Anti-inflammatory and healing action of oral gel containing borneol monoterpene in chemotherapy-induced mucositis in rats ( Rattus norvegicus )

*Correspondence: B. J. Nascimento-Júnior. Departamento de Farmácia, Faculdade de Farmácia, Universidade Federal de Pernambuco. Av. José de Sá Maniçoba, S/N – Centro – 56304-917 – Petrolina/PE, Brazil. Phone: +55 87 98807-5035; +55 87 2101-6862. E-mail: braz.jose@univasf.edu.br Anti-inflammatory and healing action of oral gel containing borneol monoterpene in chemotherapy-induced mucositis in rats (Rattus norvegicus)


INTRODUCTION
The clinical manifestations most commonly associated with treatment of cancer of the oral cavity are oral mucositis (OM), osteoradionecrosis, dry mouth, radiation caries, trismus, opportunistic infections and dysphagia, among others.OM is an important complication of oncological treatments such as head and neck radiotherapy, chemotherapy and Hematopoietic Stem Cell Transplantation (HSCT), autologous or allogeneic, having a prevalence of 75%-99%.(Simões, Castro, Cazal, 2011;Patussi et al., 2014).Therefore, the healthcare team should have a broad knowledge of all oral complications of anticancer therapy in order to improve patients´ quality of life (Sera et al., 2013).
Among the different oral manifestations, mucositis is the most common and distressing condition that develops as an acute adverse effect of chemotherapy and radiotherapy of the head and neck and is one of the leading causes of disability that limits cancer therapy dose.This complication leads to therapy alteration, dose reduction, delay, interruption or termination and can negatively affect the healing process.It is clinically characterized by erythematous and ulcerative lesions that affect the vermilion of the lips and oral mucosa (Smith et al., 2007).
Although borneol has been shown to be effective in lesions of the oral cavity, the literature reports no preclinical models demonstrating its action in oral mucositis.In order to fill this gap, the present study was conducted investigating the action of gels containing borneol in 5-Fluorouracil-induced oral mucositis using an animal model with Wistar rats.

Preparation of Aristoflex® gels containing borneol and stability tests
Aristoflex polymer was mixed with methylparaben in water at ambient temperature under stirring at 400 rpm (Heidolph RZR stirrer, Germany) for 30 minutes to complete gelation.The mixture was then incorporated into (+)-borneol powder (Sigma-Aldrich, USA) at concentrations of 1.2% and 2.4%.The gels thus obtained (30 g each, containing borneol at concentrations of 0%, 1.2% and 2.4%) were packaged, labeled and analyzed for organoleptic (appearance, color and odor) and physicochemical (pH, conductivity and viscosity) parameters and also for both primary and accelerated stability (Oliveira, 2009).

Animals
Male Wistar rats (300-350 g, 12-16 weeks, n=48) were acclimatized for six days prior to experiments and housed during the experimental period at 22±2 °C under a 12/12h light-dark cycle with access to feed and water ad libitum.After the onset of oral mucositis, the animals were tagged and randomly distributed into four subgroups of 12 animals each: G1 Group (Borneol 0%); G2 Group (Borneol 1.2%); G3 Group (Borneol 2.4%); and G4 group (Control).All animals were weighed at the beginning and end of the experiment for clinical evaluation.Another 24 animals were used in pilot tests for dose and technique adjustments, but not included in the results of the study.The project was submitted to the Research Ethics Committee on the use of animals (CEUA -UNIVASF) and was approved under permit 0001/140814 on June 2 nd , 2015.

Treatment subgroups
The G1, G2 and G3 Groups received a daily application of gels at concentrations of 0%, 1.2% and 2.4%, respectively, using disposable flexible swabs (Figure 2D).The G4 Group (Control) received topical application with distilled water.After the application, all animals were prevented from drinking water or eating for 30 minutes to allow time for the gels to act (Tanideh et al., 2014).The treatments were administered from the third to 13th day after injury with acetic acid.

Mucous removal and histology procedure
Six animals from each group were sacrificed by deep anesthesia [ketamine hydrochloride 10% (100 mg/ kg) and xylazine hydrochloride 2% (10 mg/kg) i.p., 0.5 mLper animal] on days seven and fourteen after injury.Subsequently, right cheek mucous was removed from each animal with surgical scissors and scalpel blade number 15 and placed in 10% neutral buffered formalin solution for 48h (Akgullu et al., 2015).
Specimens were dehydrated, diaphonized, embedded in paraffin and cut transversely into 5 µm-thick slices on a microtome (Leica Biosystems, Wetzlar, Germany).Mucous samples were stained with hematoxylin-eosin (HE) and Masson's trichrome.All sections were mounted on glass slides with cover slips using Entellan (Merck, Darms tadt, Germany) for microscopy.

Histological evaluation
Fields were scanned using a Leica® DM2500 microscope (Leica Biosystems, Wetzlar, Germany) under x4 and x10 objective lenses, coupled to a CELESTRON® Digital Microscope Imager Model 44421 camera (Torrance, California, United States) using the software supplied with the digital camera.The same light intensity was used in all digitalization; images were acquired in RGB format at a resolution of 2048 X 1536 pixels with JPEG extension.For quantitative analysis, the ImageJ® program was employed using the plugin "Cell Counter" and the number of inflammatory cells and amount of blood vessels stained with HE on the slides were measured.To evaluate percentage collagen, the slides stained with Masson's trichrome were photographed and then processed using ImageJ® and the Threshold plugin to measure percentage collagen at the injury site (Vilela-Goulart et al., 2008).
The semiquantitative analysis was performed by a blinded evaluator who observed inflammatory parameters including presence of cellular inflammatory infiltrate, vasodilatation, necrosis, ulceration, abscess, hemorrhage and edema.These parameters were attributed scores of 0-3, as described by Lima et al. (2005): Score 0 -normal epithelium and connective tissue without vasodilatation; absence of, or mild, cellular infiltration; absence of hemorrhagic areas, ulcerations or abscesses; Score 1mild vascular ingurgitation, re-epithelization areas; mild inflammatory infiltration with mononuclear prevalence; absence of hemorrhagic areas, edema, ulcerations or abscesses; Score 2 -moderate vascular ingurgitation, areas of hydropic epithelial degeneration, inflammatory infiltration with neutrophil prevalence, presence of hemorrhagic areas, edema and some ulcerations, absence of abscesses; Score 3 -severe vascular ingurgitation and dilatation, inflammatory infiltration with neutrophil prevalence, presence of hemorrhagic areas, edema and extensive ulceration and abscesses.

Statistical analysis
Comparisons for all variables were performed independent of day, between days, independent of group and between groups.Quantitative and semiquantitative variables were expressed as means.For comparisons, the two-way ANOVA test, followed by Tukey's post-test, was used for comparisons.In all tests, p≤0.05 was adopted for the level of rejection of the null hypothesis for significant values.

RESULTS AND DISCUSSION
The formulation developed was stable and transparent with a smooth texture, homogeneous appearance and characteristic aroma.Borneol 0% had pH = 5.0; Conductivity=100.0 mV; Viscosity=58,500 mPa.s; and centrifugation without phase separation.Borneol 1.2% had a pH = 4.7 ± 0.1; Conductivity=120.2 ± 0.5 mV; Viscosity=45,360 mPa.s; and centrifugation without phase separation.Borneol 2.4% had a pH=4.5±0.2;Conductivity=139.0±1.5 mV; Viscosity=55,560 mPa.s; and centrifugation without phase separation.Acid pH values were expected because the Aristoflex® gel base was formulated with a pH of 5.0.In a previous study, the same gel base had a pH of around 5.0, no phase change and good viscosity (Russo, Guimarães, Cardoso, 2008).Furthermore, borneol is also considered an acid pH substance (Xiao-Fei et al., 2008).Dantas et al. (2016) used a topical gel formulation with 5% borneol which had a pH of 3.95.
Group (Borneol 2.4%) had discrete hydropic degeneration, spongiosis, and partial reepithelialization in five animals.CT showed PMNs and the presence of few congested blood vessels.The G4 Group (Negative control) exhibited ulcerated areas, hydropic degeneration, spongiosis and intense exocytosis.CT had edema and necrosis, moderate congested blood vessels, as well as moderate and diffuse presence of PMNs (Figure 4).
At day 14, a statistically significant difference (p <0.05) in scores was found for the G3 group (Borneol 2.4%) compared with both the G1 (Borneol 0%) and G4 (Control) groups.This result indicated better healing for Borneol 2.4% (Figure 3).G1 Group (Borneol 0%) showed hyperkeratosis and full reepithelialization.CT had congested blood vessels, as well as moderate and diffuse presence of Mononuclear (MN) cells.The G2 Group (Borneol 1.2%) showed reepithelialization but with more blood vessels in G3.CT showed numerous clogged blood vessels and a diffuse mild chronic inflammatory process.The G3 Group (Borneol 2.4%) had a normal aspect and full reepithelialization.CT showed normal features, in addition to granulation tissue with fibroblasts, congested blood vessels and discrete, diffuse presence of MN.The G4 Group (Negative Control) had acanthosis (thickened spinous layer) with loss of integrity of basal membrane and exocytosis.Epithelialization occurred with different morphology in four animals.The CT was found to contain a large amount of granulation tissue with fibroblasts, congested blood vessels and chronic inflammatory infiltrate (Figure 5).
At seven days, the group treated with Borneol 2.4% showed a lower number of vessels than the other groups (G1, G2 and G4).The G3 Group showed statistical significance (p<0.05) when compared to the three other groups.The G2 Group (Borneol 1.2%) showed statistically significant differences when compared to both the G1 Group (Borneol 0%) and Control Groups.There were no differences between the G1 and Control Groups.At 14 days, there was statistical significance between all cross comparisons except the G1 Group and Control Group (Figure 6).
While healing depends on the neoformation of blood vessels, persistent or excessive vessels may denote the existence of chronic inflammation or fibrosis (Abbas et al., 2010).On histological analysis of the blood vessels, the Control Group showed a greater number of blood vessels in the final process of wound healing compared to the other groups (G2 and G3).This indicates that the healing process in the Control Group required a greater angiogenesis for the formation of granulation tissue (Abruceze, 2013).
The proliferation of blood vessels is stimulated by vascular endothelial growth factor (VEGF).Some factors stimulate the expression of VEGF in angiogenesis and ischemia, while increases in certain growth factors are responsible for the elevated VEGF expression in chronic inflammation and wound healing.The inflamed tissue is generally in hypoxia, where inflammation may promote angiogenesis by inducing the release of angiogenic factors leading to fibrosis (Nagy, Dvorak, Devorak, 2007).
According to a study by Dai et al. (2009), borneol showed anti-fibrotic action.
Regarding inflammatory cell count, the gels containing borneol at concentrations of 1.2% and 2.4% exhibited anti-inflammatory action at seven and 14 days, reducing the number of these cells at the injury site.The Control Group and Borneol 0% groups, at seven days, showed a much more intense inflammatory process than the treated groups (Borneol 1.2% and Borneol 2.4%), but (2013), in which the authors confirmed the action of monoterpene and suggested that the substance has therapeutic potential in painful and inflammatory diseases.Tanideh et al. (2014) tested the action of Hypericum perforatum L. extract for treating induced oral mucositis in animals.The extract of this plant contains Borneol (CIV, 2012) and was administered by gavage (systemically) and locally via an oral gel.The extract showed antiinflammatory and healing action, especially systemically.
In the present study, percentage collagen at seven days showed statistical significance on comparison of the G1 Group (Borneol 0%) with the G3 Group (Borneol 2.4%) and also on comparisons of the G2 Group and G3 Group with the G4 Group (Control).Comparison of the G2 and G3 Groups with the G4 Group also proved significant at 14 days (Figure 8).
Percentage collagen in the G2 and G3 Groups on slides stained with Masson's trichrome was higher than in both G1 and G4 Groups at 14 days.This was due to the healing effect of borneol which promoted greater formation of fibroblasts with better collagen and fibronectin production in local lesions (Figure 9).Tanideh et al. (2014) also observed increased stimulation of collagen production by topical gel Hypericum perforatum L., which helped repair the wounds, closing areas of damaged oral mucosa in the animals.This effect may be associated with the bactericidal action of borneol, which prevents secondary infection by fungi and bacteria, promoting reepithelialization and standardization of collagen fiber content (Tabanca et al., 2001;Wenqiang et al., 2006).Borneol 1.2% and Borneol 2.4% (14 days).d p<0.05:Borneol 0% vs. Borneol 2.4% (14 days) (Two-Way Analysis of Variance ANOVA, followed by Tukey's post-test).no differences were observed between the two borneol concentrations.Cell infiltrates were less intense in all groups at 14 days.Differences were observed between the G1 Group and G3 Group and also between the two gels (Borneol 1.2% and Borneol 2.4%) compared to the Control Group, but no differences between the two concentrations were evident (Figure 7).
The findings for number of inflammatory cells corroborate the results of the study by Almeida et al.All animals lost weight throughout the experiment due to the side effects of 5-fluorouracil, oral mucositis and reduction in water and feed intake.At 14 days of experimentation, the Borneol 2.4% Group had a lower percentage of weight loss relative to the other three groups (Borneol 0%, Borneol 1.2% and Control), probably due to the greater action of Borneol 2.4% in reducing inflammation and promoting wound healing (Figure 10).

CONCLUSIONS
The Borneol 2.4% Group was statistically superior to the Borneol 1.2%, Borneol 0% and Control Groups in the treatment of induced oral mucositis in rats, promoting angiogenesis control, inflammatory cell count reduction at the injury site and percentage neoformed collagen increase in reepithelialization.The G3 group (Borneol 2.4%) was FIGURE 9 -Microscopic aspects of groups at fourteen days of experimentation.G1 Group treated with gel base (Borneol 0%); G2 Group treated with gel (Borneol 1.2%); G3 Group treated with gel (Borneol 2.4%); and G4 Group (Control).Note the greater quantity of newly formed collagen in treated groups (G2 and G3).Magnification x10.Masson's trichrome.statistically superior to the other three groups on the semiquantitative evaluation, promoting improvement in reepithelialization and healing of chemotherapy-induced oral mucositis.
The confirmation of anti-inflammatory and healing action of borneol (especially Borneol 2.4%) in oral mucositis in rats renders it a good marker for predicting this activity for plant extracts rich in this substance.The study results showed that borneol has anti-inflammatory and healing action in induced oral mucositis in cheek mucosa of Wistar rats.

FIGURE 2 -
FIGURE 2 -Experimental procedure for testing.A. Placement of filter paper soaked in 50% acetic acid; B. Contact time with cheek mucosa of 60 s; C. Aspect of standardized lesion (9 mm 2 ) after filter paper removal; D. Appearance of gels used in treatments.