International Journal of Drug Development and Research

Key words

Anti-inflammatory, Ixora coccinea, Carrageenan, Paw edema, cotton pellet, Anti-histamine, Antioxidant

INTRODUCTION

Inflammation plays an important role in various diseases, such as rheumatoid arthritis, atherosclerosis and asthma, which all show a high prevalence globally. During an inflammatory response, mediators, such as pro inflammatory cytokines, including interleukin IL-1, tumour necrosis factor (TNF), interferon (INF)-γ, IL-6, IL-12, IL-18 and the granulocyte macrophage colony stimulating factor, are released; this response is antagonised by anti-inflammatory cytokines, such as IL-4, IL-10, IL-13, IFN-α and the transforming growth factor. The nuclear factor- B (NF- B) transcription factor, also plays an important role in the inflammatory response by regulating the expression of various genes encoding pro inflammatory cytokines, adhesion molecules, chemokines, growth factors, and inducible enzymes such as cyclooxygenase-2 (COX-2)[1,2]. Inducible nitric oxide synthase (iNOS) and COX-2 both stimulate the production of large amounts of pro inflammatory mediators. In chronic inflammation, the negative regulatory mechanism appears to be dysfunctional. Although inflammation is primarily a protective response (for example against microorganisms, toxins or allergens), inflammation that is chronic and uncontrolled becomes detrimental to tissues [3] .Since ancient times, in various cultures worldwide, inflammatory disorders and related diseases have been treated with plants or plant derived formulations.[4,5,6,7]. The anti-inflammatory activity of several plant extracts and isolated compounds has already been scientifically demonstrated. Ixora coccinea, Linn. (Rubiaceae) is a bushy, rounded shrub found in subtropical region of Florida. Plant is grown as ornamental plant in India. It is commonly known as Rugmini (Hindi), Vedchi (Tamil), Rangon (Bengali), flame of wood (English), Bandhaka (Sanskrit). Flowers have been reported to contain tannins, lupeol, fatty acids, β-sitosterols, cycloartenol esters and flavonoids. Flowers have reported to exhibit cytotoxic, hepatoprotective and antimicrobial activity [8]. A preliminary report [9] on the anti-inflammatory effect of ethanol extract of the leaves of I. coccinea prompted us to study this effect in detail. 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. Literature review revels that no work has been carried on the root of Ixora coccinea for antiinflammatory activity.

MATERIALS AND METHODS

PREPARATION OF PLANT EXTRACTS

Fresh Roots of I. coccinea was collected from Nanded district in January 2011 and were identified and authenticated by Professor P B Deshmukh, Taxonomist, Department of Botany, Science College Nanded. The specimen of plant (Voucher number PES’s SCN/2011/R1) submitted to the herbarium of Botany Department. The collected roots were washed under running water, air dried, and cut into small pieces and further to obtain coarse plant material. The coarse material (234 g) was macerated with ethanol for 2 days. The brownish solution was filtered, dried (12 g, yield 4.3%) and stored airtight at room temperature (30-32o C). The dried powder was dissolved in distilled water (DW) to obtain the required dosages in 1 ml solution (500, 750, 1000, or 1500 mg/kg).

EXPERIMENTAL ANIMALS

Healthy adult cross breed albino male rats (200-250 g) were used for study. The animals were kept in cages (six per cage) under standardized animal house conditions (temperature 28-31oC; photoperiod, approximately12 hrs natural light per day; relative humidity 50-55%) with continuous access to standard animal feed and water ad libtum. Before performing the experiment the ethical clearance was obtained from Institutional Animal Ethics Committee. (Reference No- 1613/PO/a/12/CPCSEA)

ANTI-INFLAMMATORY ACTIVITY CARRAGEENAN-INDUCED PAW EDEMA

The adult cross breed albino 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 1500 mg/kg of ERE, respectively. The rats of group 4, which served as the control, were treated with 1 ml of DW. The rats in the 5 group were treated with 5mg/kg indomethacin (Priya Chemical Mumbai, Maharashtra, India), the reference drug[10,11]. After 1 hr, 0.05 ml of 1% carrageenan (Priyanka Pharma/ M. K Ingredients and Specialties, Mumbai, Maharashtra, India) suspension was injected subcutaneously into the planter surface of the left hind paw [12]. The volume of the injected paw of each of these rats was measured using a plethysmometer (Orchid Scientific & Innovative India Pvt. Ltd., Nashik, Maharashtra, India) at 1 hr prior to the injection of carrageenan and 1, 2, 3, 4, and 5 hrs after the injection.

COTTON PELLET GRANULOMA

Twelve rats were randomly assigned into two groups (n=6/group). Autoclaved cotton pellets (10 mg) were implanted subcutaneously, one on each side above the scapula region, under ether anesthesia [13]. ERE 1500 mg/kg was administered orally for 7 consecutive days starting from the day of injection. On day 8, animals were killed and the pellets along with granuloma removed and dried in an oven at 60oC until a constant weight was obtained.

ANTIHISTAMINE EFFECT

Fur on left lateral side of the back of 18 rats was shaved. Twenty four hrs later, these rats were randomly assigned into three equal groups. Group 1 was treated with 1500 mg/kg of ERE orally. The other groups were treated with 0.67 mg/kg of chlorpheniramine and 1 ml of water respectively [11]. After 1 hr, these rats were subcutaneously injected with 50 μl of 200 μg/ml histamine dihydrochloride (Shanghai Golden Lili Chempharma, China) in the skin where the fur had been shaved, and 2 min later the area of the wheal formed was measured[14].

EVALUATION OF ANTIOXIDANT ACTIVITY

Antioxidant activity was assessed using thiobarbituric acid reactive substances assay [15]. Vials containing the reagents were treated in triplicate with the ERE so that the final concentrations of the extract in the various vials was 0.25, 2.5, 12.5, 18.8, 25, 50, or 125 μg/ml. Butylated hydroxyl toluene (BHT) (100 mg/ml) was used as the positive reference and DW was used as the control. The vials were mixed well and incubated at 95oC for 60 min and then allowed to cool. Butanol (5ml) was added, mixed well, and centrifuged at 1500×g for 5min.The absorbance of the butanol layer was measured at 532 nm, and the antioxidant index was calculated as follows: Antioxidant index = (1-T/C) ×100 (where T = absorbance of test and C= absorbance of control).

PHYTOCHEMICAL ANALYSIS

Phytochemical screening of the ethanolic root extract was carried out [16]. The ERE was subject to column chromatography (30cm length and 3.7 cm diameter) on reverse phase C-18 silica gel. 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 (Sigma Aldrich silica gel precoated on plastic plates and Krackeler Scientific, Incorporated, New York 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 [17. (AlCl3test: for flavonoids; diazotized para nitroaniline: for phenols; para toluene sulphonic acid: for flavonoids and steroids; SbCl3 in acetic acid test: for steroids; Libermann Burchardt spray: for triterpenoid glycosides; and Dragendorff’s reagent and iodoplatinate reagent: for alkaloids).

STATISTICAL ANALYSIS

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

RESULTS

CARRAGEENAN INDUCED PAW EDEMA

The results obtained are summarized in Table 1. As shown, all the doses of ERE 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 antiinflammatory effect was much stronger (77-90%).

COTTON PELLET GRANULOMA

The ERE caused a significant (p < 0.05) and marked inhibition (by 36.1%) of granuloma weight as compared to control (control vs. treatment: 29.2±9.6 vs. 18.6±7.1 mg).

ANTIHISTAMINE EFFECT

The ERE 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 ERE had promising (compared to BHT control) and dose dependent (r2 =0.9; p < 0.05) antioxidant activity.

PHYTOCHEMICAL ANALYSIS

Phytochemical screening of the ERE 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 AND CONCLUSION

This study examined the anti-inflammatory activity of ERE of I. coccinea in rats using the carrageenan induced paw edema test (acute inflammatory model) and cotton pellet test (chronic inflammatory model) oral administration. The results showed that the ERE has promising anti-inflammatory activity 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 [18]. In this test, development of edema (inflammatory response) is a biphasic event with a maintenance phase in between [18]. In this study ERE simultaneously inhibited all these phases. The initial phase (1-2 hrs) is primarily mediated by histamine and serotonin [18]), but platelet activating factor and arachidonic acid metabolites also play a role [19]. The ERE had strong antihistamine activity that could impair microvascular leakage induced by carrageenan [20] and thereby inhibits the initial phase of the edema test. Histamine stimulates vessel endothelial cells to increase vascular permeability [20]. Further, the ERE contained triterpenoid glycosides, many kinds of triterpenoids from angiosperms are known to impair histamine release from mast cells and exert antiinflammatory effects [21]. Such a mode of action is possible in this study as well. The edema maintained between the first and second phase (2-3 hrs) is due to kinin like substances, especially bradykinin[18]. Curtailment of this maintenance phase indicates that the ERE inhibited bradykinin release and/or its vascular permeability promoting action. The ability of the ERE of I. coccinea to inhibit the late phase of the formalin test of nociception[22] provides support to this nation. However, new research is needed to clarify this point. On the other hand, the delayed phase of the carrageenan test (3-6 hrs) has been linked to release of prostaglandins, arachidonate metabolites, neutrophil migration, release of oxygen free radicals, proteolytic enzymes, as well as other neutrophil derived mediators[19,23]. The ERE contained flavonoids and tannins. Flavonoids [25, 26] and tannins [24] impair cyclooxygynase /lipooxygenase activities that would reduce the levels of prostaglandins and other arachidonic acid metabolites. Such a mechanism may account for impairment of the late phase. In addition, the ERE showed marked and dose dependent antioxidant activity. Carrageenan paw edema is sensitive to antioxidants [19]. Thus, it may be inferred that this antioxidant is one of the mechanisms by which ERE mediates impairment in the late phase of the antiinflammatory response. The antioxidant activity of the ERE may be attributed to its flavonoids and phenols [25, 26]. On the other hand, the ERE had failed to inhibit heat induced hemolysis of rat erythrocytes in vitro [22]. This indicates that ERE cannot stabilize lysosomal membranes to inhibit the release of proteolytic enzymes; lysosomes play a major role in the inflammatory reaction [27, 28], and there is a close similarity between erythrocyte and lysosomal membrane system [27].

The cotton pellet test is considered a model for studies on chronic inflammation [13], and inflammatory granuloma is considered as a typical feature of established chronic inflammatory reaction [29]. The fact that the ERE was effective in suppressing granuloma formation in this model indicates that it may be effective in chronic inflammatory conditions. This inhibitory effect was seen when ERE was administered after the onset of inflammation, which is claimed to reflect a genuine anti-inflammatory action [30] and also shows the potential to be used as curative in different inflammatory conditions.

The previous study has shown that this ERE does not induce unacceptable side effects including gastric lesions and is well tolerated even after subchronic administration. Further, it has strong antinociceptive activity [22]. The presence of both inflammatory and antinociceptive activities in a single drug, which is devoid of major side effects, is extremely valuable, as inflammation is generally accompanied with pain[31], and currently available both steroidal and nonsteroidal allopathic anti-inflammatory drugs are associated with unacceptable and often severe side effects [31]

In conclusion, this study scientifically demonstrates, for the first time, promising anti-inflammatory activity in I. coccinea Roots. This is an important finding, both globally and locally, because inflammation is a common medical condition for which available drug therapies are poor [31]. About 3.5 to 4 billion people in the world rely on plants as sources of drugs [32], Roots of I. coccinea are noncommercial and abundantly available throughout the year.

Tables at a glance

Table 1 Table 2

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