Anti-inflammatory Activity of the Aqueous Leaf Extract of Ixora coccinea

W.D. Ratnasooriya, S.A. Deraniyagala, G. Galhena, S.S.P. Liyanage, S.D.N.K. Bathige, and J.R.A.C. Jayakody 1Department of Zoology and 2Department of Chemistry, University of Colombo, Colombo, Sri Lanka

Abstract

The aim of this study was to investigate the anti-inflammatory potential of an aqueous leaf extract (ALE) of Ixora coccinea (Rubiaceae) in rats after oral administration (500, 1000 and 1500mg/kg). This was done using the carrageenan-induced paw edema (acute inflammatory model) and cotton pellet granuloma tests (chronic inflammatory model). In the former test, ALE significantly impaired both early and late phases of the inflammatory response and also the edema maintained between the two phases. In the latter test, it significantly suppressed granuloma formation (only highest dose tested). Collectively, these data show promising anti-inflammatory activity against both acute and chronic inflammation. ALE showed strong antihistamine and antioxidant activities that can account for its anti-inflammatory potential. In addition, inhibition of prostaglandins and bradykinins may play a role.

Keywords: Antihistamine, anti-inflammatory activity, antioxidant, Ixora coccinea.

Introduction

The search for new pharmacologically active agents obtained by screening natural sources such as microbial fermentations and plant extracts has led to the discovery of many clinically useful drugs that play a major role in the treatment of human diseases. The ethnopharmacological uses as well as certain biological activities exhibited by Ixora coccinea indicate it to be a rich source of phytomedicine. Ixora coccinea Linn. (Rubiaceae)(Sinhala: ratmal; Tamil: vedchi) is a shrub (Jayaweera, 1982) with small, obovate to oval-oblong, rounded to subcordate base leaves on branched hard heavy twigs (Jayaweera, 1982). It is very common everywhere in the low country of Sri Lanka. The wide distribution in Sri Lanka has led to the extensive use of this species in the traditional system of medicine. A decoction of the roots is given for dysentery and as a sedative for hiccoughs, nausea, loss of appetite, fever, and gonorrhea. The flowers and bark are used on reddened eyes and eruptions in children (Jayaweera, 1982). Further, a decoction of the flowers is given for hemoptysis, catarrhal bronchitis, and dysmenorrhea. The leaves of I. coccinea are used in the treatment of dermatological disorders in the traditional system of medicine in Sri Lanka (Jayaweera, 1982). A preliminary report (Reene et al., 1993) on the anti-inflammatory effect of ethanol extract of the leaves of I. coccinea prompted us to study this effect in detail. We report here our investigation of the anti-inflammatory activity using the rat carrageenan-induced paw edema technique (acute inflammatory model) and cotton pellet test (chronic inflammatory model).

Materials and Methods

Collection of the herb and preparation of aqueous leaf extract (ALE)

Fresh leaves of I. coccinea were collected from Keleniya and Mirigama in the Gampaha district of Sri Lanka in August 2001 and were identified and authenticated by Professor B.A. Abeywickrama of the Botany Department of the University of Colombo. A voucher specimen (wdr/sad 1003) was deposited at the museum of the Department of Zoology. The leaves were washed under running water, air-dried, and cut into small pieces. The pieces (234g) were macerated with water and were then refluxed with 3 l of water for 2 days in a round-bottom flask fitted to a Leibig condenser. The brownish red solution was filtered and freeze-dried (12g, yield 4.3%) and stored airtight at room temperature (30-32&degreeC). The freeze-dried powder was dissolved in distilled water (DW) to obtain the required dosages in 1 ml solution (500, 750, 1000, or 1500mg/kg).

Experimental animals

Healthy adult cross-breed albino male rats (200-250g) were used in study. The animals were kept in plastic cages (six per cage) under standardized animal house conditions (temperature, 28-31&degreeC; photoperiod, approximately 12h natural light per day; relative humidity, 50-55%) with continuous access to pelleted food (Master Feed Ltd., Colombo, Sri Lanka) and tap water. Except at the time of experimental procedures, the animals were handled only during cage cleaning. All efforts were made to minimize animal suffering and to reduce the number of animals used.

Anti-inflammatory activity

Carrageenan-induced paw edema

Forty-four male rats were selected and randomly divided into five groups. The rats in groups 1, 2, and 3 were orally treated with 500, 1000, and 1500mg/kg of ALE, respectively. The rats of group 4, which served as the control, were treated with 1ml of DW. The rats in the fifth group were treated with 5 mg/kg indomethacin (Dharmasiri et al., 2002)(State Pharmaceutical Corporation, Colombo, Sri Lanka), the reference drug (Laurence & Bennett, 1992). After 1h, 0.05ml of 1% carrageenan (Sigma Chemical Company, St. Louis, MO, USA) suspension was injected subcutaneously into the planter surface of the left hindpaw as described by Winter et al. (1962). The volume of the injected paw of each of these rats was measured using a plethysmometer (Letica Scientific Instruments, Barcelona, Spain) at 1h prior to the injection of carrageenan and 1, 2, 3, 4, and 5h after the injection.

Cotton pellet granuloma

Twelve rats were randomly assigned into two groups (n = 6/group). Autoclaved cotton pellets (10mg) were implanted subcutaneously, one on each side above the scapula region, under ether anesthesia using aseptic precautions (Dhawn & Srimal, 2000). Either 1500mg/kg of ALE or 1ml DW was administered orally for 7 consecutive days starting from the day of injection. On day 8, the animals were killed and the pellets along with granuloma removed and dried in an oven at 60&degreeC until a constant weight was obtained.

Antihistamine effect

Fur on left lateral side of the back of 18 rats was shaved. Twenty-four hours later, these rats were randomly assigned into three equal groups. Group 1 was treated with 1500mg/kg of ALE orally. The other groups were treated, respectively, with 0.67mg/kg of chlorpheniramine and 1ml of water (Dharmasiri et al., 2002). After 1h, these rats were subcutaneously injected with 50μl of 200μg/ml histamine dihydrochloride (Fluka, Buchs, Switzerland) into the skin where the fur had been shaved, and 2min later the area of the wheal formed was measured (Spector, 1956).

Evaluation of antioxidant activity

Antioxidant activity was assessed using thiobarbituric acid reactive substances assay as described by Dorman et al. (1995). Vials containing the reagents were treated in triplicate with the ALE so that the final concentrations of the extract in the various vials was 0.25, 2.5, 12.5, 18.8, 25, 50, or 125μg/ml. Butylated hydroxyl toluene (BHT)(100μg/ml) was used as the positive reference and DW was used as the control. The vials were mixed well and incubated at 95&degreeC for 60min and then allowed to cool. Butanol (5ml) was added, mixed well, and centrifuged at 1500 x g for 5min. The absorbance of the butanol layer was measured at 532nm, and the antioxidant index was calculated as follows: Antioxidant index = (1 – T/C) x 100 (where T = absorbance of test and C = absorbance of control).

Evaluation of diuretic activity

This was done as described previously by us (Ediriweera & Ratnasooriya, 2002). Briefly, 12 rats were deprived of water but not food for 18h. Their urinary bladders were emptied by gentle compression of the pelvic area and by pull of their tails. Each of these rats was then orally treated with 15ml of isotonic saline (NaCl, 0.9% w/v) to impose a uniform water load. After 45min, these rats were randomly assigned into two groups (n = 6/group) and treated orally in the following manner. Group 1, 2ml of DW; group 2, 1500 mg/kg of ALE. Each of these rats was individually placed in metabolic cages and cumulative urine output was determined at hourly intervals for 5h.

Phytochemical analysis

Phytochemical screening of the aqueous extract was carried to according to Farnsworth (1988). The ALE was subject to column chromatography (30cm length and 3.7cm diameter) on reverse-phase C-18 silica gel (Fluka Chemie G, Buchs, Switzerland). The column was eluted with water, mixtures of methanol and water, methanol, mixtures of methanol and ethyl acetate, ethyl acetate, mixtures of ethyl acetate and dichloromethane, dichloromethane, mixtures of dichloromethane and hexane, and finally with hexane.

The fractions with similar thin-layer chromatography (TLC) spots were combined after inspecting under UV light. The combined fractions were subject to TLC (Aldrich silica gel precoated on plastic plates and Fluka Chemie G reverse-phase C-18 precoated glass plates). The mobile phases were 60% dichloromethane in hexane, 10% methanol in dichloromethane for normal phase chromatography, and methanol and 50% methanol in water for reverse-phase chromatography. The TLC plates were sprayed with color reagents specific for various classes of compounds (Stahl, 1965). (AlCl3test; for flavonoids; diazotized para-nitroaniline: for phenols; para-toluene sulphonic acid: for flavonoids and steroids; SbCl3 in acetic acid test: for steroids; Libermann Buchardt spray; for triterpenoid glycosides; and Dragendorff's reagent and iodoplatinate reagent: for alkaloids.)

Statistical analysis

The data are expressed as the mean &plusminus SEM. Statistical analysis was performed using Mann-Whitney U-test. Significant values were set at p&lte 0.05. Linear regression analysis was performed to assess dose-dependencies.

Results

Carrageenan-induced paw edema

The results obtained are summarized in Table 1. As shown, all the doses of ALE tested caused a significant (p < 0.05 to 0.001) and marked reduction in paw edema (28-59%) compared to control at each time point measured. Overall, this anti-inflammatory effect seemed dose-related. Indomethacin also impaired the edema formation, but this anti-inflammatory effect was much stronger (77-90%).

Cotton pellet granuloma

The ALE caused a significant (p < 0.05) and marked inhibition (by 36.1%) of granuloma weight as compared to control (control vs. treatment: 29.2&plusminus9.6 vs. 18.6&plusminus7.1mg).

Antihistamine effect

The ALE induced a significant (p < 0.05) and profound impairment (by 42.6%) of the area of wheal formed by the subcutaneous injection of histamine. This antihistamine effect was comparable to that produced by chlorpheniramine.

Antioxidant activity

As shown in Table 2, the ALE had promising (compared to BHT control) and dose-dependent (r2 = 0.9, p < 0.05) antioxidant activity.

Diuretic activity

The ALE did not induce a significant (p > 0.05) increase in cumulative urine output (control vs. treatment 4.9&plusminus0.8 vs. 4.2&plusminus1.1ml).

Phytochemical analysis

Phytochemical screening of the ALE showed the presence of quaternary base alkaloids, flavonoids, tannins/polyphenols, steroids, and/or terpenoids and saponins. Thin-layer chromatography of the methylene chloride, methylene chloride/hexane, and hexane column, fractions showed the presence of phenols (Rf 0.76, 0.12, 0.08), flavonoids (Rf 0.83, 0.7), steroids (Rf 0.09), and triterpenoid glycosides (Rf 0.64, 0.17, 0.73) and a quaternary base (Rf 0.15) on spraying with characteristic reagents.

Discussion

This study examined the anti-inflammatory activity of ALE of I. coccinea in rats using the carrageenan-induced paw edema test (acute inflammatory model) and cottonpellet test (chronic inflammatory model) after oral administration.

The results show that the ALE has promising anti-inflammatory activity both against acute (exudative phase) and chronic (proliferative phase) inflammation.

The carrageenan-induced paw edema test is widely accepted as a sensitive phlogistic tool for investigating potential anti-inflammatory agents, particularly the non-steroidal type (Vineger et al., 1969). In this test, development of edema (inflammatory response) is a biphasic event with a maintenance phase in between (Vineger et al., 1969). In this study, ALE simultaneously inhibited all these phases. The initial phase (1-2h) is primarily mediated by histamine and serotonin (Vineger et al., 1969), but platelet activating factor and arachidonic acid metabolites also play a role (Boughton-Smith et al., 1993). The ALE had strong antihistamine activity that could impair microvascular leakage induced by carrageenan (Kuriyama et al., 2000) and thereby inhibits the initial phase of the edema test. Histamine stimulates vessel endothelial cells to increase vascular permeability (Kuriyama et al., 2000). Further, the ALE contained triterpenoid glycosides. Many kinds of triterpenoids from angiosperms are known to impair histamine release from mast cells and exert anti-inflammatory effects (Janaki et al., 1999). Such a mode of action is possible in this study as well. The edema maintained between the first and second phase (2-3h) is due to kinin-like substances, especially bradykinin (Vineger et al., 1969). Curtailment of this maintenance phase indicates that the ALE had inhibited bradykinin release and/or its vascular permeability promoting action. The ability of the ALE of I. coccinea to inhibit the late phase of the formalin test of nociception (Ratnasooriya et al., 2004) provides support to this notion.
