Aegiceras corniculatum extract suppresses initial and late phases of inflammation in rat paw and attenuates the production of eicosanoids in rat neutrophils and human platelets

Journal of Ethnopharmacology 120 (2008) 248–254

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Aegiceras corniculatum extract suppresses initial and late phases of inflammation in rat paw and attenuates the production of eicosanoids in rat neutrophils and human platelets Talat Roome, Ahsana Dar ∗ , Sabira Naqvi, Shamsher Ali, Muhammad Iqbal Choudhary Pharmacology Section, H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan

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Article history: Received 25 September 2007 Received in revised form 5 July 2008 Accepted 23 August 2008 Available online 2 September 2008 Keywords: Aegiceras corniculatum Anti-inflammatory activity Rat paw edema Leukotriene 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 Aegiceras corniculatum Linn. Blanco stems extracts and their mechanism of action against various pro-inflammatory mediators 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 in vitro studies human platelets and rat neutrophils were stimulated with Ca2+ -ionophore A23187 leading to the production of various pro-inflammatory metabolites, i.e., 12-HTT, 12-HETE and LTB4 and 5-HETE which were quantified by HPLC. Results: The highly polar methanol extract (100 mg/kg) caused ∼90% reduction in the carrageenanand prostaglandin E2-induced paw edema in rats. It also caused the inhibition of cycloxygenase-1 metabolite, 12-HHT (IC50 41.1 ± 1.5 ␮g/ml) with a concomitant rise in 12-lipoxygenase metabolite, 12HETE 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 selectivity towards 12-lipoxygenase. Non-polar hexane extract also antagonized the production of 5-lipoxygenase metabolites, i.e., leukotriene B4 and 5-HETE in the rat neutrophils. Furthermore, ethyl acetate extract inhibited 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 acetate extracts 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 possess significant anti-inflammatory activity via multiple mechanisms and validate their traditional use against inflammation-related diseases. © 2008 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Aegiceras corniculatum (Linn.) Blanco (family Aegicerataceae) is a mangrove plant that grows in sheltered intertidal zones at interface between land and sea in tropical and subtropical regions. Mangrove

Abbreviations: AA, arachidonic acid; COX, cyclooxygenase; PLA2 , phospholipase A2 ; 5-,12-HETE, hydroxyeicosatetraenoic acid; 12-HHT, 12hydroxyheptadecatrienoic acid; 5-LOX, 5-lipoxygenase; LT, leukotriene; NDGA, nordihydroguaiaretic acid; NSAIDs, non-steroidal anti-inflammatory drugs; PGs, prostaglandins; PMNL, polymorphonuclear leukocytes; TXA2 , thromboxane A2 . ∗ Corresponding author. Tel.: +92 21 4824930 9; fax: +92 21 4819018 9. E-mail address: [email protected] (A. Dar). 0378-8741/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2008.08.025

forest in Pakistan is rated as 5th largest area in the world where it grows along the 240 km South Eastern coastline comprising Indus Delta. Aegiceras corniculatum is used in folklore medicine against asthma, diabetes, inflammation and rheumatism (Bandaranayake, 1998). A number of chemical constituents have been isolated from it viz. flavonoids (Rao and Bose, 1959; Zhang et al., 2005), hydroquinone, benzoquinone and its derivatives (Gomez et al., 1989; Xu et al., 2004), polyphenolic compounds (lignan and tanin), ␤-sitosterol and stigmasterol (Bandaranayake, 2002), pentacyclic triterpenes and its derivatives, and saponins (Rao and Bose, 1961; Rao, 1964; Hensens and Lewis, 1966; Zhang et al., 2005). The plant has been reported to be cytotoxic (Xu et al., 2004) and inhibits tyrosine phosphatase 1B (Wang et al., 2006). Despite its use in folklore

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

against inflammation there is no information available about its anti-inflammatory properties and this prompted us to investigate and justify its traditional use. A variety of signaling molecules such as vasoactive amines and peptide (histamine, serotonin and bradykinin), arachidonic acid metabolites (prostanoids, leukotrienes and HETEs) and oxygenderived free radicals (hydroxyl radicals and superoxide anions) have been implicated in a complex process of inflammation (Safayhi and Sailer, 1997; Nathan, 2002). Among these mediators arachidonic acid (AA) metabolites including prostanoids, leukotrienes and 12-HETE are mainly involved in the pathogenesis of inflammatory diseases such as asthma, arthritis, atherosclerosis, cancer and hyperalgesia (Bertolini et al., 2002; Werz, 2004). The non-steroidal anti-inflammatory drugs (NSAIDs) inhibiting prostanoids synthesis are commonly used in the treatment of inflammatory and rheumatic disorders. However, their clinical use is hampered due to side effects notably gastric ulceration, bronchospasm, and kidney dysfunction. Currently, a considerable interest has been generated to develop dual inhibitors of cyclooxygenases and 5-lipoxygenase pathways. These non-selective inhibitors display manifold anti-inflammatory effectiveness with negligible gastric, cardiac and renal toxicity (Celotti and Laufer, 2001). The present study describes the effect of Aegiceras corniculatum extracts on acute inflammatory models representing various stages of inflammation. It also addresses the mechanism of antiinflammatory action against histamine and serotonin release and production of eicosanoids, including prostanoids, leukotrienes and HETEs.

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acetate extract caused mortality in mice with LD50 of 850 mg/kg. Methanol extract was toxic at doses above 200 mg/kg. Depending on the aforementioned results, doses of plant extracts were selected for in vivo anti-inflammatory studies. 2.4. Paw edema in rats by various edemogens

2. Materials and methods

Animal studies were performed in accordance with the declaration of Helsinki and the European Community guidelines for the ethical handling and the use of laboratory animals and through the clearance of institutional animal use committee. Wistar and Sprague-Dawley rats of either sex (n = 3–5 and 120–180 g) from the animal house of International Center for Chemical and Biological Sciences, H.E.J. Research Institute of Chemistry, University of Karachi, Pakistan were used throughout the study. The edema was induced into their right hind paw by a single subplantar injection (50 ␮l) containing either 1% carrageenan type IV (Winter et al., 1962) or 25 IU/ml of phospholipase A2 (1.25 U/paw) (Neves et al., 1993) or 100 ␮l of 1.5% dextran (Ferreira et al., 2004) or histamine, serotonin (1.0 mg/ml) or bradykinin (0.02 mg/ml) or prostaglandin E2 (1 × 10−5 mg/ml) or leukotriene B4 (2.5 × 10−4 mg/ml) or arachidonic acid (0.5%) or 2400 IU/ml of hyaluronidase (240 U/paw) (Singh et al., 1996). The average volume of right hind paw up to the tibiotarsal articulation of each rat was measured 3–4× by using plethysmometer (Model 7150, Ugo Basile, Italy) before (time 0) and after treatment (0.5–4 h) in the presence of respective edemogens. In case of PLA2 , the edema was measured at 0.5, 1 and 1.5 h, whereas hyaluronidase-induced edema was detected after 20 min. Standard drugs, plant extracts and 10% DMSO (vehicle control) were administered intraperitoneally 30 min prior to edemogens administration.

2.1. Plant material

2.5. Carrageenan-induced peritonitis in rats

Aerial parts of Aegiceras corniculatum were collected during June 2001 from Indus Delta, Sindh, Pakistan at lowest tide and identified by a taxonomist, Dr. Surriya Khatoon from the Department of Botany, University of Karachi, Karachi, Pakistan. Voucher specimen representing the collection labeled as KHU G.H.S. No. 68219 has been deposited at herbarium of this department.

The carrageenan-induced peritonitis was carried out as described by Griswold et al. (1987). Male rats of 350–400 g, three in each group were pretreated intraperitoneally with saline or 10% DMSO or 1% Na2 CO3 (control) or standard drugs or plant extract. After 1 h of respective treatment, animals were anaesthetized with diethyl ether and immediately injected with 1% carrageenan suspension (300 ␮l) into the peritoneal cavity. After 4 h rats were sacrificed via excessive inhalation of diethyl ether fumes and 20 ml of Ca2+ and Mg2+ free phosphate buffered saline (PBS; 137 mM NaCl, 10 mM Na2 HPO4 ·7H2 O and 3 mM KHPO4 ; pH 7.3) was administered into the peritoneal cavity and peritoneal lavage was collected and centrifuged at 5000 rpm for 15 min. Cells pellets containing polymorphonuclear leukocytes (PMNL) were washed twice with PBS. In case of contamination with blood, the pellets were washed with lysis buffer (157 mM NH4 Cl, 0.1 mM Na2 EDTA and 9.5 mM KHCO3 ; pH 7.3), followed by PBS and finally re-suspended in incubation buffer (137 mM NaCl, 2.5 mM KCl, 2 mM KH2 PO4 , 5 mM Na2 HPO4 ·7H2 O and 5.5 mM ␣-d glucose anhydrous; pH 7.2). Total leukocyte count, infiltrated to the site of inflammation, was determined using haemocytometer. Giemsa-stained slides were prepared for differential cell count.

2.2. Preparation of extraction Air-dried powdered stems (9.0 kg) were soaked in 50 l hexane for 7 days. After filtration and evaporation under vacuum, hexane extract (6.0 g or 0.07%, w/w) was obtained. Powdered material left after hexane filtration was soaked in 50 l ethyl acetate for 1 week. Following the aforementioned procedure, ethyl acetate extract (8.5 g or 0.09%, w/w) was yielded and the residue was soaked in butanol. 11.0 g or 0.12% (w/w) dried butanol extract was obtained. Ultimately the remaining residue after butanol extraction was soaked in 50 l methanol and 9.8 g or 0.11% (w/w) methanol extract was collected. This extraction procedure in brief, yielded non-polar (hexane), less polar (ethyl acetate) and highly polar (methanol) extracts sequentially used for conventional phytochemical tests and pharmacological studies.

2.6. Rat peritoneal neutrophils 2.3. Acute toxicity test The toxic effect of plant extract was determined using albino mice of either sex (20–25 g, n = 10 in each group), treated with hexane and ethyl acetate extracts (1–1000 mg/kg) or methanol extract (1–500 mg/kg) intraperitoneally. Behavioral changes, gross effect and mortality, if occurred, were observed up to 7 days. The hexane extract was found to be non-toxic up to 1 g/kg, whereas ethyl

Glycogen-induced rat (350–400 g) peritoneal PMNL were collected 4 h after the injection of glycogen solution (1 g/kg body weight) in PBS as described previously by Safayhi et al. (1985). Viability was greater than 95% by the trypan blue exclusion test. The mitochondrial-dependent reduction of 4-[3-(4-iodophenyl)-2-(4nitrophenyl)-2H-5-tetrazolio]-1, 3-benzene disulfonate (WST-1) to formazan (Tan and Berridge, 2000) was used to assess the

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Table 1 Effect of Aegiceras corniculatum extracts, aspirin and naproxen on carrageenaninduced paw edema in rats Treatment (mg/kg)

3rd h of edema induction Inhibition (%)

IC50 (mg/kg)

Hexane extract 10 50 100

33 ± 15* 48 ± 7** 56 ± 11***

70 ± 2.5

EtOAc extract 10 50 100

34 ± 3* 67 ± 12*** 71 ± 7***

32 ± 0.9

MeOH extract 1 5 10

18 ± 1 45 ± 10* 59 ± 6***

7.2 ± 0.5

Aspirin 10 50 100

36 ± 5* 57 ± 10*** 63 ± 7***

48 ± 0.9

Naproxen 1 5 10

16 ± 2 26 ± 10 58 ± 7***

8.7 ± 0.2

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 represents mean ± S.E., n = 6 rats per group. *p < 0.05, **p < 0.01, ***p < 0.005 represents % inhibition with respect to control groups. IC50 values obtained from regression lines showed high coefficient of determination (r2 ≥ 0.80). EtOAc, ethyl acetate extract; MeOH, methanol extract.

possible cytotoxic effect of extracts on rat neutrophils. Differential cell counting of peritoneal exudates by 100% Giemsa stain under microscope (Nikon Eclipse, Model TE 2000-E) at a magnification of 60 × 1.5 × 2.5 showed that neutrophils were predominant cells (90 ± 5%). However, the remaining 10% comprised eosinophils (6%), macrophages (3%) and lymphocytes (1%). 2.7. 5-Lipoxygenase assay This assay was performed according to Ammon et al. (1991). For 5-lipoxygenase products, i.e., LTB4 and 5-HETE the neutrophils (5 × 106 cells/ml in incubation buffer) were prewarmed at 37 ◦ C followed by incubation with the plant extracts or drugs or 0.5% DMSO (control reaction) for 5 min. Thereafter, calcium-ionophore A23187 (1.9 ␮M) was introduced and the mixture was incubated for 2 min. It was followed by the addition of calcium chloride solution (1.8 mM) and was incubated again for 5 min at 37 ◦ C. The reaction was stopped by acidification to pH 3 with 1 ml of ice-cold methanol/1N HCl (97:3). To the reaction PGB2 (50 ng) was added as an internal standard. 5-Lipoxygenase metabolites, LTB4 and 5-HETE were eluted in 100% methanol using solid-phase extraction column (Chromabond C18, 45 ␮m, 1 ml with 100 mg matrix). They were separated and quantified by transferring 20 ␮l of sample into a Gilson HPLC, equipped with an analytical rp-HPLC column (Nucleosil C18, 5 ␮m, 250 mm × 4 mm). Isocratic elution was 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. Detection wavelength was 280 nm for both PGB2 and LTB4 and 235 nm for 5-HETE. Both reaction products were identified by comparing their retention time with respective standards. The concentration of LTB4 and 5-HETE was determined by comparing the area under the peak with those of known amounts of respective metabolites.

Fig. 1. Anti-inflammatory activity of (A) hexane, (B) ethyl acetate and (C) methanol extracts of Aegiceras corniculatum (stem) against inflammation induced by various 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 administered i.p. 30 min prior to the injection of edemogens. The values (ml) and time (in parenthesis) of maximum edema induction by edemogens were: dextran 1.1 ± 0.15 (2 h), serotonin 0.64 ± 0.03 (1 h), histamine 0.29 ± 0.02 (2 h), bradykinin 0.32 ± 0.02 (2 h), PGE2 0.59 ± 0.04 (3 h), LTB4 and AA 0.57 ± 0.05 (3 h), PLA2 0.65 ± 0.03 (1 h) and hyaluronidase 0.51 ± 0.08 (20 min).

2.8. Cyclooxygenase-1 and 12-lipoxygenase assay Cyclooxygenase-1 and 12-lipoxygenase catalyze the formation of 12-HHT and 12-HETE, respectively from endogenous arachidonic acid (Safayhi et al., 1992). Human platelets (9 × 107 platelets/ml) were preincubated at 37 ◦ C for 5 min. The reaction was performed as described for 5-lipoxygenase assay. However, the reaction was

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

Dose (mg/kg, i.p.)

Neutrophils (× 106 /ml)

Inhibition of neutrophils infiltration (%)

Hexane extract

10 25 50

6.2 ± 0.2 4.5 ± 0.3*** 2.6 ± 0.3***

15.8 ± 2.0 39.2 ± 3.9 65.0 ± 4.0

EtOAc extract

1 5 10

5.3 ± 0.3** 4.4 ± 0.2*** 3.6 ± 0.2***

28.4 ± 4.7 40.6 ± 2.1 51.4 ± 2.7

MeOH extract

100

6.6 ± 0.2

10.8 ± 3.4

Phenidone

10 50 100

6.5 ± 0.1 4.3 ± 0.2*** 3.0 ± 0.1***

12.2 ± 1.9 42.0 ± 2.8 60.0 ± 1.9

Indomethacin Naproxen

10

6.7 ± 0.2

9.5 ± 2.8

100

6.3 ± 0.2

15.0 ± 3.1

Data represents mean ± S.E., n = 3 rats per group. Significant reduction in cell infiltration is represented as **p < 0.01, ***p < 0.005 with respect to control group. In control animals cellular infiltration in response to 1% carrageenan was 7.4 ± 1.4 neutrophils × 106 /ml. In the absence of carrageenan, the animals were treated with either saline or 10% DMSO demonstrated less than 1% of neutrophils. EtOAc, ethyl acetate extract; MeOH, methanol extract.

stopped by the acidification to pH 3 with 1 ml of 1% ice-cold formic acid. Prostaglandin B2 (500 ng) was used as an internal standard. The extraction of metabolites was carried out with an equal volume of chloroform:methanol (1:1). The organic extracts were evaporated to dryness and dissolved in 100% methanol and filtered with 0.45 ␮m syringe filter (Spartan 13/0, 45 RC, Germany). Analysis of 12-HHT and 12-HETE were carried out by rp-HPLC column (Nucleosil C18, 3 ␮m, 150 mm × 4.6 mm) and UV-detection (280 nm for PGB2 and 235 nm for both 12-HHT and 12-HETE). Under similar conditions, respective standards were also used. The concentration of 12-HHT and 12-HETE was determined by comparing the area under the peaks with those of known amounts of respective metabolites. 2.9. Statistical analysis The results are represented as mean ± S.E. for n experiments. IC50 values were obtained by using simple linear regression (n = 3). In all the experiments, the data were evaluated by two-way ANOVA, whereas, in rat paw edema model repeated measures ANOVA was also employed. Multiple comparisons of data were conducted by least significant difference (LSD) and Dunnet’s t-test. p-value <0.05 was considered statistically significant. 3. Results 3.1. Effect of Aegiceras corniculatum extracts and standard drugs on rat paw edema Subplantar injection of carrageenan to rats caused inflammatory reaction (local edema) in progressive manner (1–3 h), 3 h is a crucial time to observe the anti-inflammatory activity due to the formation of substantial edema volume, followed by a decline at 4th h. Pretreatment (30 min before carrageenan) with 10, 50, 100 mg/kg (i.p.) of hexane and ethyl acetate extracts and 1, 5 and 10 mg/kg (i.p.) of methanol extract derived from Aegiceras corniculatum (stems) reduced the edema in a dose-dependent manner (Table 1). This inhibitory effect was as effective as aspirin and naproxen. Significant inhibition against inflammation was also observed at 1, 2 and 4 h by plant extracts and standard drugs (data not shown). The

Fig. 2. Effect of (A) hexane, (B) ethyl acetate and (C) methanol extracts of Aegiceras corniculatum on the production of 5-LOX metabolites, LTB4 and 5-HETE. Samples were preincubated with rat neutrophils (5 × 106 cells/ml) for 5 min followed by stimulation with calcium-ionophore A23187 (1.9 ␮M). The productions of LTB4 and 5-HETE were detected by HPLC technique. Data represents mean ± S.E. of three independent experiments assayed in duplicate. *p < 0.005 represents significant reduction in LTB4 and 5-HETE productions compared to respective control. The control values (100%) in the absence of test substances were 18.2 ± 0.58 ng/ml and 185 ± 6.8 ng/ml for LTB4 and 5-HETE, respectively (n = 40).

doses tested in this in vivo model did not cause any mortality and behavioral changes to the animals. Inhibitory effect of each extracts against inflammation was compared in the presence of various edemogens such as dextran, histamine, serotonin, bradykinin, prostaglandin E2 , leukotriene

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mode of action as anti-inflammatory agent. Methysergide and chlorpheneramine (25 mg/kg, i.p.) were used as standards in serotonin and histamine-induced paw edema model as they reduced the inflammation by 83 ± 3% and 63 ± 14%, respectively. Rofecoxib (100 mg/kg) found to be highly effective (63 ± 5%) against PGE2 induced paw edema. Nordihydroguaiaretic acid (NDGA) caused ∼70% attenuation in inflammation induced by LTB4 . Both NDGA and phenidone (100 mg/kg, i.p.) were used as reference drugs in AA-induced paw edema and exhibited 69 ± 3% and 88 ± 4% antiinflammatory response, respectively. Dexamethasone (5 mg/kg, i.p.) showed 70 ± 4% inhibition in edema induced by PLA2 . 3.2. Effect of Aegiceras corniculatum extracts and standard drugs on carrageenan-induced infiltration of leukocytes in rat peritoneum The anti-inflammatory activity of hexane, ethyl acetate and methanol extracts of the plant was also evaluated in an acute in vivo model of cell infiltration into rat peritoneal cavity induced by carrageenan. Neutrophils were the predominant inflammatory cells (>90%), whereas mononuclear cells contribute <10% to the total leukocytes population. As shown in Table 2, the infiltration of neutrophils was potently reduced by hexane and ethyl acetate extracts. The inhibition was dose related with an IC50 of 37 ± 1.1 mg/kg and 9.2 ± 0.44 mg/kg, i.p. Phenidone (IC50 75.5 ± 2.1 mg/kg, i.p.) was also active in this peritonitis model. In contrast, indomethacin (10 mg/kg, i.p.), naproxen and methanol extract of the plant (100 mg/kg, i.p.) did not inhibit the cell infiltration in this model system despite their administration at near maximally tolerated doses. 3.3. Effect of Aegiceras corniculatum extracts, nordihydroguaiaretic acid and quercetin on 5-lipoxygenase products (LTB4 and 5-HETE) formation

Fig. 3. Effect of Aegiceras corniculatum extracts on the production of COX-1 and 12LOX metabolites, 12-HHT and 12-HETE. Samples were preincubated with human platelets (9 × 107 platelets/ml) for 5 min followed by stimulation with calciumionophore A23187 (1.9 ␮M). The production of 12-HHT and 12-HETE were detected by HPLC technique. (A) Inhibitory and stimulatory effect of methanol extract on the production of 12-HHT and 12-HETE, respectively at comparable concentrations. (B) Inhibitory and stimulatory effect of hexane extract on 12-HETE and 12-HHT, respectively. (C) Inhibitory effect of ethyl acetate extract on both 12-HHT and 12-HETE. Data are mean ± S.E. of three independent experiments. *p < 0.005 represents significant change in the production of 12-HHT and 12-HETE compared to their respective control. The control values (100%) in the absence of test substances were 24.5 ± 1.6 ng/ml and 66.5 ± 3.5 ng/ml for 12-HHT and 12-HETE, respectively (n = 18).

B4 , arachidonic acid, phospholipase A2 and hyaluronidase, at the observation time in which maximum edema induced by the respective edemogens. In dextran-induced paw edema hexane and ethyl acetate extracts suppressed (∼30–59%) the edema formation dose dependently (10–100 mg/kg). PGE2 and LTB4 -induced paw edema was significantly reduced in the presence of methanol and hexane extracts, respectively by ∼40, 60 and 90% at the doses of 10, 50 and 100 mg/kg. Ethyl acetate extract exhibited most potent anti-inflammatory effect against AA in a dose-dependent manner. Fig. 1A, B and C depicts that hexane, ethyl acetate and methanol extracts attenuated the edema induced by aforementioned edemogens with varying degree of potencies and established their

The effect of Aegiceras corniculatum extracts against 5lipoxygenase activity was evaluated via detecting the inhibition of its metabolites (LTB4 and 5-HETE) synthesis from rat neutrophils triggered by A23187 using HPLC technique. 5-Lipoxygenase inhibitors, NDGA and quercetin (0.1–10 ␮M) used as a reference compounds and inhibited both 5-LOX metabolites concurrently with an IC50 of 0.23 ␮g/ml (∼0.8 ␮M) and 0.85 ␮g/ml (2.1 ␮M), respectively. Likewise, hexane and ethyl acetate extracts (0.1–10 ␮g/ml) potently reduced the formation of both LTB4 and 5-HETE concentration dependently. In the presence of hexane and ethyl acetate extracts, the corresponding IC50 value for LTB4 was calculated as 0.76 ± 0.12 ␮g/ml and 0.86 ± 0.03 ␮g/ml, whereas for 5-HETE it was 2.6 ± 0.22 ␮g/ml and 3.4 ± 0.04 ␮g/ml (Fig. 2A and B). In contrast, methanol extract did not substantially decrease the formation of LTB4 and 5-HETE (Fig. 2C). It is noteworthy that all three extracts from Aegiceras corniculatum did not cause cellular toxicity on rat neutrophils at concentrations (1–100 ␮g/ml) used as determined by the reduction of WST-1 to formazan (data not shown). 3.4. Effect of Aegiceras corniculatum extracts, aspirin and indomethacin on cyclooxygenase-1 and 12-lipoxygenase product (12-HHT and 12-HETE) formation Metabolites of cyclooxygenase-1 and 12-lipoxygenase, 12-HHT and 12-HETE synthesized and released from human platelets after stimulation with A23187 were detected using HPLC technique. Cyclooxygenase inhibitors, aspirin (1–50 ␮M) and indomethacin (1–100 nM) reduced the production of 12-HHT in a concentrationdependent manner (corresponding IC50 were 15.6 ± 0.37 ␮M or

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2.8 ␮g/ml and 10.0 ± 2.7 nM or 0.0036 ␮g/ml) with a concomitant increase in 12-HETE level at comparable concentrations. Likewise, methanol extract attenuated the production of 12-HHT with an IC50 of 41.1 ± 1.5 ␮g/ml as shown in Fig. 3A. Conversely, hexane extract caused reduction in 12-HETE formation (IC50 0.36 ± 0.12 ␮g/ml) with a concomitant increase in 12-HHT at comparable concentrations (Fig. 3B). Interestingly, ethyl acetate extract suppressed the production of both metabolites but with greater affinity for 12-HHT (IC50 0.08 ± 0.002 ␮g/ml) than that of 12-HETE (IC50 8.0 ± 0.18 ␮g/ml) as depicted in Fig. 3C.

4. Discussion The current investigation demonstrates that hexane, ethyl acetate and methanol extracts derived from Aegiceras corniculatum stems possess pronounced inhibitory activity against inflammation displaying multiple mechanism of action. Acute transient phase (initial phase) of inflammation is characterized by vasodilatation, increased in vascular permeability and interstitial edema facilitated by mediators of mast cells and plasma proteins (Di Rosa et al., 1971). Degranulation of mast cells induced by dextran causes the release of histamine and serotonin during edema formation in rat paw (Ferreira et al., 2004). They interact with their respective receptors (H1, H2 and 5HT2) residing on the endothelium of micro-vessels. Both hexane and ethyl acetate extracts significantly antagonized this response by ∼60%. Additionally, ethyl acetate extract also significantly attenuated histamine-induced paw edema similar to chlorpheneramine maleate (H1 and H2 antagonist). However, it failed to antagonize bradykinin-induced edemogenic response. Moreover, the failure of plant extracts to alter the edema produced by hyaluronidase indicates that extracts do not interfere directly with the vascular permeability, thereby, implying that the plant extracts appear to be effective in antagonizing the vascular phase of inflammation either via suppressing the release of histamine and serotonin from mast cells and/or by acting at the histaminic receptors. The production of eicosanoids derived from COX and/or 5LOX pathways was mitigated by plant extracts in in vivo model of acute inflammation in which rat paw edema during late phase (3rd–4th h) was induced by either carrageenan, PGE2 , LTB4 or AA. Both isoforms of cyclooxygenases-1 and -2, have been implicated in carrageenan-induced edema resulting in the overproduction of PGE2 . In this context, methanol extract emerged as an impressive anti-inflammatory agent as it was ∼7× better than non-selective cyclooxygenase inhibitor aspirin and equipotent to naproxen sodium. It is known that PGE2 -induced paw edema in rats enhances the expression of cyclooxygenases (preferentially COX-2) with a concomitant rise in the PGE2 levels (Nantel et al., 1999). This response was markedly diminished (92 ± 2%) in the presence of methanol extract, revealing its anti-inflammatory effect via cyclooxygenase pathway. Since hexane extract was more potent against LTB4 similar to NDGA (a selective 5-LOX inhibitor) it is apparently more selective towards the 5-LOX pathway. Likewise to phenidone, ethyl acetate extract also acts as a dual inhibitor for both cyclooxygenase and 5-lipoxygenase enzymes via attenuating AA-induced edemogenic effect. Moreover, it also suppressed the inflammation induced by either PGE2 or LTB4 in rat paw. Our in vitro results correlated reasonably well with the in vivo studies. In this regard, the methanol extract suppressed the formation of prostanoid (12-HHT) with a concomitant rise in 12-HETE levels. This phenomenon is likely to occur only after selective inhibition of COX-1 enzyme thus diverting the AA metabolism towards 12-LOX pathway, and consequently increasing the 12HETE levels. This type of corresponding pattern has also been

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exhibited by widely used anti-inflammatory drugs, such as aspirin and indomethacin (Safayhi et al., 1992; Albert et al., 2002). Moreover, methanol extract failed to inhibit the production of 5-LOX metabolites LTB4 and 5-HETE in rat neutrophils, further manifesting its selectivity towards cyclooxygenase pathway. The anti-inflammatory property of methanol extract might be related to lignans and triterpenoidal saponins reported earlier to be present in it (Rao and Bose, 1959; Hensens and Lewis, 1966). On the contrary, hexane extract concurrently decreased the LTB4 and 5-HETE levels suggesting that it inhibits a common biochemical step as catalyzed by the rate-limiting enzyme 5-LOX. Yet, another lipoxygenase, i.e., 12-LOX was also potently inhibited by hexane extract (IC50 0.36 ± 0.12 ␮g/ml) as reflected by a significant reduction in its metabolite, 12-HETE. Thus hexane extract exerts its effect on AA metabolism by inhibiting both types of lipoxygenases. However, the selectivity of hexane extract towards lipoxygenases may be due to the presence of benzoquinones (Hensens and Lewis, 1966; Gomez et al., 1989; Xu et al., 2004). This class of molecules are reputed for their high selectivity towards lipoxygenase pathway (Houghton et al., 1995; Resch et al., 1998). Thus the inhibitory effect elicited by hexane extract against LTB4 and HETEs provides a pharmacological support for the use of Aegiceras corniculatum in folk medicine against asthma. Ethyl acetate extract of plant reduced the production of eicosanoids, LTB4 and 12-HHT in rat neutrophils and human platelets, respectively with minor effect on 12-HETE thus indicating that it has a relatively high affinity for 5-lipoxygenase and cyclooxygenase-1 enzymes as compared to 12-LOX enzyme. It has been reported earlier that pentacyclic triterpenes inhibit 5-LOX activity by inducing conformational changes at AA binding site (Safayhi et al., 1995), whereas, flavonoids have been implicated as a dual inhibitor of 5-LOX and COX (Chi et al., 2001). Likewise, dual anti-inflammatory activity of ethyl acetate extract could also be attributed to the previously reported chemical constituents pentacyclic triterpenes, having oleanane skeleton and flavonoids such as quercetin, kaempferol and isorhamnetin residing in it (Rao and Bose, 1959, 1961, 1962; Rao, 1964; Zhang et al., 2005). It is worth mentioning here, ethyl acetate extract effect is comparable to most potent dual acting anti-inflammatory agents such as synthetic licofelone (Tries et al., 2002) and naturally occurring hyperforin (Albert et al., 2002). Neutrophils infiltration to the site of acute inflammation is a crucial event that aggravates the inflammatory reaction producing excessive amount of proteolytic enzymes, reactive oxygen species, eicosanoids and cytokines thereby causing tissue damage. A significant inhibitory effect of hexane (65%) and ethyl acetate (96%) extracts against infiltration of neutrophils was observed having ∼2–8× more potency than phenidone. This property of plant extracts is more likely to be due to its interaction with cellular phase of inflammation with a reasonable correlation with its antiinflammatory action in the in vivo models as described before. It is therefore, concluded that hexane, ethyl acetate and methanol extracts of Aegiceras corniculatum stems are endowed with anti-inflammatory activity mediated via diverse mechanisms of action. Methanol extract elicited its effect by the inhibition of prostanoids biosynthesis. Hexane extract was selective towards both 5- and 12-lipoxygenases by reducing the LTB4 and 12-HETE levels. However, multiple mechanisms of anti-inflammatory activity was displayed by ethyl acetate extract viz. reduction in the levels of leukotrienes, prostanoids and HETEs along with anti-histaminic action. Taken all these results together, it is suggested that hexane and ethyl acetate extracts substantially interfere with both vascular and cellular phases of acute inflammation that justify the traditional use of Aegiceras corniculatum by the local community against inflammatory diseases.

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