.
2 h) of the carrageenan model is mainly betle methanolic extract may have
mediated by histamine, serotonin, and beneficial effects together with drugs
increased synthesis of prostaglandins in the known for having a strong analgesic as well
damaged tissue surroundings. The late as anti-inflammatory effects. Thus, the
phase is sustained by prostaglandin release present study warrants further investigation
and mediated by bradykinin, leukotrienes, involving components of P. betle for
polymorphonuclear cells, and possible development of new class of
prostaglandins produced by tissue analgesic and anti-inflammatory drugs.

AJP, Vol. 3, No. 2, Spring 2013 122

 Antioxidant, analgesic and anti-inflammatory activities of Piper betle

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