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Brazilian Journal of Oral Sciences
Piracicaba Dental School - UNICAMP
EISSN: 1677-3225
Vol. 7, Num. 27, 2008, pp. 1666-1672

Brazilian Journal Oral Sciences, Vol. 7, No. 27, Oct/Dec, 2008, pp. 1666-1672

Antiinflammatory activity of ricinus communis derived polymer

Adriana Cristina Valderramas1*; Sérgio Henrique Pereira Moura2*; Maira Couto3, Silvana Pasetto5* ; Gilberto Orivaldo Chierice4; Sérgio Augusto Catanzaro Guimarães5*; Ana Claudia Bensuaski de Paula Zurron5*

1Master Degree in Oral Biology, Prorector for Research and Postgraduate Affairs,
2Undergraduate Student, Department of Biological Sciences
3Biologist, Department of Biological Sciences, Sacred Heart University, Bauru, SP, Brazil
4Professor, Institute of Chemistry of São Carlos, School of Engineering of São Carlos, University of São Paulo, São Carlos, SP, Brazil.
5Professor, Prorector for Research and Postgraduate Affairs, Sacred Heart
*University of Sagrado Coração, Bauru, SP, Brazil.
Correspondence to: Ana Claudia Bensuaski de Paula Zurron Universidade do Sagrado Coração (USC), Rua Irmã Arminda, 10-50, Jardim Brasil, 17011-160 Bauru, SP, Brasil Phone +55-14-2107-7260 Fax +55-14-2107-7254 E-mail: anabensuaski@yahoo.com.br

Received for publication: August 01, 2008 Accepted: November 11, 2008

Code Number: os08041

Abstract

Aim: The present study aimed to evaluate the antiinflammatory activity of the polymer derived from Ricinus communis and its mechanism of action.
Methods: The antiinflammatory activity was investigated in chronic and acute animal models and the mechanism of action involved in the antiinflammatory activity was determined by the in vitro phospholipase A2 (PLA2) enzyme assay.
Results: In mouse ear edema (10.0 mg/ear) and granulomatous tissue formation (500 mg/kg) models, the polymer inhibited the inflammatory response in 75.08 ± 1.80% and 61.70 ± 1.80% of the cases, respectively (p<0.001). Oral administration of the Ricinus communis polymer (500 mg/kg) inhibited 72.00 ± 1.20% of formalin-induced inflammation. Topical administration of the polymer on oral lesions of mice showed that the oral mucosa was recovered in 60.00 ± 1.40% (p<0.05) of the cases. In in vitro assay, the phospholipase A2 enzyme was inhibited by the Ricinus communis polymer (5.0 mg/mL) in a dose-dependent manner (84.60 ± 1.41%).
Conclusion: the polymer derived from Ricinus communis showed a significant antiinflammatory activity, confirming that the pharmacological mechanism involved in this antiinflammatory action was related to the inhibition of the PLA2 enzyme.

Key Words: polymer, Ricinus communis, phospholipase A2, antiinflammatory activity.

Introduction

Reconstruction of local bone defects resulting from trauma or bone tumors is a major problem in orthopedics and dental surgery. Currently, large amounts of autogenous bone grafts are used to manage these clinical situations. Although autogenous bone grafts, which combine osteoconductive, osteogenic and osteoinductive properties, are the gold standard for treating bone defects, only a few donor sites in the skeletal system are appropriate for supplying autogenous bone1 .

Polymers are the most versatile class of biomaterials, being extensively applied in medicine and biotechnology2 . A natural polyurethane resin obtained by polymerization of the polyester polyol is derived from the castor beam plant (Ricinus communis ), which native of tropical regions3 .

According to Kojima et al.3 , this polymer has been developed for bone repair because of its capacity to stimulate new fibroblast formation and its progressively replacement by bone around and inside the material’s porosities material with no late inflammatory response and no signs of systemic toxic effects.

The methanolic extract obtained from Ricinus communis root showed a significant antiinflammatory and free radical scavenging activity4 . The pharmacological activity may be due to the presence of phytochemicals in the plant extract, like flavonoids, alkaloids and tannins, which have various biological actions4 .

In Endodontics, a polymer detergent derived from Ricinus communis has been developed for use as a root canal irrigant5-8. The polymer detergent has similar antimicrobial activity to that of 0.5% sodium hypochlorite when used for irrigation of necrotic root canals5-8 . Although sodium hypochlorite is still the most widely used root canal irrigant, studies have searched for alternative solutions and therapeutic resources that might improve the success rate of endodontic treatment8 .

Barros et al.1 reported that polymers offer the advantage that has biocompatibility with dental tissue, is osteoconductive and osteogenic, and provide space for the formation of new bone. The Ricinus communis polymer showed fibroblastic neoformation progressively replaced by bone around and inside the porosities of material with absence of late inflammation reaction1 .

Specific inhibitors of phospholipase A2 (PLA2) have been sought for a variety of purposes. Since this enzyme is believed to control a number of processes ranging from mobilization of eicosanoids to metabolism of phospholipids, these inhibitors could be useful for controlling inflammatory processes such as rheumatoid arthritis, asthma and psoriasis9. Enzyme inhibition assays are important tools in the search for new drugs, and so the PLA2 assay could determine the mechanism of action of Ricinus communis polymer in the antiinflammatory study. The present study investigated the acute and chronic antiinflammatory activity of the polymer obtained from Ricinus communis in mouse ear edema, formation of granulomatous tissue in rats, formalin-induced paw edema in mice and in oral mucosa lesions in mice. The antiinflammatory mechanism of action of Ricinus communis was assessed using the PLA2 enzyme assay. Morphological analyses were also performed by means of topical antiinflammatory activity tests.

Material and Methods

Animals

Fasted male Wistar rats (200-250 g) and male Swiss mice (20-25 g) obtained from the Central Animal House of Sacred Heart University (USC) were used. The animals had free access to tap water. Experimental protocols were approved by the institutional Ethics Committee and were conducted according to recommendations of the Canadian Council on Animal Care10. All experiments were performed in the morning, according to current guidelines for laboratory animal care and ethical guidelines for the investigation of experimental inflammation in conscious animals11 .

Chemical Agents and Reagents

A natural detergent obtained from Ricinus communis was used in this research. This substance was produced by the Group of Analytic Chemistry and Technology of Polymers from University of São Paulo, São Carlos, SP, Brazil. The technology created at the Institute of Chemistry of São Carlos produced a polymer that had characteristics of a bone substitute in Brazil12 .

Other substances were also used in the experiments: arachidonic acid, hydrocortisone, indomethacin, dexamethasone, formalin, sodium taurocholate, calcium chloride, phosphatidylcholine dipalmitate, phospholipase A2 and sodium hydroxide (Sigma-Aldrich Corp., St Louis, MO, USA), acetone, propolix (Herbarium Laboratório Botânico Ltda, Colombo, PR, Brazil.), tween 80, ethyl alcohol and xylene (Merck KGaA, Darmstadt, Germany), xylazine hydrochloride (Bayer S/A, São Paulo, SP, Brazil), ketamine (Laboratórios Pfizer Ltda, São Paulo, SP, Brazil).

Acute Toxicity in Animals

The acute toxicity of the Ricinus communis polymer was investigated by intraperitoneal and oral administration in male mice (n=50) weighing 20-25 g. In this assay, increasing doses of the test substance were orally and intraperitoneally administered to groups of 10 animals per dose (100, 300, 500, 700, 1,000 mg/kg). The animals were observed for 14 days and the mortality rate was recorded13 .

Mouse Ear Edema Induced by Arachidonic Acid

The in vivo antiinflammatory activity of Ricinus communis polymer was assessed in the mouse ear edema model using arachidonic acid to induce inflammation14 . Control mice received only the irritant agents, whereas experimental mice also received the Ricinus communis polymer (5 and 10.0 mg/ear) applied topically together with the irritant agent. Arachidonic acid was dissolved in acetone at concentrations of 10 mg/mL and each mouse received 0.5 μg/ear of arachidonic acid on the left ear. The drugs were applied topically to the inner surface of the ear with an automatic pipette in a volume of 5 μL of arachidonic acid. The right ear (control) received 20 μL of acetone (vehicle). The mice were killed by cervical dislocation 1h after treatment with arachidonic acid. Each ear was removed with a metal punch (6 mm diameter disc) and the edema was calculated by subtracting the weight of the right ear (control) from the left treated ear. Hydrocortisone (217 μg/ ear) was used as a positive control.

Drug effects were expressed as percentage of inhibition, according to the following equation: [weight of left minus right control ears] - [weight of left minus right treated ears] X 100¸ weight of left control ear

Phospholipase Activity

The inhibition of PLA2 activity (purified from Apis mellifera bee venom) by the polymer obtained from Ricinus communis was assayed by measuring the decrease in the pH of the incubation mixture using a pH electrode in a closed stirring chamber.

The test substance was incubated for 30 min with the PLA2 enzyme and added to the assay medium containing 4 mM sodium taurocholate, 12 mM calcium chloride and 7 mM phosphatidylcholine dipalmitate (Sigma-Aldrich Corp.). This technique is reliable under pH 5.015. In the present study, the mean initial pH of the phospholipids mixture was 8.0. Positive controls were set up using purified PLA2 (0.33 μg/mL) from bee venom. The total incubation volume was 2.5 mL. The four different concentrations of the polymer used in these in vitro experiments were 2.5, 3.0, 3.5 and 5.0 mg/mL. The PLA2 inhibition was calculated according to the following equation: [D pH (treated/min)¸ D pH (control/60 min)] x100 = % enzymatic reaction

Granulomatous Tissue Formation

Cotton rolls (Johnson and Johnson, New Brunswick, NJ, USA) were cut into 5-mm pieces and sterilized in groups of four pellets (160 mg). Rats were anesthetized with xylazine hydrochloride (50 mg/kg) and ketamine hydrochloride (180 mg/kg) and then the pellets were implanted subcutaneously into four symmetrically distributed positions in the abdomen16-17 .

Two hours after implanting the cotton pellets, the animal groups (n=10 each) were treated orally by gastric gavage with tween (10 mL/kg), dexamethasone (0.2 mg/kg), and the Ricinus communis polymer at doses of 250 and 500 mg/kg. Daily application of these substances continued for 6 days. On the 7th day, the animals were killed by cervical dislocation, the cotton pellets removed, dried (60°C) and weighed. The difference between the initial and final dry weight corresponded to the weight of the granulomatous tissue formed.

Formalin-Induced Paw Edema in Mice

The method applied was similar to that described by Henriques et al.18. Groups of male animals were treated orally by gastric gavage with tween (10 mL/kg), indomethacin (30 mg/kg) used as positive controls or with the Ricinus communis polymer (250 and 500 mg/kg, p.o. respectively). The polymer was administered 30 min before the injection of 2% formalin in PBS (30 μL/paw) into the sub plantar area of the left hind paw. Paw volume was measured 4 h after formalin injection. Edema was calculated as the difference (μL) between the injected and control paw. The area under the curve (AUC) versus Ä paw volume was calculated for each animal and edema was expressed as the mean ± SEM of AUC.

Effect of Ricinus communis in Oral Mucosa Lesions of Mice

Male Swiss mice were used in this study. An ulcerated lesion was produced with topical application of NaOH (40%) in the oral mucosa of anesthetized animals19-20 . Lesions of the experimental group were treated with a daily topical administration of the polymer derived from Ricinus communis at dose of 250 and 500 mg/kg. Lesions of negative and positive control animal groups were treated with a solution of tween 80 and a propolis solution (propolix 11%), respectively. Fourteen days later, the animals were sacrificed. The ulcerated lesions were counted and classified according to level of severity presented21

Histological Analysis

Tissues obtained from the ears and oral mucosa lesions of mice were fixed in Bouin for 24 h, then dehydrated through ascending concentrations of ethyl alcohol, cleared with xylene, embedded in Histosec (Merck; 11609), and prepared for microtomy. Eight-micrometer-thick sections were then deparaffinized and rehydrated through

Antiinflammatory activity of ricinus communis derived polymer

descending concentrations of ethyl alcohol. These samples were stained with hematoxylin and eosin for morphological analysis, using hematoxylin for 15 min. The samples were washed in running water for 10 min and sections turned from red to blue. Sections were stained with eosin for 10 min and washed in water, dehydrated, placed in 95% alcohol, cleared and mounted in resin22. Photomicrographs were obtained with an Axiophot in Photomicroscope (D7082; Carl Zeiss, Jena, Germany). The lesions were counted by histomorphometry analysis.

Statistical Analysis

Results were reported as means ± SEM and were analyzed statistically by analysis of variance followed by Tukey’s test. P values of less than 0.05 were considered significant.

Results

Acute Toxicity in Animals

Ricinus communis polymer at doses of up to 1,000 mg/kg did not cause any mortality in male albino mice (n=30) during 14 days after intraperitoneal injection and oral administration.

Mouse Ear Edema Induced by Arachidonic Acid

The mouse ear edema reached a maximum at 1h after arachidonic acid application. The Ricinus communis polymer significantly inhibited swelling, considerably reducing the vascular permeability response to arachidonic acid application. The polymer (5.0 and 10.0 mg/ear) inhibited the inflammation induced by arachidonic acid in a concentration-dependent manner in 54.40 ± 1.60% and 75.08 ± 1.80% (p<0.001, respectively) (Table 1; Figure 1). The positive control, hydrocortisone (217 μg/ ear) inhibited 77.00 ± 1.20% of the topical inflammation (p<0.001). Histologically, the topical application of arachidonic acid caused neutrophil accumulation (Figure 1A). In this experiment, a rapid, albeit transient edema, was induced accompanied by erythema.

Ricinus communis polymer application immediately after the irritant agent inhibited acute inflammation caused by arachidonic acid. In this treatment, neutrophils were absent (Figure 1B), confirming the antiinflammatory effect mentioned by Ricinus communis polymer.

Phospholipase Activity

At concentrations up to 5.0 mg/mL, the polymer obtained from Ricinus communis had a significant inhibitory activity against PLA2 enzyme (0.33 μg/mL bee venom) (Table 2). The polymer inhibited the PLA2 activity around 84.60 ± 1.41% (p<0.05). These results showed that the possible mechanism of action of Ricinus communis involved in its antiinflammatory activity would occur by PLA2 inactivation, that releases arachidonic acid during the beginning of the inflammatory cascade.

Granulomatous Tissue Formation

Treatment for 6 days with dexamethasone and the polymer of R. commus inhibited the formation of granulomatous tissue induced by implantation of subcutaneous cotton pellets into the abdominal region (Table 3). The polymer at the dose of 250 and 500 mg/kg inhibited the inflammatory process by 55.70 ± 1.20 and 61.70 ± 1.80%, respectively (p<0.001). Dexamethasone was effective in inhibiting inflammation by 72.30 ± 1.60% (p<0.001).

Formalin-Induced Paw Edema in Mice

Intradermal injection of formalin (1%, 20 μL), into one of the hind paw of normal rats (control group-treated orally with tween) caused a local inflammatory response, which reached a maximum intensity of edema at hour 4 after application of the phlogistic agent.

Ricinus communis polymer (250 and 500 mg/kg, p.o.) significantly decreased paw swelling by 61.80 ± 1.30% and 72.00 ± 1.20%, respectively (Table 4, p<0.001). Treatment of the animals with indomethacin (30 mg/kg), the reference antiinflammatory, significantly reduced the intensity of edema induced by formalin around 80.00 ± 2.00% (p<0.001).

Effect of Ricinus communis in Oral Mucosa Lesions of Mice

In experiments of oral mucosa lesions induced by sodium hydroxyl 40%, the polymer obtained of Ricinus communis (250 and 500 mg/kg) healed respectively 35.00 ± 1.20% and 60.00 ± 1.40% of the oral mucosa lesion (p<0.05) (Table 5; Figure 2). The positive control (propolix) healed 78.00 ± 1.60 (p<0.05). When only NaOH was applied topically, it was observed that the surface epithelium was destroyed (Figure 2A). In Figure 2B, the oral lesion was healed and the surface epithelium was recovered in mice treated with the Ricinus communis polymer.

Discussion

The development of polyurethane resins derived from Ricinus communis widened the scopes in different fields of medical and dental research, as they were shown to have biocompatibility and potential applicability in several areas8. In Endodontics, a detergent derived from Ricinus communis has been proven to have similar antimicrobial activity as that of 0.5% sodium hypochlorite when used for irrigation of necrotic root canals5-6 .

In the present study, we examined the antiinflammatory activity of the polymer obtained from seeds of Ricinus communis. This substance is a detergent derived from castor bean oil, has similar antimicrobial activity to that of 0.5% sodium hypochlorite when used for irrigation of necrotic root canals, is biocompatible with the periapical tissues, increases dentinal permeability and has similar ability to remove smear layer from the root canals as that of 17% EDTA8 .

Studying the extract of leaves and root of Ricinus communis, Ilavarasan et al.4 observed a significant antiinflammatory activity in rats, possibly due to the presence of flavonoids, alkaloids and tannins present in the plant extract. It was also reported that flavonoids obtained from Ricinus communis root extract would explain its free radical scavenging activity. The excessive generation of reactive oxygen species (ROS) leads to a variety of pathological processes, such as inflammation, diabetes, hepatic damage and cancer4 .

Inflammation is generally defined as the response of living tissue to an injurious stimulus23. The usual features of inflammation include the activation of epithelial cells and resident macrophages, and the recruitment and activation of neutrophils, eosinophils, monocytes and lymphocytes24 . Leukotriene and prostaglandin synthesis is involved in arachidonic acid-induced ear edema25. Chemically induced edema represents an acute local inflammation eliciting a complex series of physiological events involving many processes in which components of a plant extract may interact, inhibiting kinins and prostaglandins on vascular permeability that appear to be involved with inflammatory process26-27 . Acute and chronic inflammatory processes can be induced by several means, and antiinflammatory agents exert their effects through different manners28-29. For screening of new antiinflammatory compounds, the croton oil-induced mouse ear edema assay is widely used in conjunction with the in vitro phospholipase A2 assay30-31. Enzyme inhibition assays are important tools in the search for new drugs. Is has been established that the inflammation induced by arachidonic acid involves an increase in PLA2 activity, which, in turn, leads to the release of arachidonic acid and subsequent biosynthesis of leukotrienes and prostaglandins, thus also involving the lipooxygenase pathway32 .

Bresnick et al.33 stated that PLA2 catalyzes the sn-2 hydrolysis of phospholipids releasing free fatty acids, predominantly arachidonic acid and lysophospholipids. These products can have biological actions or be further metabolized to form a variety of proinflammatory lipid mediators including prostaglandins, leukotrienes plateletactivating factor and thus the inhibition of PLA2 by pharmacological agents should have led to an antiinflammatory effect.

Glycosides obtained from Ipomoea imperati also showed activity in both tests mentioned above, reducing mouse ear edema induced and inhibiting bee-venom phospholipase A2 (PLA2) activity32 .

The cotton pellet-induced granuloma was used as a chronic model to evaluate the antiinflammatory effects of natural products and have good predictive value for screening antiinflammatory agents34. This method is a suitable test for assessing the antiinflammatory activity drugs and widely used to evaluate the transudative and proliferative components of chronic inflammation35. In chronic inflammatory states, the efficacy of antiinflammatory agents can be indicated by inhibiting the increase in fibroblasts and the infiltration of neutrophils and exudation36-37 .

When macrophages, epithelioid cells and multinucleate giant cells predominate at the site of inflammation, the lesion is named nodular chronic inflammation or granuloma. The fluid absorbed by the pellet greatly influences the wet weight of the granuloma. Dry weight correlates well with the amount of granulomatous tissue formed38 .

Catanzaro Guimarães et al.38 reported that the inflammatory process is represented by edema, which is formed in the early stages of granuloma development, or as a result of immune reactions participating in the pathogenesis of these lesions. These reactions originate immune complexes that activate the complement system to generate vasodilatator mediators. The latter mechanism is represented by cells, collagen fiber and newly formed bloods vessels38 .

The formalin’s test is a well-known model of chemically induced inflammation and nociception39. The intra-plantar injection of diluted formaldehyde causes inflammation that release mediators such as bradykinin, histamine, sympathomimetic amines, tumor necrosis factor-á and interleukins40-43. In addition to these mediators, local prostaglandin levels are responsible for the progress of nociception and are targeted by most non-steroidal antiinflammatory drugs43 .

In the formalin assay, edema that is maintained during the plateau phase occurs due to kinin-like substances. Later, the swelling phase prevails due to the release of prostaglandin-like substance44. Thereby, the formalin assay is well suited for the comparative bioassay of antiinflammatory agents since the relative potency estimates obtained for most drugs tend to reflect clinical experience45 . In experiments of oral mucosa lesions induced by sodium hydroxyl 40%, the Ricinus communis polymer was tested. Ulcers are defined as local defects on the surface of tissues or organs. These defects are produced by loss of surface epithelium with exposure of connective tissue. Contact of the mucous membrane with physical or chemical agents represents the main source of mouth ulcers46. Oral ulcers are one of the most common complaints in the dental practice and are usually caused by mechanical trauma induced by ill-fitted dentures, orthodontic appliances and fractured crowns and restorations47 .

With major loss of cells involved in oral lesion formation, the inflammatory and repair processes occur by secondary union and involve a series of vascular, cellular, neurological and humoral events. Ulcer healing is a dynamic process of filling mucosal defects with proliferating and migrating of epithelial cells as well as connective tissue, resulting in the reconstruction of the mucosal architecture48 .

In conclusion, the findings of the present study showed a potent antiinflammatory activity of Ricinus communis polymer, this antiinflammatory effect being related to the phospholipase A2 enzyme inhibition. This study establishes the therapeutic rationale of using Ricinus communis polymer in various inflammatory events, which has antimicrobial activity and has been used for root canal irrigation.

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