|Year : 2009 | Volume
| Issue : 2 | Page : 80-90
Anti-inflammatory activity of ixora coccinea methanolic leaf extract
SM Handunnetti1, RR Kumara1, SA Deraniyagala2, WD Ratnasooriya3
1 Institute of Biochemistry, Molecular Biology and Biotechnology, University of Colombo, Sri Lanka
2 Department of Chemistry, Faculty of Science, University of Colombo, Sri Lanka
3 Department of Zoology, Faculty of Science, University of Colombo, Sri Lanka
|Date of Submission||20-Jan-2009|
|Date of Acceptance||11-Feb-2009|
|Date of Web Publication||2-Jan-2010|
S M Handunnetti
Institute of Biochemistry, Molecular Biology and Biotechnology, University of Colombo
Source of Support: None, Conflict of Interest: None
| Abstract|| |
The anti-inflammatory activity of methanolic leaf extract (MLE) of Ixora coccinea Linn. (Rubiaceae) was investigated in this study. MLE showed dose-dependent anti-inflammatory activity in carrageenan-induced rat paw edema model (r = 0.7; P<0.01). MLE at a dose of 500, 1000, and 1500 mg/kg showed maximum inhibition of edema 36.7, 46.5, and 64.5% respectively (P<0.01). Oral administration of MLE of rats at a dose of 1500 mg/kg significantly inhibited peritoneal phagocytic cell infiltration (45.9%; P<0.05), impaired nitric oxide (NO) production in peritoneal cells (40.8%; P<0.01) and showed antihistamine activity (54.9%; P<0.01). In vitro treatment of rat peritoneal cells with MLE inhibited NO production dose-dependently (82.2% at 400 μg/ml, r = 0.99; P<0.05). MLE also possessed significant, dose-dependent in vitro anti-oxidant activity (r = 0.88; P<0.01; IC50 value = 8.0 μg/ml), membrane stabilizing activity (r = 0.81; P<0.01; IC50 value = 6.4 ng/ml) and lipid peroxidation activity (36.7% at 250 μg/ml; P<0.01). Thirty-day oral treatment of rats with 1500 mg/kg did not show any adverse signs of toxicity or behavioral changes. These results suggest that anti-inflammatory activity of I. coccinea is mediated via inhibition NO production, phagocytic cell infiltration, anti-histamine effect, scavenging of free radicals, membrane stabilizing activity and lipid peroxidation.
Keywords: anti-inflammatory activity, anti-oxidant, cell infiltration, Ixora coccinea, membrane stabilization, nitric oxide
|How to cite this article:|
Handunnetti S M, Kumara R R, Deraniyagala S A, Ratnasooriya W D. Anti-inflammatory activity of ixora coccinea methanolic leaf extract. Phcog Res 2009;1:80-90
|How to cite this URL:|
Handunnetti S M, Kumara R R, Deraniyagala S A, Ratnasooriya W D. Anti-inflammatory activity of ixora coccinea methanolic leaf extract. Phcog Res [serial online] 2009 [cited 2021 Jan 25];1:80-90. Available from: http://www.phcogres.com/text.asp?2009/1/2/80/58136
| Introduction|| |
Medicinal remedies based on herbs were widely used before the advent of modern pharmacology. Presently about 80% of the world's population relies mainly on medicinal plants as a source of remedies for treatment of disease  . In Sri Lanka, a wide variety of plants are used in both Ayurveda and traditional medicine for anti-inflammatory effects  . Ixora coccinea Linn. (Rubiaceae) commonly known as rathmal in Sinhalese and vedchi in Tamil is one of these plants. It is a shrub with small obvate to oval-oblong, rounded to subcordate base leaves on branched hard heavy twigs  . Different plant parts of I. coccinea are used for treatment of various disease conditions some of which are associated with inflammation. A decoction of the flowers is given for haemophytis, acute bronchitis and dysmenorrhoea  . Further, the flowers and bark are used on reddened eyes and eruptions in children. A decoction of the root is given for dysentery, loss of appetite, fever, and gonorrhea, and as a sedative for hiccoughs and nausea. The leaves are used for dermatological disorders in traditional systems of medicine in Sri Lanka  .
Previous studies have reported anti-inflammatory effects of aqueous leaf extract of I. coccinea using both acute and chronic inflammatory models  . The aqueous leaf extract was also shown to possess anti-histamine and antinociceptive activities  . Lupeol isolated from the petroleum ether fraction of ethanol extract of leaves was shown to have anti-inflammatory activity in carrageenan- induced rat paw edema assay  . In this study we investigated the in vivo anti-inflammatory activity of methanolic leaf extract (MLE) of I. coccinea using the carrageenan-induced rat paw edema model and it shows potent anti-inflammatory activity. We demonstrate here for the first time, the regulatory effects of leaf extracts of I. coccinea on inflammatory mediators such as nitric oxide. In addition this study gives insight on the underlying mechanism(s) of anti-inflammatory activity, the effect of MLE on immunephagocytic cell infiltration, scavenging of free radicals, lipid peroxidation and membrane stabilizing activity.
| Materials and Methods|| |
Ascorbic acid, bovine serum albumin (BSA), carrageenan, 1,1-dipheny1-2-picrylhydrazil (DPPH), 6-Hydroxy 2, 5, 7, 8 - tetramethylchroma n-2-carboxylic acid (Trolox), lipopolysaccaride (LPS), NaNO 2 Neutral Red, N-(1- naphthyl) ethylenediamine hydrochloride, N-monomethylL-arginine acetate salt (NMMA; nitric oxide synthase inhibitor), sulphanilamide, thiobarbituric acid (TBA) were purchased from Sigma Aldrich (St. Louis, MO, USA). Gum acacia (GA) and histamine dihydrochloride were purchased from Fluka, (Buchs, Switzerland) and Avondale Laboratories Ltd (Banburg, U.K.) respectively. RPMI 1640 medium was obtained from GIBCO BRL, Life Technologies (Paisley, Scotland). Indomethacin, prednisolone, aspirin, chloropheniramine were purchased from the State Pharmaceutical Corporation (Colombo, Sri Lanka). Randox assay kits were purchased from Randox Laboratories Ltd. (Antrim, UK). All other chemicals and reagents were of analytical grade.
Preparation of plant material and extraction
Leaves of I. coccinea were collected in October 2005 from Palpola area in the Kalutara district of Sri Lanka. Identification and authentication of the plant was done by Dr. S. Ranwala, Department of Botany, University of Colombo. The voucher specimen (rrk/lc/ool) was deposited at the Institute of Biochemistry, Molecular Biology and Biotechnology, University of Colombo.
The air dried leaves (100 g) were boiled in 3 L of methanol for 16 h. Methanolic leaf extract (MLE) was concentrated under reduced pressure at 40°C. Methanol was completely removed, the product was freeze dried and a brown color hygroscopic residue was obtained (yield 14.5%).
Adult albino Wistar rats (8-an weeks, 150-200 g) were obtained from the Medical Research Institute, Colombo, Sri Lanka and kept in the animal house, Department of Zoology, University of Colombo, under standard conditions (temperature: 28 ± 2°C; photoperiod: 12 h natural light and 12 h dark; humidity: 50 ± 2%) with free access to food pellets (Finisher Feed, Ceylon Grain Elevators, Colombo, Sri Lanka) and tap water. Ethical clearance for this study was obtained from the Research, Ethics and Higher Degrees committee of the Institute of Biochemistry, Molecular Biology and Biotechnology, University of Colombo.
Assay for carrageenan-induced rat paw edema
MLE was resuspended in 1% gum acacia (GA) and anti-inflammatory activity of MLE was determined as described previously  using the rat paw-edema assay and the same doses  . Forty eight rats were randomly divided into six groups labeled 1 to 6. Groups 1, 2 and 3 were orally administered with three different doses of MLE, 500, 1000, and 1500 mg/kg respectively (n = 8/group). Fourth group (n = 8) was treated with 1 ml of 1% GA and served as the control for groups a. to 3. Fifth group (n = 8) was treated with indomethacin (5 mg/kg) which was used as the reference drug and the sixth group was treated with 1 ml of distilled water (DW) (n = 8) and served as the control for group 5. After 1 h, 0.1 ml of 1% carrageenan suspension in saline was injected subcutaneously into the planter surface of the left hind paw of animals under the mild ether anesthesia. The volume of the paw up to ankle joint was measured 1 h prior to the injection (V o ) and hourly up to 5 h (V t ) after the injection of carrageenan, using a plethysmometer (Panlab s.I., Barcelona, Spain). The percentage inhibition of edema was calculated using these paw volumes, with respect to their controls.
Assay for infiltration of rat peritoneal cells
The effect of MLE on infiltration of rat immune cells to the peritoneal cavity was assessed by a modification  of a previously described method for isolation of peritoneal cells  . Two groups of rats (n = 6/group) were orally administered with MLE (1500 mg/kg) and prednisolone (10 mg/kg) respectively. Two control groups of rats (n = 6/group) were treated with 1 ml of 1% GA and 1 ml of DW. After 1 h, carrageenan was injected into the peritoneal cavity mg/ml in Phosphate Buffered Saline (PBS), pH 7.4; 5 mg/kg). Sterile PBS (40 ml) was injected 2 h later, and 5 min after this injection, 35 ml of peritoneal fluid was drained using a 18 G cannular. These procedures were performed under ether anesthesia. Peritoneal fluid was centrifuged at 150 g for 10 min at 4°C. The supernatant was removed and the peritoneal cells were resuspended in 1 ml of PBS. A 50 ml aliquot of the cell suspension was mixed with 10 ml of 1% Neutral Red to visualize the phagocytic cells. Total cell and phagocytic/macrophage counts were made using a haemocytometer.
Assay for nitric oxide production by peritoneal cells
Nitric oxide (NO) production by rat peritoneal cells was determined by measuring nitrite in culture supernatants using Griess reagent as described previously  . Nitrite concentrations in cell free supernatants measured by Griess reaction according to the protocol of Steuhr and Nathan (1989)  , serve as a reflection of NO production. The effect of MLE on NO production by rat peritoneal cells was assessed under in vitro and in vivo conditions described below, i) In vitro: Treatment of peritoneal cells isolated from healthy rats not exposed to MLE in vivo, with MLE and ii) In vivo: Assessing the inhibitory effects of the peritoneal cells collected from rats treated with MLE orally.
Four groups of rats (n=6/group) were orally treated with MLE (1500 mg/kg), the reference drug, prednisolone (10 mg/kg) and their respective controls (1 ml of 1% gum acacia (GA) and 1 ml of DW) as described above , , and peritoneal cells that were exposed to MLE in vivo were obtained to assay for NO production.
In vitro treatment of MLE was performed using a modification of the previously described method  . Peritoneal cells were collected from rats that were injected intraperitoneally with carrageenan (5 mg/kg) and then treated in vitro with 100, 200, and 400 μg/ml of MLE in RPMI 1640 medium supplemented with 1% BSA for 30 min at 37°C. Peritoneal cells were treated with 1 mM NMMA in RMPI 1640 medium as positive control. Cells were centrifuged at 150 g for 2 min and resuspended in culture medium containing 1% bovine serum albumin and cultured for 24h. The viability of cells after 30 min incubation with MLE and after 24 h in culture in culture medium was assessed by Trypan blue exclusion test  .
Assay for nitrite
To assay the NO production by rat peritoneal cells, cells from each animal (n = 6) were plated in g6 well tissue culture plates at 1x10 6 cells/ml in RPMI 1640 medium supplemented with 1% BSA and incubated at 37°C in CO, incubator (5% CO 2 ±95% air) (Sanyo Electric Co. Ltd., Osaka, Japan). After 24 h, culture supernatant was aspirated from each well, centrifuged at 10,000 g for 10 min and clear supernatant was assessed for nitrite production. For quantification of nitrite, 100 ml of culture supernatant was mixed with an equal volume of Griess reagent (equal mixture of 1% Sulphanilamide in 5% phosphoric acid and 0.1% N-(inaphthyl) ethylenediamine hydrochloride in DW), kept at room temperature (25°C) for 15 min and optical density (OD) was read at 540 nm in a ELISA plate reader (ELX 800, BioTek Instruments INC, Winooski, VT, USA). The nitrite concentration was calculated using calibration curve between 0.7-100 μM NaNO 2 .
In vivo assay for anti-histamine activity
This assay was performed as described previously  . Fur on left lateral side of the back of these rats was removed. Twenty-four h later, these rats were randomly assigned in to four groups (n = 6/group) and orally administered with MLE in 1% GA (1500 mg/kg), chlorphenira mine (0.67 mg/kg) 1 ml of 1% GA and 1 ml of distilled water. After 1 h, these rats were subcutaneously injected with 50 μI of 200 μg/ml histamine dihydrochloride in saline in to the skin where the fur had been shaved, and 2 min later the area of the wheal formed was measured. Anti-histamine activity was calculated compared to the respective controls.
Assay for in vitro anti-oxidant activity
In vitro anti-oxidant activity was assessed by DPPH method as described previously  . DPPH solution (20 μg/ml) was prepared using methanol and OD value was adjusted to 0.7 at 53.7 nm. Trolox (25 μg/ml) solution was prepared to calculate the Trolox equivalent for MLE concentrations. A dilution series of MLE was prepared using PBS at concentrations of 10, 100, 250, 500, 1000, and 2500 μg/ml. OD 517 value of samples were measured 5 min after mixing 300 μl of MLE dilution and 300 μl of DPPH solution. PBS was used as the control and ascorbic acid was used as the positive control. Percentage inhibition of DPPH free radical scavenging was calculated based on the control reading, which contained DPPH and PBS without any extract using the following equation:
% Scavenging Activity = [(OD) control -OD sample /OD control ] x 100
Anti oxidant activity of the MLE was expressed as IC 50 . IC 50 value was defined as the concentration (in μg/ml) of MLE that inhibit the scavenging of DPPH radicals by 50%. Trolox equivalents for MLE was also derived from a standard Trolox curve (3.3.-20 mM)  .
Assay for membrane stabilizing activity
This assay was performed using a modification of the heat-induced hemolysis of rat erythrocytes as described previously  . A tenfold dilution series of MLE was made using PBS for concentrations from 1 mg/ml to 0.001 μg/ml. Dilutions of aspirin was also was made for the same concentrations and used as the reference drug. One ml of PBS was used as control. Twenty μl of rat blood was added to each tube containing 1 ml of test, standard drug and control samples. All dilutions of MLE, aspirin and GA were made in triplicates. Samples were first incubated at 37°C for 15 min. The modifications included an additional centrifugation step after this initial incubation. Cell suspensions were centrifuged at 1500 g for 3 min, the supernatants were removed and the cells were resuspended in 1 ml of PBS. This centrifugation step removed the color originating from the MLE itself which subsequently interference with the OD 540 measurement. The samples were then incubated at 54°C for 25 min to initiate heat-induced hemolysis and centrifuged at 1500 g for 5 min. Supernatants (200 μl) were transferred into an ELISA plate and the OD value was measured at 540 nm. Percent inhibition of haemolysis was calculated with respect to the controls and IC 50 values were derived.
Percent inhibition of haemolysis = [(OD) control -OD sample /OD control ] x100
In vitro assay for lipid peroxidation activity
Lipid peroxidation activity was assessed using TBA reactive substances assay as described by Dorman et al. (1995)  . The concentrations of MLE used were 15.75, 31.25, 62.5, 125, and 250 μg/ml. Ascorbic acid (100 μg/ml) was used as the positive control. OD value was measured at 540 nm and the lipid peroxidation activity was calculated with respect to the control.
Phytochemical analysis and determination of metal ions
Qualitative analysis for tannins, phlobatannins, saponin, flavonoids steriods, terpenoids (Salkowski test) and cardiac glycosides (Keller-Killani test) were carried out as described previously  . Metal ions (K, Ca, Mg, Na, Fe, Zn, Cr, Ni, Mn, Cu, Cd, Hg) were quantified as previously described  . Air-dried plant materials were dried in an electric oven at 105°C until constant weight was reached. Half a gram was digested with 10 ml of concentrated H 2 SO 4 , and 7.5 ml of conc. HNO 3 for 2 h at 450°C on an electric digester. Solution was brought to room temperature and a few drops of H 2 O 2 were added until it become colorless. The colorless solution was filtered and diluted up to 50.00 ml. Absorbance was measured using Atomic Absorption Spectrophotometer (GBC Scientific Equipment, USA) and concentrations of metal ions were calculated using respective calibration curves.
Evaluation of toxicity
Rats (n = 6/group) were treated either with 1500 mg/kg/day of MLE or a. ml of 1% GA daily for 30 consecutive days. After the oral treatment, rats were observed for overt clinical signs of acute toxicity or stress during the period of treatment. Rats were weighed prior to the start of the experiment and on day 1 of post-treatment. On day 1 of post-treatment, 1 ml of blood was obtained from the tail under mild ether anesthesia and serum was separated. The red blood cell (RBC) and white blood cell (WBC) counts were made using fresh blood as described previously  . Serum concentrations of albumin, creatinine, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), formally referred to as glutamic pyruvic transaminase (SGPT) and glutamic oxaloacetic transaminase (SGOT) respectively, and urea were determined using Randox assay kits. Na and K were determined using Atomic Absorption Spectrophotometer (GBC Scientific Equipment, USA).
Data are presented as the mean ± standard error of mean (SEM). Statistical analyses used one-way analysis of variance (ANOVA) to account for the different treatment doses and were complemented with unpaired t-test. Differences were considered statistically significant at P < 0.05. Statistical analysis was performed using SPSS version 15.0.
| Results|| |
Inhibition of carrageenan-induced paw edema by MLE
As shown in [Figure 1], compared with the 1% GA control, treatment with MLE at 1500, 1000 and 500 mg/kg showed significant inhibition of paw edema in a dose-dependent manner (r = 0.7; P < 0.01). Maximum inhibition of paw edema was recorded as 64.5 ±3.2%, 46.5 ±3.7% and 36.7 ±5.5% respectively at 2 h (mean ±SEM) (P < 0.01). Inhibition of paw edema was significant up to 5 th h at 1500 mg/kg (P < 0.05) and up to 4 th h in the other two doses (P < 0.05). The reference drug, indomethacin showed significant inhibition (59.7 ±7.1%) up to 2 h (P < 0.01). Although the inhibition declined, it was significant up to the 5 th h (P < 0.05). In contrast to the two lower doses of MLE (1000 and 500 mg/kg), the highest dose of MLE (1500 mg/kg) showed a different pattern of inhibition which was similar to that of indomethacin, especially with respect to the anti-inflammatory activity up to 2 h.
Infiltration of rat peritoneal cells
Infiltration of total peritoneal cells was significantly inhibited (47.7 ±3.8%) by MLE compared to the GA control (P < 0.05) [Figure 2]. Of the two reference drugs used, indomethacin was not effective (18.8 ±10.4% inhibition) where as prednisolone significantly inhibited infiltration of peritoneal cells (91.3 ±4.61%; P < 0.05). Differential counts of peritoneal cells of the control rats showed 54.9 ±1.2% of phogocytic cells. Compared to the control rats, significant reductions were observed in the phagocytic cell counts in both MLE and prednisolone treated rats (50.5 ±9.4% and 91.2 ±1.8% reduction; P < 0.05 and P <0.01 respectively). .
Inhibition of nitric oxide production
Peritoneal cells obtained from rats given oral treatment of MLE showed significant inhibition of their NO production (40.8 ±4.8%; P < o.00i) [Figure 3]A. Oral treatment of rats with prednisolone, the reference drug, also inhibited the NO production significantly (63.5 ±4.8%; P < 0.001).
Assay for in vitro cytotoxicity of the MLE using the Trypan blue test showed comparable viable cells counts in MLE concentrations less than 800 μg/ml (85.6 ±0.58% in 400 μg/ml) to that of normal culture medium (87.1 ±0.65%.) These viable cell counts obtained after 30 min incubations were also reflected in the cell counts taken after 24 h in culture (78.6 ±0.39% and 79.6 ±1.04 respectively) indicating that MLE concentrations of 400 μg/ml or lower were not cytotoxic to peritoneal cells and are suitable for the in vitro treatment to assay for NO inhibitory activity.
Peritoneal cells obtained from carrageenan-treated rats had produced 50.1 μM of NO in vitro where as the cell free medium showed very low levels of NO [Figure 3]B indicating minimal background levels of nitrite in culture supernatants. In vitro MLE treatment of peritoneal cells demonstrated a dose-dependent inhibition of NO production (r = 0.99; P < 0.05). Maximum inhibition by MLE was observed at 400 μg/ml (82.2 ±1.2%; P < 0.01). NMMA, which is a nitric oxide synthase inhibitor also showed a comparable inhibition of NO production (70.1 ±2.6%; P < 0.01).
Anti - oxidant activity of MLE
Percentage scavenging of MLE increased dose-dependently upto 100 μg/ml (r = 0.88; P < 0.01) and thereafter reached a plateau [Table 1]. The IC 50 values for MLE and ascorbic acid were found to be 8.0 μg/ml and 4.g μg/ml respectively. The corresponding Trolox equivalents for MLE and ascorbic acid were 19.3 and 13.4 μM respectively.
Membrane stabilizing activity
In the modified assay in which the MLE dilutions were removed by centrifugation following the initial incubation at 37°C, so that the color of the MLE would not interfere with the final OD 540 reading, a very clear pattern of increase in inhibition of haemolysis was observed with the increasing MLE concentrations [Figure 4]. With the increasing MLE concentrations, the inhibition of haemolysis increased dose dependently (r = 0.81; P < 0.01) up to 100 μg/ml and thereafter reached a plateau indicating constant higher inhibitions maintained at high MLE concentrations (P < 0.01). The IC 50 values for membrane stabilizing activity for MLE and aspirin were 6.4 ng/ml and 0.25 ng/ml respectively.
Assay foranti-histamine effect
MLE had induced a significant anti-histamine effect (54.9 6.5%; P < 0.01), which was comparable to that of the reference drug, chloropheniramine (60.0 ±5.6%; P < 0.01).
In vitro lipid peroxidation activity of MLE
The lipid peroxidation activity (36.7 ±1.8%) detected at 250 μg/ml of MLE decreased to 15.3 ±2.8% at 125 μg/ml (P < 0.01). The standard drug, ascorbic acid showed a 52.1 ±2.2% activity at 100 μg/ml (P < 0.01).
Treatment with 1500 mg/kg/day of MLE for 30 days failed to produce any overt clinical signs of toxicity or stress. MLE treatment did not significantly alter the body weights (control vs MLE: 284.8 ±3.9 versus 283.8 ±4.0 g), serum levels of creatinine (control vs MLE; 2.41 ±0.03 mg/dl vs 2.30 0.02) and albumin: 3.31 ±0.53 g/dl vs 3.93 ±0.05 g/dl, Na (control vs MLE; 7256.57 ±143.7 ppm vs 7125.43 ±27.4 PPm), K (control vs MLE; 296.14 ±13.41 ppm versus 284.43 ±4.11 ppm) or hematological parameters (RBC count: control vs MLE; 3.78 ±0.14 x 10 6 vs 3.98 ±0.22 x 10 6 ; WBC count: 10.0 ±0.51 x 10 3 vs 9.9 ±0.24 x 10 3 ). Serum levels of AST and ALT were elevated following MLE treatment (AST: control vs MLE; 16.07 ±0.17 U/I vs 22.65 ±1.35 U/I; ALT: 14.08 ±0.54 vs 18.40 ±0.51 U/I; urea: 42.21 ±1.46 mg/dI vs 48.51 ±0.57 mg/dl) (P < 0.01), however, these levels were within the normal range of in-house animals  .
Analysis for phytochemicals and metal ions
Qualitative phytochemical screening showed the presence of alkaloids, saponins, tannins, phlobatanins, steroids, flavonoids, terpenoids and cardiac glycosides. The metal ion content in I. coccinea leaves were K: 20.634 ±4.472 mg/g, Ca: 16.139 ±1.996 mg/g; Mg: 1.731 ±0.808 mg/g; Na: 0.530, 0.028 mg/g; Fe: 0.293 ±0.034 mg/g; Zn: 0.019 ± 0.016 mg/g and Cr/Ni/Mn/Cu/Cd/Hg < 0.0001 mg/g.
| Discussion|| |
This study examined the anti-inflammatory activity of MLE of I. coccinea using the carrageenan-induced rat paw edema assay and the results show a dose-dependent in vivo anti-inflammatory activity. These results are consistent with our previous findings using aqueous leaf extracts of I. coccinea  . This study also demonstrated for the first time the inhibitory effects of leaf extracts of I. coccinea on nitric oxide which is a key inflammatory mediator and other activities such as infiltration of phagocytic cells, membrane stabilization and scavenging of free radicals.
The paw edema assay for anti-inflammatory activity showed some contrasting differences with MLE especially during the early phase of the assay compared to the previously described findings with the aqueous leaf extract of I. coccinea  . High concentration of MLE (1500 mg/kg) showed the significantly higher inhibition of paw edema one hour after carrageenan injection compared to the lower concentrations of the MLE (500 and 1000 mg/kg). This may be due to the presence of different anti-inflammatory components in the two extracts prepared using methanol and water having different polarities. Further studies are required to separate the anti-inflammatory active components from these solvent extracts.
The anti-inflammatory effects of the MLE reached its higher levels during the period between 1-2 hours. Cyclooxygenase (COX) inhibitors have been shown to inhibit the edema during this phase  and it is possible that inhibition of arachidonic acid metabolites is involved in the anti-inflammatory activity of MLE. The fact that the strong inhibition by MLE during 1-2 hours overlaps that by indomethacin supports this notion. Further, as shown previously  and in this study, I. coccinea contains flavonoids and tannins. Flavonoids and tannins have been shown to impair cyclooxygenase/lipoxygenase activities that would reduce the levels of prostaglandins and other arachidonic acid metabolites ,, .
Migration or the infiltration of immune cells to the site of inflammation is an important processe that take place in an inflammatory response. Experimental systems such as carrageenan-induced pleurisy in rats have been used to study cell migration process and the inhibitory effects of drugs on cell migration  . In the present study, we used a recently developed in vivo assay, ie., carrageenan-induced infiltration of rat peritoneal cells  to assess the inhibitory effects of MLE on immune cell infiltration. Our findings show significant inhibition of rat peritoneal cell infiltration which indicate that MLE of I. coccinea has potent leukocyte infiltration/migration inhibitory activity.
The anti-inflammatory activity in the paw edema assay showed that MLE has significant inhibitory activity up to the 4 th hour. Previous studies have shown that mobilization of phagocytic cells, neutrophils, and monocytes/macrophages to the site of inflammation is a characteristic feature of this late phase  apart from other events linked to the release of oxygen free radicals , and nitric oxide  . This inhibitory effect of MLE on cell migration and mobilization in vivo as shown in the infiltration of rat peritoneal cell assay suggest that similar inhibitory effect of MLE on immune cell migration could have contributed to the curtailment of inflammation in vivo, in the rat paw edema assay. Further, prednisolone, the reference drug used showed a significantly higher inhibition of peritoneal cell infiltration suggesting that cell infiltration being inhibited by phospholipase 2 activity at an earlier step of processing of arachidonate metabolites or by arachidonate cyclooxygenase (COX) inhibitors. Recent studies have shown either a low  or no effect , of COX inhibitors on migration of leukocytes in different experimental models. It is possible that MLE of I. coccinea may inhibit cell infiltration by affecting the arachidonate pathway at steps other than the COX pathway or affecting other activities such as those of leucocyte chemotactic factors and monocyte specific chemotactic factors  .
The present study also showed a strong in vivo antihistamine activity of the MLE which was detected within 2 min of histamine injection which is consistent with the previous findings on aqueous leaf extracts of I. coccinea  . The activity of inflammatory mediators such as histamine, serotonin, and arachidonic acid metabolites has been highlighted during this early phase  . Histamine released from mast cells is known to stimulate endothelial cells to increase vascular permeability  . It is possible that this anti-histamine activity of the MLE could at least in part contributed to the impairment of carrageenan-induced microvascular leakage in the paw edema assay reflecting on the anti-inflammatory mechanisms operating during the early phase. Triterpenes from angiosperms are known to impair histamine release from mast cells and exert anti-inflammatory activity  and phytochemical analysis of I. coccinea leaves performed in this study as well as in a previous study  has shown the presence of triterpenoids. MLE also showed marked and dose-dependent anti-oxidant activity. Carrageenan-induced paw edema is sensitive to anti-oxidants  . This is likely to be another mechanism by which MLE mediates impairment of late phase of the anti-inflammatory response. The anti-oxidant activity of the MLE could be due to the presence of flavonoids and phenols , . A recent study has shown anti-oxidant activity of flowers of I. coccinea and suggests that the anti-oxidant activity involves the presence of hydrophilic phenolics  . In addition, MLE also showed membrane stabilizing activity in the heat-induced hemolysis of rat erythrocytes in vitro. Although the previous study had not shown significant membrane stabilizing activity in ALE of I. coccinea  , the present study showed very high activity in MLE. This indicates that the MLE could stabilize the lysosomal membranes to inhibit the release of proteolytic enzymes; lysosomes play a major role in the inflammatory reaction , . There is a close similarity between erythrocytes and lysosomal membrane system  .
An important finding of this study is that MLE showed a significant inhibitory effect on NO production by rat peritoneal cells. This was shown by inhibitory effect on NO production of peritoneal cells following in vivo treatment of rats with MLE as well as direct effect on peritoneal cells, following in vitro treatment with MLE. Nitric oxide is an important mediator in an inflammatory response  . Several compounds such as sesquiterpene lactone from Artemesia princes Pampan (Asteraceae), flavin 3,3'-digallate which is a polyphenol from Black tea and resveratrol which is a naturally occurring flavinoid from grapes and grapefruits have been shown to inhibit the inducible nitric oxiside synthase (iNOS) expression  . These results emphasis the importance of conducting further studies to elucidate the specific mechanisms of NO inhibitory activity of MLE of I. coccinea. This study also showed that the lipid peroxidation activity of MLE decreased proportionately with MLE concentration. This has some implications on the activity of MLE in vivo. The interaction of NO with the super oxide anion gives rise to peroxynitrite which is a highly potent oxidant that damages the tissue in inflammation. Previous studies have shown that peroxynitrite-dependent lipid peroxidation can be regulated by high concentrations of NO  . Dose-dependent inhibition of NO production by MLE observed in the present study suggests that the peroxynitrite-dependent lipid peroxidation could also be reduced by MLE.
Thirty day (chronic) treatment of MLE was well tolerated, there were no deaths and it did not induce any unacceptable side effects or any overt signs of clinical toxicity. The biochemical analysis also did not indicate any hemotoxicity, nephrotoxicity or hepatotoxicity. The analysis of metal ions in leaves of I. coccinea indicated the absence of toxic, heavy metal such as Cr, Cd, and Hg.
In conclusion, this study has shown promising anti-inflammatory activity in the methanolic leaf extracts of I. coccinea. Compared to the previous study on aqueous leaf extracts of I. coccinea  , the present study has demonstrated specific mechanisms of the anti- inflammatory activity mediated by inhibition of peritoneal immune cell infiltration and NO production and also membrane stabilizing activity and anti-oxidant activity. Findings of the present study also corroborate the use of I. coccinea in traditional medicinal practice in Sri Lanka for treatment of diseases associated with inflammation.
| Acknowledgements|| |
Authors wish to thank J.R.A.C. Jayakody, Department of Zoology, Faculty of Science, University of Colombo for
technical assistance. Authors thank E. H. Karunanayake and K. H. Tennekoon for valuable advice during this study and critically reviewing the manuscript. Financial assistance from grant No 2005/NSF/HS/15 of the National Science Foundation and grant No 05-52 of the National Research Council, Sri Lanka are acknowledged.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]