Journal of Ethnopharmacology 120 (2008) 248–254

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Journal of Ethnopharmacology

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Aegiceras corniculatum extract suppresses initial and late phasesof inflammation in rat paw and attenuates the productionof eicosanoids in rat neutrophils and human platelets

Talat Roome, Ahsana Dar ∗, Sabira Naqvi,Shamsher Ali, Muhammad Iqbal ChoudharyPharmacology Section, H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences,University of Karachi, Karachi 75270, Pakistan

a r t i c l e i n f o

Article history:Received 25 September 2007Received in revised form 5 July 2008Accepted 23 August 2008Available online 2 September 2008

Keywords:Aegiceras corniculatumAnti-inflammatory activityRat paw edemaLeukotriene B4 (LTB4)5- and 12-hydroxyeicosatetraenoic acid(HETE)12-Hydroxyheptadecatrienoic acid(12-HHT)

a b s t r a c t

Aim of the study: The present study is designed to explore the anti-inflammatory potential of Aegicerascorniculatum Linn. Blanco stems extracts and their mechanism of action against various pro-inflammatorymediators and to validate its traditional use against inflammatory diseases.Materials and methods: Rat paw edema and peritonitis models were employed for in vivo studies. For invitro studies human platelets and rat neutrophils were stimulated with Ca2+-ionophore A23187 leadingto the production of various pro-inflammatory metabolites, i.e., 12-HTT, 12-HETE and LTB4 and 5-HETEwhich were quantified by HPLC.Results: The highly polar methanol extract (100 mg/kg) caused ∼90% reduction in the carrageenan-and prostaglandin E2-induced paw edema in rats. It also caused the inhibition of cycloxygenase-1metabolite, 12-HHT (IC50 41.1 ± 1.5 �g/ml) with a concomitant rise in 12-lipoxygenase metabolite, 12-HETE in A23187 stimulated human platelets. Conversely, the non-polar hexane extract attenuated (IC50

0.36 ± 0.12 �g/ml) 12-HETE formation with a parallel rise in 12-HHT, thereby displaying a selectivitytowards 12-lipoxygenase. Non-polar hexane extract also antagonized the production of 5-lipoxygenasemetabolites, i.e., leukotriene B4 and 5-HETE in the rat neutrophils. Furthermore, ethyl acetate extract inhib-ited both COX and 5-LOX with a marked decline in the production of 12-HHT (IC50 0.08 ± 0.002 �g/ml)and LTB4 (IC50 0.86 ± 0.03 �g/ml), respectively. The anti-inflammatory effect of hexane and ethyl acetateextracts was also reflected by the diminution of carrageenan-induced cell infiltration in rat peritoneum.Additionally, plant extracts caused ∼60% suppression in dextran-induced paw edema implying that they

also ameliorate histamine and serotonin release.Conclusion: Hexane, ethyl acetate and methanol extracts derived from Aegiceras corniculatum possesssignificant anti-inflammatory activity via multiple mechanisms and validate their traditional use against

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. Introduction

Aegiceras corniculatum (Linn.) Blanco (family Aegicerataceae) is aangrove plant that grows in sheltered intertidal zones at interface

etween land and sea in tropical and subtropical regions. Mangrove

Abbreviations: AA, arachidonic acid; COX, cyclooxygenase; PLA2,hospholipase A2; 5-,12-HETE, hydroxyeicosatetraenoic acid; 12-HHT, 12-ydroxyheptadecatrienoic acid; 5-LOX, 5-lipoxygenase; LT, leukotriene; NDGA,ordihydroguaiaretic acid; NSAIDs, non-steroidal anti-inflammatory drugs; PGs,rostaglandins; PMNL, polymorphonuclear leukocytes; TXA2, thromboxane A2.∗ Corresponding author. Tel.: +92 21 4824930 9; fax: +92 21 4819018 9.

E-mail address: ahsanadar@hotmail.com (A. Dar).

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378-8741/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2008.08.025

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orest in Pakistan is rated as 5th largest area in the world where itrows along the 240 km South Eastern coastline comprising Induselta.

Aegiceras corniculatum is used in folklore medicine againststhma, diabetes, inflammation and rheumatism (Bandaranayake,998). A number of chemical constituents have been isolatedrom it viz. flavonoids (Rao and Bose, 1959; Zhang et al., 2005),ydroquinone, benzoquinone and its derivatives (Gomez et al.,989; Xu et al., 2004), polyphenolic compounds (lignan and tanin),

-sitosterol and stigmasterol (Bandaranayake, 2002), pentacyclic

riterpenes and its derivatives, and saponins (Rao and Bose, 1961;ao, 1964; Hensens and Lewis, 1966; Zhang et al., 2005). The plantas been reported to be cytotoxic (Xu et al., 2004) and inhibits tyro-ine phosphatase 1B (Wang et al., 2006). Despite its use in folklore

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gainst inflammation there is no information available about itsnti-inflammatory properties and this prompted us to investigatend justify its traditional use.

A variety of signaling molecules such as vasoactive amines andeptide (histamine, serotonin and bradykinin), arachidonic acidetabolites (prostanoids, leukotrienes and HETEs) and oxygen-

erived free radicals (hydroxyl radicals and superoxide anions) haveeen implicated in a complex process of inflammation (Safayhind Sailer, 1997; Nathan, 2002). Among these mediators arachi-onic acid (AA) metabolites including prostanoids, leukotrienesnd 12-HETE are mainly involved in the pathogenesis of inflam-atory diseases such as asthma, arthritis, atherosclerosis, cancer

nd hyperalgesia (Bertolini et al., 2002; Werz, 2004).The non-steroidal anti-inflammatory drugs (NSAIDs) inhibit-

ng prostanoids synthesis are commonly used in the treatmentf inflammatory and rheumatic disorders. However, their clin-cal use is hampered due to side effects notably gastriclceration, bronchospasm, and kidney dysfunction. Currently,

considerable interest has been generated to develop dualnhibitors of cyclooxygenases and 5-lipoxygenase pathways. Theseon-selective inhibitors display manifold anti-inflammatory effec-iveness with negligible gastric, cardiac and renal toxicity (Celottind Laufer, 2001).

The present study describes the effect of Aegiceras cornicula-um extracts on acute inflammatory models representing varioustages of inflammation. It also addresses the mechanism of anti-nflammatory action against histamine and serotonin release androduction of eicosanoids, including prostanoids, leukotrienes andETEs.

. Materials and methods

.1. Plant material

Aerial parts of Aegiceras corniculatum were collected during June001 from Indus Delta, Sindh, Pakistan at lowest tide and identi-ed by a taxonomist, Dr. Surriya Khatoon from the Department ofotany, University of Karachi, Karachi, Pakistan. Voucher specimenepresenting the collection labeled as KHU G.H.S. No. 68219 haseen deposited at herbarium of this department.

.2. Preparation of extraction

Air-dried powdered stems (9.0 kg) were soaked in 50 l hexaneor 7 days. After filtration and evaporation under vacuum, hex-ne extract (6.0 g or 0.07%, w/w) was obtained. Powdered materialeft after hexane filtration was soaked in 50 l ethyl acetate for

week. Following the aforementioned procedure, ethyl acetatextract (8.5 g or 0.09%, w/w) was yielded and the residue wasoaked in butanol. 11.0 g or 0.12% (w/w) dried butanol extract wasbtained. Ultimately the remaining residue after butanol extractionas soaked in 50 l methanol and 9.8 g or 0.11% (w/w) methanol

xtract was collected. This extraction procedure in brief, yieldedon-polar (hexane), less polar (ethyl acetate) and highly polarmethanol) extracts sequentially used for conventional phytochem-cal tests and pharmacological studies.

.3. Acute toxicity test

The toxic effect of plant extract was determined using albino

ice of either sex (20–25 g, n = 10 in each group), treated with hex-

ne and ethyl acetate extracts (1–1000 mg/kg) or methanol extract1–500 mg/kg) intraperitoneally. Behavioral changes, gross effectnd mortality, if occurred, were observed up to 7 days. The hex-ne extract was found to be non-toxic up to 1 g/kg, whereas ethyl

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acology 120 (2008) 248–254 249

cetate extract caused mortality in mice with LD50 of 850 mg/kg.ethanol extract was toxic at doses above 200 mg/kg. Depending

n the aforementioned results, doses of plant extracts were selectedor in vivo anti-inflammatory studies.

.4. Paw edema in rats by various edemogens

Animal studies were performed in accordance with the decla-ation of Helsinki and the European Community guidelines for thethical handling and the use of laboratory animals and throughhe clearance of institutional animal use committee. Wistar andprague-Dawley rats of either sex (n = 3–5 and 120–180 g) fromhe animal house of International Center for Chemical and Bio-ogical Sciences, H.E.J. Research Institute of Chemistry, Universityf Karachi, Pakistan were used throughout the study. The edemaas induced into their right hind paw by a single subplantar injec-

ion (50 �l) containing either 1% carrageenan type IV (Winter et al.,962) or 25 IU/ml of phospholipase A2 (1.25 U/paw) (Neves et al.,993) or 100 �l of 1.5% dextran (Ferreira et al., 2004) or histamine,erotonin (1.0 mg/ml) or bradykinin (0.02 mg/ml) or prostaglandin2 (1 × 10−5 mg/ml) or leukotriene B4 (2.5 × 10−4 mg/ml) or arachi-onic acid (0.5%) or 2400 IU/ml of hyaluronidase (240 U/paw)Singh et al., 1996). The average volume of right hind paw up tohe tibiotarsal articulation of each rat was measured 3–4× by usinglethysmometer (Model 7150, Ugo Basile, Italy) before (time 0)nd after treatment (0.5–4 h) in the presence of respective ede-ogens. In case of PLA2, the edema was measured at 0.5, 1 and 1.5 h,hereas hyaluronidase-induced edema was detected after 20 min.

tandard drugs, plant extracts and 10% DMSO (vehicle control)ere administered intraperitoneally 30 min prior to edemogens

dministration.

.5. Carrageenan-induced peritonitis in rats

The carrageenan-induced peritonitis was carried out asescribed by Griswold et al. (1987). Male rats of 350–400 g, three

n each group were pretreated intraperitoneally with saline or 10%MSO or 1% Na2CO3 (control) or standard drugs or plant extract.fter 1 h of respective treatment, animals were anaesthetized withiethyl ether and immediately injected with 1% carrageenan sus-ension (300 �l) into the peritoneal cavity. After 4 h rats wereacrificed via excessive inhalation of diethyl ether fumes and 20 mlf Ca2+ and Mg2+ free phosphate buffered saline (PBS; 137 mM NaCl,0 mM Na2HPO4·7H2O and 3 mM KHPO4; pH 7.3) was adminis-ered into the peritoneal cavity and peritoneal lavage was collectednd centrifuged at 5000 rpm for 15 min. Cells pellets contain-ng polymorphonuclear leukocytes (PMNL) were washed twice

ith PBS. In case of contamination with blood, the pellets wereashed with lysis buffer (157 mM NH4Cl, 0.1 mM Na2EDTA and.5 mM KHCO3; pH 7.3), followed by PBS and finally re-suspended

n incubation buffer (137 mM NaCl, 2.5 mM KCl, 2 mM KH2PO4,mM Na2HPO4·7H2O and 5.5 mM �-d glucose anhydrous; pH 7.2).otal leukocyte count, infiltrated to the site of inflammation, wasetermined using haemocytometer. Giemsa-stained slides wererepared for differential cell count.

.6. Rat peritoneal neutrophils

Glycogen-induced rat (350–400 g) peritoneal PMNL wereollected 4 h after the injection of glycogen solution (1 g/kg body

eight) in PBS as described previously by Safayhi et al. (1985). Via-ility was greater than 95% by the trypan blue exclusion test. Theitochondrial-dependent reduction of 4-[3-(4-iodophenyl)-2-(4-

itrophenyl)-2H-5-tetrazolio]-1, 3-benzene disulfonate (WST-1)o formazan (Tan and Berridge, 2000) was used to assess the

250 T. Roome et al. / Journal of Ethnopharmacology 120 (2008) 248–254

Table 1Effect of Aegiceras corniculatum extracts, aspirin and naproxen on carrageenan-induced paw edema in rats

Treatment (mg/kg) 3rd h of edema induction

Inhibition (%) IC50 (mg/kg)

Hexane extract10 33 ± 15* 70 ± 2.550 48 ± 7**100 56 ± 11***

EtOAc extract10 34 ± 3* 32 ± 0.950 67 ± 12***100 71 ± 7***

MeOH extract1 18 ± 1 7.2 ± 0.55 45 ± 10*10 59 ± 6***

Aspirin10 36 ± 5* 48 ± 0.950 57 ± 10***100 63 ± 7***

Naproxen1 16 ± 2 8.7 ± 0.25 26 ± 1010 58 ± 7***

The maximum value of paw edema volume in control animals was 0.65 ± 0.02 ml(treated with 0.9% saline or 10% DMSO; n = 41 rats) detected at 3rd h. Data repre-sents mean ± S.E., n = 6 rats per group. *p < 0.05, **p < 0.01, ***p < 0.005 represents %isM

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Fig. 1. Anti-inflammatory activity of (A) hexane, (B) ethyl acetate and (C) methanolextracts of Aegiceras corniculatum (stem) against inflammation induced by vari-ous edemogens. Data represent the mean ± S.E., n = 3–5 rats per group. *p < 0.05,***p < 0.005 with respective control group. Plant extracts (100 mg/kg) were admin-istered i.p. 30 min prior to the injection of edemogens. The values (ml) and time (inp((h

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nhibition with respect to control groups. IC50 values obtained from regression lineshowed high coefficient of determination (r2 ≥ 0.80). EtOAc, ethyl acetate extract;eOH, methanol extract.

ossible cytotoxic effect of extracts on rat neutrophils. Differentialell counting of peritoneal exudates by 100% Giemsa stain undericroscope (Nikon Eclipse, Model TE 2000-E) at a magnification

f 60 × 1.5 × 2.5 showed that neutrophils were predominant cells90 ± 5%). However, the remaining 10% comprised eosinophils6%), macrophages (3%) and lymphocytes (1%).

.7. 5-Lipoxygenase assay

This assay was performed according to Ammon et al. (1991).or 5-lipoxygenase products, i.e., LTB4 and 5-HETE the neutrophils5 × 106 cells/ml in incubation buffer) were prewarmed at 37 ◦Collowed by incubation with the plant extracts or drugs or 0.5%MSO (control reaction) for 5 min. Thereafter, calcium-ionophore23187 (1.9 �M) was introduced and the mixture was incubated

or 2 min. It was followed by the addition of calcium chlorideolution (1.8 mM) and was incubated again for 5 min at 37 ◦C.he reaction was stopped by acidification to pH 3 with 1 ml ofce-cold methanol/1N HCl (97:3). To the reaction PGB2 (50 ng)as added as an internal standard. 5-Lipoxygenase metabolites,

TB4 and 5-HETE were eluted in 100% methanol using solid-phasextraction column (Chromabond C18, 45 �m, 1 ml with 100 mgatrix). They were separated and quantified by transferring 20 �l

f sample into a Gilson HPLC, equipped with an analytical rp-HPLColumn (Nucleosil C18, 5 �m, 250 mm × 4 mm). Isocratic elutionas performed with methanol:water (74:26) containing 0.007%

v/v) trifluoroacetic acid (TFA), pH 4.8 at a flow rate of 1 ml/min.

etection wavelength was 280 nm for both PGB2 and LTB4 and35 nm for 5-HETE. Both reaction products were identified byomparing their retention time with respective standards. The con-entration of LTB4 and 5-HETE was determined by comparing therea under the peak with those of known amounts of respectiveetabolites.

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arenthesis) of maximum edema induction by edemogens were: dextran 1.1 ± 0.152 h), serotonin 0.64 ± 0.03 (1 h), histamine 0.29 ± 0.02 (2 h), bradykinin 0.32 ± 0.022 h), PGE2 0.59 ± 0.04 (3 h), LTB4 and AA 0.57 ± 0.05 (3 h), PLA2 0.65 ± 0.03 (1 h) andyaluronidase 0.51 ± 0.08 (20 min).

.8. Cyclooxygenase-1 and 12-lipoxygenase assay

Cyclooxygenase-1 and 12-lipoxygenase catalyze the formationf 12-HHT and 12-HETE, respectively from endogenous arachidoniccid (Safayhi et al., 1992). Human platelets (9 × 107 platelets/ml)ere preincubated at 37 ◦C for 5 min. The reaction was performed

s described for 5-lipoxygenase assay. However, the reaction was

T. Roome et al. / Journal of Ethnopharmacology 120 (2008) 248–254 251

Table 2Effect of Aegiceras corniculatum extracts, phenidone, indomethacin and naproxen onthe infiltration of neutrophils into the site of carrageenan-induced inflammation

Treatment Dose (mg/kg, i.p.) Neutrophils(× 106/ml)

Inhibition of neutrophilsinfiltration (%)

Hexane extract 10 6.2 ± 0.2 15.8 ± 2.025 4.5 ± 0.3*** 39.2 ± 3.950 2.6 ± 0.3*** 65.0 ± 4.0

EtOAc extract 1 5.3 ± 0.3** 28.4 ± 4.75 4.4 ± 0.2*** 40.6 ± 2.1

10 3.6 ± 0.2*** 51.4 ± 2.7

MeOH extract 100 6.6 ± 0.2 10.8 ± 3.4

Phenidone 10 6.5 ± 0.1 12.2 ± 1.950 4.3 ± 0.2*** 42.0 ± 2.8

100 3.0 ± 0.1*** 60.0 ± 1.9

Indomethacin 10 6.7 ± 0.2 9.5 ± 2.8

Naproxen 100 6.3 ± 0.2 15.0 ± 3.1

Data represents mean ± S.E., n = 3 rats per group. Significant reduction in cell infil-tration is represented as **p < 0.01, ***p < 0.005 with respect to control group. Incontrol animals cellular infiltration in response to 1% carrageenan was 7.4 ± 1.4nea

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Fig. 2. Effect of (A) hexane, (B) ethyl acetate and (C) methanol extracts of Aegicerascorniculatum on the production of 5-LOX metabolites, LTB4 and 5-HETE. Sampleswere preincubated with rat neutrophils (5 × 106 cells/ml) for 5 min followed bystimulation with calcium-ionophore A23187 (1.9 �M). The productions of LTB4 and5-HETE were detected by HPLC technique. Data represents mean ± S.E. of threeirc1

eutrophils × 106/ml. In the absence of carrageenan, the animals were treated withither saline or 10% DMSO demonstrated less than 1% of neutrophils. EtOAc, ethylcetate extract; MeOH, methanol extract.

topped by the acidification to pH 3 with 1 ml of 1% ice-cold formiccid. Prostaglandin B2 (500 ng) was used as an internal standard.he extraction of metabolites was carried out with an equal volumef chloroform:methanol (1:1). The organic extracts were evapo-ated to dryness and dissolved in 100% methanol and filtered with.45 �m syringe filter (Spartan 13/0, 45 RC, Germany). Analysis of2-HHT and 12-HETE were carried out by rp-HPLC column (Nucle-sil C18, 3 �m, 150 mm × 4.6 mm) and UV-detection (280 nm forGB2 and 235 nm for both 12-HHT and 12-HETE). Under similaronditions, respective standards were also used. The concentra-ion of 12-HHT and 12-HETE was determined by comparing therea under the peaks with those of known amounts of respectiveetabolites.

.9. Statistical analysis

The results are represented as mean ± S.E. for n experiments.C50 values were obtained by using simple linear regression (n = 3).n all the experiments, the data were evaluated by two-way ANOVA,

hereas, in rat paw edema model repeated measures ANOVA waslso employed. Multiple comparisons of data were conducted byeast significant difference (LSD) and Dunnet’s t-test. p-value <0.05as considered statistically significant.

. Results

.1. Effect of Aegiceras corniculatum extracts and standard drugsn rat paw edema

Subplantar injection of carrageenan to rats caused inflammatoryeaction (local edema) in progressive manner (1–3 h), 3 h is a crucialime to observe the anti-inflammatory activity due to the forma-ion of substantial edema volume, followed by a decline at 4th h.retreatment (30 min before carrageenan) with 10, 50, 100 mg/kgi.p.) of hexane and ethyl acetate extracts and 1, 5 and 10 mg/kg (i.p.)

f methanol extract derived from Aegiceras corniculatum (stems)educed the edema in a dose-dependent manner (Table 1). Thisnhibitory effect was as effective as aspirin and naproxen. Signifi-ant inhibition against inflammation was also observed at 1, 2 andh by plant extracts and standard drugs (data not shown). The

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ndependent experiments assayed in duplicate. *p < 0.005 represents significanteduction in LTB4 and 5-HETE productions compared to respective control. Theontrol values (100%) in the absence of test substances were 18.2 ± 0.58 ng/ml and85 ± 6.8 ng/ml for LTB4 and 5-HETE, respectively (n = 40).

oses tested in this in vivo model did not cause any mortality andehavioral changes to the animals.

Inhibitory effect of each extracts against inflammation wasompared in the presence of various edemogens such as dextran,istamine, serotonin, bradykinin, prostaglandin E2, leukotriene

252 T. Roome et al. / Journal of Ethnophar

Fig. 3. Effect of Aegiceras corniculatum extracts on the production of COX-1 and 12-LOX metabolites, 12-HHT and 12-HETE. Samples were preincubated with humanplatelets (9 × 107 platelets/ml) for 5 min followed by stimulation with calcium-ionophore A23187 (1.9 �M). The production of 12-HHT and 12-HETE were detectedby HPLC technique. (A) Inhibitory and stimulatory effect of methanol extract on theproduction of 12-HHT and 12-HETE, respectively at comparable concentrations. (B)Inhibitory and stimulatory effect of hexane extract on 12-HETE and 12-HHT, respec-tively. (C) Inhibitory effect of ethyl acetate extract on both 12-HHT and 12-HETE. Dataare mean ± S.E. of three independent experiments. *p < 0.005 represents significantcta

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hange in the production of 12-HHT and 12-HETE compared to their respective con-rol. The control values (100%) in the absence of test substances were 24.5 ± 1.6 ng/mlnd 66.5 ± 3.5 ng/ml for 12-HHT and 12-HETE, respectively (n = 18).

4, arachidonic acid, phospholipase A2 and hyaluronidase, at thebservation time in which maximum edema induced by the respec-ive edemogens. In dextran-induced paw edema hexane and ethylcetate extracts suppressed (∼30–59%) the edema formation doseependently (10–100 mg/kg). PGE2 and LTB4-induced paw edemaas significantly reduced in the presence of methanol and hex-

ne extracts, respectively by ∼40, 60 and 90% at the doses of 10,0 and 100 mg/kg. Ethyl acetate extract exhibited most potent

nti-inflammatory effect against AA in a dose-dependent manner.ig. 1A, B and C depicts that hexane, ethyl acetate and methanolxtracts attenuated the edema induced by aforementioned ede-ogens with varying degree of potencies and established their

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macology 120 (2008) 248–254

ode of action as anti-inflammatory agent. Methysergide andhlorpheneramine (25 mg/kg, i.p.) were used as standards in sero-onin and histamine-induced paw edema model as they reducedhe inflammation by 83 ± 3% and 63 ± 14%, respectively. Rofecoxib100 mg/kg) found to be highly effective (63 ± 5%) against PGE2-nduced paw edema. Nordihydroguaiaretic acid (NDGA) caused70% attenuation in inflammation induced by LTB4. Both NDGAnd phenidone (100 mg/kg, i.p.) were used as reference drugs inA-induced paw edema and exhibited 69 ± 3% and 88 ± 4% anti-

nflammatory response, respectively. Dexamethasone (5 mg/kg,.p.) showed 70 ± 4% inhibition in edema induced by PLA2.

.2. Effect of Aegiceras corniculatum extracts and standard drugsn carrageenan-induced infiltration of leukocytes in rateritoneum

The anti-inflammatory activity of hexane, ethyl acetate andethanol extracts of the plant was also evaluated in an acute in

ivo model of cell infiltration into rat peritoneal cavity inducedy carrageenan. Neutrophils were the predominant inflamma-ory cells (>90%), whereas mononuclear cells contribute <10%o the total leukocytes population. As shown in Table 2, thenfiltration of neutrophils was potently reduced by hexane andthyl acetate extracts. The inhibition was dose related with anC50 of 37 ± 1.1 mg/kg and 9.2 ± 0.44 mg/kg, i.p. Phenidone (IC505.5 ± 2.1 mg/kg, i.p.) was also active in this peritonitis model. Inontrast, indomethacin (10 mg/kg, i.p.), naproxen and methanolxtract of the plant (100 mg/kg, i.p.) did not inhibit the cell infil-ration in this model system despite their administration at near

aximally tolerated doses.

.3. Effect of Aegiceras corniculatum extracts,ordihydroguaiaretic acid and quercetin on 5-lipoxygenaseroducts (LTB4 and 5-HETE) formation

The effect of Aegiceras corniculatum extracts against 5-ipoxygenase activity was evaluated via detecting the inhibitionf its metabolites (LTB4 and 5-HETE) synthesis from rat neu-rophils triggered by A23187 using HPLC technique. 5-Lipoxygenasenhibitors, NDGA and quercetin (0.1–10 �M) used as a referenceompounds and inhibited both 5-LOX metabolites concur-ently with an IC50 of 0.23 �g/ml (∼0.8 �M) and 0.85 �g/ml2.1 �M), respectively. Likewise, hexane and ethyl acetate extracts0.1–10 �g/ml) potently reduced the formation of both LTB4 and-HETE concentration dependently. In the presence of hexane andthyl acetate extracts, the corresponding IC50 value for LTB4 wasalculated as 0.76 ± 0.12 �g/ml and 0.86 ± 0.03 �g/ml, whereas for-HETE it was 2.6 ± 0.22 �g/ml and 3.4 ± 0.04 �g/ml (Fig. 2A and). In contrast, methanol extract did not substantially decrease the

ormation of LTB4 and 5-HETE (Fig. 2C). It is noteworthy that allhree extracts from Aegiceras corniculatum did not cause cellularoxicity on rat neutrophils at concentrations (1–100 �g/ml) useds determined by the reduction of WST-1 to formazan (data nothown).

.4. Effect of Aegiceras corniculatum extracts, aspirin andndomethacin on cyclooxygenase-1 and 12-lipoxygenase product12-HHT and 12-HETE) formation

Metabolites of cyclooxygenase-1 and 12-lipoxygenase, 12-HHT

timulation with A23187 were detected using HPLC technique.yclooxygenase inhibitors, aspirin (1–50 �M) and indomethacin1–100 nM) reduced the production of 12-HHT in a concentration-ependent manner (corresponding IC50 were 15.6 ± 0.37 �M or

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.8 �g/ml and 10.0 ± 2.7 nM or 0.0036 �g/ml) with a concomitantncrease in 12-HETE level at comparable concentrations. Likewise,

ethanol extract attenuated the production of 12-HHT with an IC50f 41.1 ± 1.5 �g/ml as shown in Fig. 3A. Conversely, hexane extractaused reduction in 12-HETE formation (IC50 0.36 ± 0.12 �g/ml)ith a concomitant increase in 12-HHT at comparable concen-

rations (Fig. 3B). Interestingly, ethyl acetate extract suppressedhe production of both metabolites but with greater affinityor 12-HHT (IC50 0.08 ± 0.002 �g/ml) than that of 12-HETE (IC50.0 ± 0.18 �g/ml) as depicted in Fig. 3C.

. Discussion

The current investigation demonstrates that hexane, ethylcetate and methanol extracts derived from Aegiceras corniculatumtems possess pronounced inhibitory activity against inflammationisplaying multiple mechanism of action.

Acute transient phase (initial phase) of inflammation is char-cterized by vasodilatation, increased in vascular permeabilitynd interstitial edema facilitated by mediators of mast cells andlasma proteins (Di Rosa et al., 1971). Degranulation of mast cells

nduced by dextran causes the release of histamine and sero-onin during edema formation in rat paw (Ferreira et al., 2004).hey interact with their respective receptors (H1, H2 and 5HT2)esiding on the endothelium of micro-vessels. Both hexane andthyl acetate extracts significantly antagonized this response by60%. Additionally, ethyl acetate extract also significantly atten-ated histamine-induced paw edema similar to chlorpheneraminealeate (H1 and H2 antagonist). However, it failed to antagonize

radykinin-induced edemogenic response. Moreover, the failuref plant extracts to alter the edema produced by hyaluronidasendicates that extracts do not interfere directly with the vascularermeability, thereby, implying that the plant extracts appear to beffective in antagonizing the vascular phase of inflammation eitheria suppressing the release of histamine and serotonin from mastells and/or by acting at the histaminic receptors.

The production of eicosanoids derived from COX and/or 5-OX pathways was mitigated by plant extracts in in vivo modelf acute inflammation in which rat paw edema during latehase (3rd–4th h) was induced by either carrageenan, PGE2, LTB4r AA. Both isoforms of cyclooxygenases-1 and -2, have beenmplicated in carrageenan-induced edema resulting in the over-roduction of PGE2. In this context, methanol extract emerged asn impressive anti-inflammatory agent as it was ∼7× better thanon-selective cyclooxygenase inhibitor aspirin and equipotent toaproxen sodium. It is known that PGE2-induced paw edema in ratsnhances the expression of cyclooxygenases (preferentially COX-2)ith a concomitant rise in the PGE2 levels (Nantel et al., 1999).

his response was markedly diminished (92 ± 2%) in the pres-nce of methanol extract, revealing its anti-inflammatory effect viayclooxygenase pathway. Since hexane extract was more potentgainst LTB4 similar to NDGA (a selective 5-LOX inhibitor) it ispparently more selective towards the 5-LOX pathway. Likewiseo phenidone, ethyl acetate extract also acts as a dual inhibitor foroth cyclooxygenase and 5-lipoxygenase enzymes via attenuatingA-induced edemogenic effect. Moreover, it also suppressed the

nflammation induced by either PGE2 or LTB4 in rat paw.Our in vitro results correlated reasonably well with the in vivo

tudies. In this regard, the methanol extract suppressed the forma-

ion of prostanoid (12-HHT) with a concomitant rise in 12-HETEevels. This phenomenon is likely to occur only after selectivenhibition of COX-1 enzyme thus diverting the AA metabolismowards 12-LOX pathway, and consequently increasing the 12-ETE levels. This type of corresponding pattern has also been

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xhibited by widely used anti-inflammatory drugs, such as aspirinnd indomethacin (Safayhi et al., 1992; Albert et al., 2002).oreover, methanol extract failed to inhibit the production of

-LOX metabolites LTB4 and 5-HETE in rat neutrophils, furtheranifesting its selectivity towards cyclooxygenase pathway. The

nti-inflammatory property of methanol extract might be relatedo lignans and triterpenoidal saponins reported earlier to be presentn it (Rao and Bose, 1959; Hensens and Lewis, 1966).

On the contrary, hexane extract concurrently decreased the LTB4nd 5-HETE levels suggesting that it inhibits a common biochemicaltep as catalyzed by the rate-limiting enzyme 5-LOX. Yet, anotheripoxygenase, i.e., 12-LOX was also potently inhibited by hexanextract (IC50 0.36 ± 0.12 �g/ml) as reflected by a significant reduc-ion in its metabolite, 12-HETE. Thus hexane extract exerts its effectn AA metabolism by inhibiting both types of lipoxygenases. How-ver, the selectivity of hexane extract towards lipoxygenases may beue to the presence of benzoquinones (Hensens and Lewis, 1966;omez et al., 1989; Xu et al., 2004). This class of molecules are

eputed for their high selectivity towards lipoxygenase pathwayHoughton et al., 1995; Resch et al., 1998). Thus the inhibitory effectlicited by hexane extract against LTB4 and HETEs provides a phar-acological support for the use of Aegiceras corniculatum in folkedicine against asthma.Ethyl acetate extract of plant reduced the production of

icosanoids, LTB4 and 12-HHT in rat neutrophils and humanlatelets, respectively with minor effect on 12-HETE thus indi-ating that it has a relatively high affinity for 5-lipoxygenase andyclooxygenase-1 enzymes as compared to 12-LOX enzyme. It haseen reported earlier that pentacyclic triterpenes inhibit 5-LOXctivity by inducing conformational changes at AA binding siteSafayhi et al., 1995), whereas, flavonoids have been implicateds a dual inhibitor of 5-LOX and COX (Chi et al., 2001). Likewise,ual anti-inflammatory activity of ethyl acetate extract could alsoe attributed to the previously reported chemical constituentsentacyclic triterpenes, having oleanane skeleton and flavonoidsuch as quercetin, kaempferol and isorhamnetin residing in it (Raond Bose, 1959, 1961, 1962; Rao, 1964; Zhang et al., 2005). It isorth mentioning here, ethyl acetate extract effect is comparable toost potent dual acting anti-inflammatory agents such as synthetic

icofelone (Tries et al., 2002) and naturally occurring hyperforinAlbert et al., 2002).

Neutrophils infiltration to the site of acute inflammation iscrucial event that aggravates the inflammatory reaction pro-

ucing excessive amount of proteolytic enzymes, reactive oxygenpecies, eicosanoids and cytokines thereby causing tissue damage.

significant inhibitory effect of hexane (65%) and ethyl acetate96%) extracts against infiltration of neutrophils was observed hav-ng ∼2–8× more potency than phenidone. This property of plantxtracts is more likely to be due to its interaction with cellularhase of inflammation with a reasonable correlation with its anti-

nflammatory action in the in vivo models as described before.It is therefore, concluded that hexane, ethyl acetate and

ethanol extracts of Aegiceras corniculatum stems are endowedith anti-inflammatory activity mediated via diverse mechanisms

f action. Methanol extract elicited its effect by the inhibition ofrostanoids biosynthesis. Hexane extract was selective towardsoth 5- and 12-lipoxygenases by reducing the LTB4 and 12-HETE

evels. However, multiple mechanisms of anti-inflammatory activ-ty was displayed by ethyl acetate extract viz. reduction in the levelsf leukotrienes, prostanoids and HETEs along with anti-histaminic

ction. Taken all these results together, it is suggested that hexanend ethyl acetate extracts substantially interfere with both vascularnd cellular phases of acute inflammation that justify the tradi-ional use of Aegiceras corniculatum by the local community againstnflammatory diseases.

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cknowledgement

Mr. Tahir Qureshi, Director, International Union of Conservationf Nature (IUCN), Pakistan, is acknowledged for his cooperation torovide mangrove plant, Aegiceras corniculatum from Indus Delta,indh, Pakistan and sharing knowledge about its folk use.

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