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Journal of Ethnopharmacology 135 (2011) 654–661

Contents lists available at ScienceDirect

Journal of Ethnopharmacology

journa l homepage: www.e lsev ier .com/ locate / je thpharm

tudies on anti-inflammatory and analgesic activities of betel nut in rodents

hagufta Khana, Malik Hassan Mehmooda,∗, Anita Naushir Akbar Ali a,d, Fahad Shabbir Ahmedb,hsana Darc, Anwarul-Hassan Gilania

Natural Product Research Division, Department of Biological and Biomedical Sciences, the Aga Khan University Medical College, Karachi 74800, PakistanJinnah Medical and Dental College, Karachi, PakistanPharmacology Section, HEJ Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, PakistanThe current address: Department of Anesthesiology, University of Arkansas for Medical Sciences, 4301 W Markham St., Little rock, AR 72205, USA

r t i c l e i n f o

rticle history:eceived 27 May 2010eceived in revised form 12 March 2011ccepted 29 March 2011vailable online 9 April 2011

eywords:etel nutnti-inflammatorynalgesicyclooxygenase inhibitoripoxygenase inhibitornti-oxidant

a b s t r a c t

Ethnopharmacological relevance: Areca catechu, commonly known as betel nut, is very famous for itsmedicinal use in multiple disorders. It is also popular as a remedy against inflammatory disorders in theUnani (Greco-Arab) system of medicine.Objective of the study: This study was aimed at investigating the anti-inflammatory and analgesic activitiesof the crude extract of Areca catechu and its respective fractions.Materials and methods: Paw edema, formalin-induced nociception and acetic acid-induced writhingassays were carried out in vivo. Free radical scavenging activity of the plant extract was performed in vitro.Results: Preliminary experiments using a single dose (100 mg/kg) of Areca catechu and its respectivefractions demonstrated an anti-inflammatory effect on carrageenan-induced edema in mice and rats,the aqueous fraction being distinctly more effective. When studied on prostaglandin E2 (PGE2), arachi-donic acid, histamine, or serotonin (5HT)-induced edema in rats, Areca catechu and its aqueous fractionmarkedly repressed only the PGE2 and arachidonic acid-induced inflammation. When studied for anal-gesic activity, the crude extract and its aqueous fraction produced a dose-dependent (10–100 mg/kg)

inhibitory effect on formalin-induced nociception in mice and acetic acid-induced writhing in rats, sim-ilar to aspirin. In DPPH assay, Areca catechu and its aqueous fraction exhibited free radical scavengingactivity with respective IC50 values of 5.34 �g/ml (4.93–5.78, CI; 95%, n = 5) and 7.28 �g/ml (6.04–7.95,n = 4), like that of rutin with IC50 value of 4.75 �g/ml (3.89–5.42, n = 4).Conclusion: These results indicate the anti-inflammatory and analgesic effects of Areca catechu and providea rationale for its medicinal use in inflammatory disorders.

. Introduction

Areca catechu Linn. is a member of the Palmaceae family. Its known as Areca nut/Betel nut in English. In Pakistan, Betelut is referred to as Chalia or Supari (Usmanghani et al., 1997).

t is distributed throughout the world and widely cultivated inouth-Asian countries (Gupta and Warnakulasuriya, 2002). Forenturies, people living in different areas of the world haveeen using it for masticatory purposes. Betel nut is used inolk medicine for the treatment of multiple disorders, such asnflammation (gingivitis, conjunctivitis, edema, etc.), flatulence,

iarrhea and dysentery, hyper-emesis in pregnancy, dysuria andigh blood pressure (Nadkarni, 1976; Duke et al., 2002). It alsoossesses astringent, analgesic and aphrodisiac properties (Kapoor,

∗ Corresponding author. Tel.: +92 213 4864493; fax: +92 213 493 4294/2095.E-mail addresses: shagufta.khan@aku.edu (S. Khan), hassan.mehmood@aku.edu

M.H. Mehmood).

378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2011.03.064

© 2011 Elsevier Ireland Ltd. All rights reserved.

1990). Areca catechu powder has been applied topically as ananti-inflammatory remedy or the nut is made into ash for localapplication. It is also used as collyrium, in a more refined form,to get relief in conjunctivitis and epiphoria (Usmanghani et al.,1997). Moreover, a number of compound preparations contain-ing Areca catechu such as Ma’jun Supari and Ma’jun Mochrus areapplied topically in wound healing (Awan, 1960; Usmanghani et al.,1997).

Betel nut is reported to contain many chemical entities includ-ing, alkaloid (arecaidine, arecoline, guvacine and guvacoline),carbohydrates, fats, proteins and tannins (Farnsworth, 1976), �-sitosterol, gallic-acid and thiamine (Duke, 1992).

Arec catechu has been widely studied for its antiovula-tory and abortifacient (Shrestha et al., 2010), hepatoprotective(Pithayanukul et al., 2009), cardiac-suppressive (Ghayur and Gilani,

2007), anti-oxidant (Lai et al., 2007), antibacterial (Wang andHuang, 2005), cholinomimetic and acetylcholinesterase inhibitory(Gilani et al., 2004), hypocholestrolemic (Park et al., 2002), plateletaggregation modulatory (Jeng et al., 2002), anti-aging (Lee and Choi,

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S. Khan et al. / Journal of Ethno

999), anti-depressant (Dar et al., 1997) and memory enhancingctivities (Smythies, 1977).

On the other hand, there are some reports showing dose-relatedeneficial and ill-effects of areca nut extract and its one of the alka-

oids, i.e., arecoline (Lee and Choi, 1999; Chang et al., 2004; Huangt al., 2010; Liu et al., 2011). In contrast, it has also been reportedhat the alkaloids of betel nut are not the cause of cancer but thathe excessive exposure of lime, which is one of the principal ingre-ients of betel quid (areca nut, lime, leaf) contributes much to thisSmythies, 1977; Kashyap et al., 2008). Moreover, different studiesave shown the contaminated and aflatoxin infected nut chewings a major cause of numerous diseases in third-world countriesRaisuddin and Misra, 1991; Srivatanakul et al., 1991). This is alsoommon with other medicinal plants such as, tea; while the tan-ins in tea are known to be carcinogenic, the proven usefulness ofolyphenols is unquestionable for their cardiovascular protectiveffects (Weisburger and Chung, 2002).

Despite the presence of some reports on the pro/anti-nflammatory and anti-oxidant activities of Areca catechu extract ofome other regions (Lee and Choi, 1999; Chang et al., 2004; Huangt al., 2010), no study is available on Areca catechu of Pakistani ori-in demonstrating its anti-inflammatory or analgesic properties,ven though, in the traditional system of medicine, it has beensed to relieve inflammation and pain for a long time (Kapoor,990; Duke et al., 2002). This investigation demonstrated the anti-

nflammatory effect of betel nut, mediated through cyclooxygenasend lipoxygenase inhibitory pathways, and through degradationnd/or inactivation of prostaglandin E2 (PGE2), for its per-eived medicinal use in inflammatory disorders. Moreover, thistudy also provides the first evidence for its analgesic effect inodents.

. Materials and methods

.1. Plant material and preparation of the crude extract

Areca catechu nuts were purchased from a local market anduthenticated by Mr. Abrar from the Department of Botany, Uni-ersity of Karachi. A voucher sample was submitted to the Karachiniversity Herbarium (KUH) with reference no. 67278. Betel nuts

1 kg) were ground into powder and soaked for a period of 6 daysn 70% methanol. The filtrate was evaporated to produce a darkrown pasty extract using a rotary evaporator (Buchii model RE-11, Switzerland) under reduced pressure, and producing 266 g ofreca catechu extract (26.6%). The crude extract (241 g) was par-itioned between water and hexane (1:1). Evaporation of hexaneroduced a gum-like material (hexane fraction, weighing 0.5 g). Thequeous layer was further partitioned with chloroform, n-butanol,ichloromethane and ethyl acetate respectively, and obtained 12 ghloroform fraction, 18 g n-butanol fraction, 15 g dichloromethaneraction, 8 g ethyl acetate fraction. The remaining aqueous layerielded 180 g as aqueous fraction (Dar and Khatoon, 2000). How-ver, 7.5 g of the extract was lost during the process of fractionation,hich is not more than 3% of the total extract used for fraction-

tion. Moreover, the chloroform, n-butanol and dichloromethaneractions were not found to be effective in any of the assays carriedut in this study.

.2. Phytochemical analysis

Preliminary phytochemical screening of the crude extract and

ts aqueous fraction was carried out qualitatively for the presence oflkaloids, anthraquinones, coumarins, flavonoids, saponins, sterols,annins and terpenes by the following standard methods (Edeogat al., 2005).

acology 135 (2011) 654–661 655

2.3. Drugs and animals

Acetic acid, arachidonic acid, caffeic acid, chlorpheniraminemaleata, 1,1-diphenyl-2 picrylhydrazyl, formalin, histamine phos-phate, lambda carrageenan, methysergide maleate, prostaglandinE2 (PGE2), serotonin and rutin were purchased from Sigma–Aldrich,St. Louis, Missouri, USA, while aspirin was purchased from Reckittand Colman, Pakistan. Oral treatments were given in a volume of5 ml/kg and 10 ml/kg to rats and mice, respectively. All the testcompounds were solubilized in physiological saline except organicfractions (hexane and ethyl acetate), which were dissolved in 10%DMSO.

BALB/c mice (20–30 g) and Sprague Dawley (SD) rats(120–180 g) of either sex, provided by the animal house ofthe Aga Khan University, Karachi, were used. All animals were keptunder standard conditions with normal light cycle (12 h), with freeaccess to food and water. These experiments were carried out inaccordance with ethical guidelines of the Institute of LaboratoryAnimal Resources, Commission on Life Sciences, National ResearchCouncil (NRC, 1996).

2.4. Carrageenan-induced paw edema in mice

The anti-inflammatory activity of Areca catechu and its frac-tions were determined using the model of carrageenan-inducedpaw edema (Winter et al., 1962). Mice of either sex were dividedinto six groups (n = 6 each). All mice were orally treated with saline(negative control) or aspirin (positive control) or Areca catechuextract or hexane fraction or ethyl acetate fraction or aqueous frac-tion at a dose of 100 mg/kg. Following 30 min of oral treatment,30 �l of 1% carrageenan was administered into the sub-plantarside of the right hind paw of all the animals. After 3rd h ofcarrageenan administration, all animals were killed by cervical dis-location and their hind paws were immediately amputated at theankle joint and weighed simultaneously. The weight of the lefthind paw (control) was subtracted from the weight of the righthind paw (test) to obtain the weight of the resultant edema. Per-cent inhibition of paw edema was calculated using the followingformula:

EW = EWC − EWT

EW = edema weight,EWC = edema weight of right paw (control),EWT = edema weight of left paw (test).

Percent inhibition = EWC − EWT

EWC× 100

2.5. Carrageenan-induced paw edema in rats

SD rats of either sex were divided into ten different groups(n = 6 each) and were treated orally with saline (negative con-trol), different doses (10, 50 and 100 mg/kg) of aspirin (positivecontrol) or Areca catechu or its aqueous fraction. After 30 min oftreatment, 50 �l of 1% carrageenan was administered into thesub-plantar side of the right hind paw. The volume of the righthind paw was measured (Winter et al., 1962) using a plethys-mometer (IITC Inc. Life Sciences, Woodland Hills, CA). The pawvolume was measured before and at 1st, 2nd, 3rd and 4th h

after carrageenan administration. The edema volume of paw andpercent inhibition of edema were calculated using the followingformulas:

EVC = PVA − PVI

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56 S. Khan et al. / Journal of Ethno

EV = edema volume,PVI = paw volume before carrageenan administration (i.e. initialpaw volume).PVA = paw volume after carrageenan administration.

ercent inhibition = EVC − EVT

EVC× 100

EVC = edema volume of control animal,EVT = edema volume of test animal.

.6. Prostaglandin E2-induced paw edema in rats

Rats (SD) of either sex were divided into eight different groupsn = 6 each). Rats were treated intraperitoneally with saline (con-rol) or Areca catechu extract (10, 50 and 100 mg/kg) or its aqueousraction (10, 50 and 100 mg/kg) or aspirin. After 30 min of treat-

ent, 100 �l of prostaglandin E2 (0.01 �g/ml) was administerednto the sub-plantar side of the right hind paw of each rat and theaw edema was determined (Parmar and Gosh, 1978).

.7. Arachidonic acid-induced paw edema in rats

Rats (SD) of either sex were divided in five different groupsn = 6 each). Rats were treated intraperitoneally with saline (con-rol) or aspirin or caffeic acid or areca extract or aqueous fraction100 mg/kg). After 30 min of treatment, 100 �l of 0.5% arachi-onic acid was administered into the sub-plantar side of the rightind paw of each rat, and the edema volume was determined asescribed by Di Martino et al. (1987).

.8. Histamine induced paw edema in rats

In this assay, SD rats were divided into four groups (n = 6 each),hich were orally treated with saline (negative control) or chlor-heniramine (positive control 25 mg/kg), or Areca catechu extract100 mg/kg) or its aqueous fraction (100 mg/kg). After 30 min of oraldministration, 100 �l (1 mg/ml) of histamine was injected into theub-plantar side of the right hind paw of the rats and paw edemaas determined (Parmar and Gosh, 1978).

.9. Serotonin induced paw edema in rats

Rats (SD) were divided into the four groups (n = 6 each); whichere orally treated with saline (negative control) or methysergide

positive control, 25 mg/kg) or Areca catechu extract (100 mg/kg)r its aqueous fraction (100 mg/kg). After 30 min of oral admin-stration, 100 �l (1 mg/ml) of serotonin was injected into theub-plantar side of the right hind paw of rats and paw edema wasetermined (Parmar and Gosh, 1978).

.10. Formalin-induced nociception in mice

The antinociceptive activity of test material was assessed usinghe formalin assay (Hunskaar and Hole, 1987). A total of ten dif-erent groups of mice (n = 6 each) were treated orally with salinecontrol) or different doses (10, 50 and 100 mg/kg) of aspirin orreca catechu extract or its aqueous fraction. After 1 h of oralreatment, 20 �l of 1% formalin was injected into the sub-plantarpace of the right hind paw and then the animals were placed intransparent plastic observation chamber. The duration of paw

icking was recorded between 0-5 min (1st phase) and 15-45 min2nd phase). Percent inhibition of nociception during 1st and 2ndhases was determined by comparing the paw licking time withhe respective positive control.

acology 135 (2011) 654–661

2.11. Acetic acid-induced writhing in mice

The test was performed as described earlier (Collier et al.,1968). In this experiment the mice were divided into ten differentgroups (n = 6 each), which were treated orally with saline (nega-tive control) or different doses (10, 50 and 100 mg/kg) of aspirin(positive control) or Areca catechu extract or its aqueous fraction.After 30 min of the oral treatment, 0.8% acetic acid was adminis-tered through intra-peritoneal route. Immediately after injection ofacetic acid, each animal was placed in a transparent plastic observa-tion chamber and the number of writhing movements was countedfor 30 min commencing after injection of acetic acid. Percent inhi-bition of the number of writhing movements was calculated andcompared with positive control.

2.12. Free radical scavenging activity

Free radical scavenging activity was determined by followingthe previously performed assay with a slight modification (Lucianaet al., 2001). The experiment was divided into two groups, thepositive control group was treated with rutin and the experimentgroups were treated with Areca catechu extract or its aqueousfraction (1 mg/ml). Plant extract and its aqueous fraction werediluted to final concentrations of 1, 5, 10 and 15 �g/ml in methanoland finally the negative control group, methanol (300 �l) alongwith test solution (800 �l of methanol) was used as a blank. Thereaction mixture containing 800 �l of test sample and 300 �l of1,1-diphenyl-2-picryl-hydrazil (DPPH) was incubated at room tem-perature for 30 min, and the absorbance was measured at 518 nm.The absorbance of the test sample was subtracted from that of theblank and the percent antioxidant activity was calculated. Eachexperiment was performed in duplicates. The percentage inhibitionwas calculated by the following equation:

% DPPH radical inhibition

= 1 − (control absorbance − sample absorbance)control

× 100

The DPPH solution was used as a control. Theconcentration–response curves were plotted as concentrationof extracts in �g/ml against percent of free radical scavengingactivity for the calculation of EC50 values along with ±SEM.

2.13. Data analysis

Results are expressed as the mean ± SEM (n = number of obser-vations) and the median inhibitory concentration (IC50) valueswith 95% confidence intervals. p < 0.05 was considered signifi-cantly different using one-way ANOVA followed by Dunnett’stest or unpaired t-test. The concentration–response curves (CRCs)were analyzed by non-linear regression using GraphPAD program(GraphPAD, San Diego, California, USA).

3. Results

3.1. Phytochemical analysis

Preliminary phytochemical analysis revealed the presence ofalkaloids, flavonoids, saponins, sterols, tannins and terpenes inAreca catechu extract and in its aqueous fraction.

3.2. Carrageenan-induced paw edema in mice

Areca catechu extract, its aqueous (A. fraction), ethyl acetate(E. fraction) and hexane (H. fraction) fractions showed significantreduction against carrageenan-induced paw edema in mice with

S. Khan et al. / Journal of Ethnopharm

Fig. 1. Effects of Areca catechu (A.Catechu) extract its hexane, ethyl acetate andaqueous fractions on carrageenan-induced paw edema in mice. Each bar repre-ssb

pcic

3

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TE

E*

TE

E*

ents mean ± SEM of 6 determinations. *#p < 0.05, **p < 0.01, ###***p < 0.001; * versusaline control, while # versus positive control of aspirin (one-way ANOVA, followedy Dunnett’s test or unpaired t-test).

ercent inhibition of 52 ± 3.8, 76 ± 4.6, 33 ± 3.5, 16 ± 2.4 of 100% ofarageenon control, similar to aspirin (Fig. 1). The order of activ-ty in the test substance was observed as aqueous fraction > Arecaatechu extract > ethyl acetate fraction > hexane fraction.

.3. Carrageenan-induced paw edema in rats

Areca catechu extract and its aqueous fraction demonstratedose (50 and 100 mg/kg) and time (2–4 h)-dependent reduc-ion against carrageenan-induced paw edema in rats. Theercent inhibitory order of edema at maximum tested dose

able 1ffect of Areca catechu extract and its aqueous fraction on carrageenan-induced paw edem

Treatment Dose (mg/kg) Percent inhibition of ed

1 h

Areca catechu extract 10 4.8 ± 0.44n.s.

Aqueous fraction 10 6.3 ± 2.43n.s.

Aspirin 10 4.1 ± 1.79n.s.

Areca catechu extract 50 9.4 ± 1.10n.s.

Aqueous fraction 50 8.7 ± 2.37n.s.

Aspirin 50 7.4 ± 2.68n.s.

Areca catechu extract 100 9.0 ± 1.66n.s.

Aqueous fraction 100 7.2 ± 2.01n.s.

Aspirin 100 11.8 ± 2.93n.s.

ach value shown represents mean ± SEM (n = 4–6).*p < 0.01, ***p < 0.001 and n.s. (non-significant) versus only carrageenan treated group (o

able 2ffect of Areca catechu extract and its aqueous fraction on prostaglandin E2-induced paw

Treatment Dose (mg/kg) Percent inhibition

1 h

Areca catechu extract 10 1.5 ± 0.64n.s.

Aqueous fraction 10 1.9 ± 0.2n.s.

Areca catechu extract 50 15.2 ± 1.31*Aqueous fraction 50 51.2 ± 2.1***

Areca catechu extract 100 62 ± 1.35***Aqueous fraction 100 86 ± 3.63***

ach value shown represents mean ± SEM (n = 4–6).p < 0.05, ***p < 0.001 and n.s. (non-significant) versus only prostaglandin E2 treated grou

acology 135 (2011) 654–661 657

(100 mg/kg) was aqueous fraction > Areca catechu extract > aspirin(Table 1).

3.4. Prostaglandin E2-induced paw edema in rats

Areca catechu extract and its aqueous fraction demonstrateddose-dependent (50 and 100 mg/kg) and persistent inhibitionagainst prostaglandin E2 (PGE2)-induced paw edema in rats as seenin Table 2, while a separate group of mice pretreated with aspirin(100 mg/kg) did not show any protective effect on PGE2)-inducedrat paw edema (data not shown).

3.5. Arachidonic acid-induced paw edema in rats

Areca catechu extract and its aqueous fraction showed markedinhibition of arachidonic acid-induced paw edema in rats at a doseof 100 mg/kg, similar to caffeic acid (Table 3), while it showed noeffect on lower doses (data not shown). The percent inhibitory orderof edema at 100 mg/kg was caffeic acid ≥ aqueous fraction > Arecacatechu extract (Table 3).

3.6. Histamine-induced paw edema in rats

When tested against histamine-induced paw edema, Areca cat-echu and its aqueous fraction (100 mg/kg) failed to produce anysignificant inhibition of edema when compared with the inhibitoryeffect of chlorpheniramine (Table 4).

3.7. Serotonin-induced paw edema in rats

There was no consequential inhibition of edema in this assayeither by Areca catechu extract or with its aqueous fraction(100 mg/kg) when compared with methysergide treated group(Table 5).

a in rats.

ema volume

2 h 3 h 4 h

23.7 ± 1.97** 25.4 ± 1.09** 21.8 ± 1.77**32.2 ± 1.52** 30.1 ± 2.99** 29.6 ± 2.68**

6.7 ± 2.14n.s. 5.4 ± 1.59n.s. 4.2 ± 1.69n.s.

40.8 ± 1.96** 42.6 ± 1.87** 43.1 ± 1.69**62.4 ± 1.72*** 63.7 ± 0.58*** 62.0 ± 0.94***10.8 ± 1.90n.s. 12.3 ± 2.98n.s. 8.4 ± 2.39n.s.

59.5 ± 1.80*** 60.4 ± 1.05*** 61.1 ± 2.41***80.2 ± 0.76*** 83.4 ± 0.28*** 84.0 ± 0.19***47.2 ± 1.91** 45.6 ± 2.01** 43.9 ± 2.47**

ne-way ANOVA, followed by Dunnett’s test).

edema in rats.

of edema volume

2 h 3 h 4 h

2 ± 0.41n.s. 1.2 ± 0.63n.s. 3 ± 0.70n.s.

2.5 ± 0.41n.s. 3 ± 0.58n.s. 2.9 ± 0.58n.s.

18 ± 2.29* 16 ± 1.5* 17 ± 2.05*49 ± 1.65*** 52 ± 1.3*** 55 ± 1.94***

61 ± 2.87*** 63 ± 1.3*** 62 ± 1.2***86 ± 1.82*** 88 ± 3.03*** 87 ± 2.94***

p (one-way ANOVA, followed by Dunnett’s test).

658 S. Khan et al. / Journal of Ethnopharmacology 135 (2011) 654–661

Table 3Effect of Areca catechu extract and its aqueous fraction on arachidonic acid-induced paw edema in rats.

Treatment Dose (mg/kg) Percent inhibition of edema volume

1 h 2 h 3 h 4 h

Areca catechu extract 100 17 ± 1.2* 19 ± 2.41* 23 ± 0.32** 24 ± 1.04**Aqueous fraction 100 47 ± 1.21*** 48 ± 1.55*** 49 ± 1.07*** 50 ± 0.75***Caffeicacid 100 63 ± 2.32*** 70 ± 0.4*** 71 ± 1.31*** 73.2 ± 1.9***Aspirin 100 0.75 ± 0.5n.s. 2 ± 0.62n.s. 2 ± 0.63n.s. 1 ± 0.5n.s.

Each value shown represents mean ± SEM (n = 4–6).*p < 0.05, **p < 0.01, ***p < 0.001 and n.s. (non-significant) versus only arachidonicacid treated group (one-way ANOVA, followed by Dunnett’s test).

Table 4Effect of Areca catechu extract and its aqueous fraction on histamine-induced paw edema in rats.

Treatment Dose (mg/kg) Percent inhibition of edema volume

1 h 2 h 3 h 4 h

Areca catechu extract 100 4.3 ± 1.70n.s. 2.7 ± 0.39n.s. 4.8 ± 1.09n.s. 5.1 ± 0.98n.s.

Aqueous fraction 100 6.1 ± 2.11n.s. 3.2 ± 1.25n.s. 4.6 ± 1.83n.s. 3.0 ± 1.71n.s.

Chlorpheniramine 25 71.9 ± 2.99*** 68.2 ± 2.74*** 68.4 ± 2.39*** 67.2 ± 2.91***

Each value shown represents mean ± SEM (n = 4–6).***p < 0.001 and n.s. (non-significant) versus only histamine treated group (one-way ANOVA, followed by Dunnett’s test).

Table 5Effect of Areca catechu extract and its aqueous fraction on serotonin-induced paw edema in rats.

Treatment Dose (mg/kg) Percent inhibition of edema volume

0.5 h 1 h 2 h 3 h

Areca catechu extract 100 2 ± 0.70n.s. 5.3 ± 1.31n.s. 6.8 ± 2.11n.s. 6.7 ± 2.13n.s.

Aqueous fraction 100 9.9 ± 2.88n.s. 7.3 ± 1.95n.s. 8.1 ± 2.89n.s. 8.8 ± 2.75n.s.

Methysergide 25 79.5 ± 2.63*** 77.9 ± 2.93*** 78.1 ± 2.56*** 83.4 ± 1.16***

Each value shown represents mean ± SEM (n = 4–6).***p < 0.001 and n.s. (non-significant) versus only serotonin treated group (one-way ANOVA, followed by Dunnett’s test).

Table 6Effect of Areca catechu extract and its aqueous fraction on formalin-induced pawnociception in mice.

Treatment Dose (mg/kg) Percent inhibition of nociception

1st phase 2nd phase

Areca catechu extract 10 0 10.5 ± 1.37*Aqueous fraction 10 0 14.6 ± 0.12*Aspirin 10 0 5.2 ± 1.04n.s.

Areca catechu extract 50 8.4 ± 1.69n.s. 25.5 ± 1.19**Aqueous fraction 50 7.5 ± 2.88n.s. 46.0 ± 0.55**Aspirin 50 3.1 ± 2.79n.s. 15.3 ± 1.60*

Areca catechu extract 100 9.0 ± 2.99n.s. 46.3 ± 0.18**Aqueous fraction 100 8.6 ± 2.73n.s. 78.8 ± 1.10***Aspirin 100 10.2 ± 2.98n.s. 52.9 ± 2.18***

Each value shown represents mean ± SEM (n = 4–6).*t

3

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3

dw

Table 7Effect of Areca catechu extract and its aqueous fraction on acetic acid-inducedabdominal writhings in mice.

Treatment Dose (mg/kg) Percent inhibition ofabdominal writhings

Areca catechu extract 10 12.4 ± 1.58*Aqueous fraction 10 22.1 ± 1.19**Aspirin 10 5.1 ± 1.46n.s.

Areca catechu extract 50 39.1 ± 0.74**Aqueous fraction 50 50.1 ± 1.94***Aspirin 50 27.9 ± 1.83**

Areca catechu extract 100 65.2 ± 1.41***Aqueous fraction 100 80.1 ± 1.07***Aspirin 100 49.3 ± 0.22***

antioxidant activities of Areca catechu extract and its aqueous frac-

p < 0.05, **p < 0.01, ***p < 0.001 and n.s. (non-significant) versus only farmalinreated group (one-way ANOVA, followed by Dunnett’s test).

.8. Formalin-induced nociception in mice

In this inquisition, no effect was observed in the 1st phasef all doses of the test materials. However, in 2nd phase of thexperiment, a marked dose-dependent inhibition in writhings wasbserved at all doses of Areca catechu extract and its aqueous frac-ion, similar to aspirin (Table 6).

.9. Acetic acid-induced writhing in rats

In this assay Areca catechu extract and its aqueous fraction pro-uced dose-dependent inhibition of acetic acid-induced abdominalrithing in rats, in a similar way to aspirin (Table 7).

Each value shown represents mean ± SEM (n = 4–6).*p < 0.05, **p < 0.01, ***p < 0.001 and n.s. (non-significant) versus only acetic acidtreated group (one-way ANOVA, followed by Dunnett’s test).

3.10. Free radical scavenging activity

Areca catechu extract and its aqueous fraction showedconcentration-dependent (0.3–30 �g/ml) free radical scavengingactivity with respective IC50 values of 5.34 �g/ml (4.93–5.78, CI;95%, n = 5) and 7.28 �g/ml (6.04–7.95, n = 4), like that of rutin withIC50 value of 4.75 �g/ml (3.89–5.42, n = 4) as shown in Fig. 2.

4. Discussion

This study reports the anti-inflammatory, antinociceptive and

tion using in vivo and in vitro assays. Carrageenan-induced pawedema test is a well-established animal model of inflammation todetect the anti-inflammatory activity of test materials. Our pre-

S. Khan et al. / Journal of Ethnopharm

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ig. 2. Free radical scavenging activity of Areca catechu (A.Catechu) extract, its aque-us fraction and rutin in DPPH assay.

iminary studies in mice showed that Areca catechu extract, itsexane, ethyl acetate and aqueous fractions demonstrated a sig-ificant inhibition against carrageenan-induced edema, similar tospirin, the aqueous fraction being distinctly the most effective.his might suggest additional anti-inflammatory mechanism(s)long with the involvement of cyclooxygenase (COX) inhibitoryathway. These findings are consistent with the earlier studieshowing anti-inflammatory potential of betel nut extract (Lee andhoi, 1999; Chang et al., 2004; Huang et al., 2010). On the basis ofhese findings, plant extract and its fractions were further testedn acute inflammatory model of rat paw edema. In control animals,

aximum edema induction by carrageenan was evident at the 2ndof administration reaching a plateau at the 3rd h followed bydecline at the 4th h of observation, which is also in line with

he previous study (Mazzanti and Braghiroli, 1994). Areca catechuxtract and its aqueous fraction reduced edema volume in a dosend time-related manner, indicating the anti-inflammatory effectf the plant extract and its aqueous fraction. The inhibition of edemabserved was more pronounced in the later phase than in the ear-ier phase, which was similar to the effect of aspirin, a prototypef non-steroidal anti-inflammatory drugs (NSAIDs) (Burke et al.,006), indicating that the anti-inflammatory effect of Areca catechu

s possibly mediated through a cyclooxygenase enzyme inhibitoryathway. Moreover, hexane and ethyl acetate fractions of Areca cat-chu did not cause significant inhibition of edema in rats (data nothown), hence were not studied further. The difference in the anti-nflammatory effect of hexane and ethyl acetate fractions in micend rats may be due to species selectivity, as species-specific bio-ogical activities are known to exist (Ghayur et al., 2005; Ghayurnd Gilani, 2006).

Inflammation is characterized by the accumulation of a vari-ty of mediators at the site of the injury or infection. Carrageenannduces paw edema biphasically: the initial phase extending from

to 2.5 h, predominantly results due to the release of histamine,erotonin and bradykinin, and the later phase begins due to thever-production of prostaglandins such as PGE2 in tissue (Diosa, 1974). However, COX enzyme is known to play a key role

n the development of the later phase of inflammation in thearrageenan-induced edema model by converting arachidonic acidnto prostaglandins. This enzyme is considered to be a knownarget for a variety of NSAIDs such as aspirin, which inhibits rataw edema at the later phase following carrageenan injection.

hen tested in rats, Areca catechu extract and its aqueous fraction

emonstrated dose and phase-dependent inhibition of edema athe 2nd h with maximum respective inhibition of 59.5 ± 1.80 and0.2 ± 0.76% in a pattern similar to that of aspirin (47.2 ± 1.91%).

acology 135 (2011) 654–661 659

The aqueous fraction was more potent than the crude extract andaspirin. Separate sets of experiments were conducted to determinethe effect of Areca catechu extract and its aqueous fraction againsthistamine or serotonin-induced inflammation, where they failedto inhibit the edemogenic effect of histamine or serotonin. Thesedata indicate that the betel nut extract and its aqueous fractionare unlikely to interfere with histamine or serotonin release atthe initial phase of inflammation. Thus, it can be speculated thatthe inhibition of edema by the extract and its aqueous fractionat the later phase of inflammation-induced by carrageenan maybe mediated possibly through the inhibition of prostaglandin syn-thesis similar to aspirin. However, the stronger anti-inflammatoryresponse of the aqueous fraction than the parent extract and aspirinindicates the presence of some additional constituents, involvingdifferent mode(s) of action, in the aqueous fraction with rela-tively better efficacy. In our further attempt to explore the possibleexplanation, in addition to the COX-inhibitory effect of Areca cate-chu, we observed the prostaglandin degrading and/or inactivatingactions of the crude extract and its aqueous fraction, which wasevident by their inhibitory effect on PGE2-induced rat paw edema,while aspirin was found to be ineffective in this model. Moreover,the crude extract and its aqueous fraction showed a lipoxygenaseinhibitory property by exhibiting an anti-inflammatory effect onarachidonic acid-induced rat paw edema, similar to caffeic acid, alipoxygenase inhibitor (Sud’ina et al., 1993), but aspirin was foundinactive, as the arachidonic acid-induced paw edema is highly sen-sitive to inhibitors of the lipoxygenase pathway and is resistantto selective COX inhibitors (Di Martino et al., 1987). These datademonstrate the anti-inflammatory effect of Areca catechu extractand its aqueous fraction mediated though multiple pathways.

Chang et al. (2004) showed the role of Areca catechu extract andarecoline in the pathogenesis of oropharyngeal cancer by activa-tion of MEK1/ERK/c-Fos pathway. However, there are controversiesin the available literature on Areca catechu extract such as thefact that Chang et al. (2004) showed the dose-related pro andanti-inflammatory effects Areca catechu extract and arecoline in dif-ferent epithelial cells, while at the same time, arecoline, an activeconstituent of Areca catechu was found to have a blunting effect oninflammatory mediators, such as prostaglandin E2 and interleukin-6. Similarly, multiple studies (Sundqvist et al., 1989; Dave et al.,1992; Jeng et al., 2001) conducted in different cell lines, have alsoshown the cytotoxic and genotoxic effects of the crude extract andarecoline. On the other hand, Lee and Choi (1999) and Huang et al.(2010) have reported the anti-inflammatory (carrageenan-inducedinflammation in rats) and anti-oxidant (biochemical assays) effectsof Areca catechu extract, showing that despite harmful effects ofAreca catechu extract, it still has sufficient potential to be studied.Our study, being in line with the previous reports (Lee and Choi,1999; Huang et al., 2010), provided additional information in thefollowing ways:

1. Species-specific anti-inflammatory effects of the crude extractand its respective fractions which were shown in mice and rats.

2. Anti-inflammatory effect of Areca catechu extract and its aqueousfraction mediated though multiple pathways i.e. cyclooxyge-nase and lipoxygenase inhibition of arachidonic acid metabolism(dual inhibitor property) and its additional effect causing degra-dation and/or inactivation of prostaglandins (PGE2).

3. It has also been identified that the aqueous fraction was found tobe most effective in its anti-inflammatory and analgesic effectsas compared to the parent extract and rest of the tested frac-tions, hence, the aqueous fraction could be studied further for

the isolation of some pure compound(s).

The NSAIDs are also used as analgesics because prostaglandinsare pro-inflammatory mediators contributing to the signs and

6 pharm

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60 S. Khan et al. / Journal of Ethno

ymptoms of inflammation such as pain (Crofford, 2000). Arecaatechu extract was thus assessed for the first time for its possiblenti-nociceptive effect using two different in vivo models: the aceticcid (AA)-induced writhings in mice and the formalin-induced lick-ng in rats.

When tested in AA-induced writhing assay, Areca catechuxtract and its aqueous fraction exhibited dose-dependent anal-esic effects, the aqueous fraction being more potent than theositive control, aspirin. AA induces inflammatory pain by causingapillary permeability and liberating endogenous substances thattimulate pain nerve endings (Raj, 1996). While, NSAIDs cause inhi-ition of COX enzyme in peripheral tissues and interfere with theransduction mechanism of primary afferent nociceptors (Fields,987).

In the formalin-induced licking (nociception) model, the irri-ant produces pain in two phases: the 1st phase extends from-5 min which is mediated by the central nervous system viairect activation of peripheral nociceptors, as reflected by a rise

n levels of substance-P or bradykinin, whereas the 2nd phase15–45 min) is mediated through the release of a variety ofnflammatory mediators including bradykinin and prostaglandinst the peripheral site, causing pain perception (Shibata et al.,989), which is known to be inhibited by aspirin. On thether hand, opioid receptor agonists, such as morphine exertn analgesic effect via inhibiting the initial phase of formalin-nduced nociception (Souza et al., 1998). Pretreatment of animals

ith Areca catechu extract or its aqueous fraction inhibitedhe 2nd phase of formalin-induced nociception at maximumested dose by 46.3 ± 0.18 and 78.8 ± 1.10% respectively, simi-ar to aspirin (52.9 ± 2.18%). The order of potency was: aqueousraction > Areca catechu extract > aspirin. This suggests that Arecaatechu extract and its aqueous fraction possess analgesic effectossibly mediated through the inhibition of prostaglandin synthe-is.

These results show the anti-inflammatory and analgesic effectsf Areca catechu involving different mode(s) of action with betterfficacy than aspirin. Stronger effects of Areca catechu than aspirinre also in line with the proven concept that medicinal plants pos-ess a combination of constituents, involving different mode(s) ofctions, offering synergistic or side-effect nullifying effects (Gilanind Rahman, 2005).

Generation of reactive oxygen species (ROS) is associated withhe pathogenesis of multiple diseases such as atherosclerosis, dia-etes, cancer, arthritis and the aging process. Inflammation is also aomplex process and ROS play an important role in the pathogen-sis of inflammatory diseases (Conner and Grisham, 1996). Thusntioxidants, which can scavenge ROS, are expected to improvenflammatory disorders. The free radical scavenging activity of thextract and its aqueous fraction was evaluated on the basis ofts ability to scavenge the synthetic DPPH. Areca catechu extractnd its aqueous fraction exhibited significant antioxidant activ-ty, similar to rutin. Free radicals, such as hydroxyl radicals haveeen demonstrated to be a contributing factor in the tissue injurynd modulation of pain (Khalil et al., 1999). Thus, a substancehich either inhibits the generation of free radical enzymes orirectly scavenges the reactive free radicals to remove excessree radicals effectively can also prevent inflammation throughts antioxidant effect. The observed antioxidant effect of Arecaatechu extract and its aqueous fraction is also consistent withhe previous study (Lee and Choi, 1999). However, we found atronger anti-oxidant activity of the extract as compared to ear-ier report of Lee and Choi (1999), which might be due to the

ffect of environmental and regional factors which are knowno influence the expression of phytochemical constituents in theame plants grown in different areas (Harborne, 1993; Lee et al.,007).

acology 135 (2011) 654–661

5. Conclusion

Areca catechu extract and its aqueous fraction possess anti-inflammatory and analgesic activities mediated possibly throughcyclooxygenase and lipoxygenase inhibitory pathways, and bydegradation and/or inactivation of prostaglandin E2 (PGE2). Thesefindings suggest that the areca extract and its aqueous fractionhave good correlations with the medicinal use of Areca catechuin inflammatory disorders in the Unani (Greco-Arab) system ofmedicine. However, further bioassay-directed fractionation stud-ies are required using a more potent aqueous fraction to identifythe active compound(s) and its/their exact mode(s) of action.

Acknowledgement

We would like to thank the Professor Atta-ur-Rahman’s researchgroup for the preparation of Areca catechu extract and its fractions.

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