Topical Application of Orabase Bromelain Gel Reduces Inflammatory Process, Bone Loss and Steatosis in Experimental Periodontitis

Abstract

This study aimed to develop a bromelain-based gel and evaluate its effects in a ligature-induced periodontitis model in rats. The bromelain-based gel was formulated in two concentrations (1% and 10%). Ten animals were used per group: Control, Periodontitis, Periodontitis + Neutral Gel, Periodontitis + 1% Bromelain Gel, Periodontitis + 10% Bromelain Gel and Periodontitis + 2% Chlorhexidine Gel. The clinical parameters of tooth mobility (TM), gingival bleeding index (GBI) and probing pocket depth (PPD) were evaluated and, after euthanasia, gingival and liver tissue were collected by biopsy for biochemical evaluation of myeloperoxidase (MPO), malonaldehyde (MDA), glutathione (GSH) and from the jaws for morphometric analysis of alveolar bone height. The clinical parameters of MT, GBI and PPD of the groups treated with bromelain gel showed a significant reduction (MT = 67.04%, GBI = 67.72% and PPD = 41.22%) compared to the group with periodontitis. The results for MPO dosage in the gingival tissue showed statistically significant differences between the groups when compared to the group with periodontitis, MDA and GSH showed promising results in the groups treated with gels in the dosages of gingival and liver tissue. The results for ABL showed significant differences when compared to the groups that received gel treatment. Therefore, the bromelain orabase gel evaluated in periodontitis showed a positive response for the following parameters TM, GBI and PPD, MPO, MDA and ABL of the oral tissues, preventing alveolar bone resorption caused by the disease, highlighting the potential as an adjuvant treatment of periodontitis, in addition to reducing the systemic effects on the liver tissue.

Keywords:
Periodontal disease; Bone biology; Periodontal medicine; Inflammation

HIGHLIGHTS

Bromelain gel reduces periodontitis

Ligation-induced periodontitis reduces liver damage in rats

Bromhexine gel is effective in reducing steatosis

INTRODUCTION

Periodontitis is a multifactorial chronic inflammatory disease that affects millions of people worldwide, contributes significantly to the global burden of oral diseases, with its severe form being the sixth most prevalent condition, estimated at 7% to 11% of the global adult population [¹-3]. The disease is caused by the accumulation of bacteria in the subgingival sulcus with an immune-inflammatory response of the body. Several cytokines, growth factors, enzymes and inflammatory cells act in a specific way resulting in progressive aggression to periodontal tissues [⁴]. Gingivitis, the least aggressive form of periodontal disease, is caused by bacterial biofilm (dental plaque) that accumulates on teeth adjacent to the gingival tissue [⁵]. However, gingivitis does not affect the underlying supporting structures of the teeth and is reversible [⁶].

Periodontitis is an aggravation of untreated gingivitis that results in loss of connective tissue and bone support and is a major cause of tooth loss in adults. In addition to the pathogenic microorganisms present in the biofilm, genetic and environmental factors, mainly smoking, contribute to the onset of the disease [⁷]. Genetic, dermatological, hematological and neoplastic disorders can also have periodontal manifestations [⁸]. Common forms of periodontitis have also been associated with complications in the general health of the organism, such as adverse pregnancy outcomes, diabetes, metabolic syndrome, stroke, respiratory diseases [⁹-¹¹] and liver disease [¹²,¹³].

Bacterial species are necessary to initiate and maintain these responses that lead to destruction. However, the central role of the pathogenesis of the disease is played by the imbalance of the innate and adaptive responses of the host [¹⁴]. Lipopolysaccharides (LPS) derived from microorganisms induce an inflammatory infiltrate with recruitment of polymorphonuclear neutrophils, macrophages and release of tumor necrosis factor (TNF), interleukins 1 and 17, among others, and prostaglandin E2 [¹⁵]. Released cytokines act on inflammation and bone resorption [¹⁶].

Currently, therapy for periodontitis consists of scraping and smoothing the roots to remove plaque, as well as applying antibiotics that are designed to disinfect adjacent periodontal root surfaces [¹⁷]. These therapies have proven long-term efficacy, despite not being functional in all patients. Research has emphasized the management of tissue inflammation, however, advances in the understanding of bone metabolism are opening new avenues of understanding of pathological bone loss in periodontitis. This knowledge, coupled with the development of new drugs that can inhibit bone tissue loss/destruction, offers us opportunities to target adjuvant treatment for periodontitis [¹⁸].

Previous studies have found that bromelain (derived from Ananas comosus) at a dose of 15mg/kg intraperitoneally was effective in reducing concentrations of malonaldehyde (MDA) and increased levels of glutathione (GSH), demonstrating antioxidant potential. In addition, bromelain has been shown to inhibit the growth of microorganisms involved in the progression of periodontitis and to reduce neutrophil chemotaxis for the periodontal site by 40% [²,¹⁹]. Bromelain is the general term used for a group of proteolytic enzymes that are commonly associated with more specific endopeptidases present in the tissue of the Bromeliaceae plant family. The most prominent plant belonging to this family is the pineapple (Ananas comosus) [¹⁹].

In view of the results observed so far for bromelain, it has shown to be promising as a target for the investigation of its osteogenic, anti-inflammatory and oxidative stress effects on periodontal tissues. With this in mind, this study aimed to develop a bromelain-based gel and evaluate its effects on periodontal tissues in a ligature-induced periodontitis model in rats.

MATERIAL AND METHODS

Bromelain-based gel formulation

The bromelain-based gel was formulated in two concentrations 1% (0.05 g of Bromelain) and 10% (0.5 g of Bromelain) [²⁰], to composition of a 10 g gel, Natrosol® (Grandbox, Itaquaquecetuba, SP, Brazil) was used as a gelling agent. To obtain the Natrosol gel, the water was initially heated until it reached a temperature of 60º C, then the polymer was added without exceeding 70º C until the gel was completely formed. Natrosol has a high tolerance to acids. dermatologically tested and hypoallergenic. Formulated with natural polymer hydroxyethylcellulose, commonly indicated for the incorporation of active ingredients that lead to a decrease in the pH of the formulation, used in the preparation of various formulas with the incorporation of chemically compatible active ingredients. Bromelain was obtained commercially from the company Sigma and added in the aforementioned proportions to the gel (Bromelain from the pineapple stem, Sigma-Aldrich®, São Paulo, SP, Brazil).

Determination and correction of pH

The pH was measured using the BEL W3B Potentiometer, with electrode and temperature sensor previously calibrated in buffer solution 7.0 and 4.0, at a temperature of 25±0.5ºC. The electrode was introduced directly into the formulation, and the analysis was carried out in triplicate [²¹].

Quantification of bromelain by UV-vis

For the quantification of bromelain, a standard solution in water of bromelain was initially obtained at a concentration of 10 mg/mL, then the sample was diluted to concentrations of 1; 0.8; 0.6; 0.4 and 0.2 mg/mL to construct the analytical curve, using water as a blank. The analytical curve was constructed from sample readings on a UV-VIS Spectrophotometer (Model 1800, Shimadzu). Readings were performed according to the protocol adapted from Velloso and coauthors [²²].

Animal experimentation

Periodontitis was induced in male rats (Rattus Norvegicus, Wistar) weighing between 150.0 and 180.0 g. All animal handling procedures were carried out in accordance with the institutional standards designated by the UFPI Animal Research Ethics Committee (nº 385/2017). 10 animals were used per group, separated into 6 groups as follows: Control (no induction, no treatment), Periodontitis (with ligation, no treatment), Periodontitis + Gel containing only the excipient (Neutral gel), Periodontitis + Bomelain 1 gel %, Periodontitis + 10% Bromelain gel and Periodontitis + 2% Chlorhexidine gel (positive control).

Periodontitis Model

The periodontitis model was induced following already standardized models [²³]. The animals underwent the periodontitis induction procedure (ligature insertion), with prior intramuscular anesthesia with 35 mg/kg of Ketamine (Francotar-Virbac®, Roseira, SP, Brazil) and 15 mg/kg of Xylazine (Francotar-Virbac ®, Roseira, SP, Brazil) according to the manufacturer's proportion. After immobilization, a 3-0 nylon thread (Shalon®, Goiânia, GO, Brazil) was positioned around the lower first molars to help prevent the accumulation of microorganisms.

Application of gels

The gels were applied topically with a fine-tipped syringe around the lower first molars, starting treatment on the same day as periodontitis induction and, subsequently, once a day for 15 days at a defined time.

Gingival Bleeding Index (GBI)

The scores for the presence of inflammation, color change, edema, presence of ulcer and hemorrhage were evaluated, leading to scores from 0 to 5 according to Liu and coauthors [²⁴].

Probing Pocket Depth (PPD)

Three points were evaluated and used for measurement (mesiobuccal, distal-buccal and midbuccal), with a periodontal probe (0.2 mm radius tip) [²⁵].

Tooth Mobility (TM)

The mobility of the first molars was evaluated, whose results were divided into scores: when there was physiological mobility (0), mild (1), moderate (2) and intense (3)[²⁶].

Evaluation of Alveolar Bone Loss (ABL)

The mandibles were stained with a 1% aqueous methylene blue solution (Dinâmica®)^‡. The ABL was used to delimit the enamel-cement joint (ECJ), where the images of each hemimandible on its lingual surface were captured with the aid of a magnifying glass (Nova Optical Systems)^§ at 30x magnification. Two properly calibrated examiners blinded to the knowledge of the groups performed the measurements using ImageJ image analysis software (ImageJ v.1.48 Media Cybernetics)[²⁷].

Myeloperoxidase (MPO) activity

Tissue samples were weighed (minimum of 25 mg) and dosage was performed to verify the neutrophilic infiltrate. Results were reported as units of MPO per milligram of tissue [²⁵].

MDA Levels

The dosage of malondialdehyde levels was determined by the method of Uchiyama and Mihara [²⁶] to evaluate the antioxidant activity of bromelain.

Liver tissue GSH

The protocol used to determine GSH levels in liver tissues was carried out according to Chaves and coauthors [²⁵]

Statistical analysis

Data were plotted and analyzed using GraphPad Prisma version 7.0®. Results were expressed as mean ± standard deviation and/or median and quartile range. The difference between the three groups was calculated using the test of variance (ANOVA) and the Student-Newman-Keuls and Turkey test for parametric data. For non-parametric data, the difference between groups was calculated using the Kruskal-Wallis test followed by the Dunn test for multiple comparisons. Values of p<0.05 were considered statistically significant.

RESULTS

The ligature-induced periodontitis model in rats was effective in triggering the inflammatory process, as demonstrated by our data, developing edema, gingival inflammation, loss of tooth attachment and mainly bone resorption. Treatment with bromelain orabase gel was started on the same day after induction of periodontitis. The rats were divided into four groups and treated with different concentrations of gels (Bromelain 1%, Bromelain 10%, Chlorhexidine 2% and Neutral gel) during the 15 experimental days, the Control group served to evaluate the parameters in relation to the groups that had induced periodontitis in the right and left lower first molars.

Obtaining gels and pH correction

The gel formulation showed good adhesion to the oral mucosa and stability during the experimental days. The potentiometer electrode was inserted directly into the samples in order to check the pH of the formulations. The pH of the formulations before and after their adjustment using 10% citric acid resulted in: pH before ± SD - 5.66 ± 0.15; pH after ± SD - 4.46 ± 0.58.

Quantification of bromelain by UV-vis

After the linear regression analysis for the Bromelain analytical curve, the equation of the regression line from x to y was obtained, where y = 0.4485x + 0.0293, in which the coefficient of determination (R2) for the curve was of 0.9994, being above 0.99, which is the minimum value required by ANVISA (2017). Furthermore, the slope (a) is non-zero. The value of the limit of quantification was 0.0312 mg/mL, which is the smallest amount of the analyte that can be quantified accurately and precisely, and the limit of detection was 0.0473 mg/mL, which is the smallest amount of the analyte that can be detected.

GBI, PPD, TM

The periodontitis induction model was confirmed by an experienced examiner who performed blind analysis of the regions around the lower first molars on both sides of each animal. There was a significant difference in SSI with a reduction of about 64% in the groups treated with 1% and 10% bromelain (Control, 0.44 ± 0.51; periodontitis, 3.77 ± 0.43; periodontitis + neutral gel, 3, 50 ± 0.52; Periodontitis + 1% bromelain, 1.39 ± 0.61; Chlorhexidine, 1.50 ± 0.51 P<0.05), when compared with the periodontitis and neutral gel group (Figure 1A ). The data from the PPD analysis show an improvement, that is, a decrease in the probing depth index in the groups: Bromelain 1% = 48.98%; 10% bromelain = 41.22%; Chlorhexidine 2% = 47.75% (Control, 0.59 ± 0.20; Periodontitis, 2.45 ± 0.60; Periodontitis + neutral gel, 2.13 ± 0.43; Periodontitis + 1% bromelain, 1.25 ± 0.50; Periodontitis + 10% bromelain, 1.44 ± 0.48; Chlorhexidine, 1.28 ± 0.57 P < 0.05) when compared to the periodontitis group (Figure 1B). The TM evaluation showed that the 10% bromelain group had the best result with a reduction of 67.04% (Control, 0.25 ± 0.45; Periodontitis, 2.7 ± 0.47; Periodontitis + neutral gel, 2 .25 ± 0.68; Periodontitis + 1% bromelain, 1.3 ± 0.47; Chlorhexidine, 1.25 ± 0.44; P < 0.05) when compared to the periodontitis group (Figure 1C).

Figure 1
Oral parameters, means and standard deviation of analyses: GBI, PPD, TM.

Gingival MPO and MDA

The results of the assessment of tissue neltrophil infiltration, (MPO, nmol/mg) in the gingival tissue biopsy samples showed statistically significant differences. The 10% Bromelain gel group had the lowest MPO dosage value (52.70% reduction) when compared to the Periodontitis group. (Control, 5.04 ± 1.53; Periodontitis, 11.10 ± 2.38; Periodontitis + Neutral gel, 9.17 ± 4.17; Periodontitis + 1% Bromelain, 5.53 ± 3.05; Chlorhexidine, 5.44 ± 2.29 P<0.05) (Figure 2A).

The result of lipid peroxidation (MDA) of the gingival tissue showed that there was a statistically significant difference between the treated and Periodontitis groups (P<0.05). There was no significant difference between the 1% Bromelain, 10% Bromelain and 2% Chlorhexidine groups. (Control, 25.60 ± 10.53; Periodontitis, 38.18 ± 19.05; Periodontitis + Neutral gel, 34.00 ± 11.90; Periodontitis + 1% Bromelain, 27.75 ± 6.96; Periodontitis + 10% Bromelain, 28.44 ± 5.46 Chlorhexidine, 25.40 ± 6.96;) (Figure 2B).

Figure 2
Oral parameters, means and standard deviation of the analyses: MPO and MDA.

ABL assessment

The results for ABL showed significant differences when the groups that received treatment with gel (Periodontitis + neutral gel, 2.75 ± 1.47; Periodontitis + bromelain 1%, 2.98 ± 0.74; Periodontitis + bromelain 10%, 2,69 ± 1.21; Chlorhexidine, 2.89 ± 1.21 P <0.05) were compared to periodontitis (Figure 3).

Figure 3
Means and standard deviation of alveolar bone height values of lower molars.

MDA and hepatic GSH

The results for MDA dosage showed significant differences when comparing the groups that received gel treatment with the periodontitis group (Control, 5.12 ± 02.59; Periodontitis, 7.77 ± 06.33; Periodontitis + Neutral gel, 6 .26 ± 03.23; Periodontitis + Bromelain 1%, 5.12 ± 03.09; Periodontitis + Bromelain 10%, 5.25 ± 02.81; Chlorhexidine 2%, 5.28 ± 02.96, p <0 .05) (Figure 4). The results for GSH dosage also showed significant differences when comparing the groups that received gel treatment with the periodontitis group (Control, 467.6 ± 73.28; Periodontitis, 199.9 ± 49.44; Periodontitis + Neutral gel, 339.2 ± 49.44; Periodontitis + Bromelain 1%, 476.5 ± 47.5; Periodontitis + Bromelain 10%, 453.4 ± 40.16; Chlorhexidine 2%, 269.1 ± 64.62, p < 0.05), where the untreated group presented a lower amount of this endogenous antioxidant (Figure 4).

Figure 4
Means and standard deviation of liver tissue MDA and GSH values.

DISCUSSION

This study is a pioneer in the literature that verified the action of bromelain orabase gel in a model of ligature-induced periodontitis in rats. The results indicated that bromelain gels are viable for use on the oral mucosa and showed good release of the active ingredient, interfering with the disease, improving important clinical parameters, reducing bone resorption and systemic effects on liver tissue.

There are already studies that demonstrate the anti-inflammatory effect of bromelain both in vitro [²⁸] and in vivo [²]. The present study also evaluated the effects of bromelain in the form of orabase gel in a model of periodontitis induced in rats. It is known that periodontitis leads to alveolar bone loss and is a concern among professionals, as it presents high rates of tooth loss in affected individuals, being characterized as an important public health diseases [²⁹]. The use of antibiotics, together with scaling and root straightening to remove plaque and dental calculus, are the current therapeutic measures [³⁰]. Other surgical techniques and guided tissue regeneration are also used, but the results in the regenerative process are quite limited when compared to alveolar bone, which motivates the search for new methods, techniques and products with the potential to mitigate the damage caused by periodontitis [³¹].

The clinical parameters of gingival bleeding index, probing depth index and tooth mobility were evaluated before euthanasia, where the results obtained by statistical analysis showed significant differences between the groups. These differences showed a decrease in the rates of the treated groups, when compared to the periodontitis group, preventing its progression. Similar findings were demonstrated by other authors who used gels in the treatment of periodontitis, Souza Filho and coauthors [³¹] they used cashew gum polysaccharide gel as an adjuvant treatment, reducing clinical inflammatory parameters and alveolar bone loss.

GBI, PPD and TM are inflammatory clinical parameters caused by infection caused by the presence of bacterial biofilm in the subgingival groove. Studies have shown that bromelain has a synergistic effect when administered together with antibiotics, mainly amoxicycline, which is the first choice of antibiotic therapy in periodontics, especially when it is not possible to perform microbiological susceptibility testing, and these two mechanisms can be explored when aims to further unveil the benefits of bromelain against specific infections [³²]. Such clinical parameters may also have been reduced, due to the anti-inflammatory potential reported by other authors, with activity in pro-inflammatory cytokines, such as gamma interferon (IFN-γ), tumor necrosis factor (TNF-α) and stimulatory factors. of granulocyte-macrophages (GMCSF), which are cytokines involved in the development of periodontitis [¹⁹].

The inhibitory mechanism of bromelain in LPS-induced inflammation was shown to cause a decrease in the secretion of cytokines, compared to fruit-derived bromelain, stem-derived bromelain showed a greater amount of protease and proteolytic action [³³]. The anti-inflammatory activity of bromelain gel may also be involved in reducing the expression of COX-2, which is an important mediator of inflammation, and in the rapid reexpression of CD62L/L-selectin molecules, where these findings confirm the decrease in migration neutrophilic activity for periodontal tissue [²,⁴].

The results showed that the bromelain orabase gel was effective in reducing the levels of neutrophil infiltration, which when statistically analyzed had a decrease in the bromelain 1% groups of 50.18% and in the bromelain 10% groups of 52.70% in the gingival tissue when compared to the periodontitis group. These data were confirmed by the MPO dosage, similar to the study by Sharma and Sharma [³⁴], where a pre-treatment with bromelain showed a reduction of up to 78.25% in MPO activity in a carrageenan-induced inflammation assay, the results confirmed that bromelain improved inflammation by inhibiting trans-endothelial migration and MPO release, facilitating faster apoptosis of neutrophils.

MDA is a presumptive biomarker for lipid peroxidation in living organisms, commonly used to evaluate lipoperoxidation generated by the degradation of organelles and cell membrane phospholipids, where studies have reported that lipid peroxidation produced by periodontal inflammation, as observed in our animal model through from gingival levels of MDA, it also diffuses into the bloodstream, generating a systemic effect and causing pathology in other organs [²⁷,³⁴,³⁵].

Our results showed a significant difference between the groups treated with 1% bromelain, 10% bromelain and 2% chlorhexidine gels, with a reduction of 25.51%, 27.32%, and 33.47%, respectively, indicating that the treated group with 10% bromelain, a better reduction in the inflammatory process was achieved, corroborating the work of Alves and coauthors [²], where bromelain was able to reduce oxidative stress caused by periodontitis. Antioxidant agents reduce oxidative stress by terminating the oxidative chain reaction, which inhibits oxidative damage. Samples of aqueous and ethanolic extracts were effective in the scavenging activities of ABTS and DPPH radicals and reducing power in the study by Huang and coauthors [³⁶]. The antioxidant activities of the aqueous extracts of the stem were more effective with little or no side effects.

Regarding the jaws, when statistically evaluated, they showed statistically significant differences, with a reduction (bromelain 1% = 17.45%; bromelain 10% = 25.48%; chlorhexidine 2% = 19.94%) when compared to the periodontitis group. In this result, bromelain gel at a concentration of 10% showed better percentages than the gel commonly used in dental clinics. The results from bromelain orabase gels corroborated Yuan and coauthors [³⁷], Secor and coauthors [²⁸], Chakraborty [³⁸] and Alves and coauthors [²], who identified bromelain as an anti-inflammatory agent, and due to this action, it acts by reducing the release of inflammatory mediators whose effects on osteoclasts are indirect and stimulate bone resorption. Another important effector pathway may be the increase in the proteolytic effect of serine proteinases, such as proteinase 3 (PR3) and neutrophil elastase, through the oxidative inactivation of their main inhibitor a1-antitrypsin (a1-AT) [³⁹], Madkhali, Hussein, Alnahdi [²⁰] and Silva and coauthors [¹⁹] hypothesized that the anti-inflammatory effects of bromelain may occur due to its proteolytic activity on the surface of cells, mainly leukocytes, present in the microenvironment.

Bromelain gels showed efficacy compared to chlorhexidine, according to the study by Lucena and coauthors [⁴⁰], a comparative study of a plant extract showing its therapeutic efficacy in relation to chlorhexidine. Regarding substantivity, in an in vitro study, it was observed that chlorhexidine remained in the dentin of the root canal in effective antimicrobial portions for a short period of time.

Oxidation in biological systems is related to the development of pathologies in humans. The results of measuring MDA and GSH in liver tissue demonstrated that systemic effects on the liver were reduced. Some studies have shown that bromelain is a proteolytic enzyme that has the ability to cleave internal peptide bonds in proteins, being called endopeptidases. The use of endopeptidases favors the generation of peptides with different amino acid sequences. This process is considered positive from a biotechnological point of view, as intrinsic factors of peptides such as size, amino acid arrangement, electrical charge and molecular mass influence their bioactivity [¹³]. We hypothesize that bromelain may have reduced the harmful effects that periodontitis causes to liver tissue due to its enzymatic activity or due to its potential to reduce the inflammatory clinical parameters of the disease, which consequently minimized systemic damage to the body [¹²].

CONCLUSION

Bromelain orabase gels evaluated in ligature-induced periodontitis in rats showed a positive response for the following parameters: GBI and PPD, TM, MPO and MDA of oral tissues, preventing alveolar bone resorption caused by the disease, highlighting its potential as an adjuvant treatment for periodontitis, as well as reducing systemic effects on liver tissue, demonstrating that it may also have an antioxidant effect.

Acknowledgements

We would like to thank the Federal University of Piauí for all the assistance provided to the Doctor/author of this work, and also the Federal University of Delta do Parnaiba for providing the laboratory and materials necessary to carry out the experiments.

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• Funding:
  This research received no external funding.

Edited by

• Editor-in-Chief:
  Paulo Vitor Farago

• Associate Editor:
  Fábio André dos Santos

Publication Dates

• Publication in this collection
  03 Feb 2025
• Date of issue
  2025

History

• Received
  29 Apr 2024
• Accepted
  09 Oct 2024