A new release nanosystem mucoadhesive gel of Brazilian Red Propolis-containing chitosan: in vitro Citoxicity and Antimicrobial test. In vivo ligature induced periodontitis model- propolis treatment in rats

How to cite this article: Cinthia Figueiredo Oliveira, Tatiana Gomes Ribeiro,Flávio Henrique de Abreu Rosa,Adélia Cristina Souza Araújo,Rafael Tomaz Gomes,Rachel Ol ivei ra Cast i lho,André Augusto Gomes Faraco,Mirian Tereza Paz Lopes,Vagner Rodrigues Santos. A new release nanosystem mucoadhesive gel of Brazilian Red Propolis -containing chitosan: in vitro Citoxicity and Antimicrobial test. In vivo ligature induced periodontitis modelpropolis treatment in rats.International Journal of Traditional and Complementary Medicine 2022, 7:40.


Introduction
Periodontal disease has a great prevalence in the world population and is a major cause of tooth loss in adults. The clinical signs of Chronic Periodontitis are chronic gingivitis caused by dental biofilm or plaque above and subgingival, periodontal pocket, insertion loss concurrently with the resorption of the alveolar bone of tooth support [1] .
There is evidence, supported by previous studies that Chronic Periodontitis is the evolution of a chronic untreated gingivitis, associated with biofilm buildup of plaque and tartar and a specific microbiota. Microbiological studies have observed the presence of Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens, Fusobacterium nucleatum, Eikenella corrodens, Tanerella forsythia, Aggregatibacter actinomycetemcomitans, Enterococcus faecalis, and in some cases of refractory periodontitis, Candida albicans among other less common species [2] .The antimicrobial effectiveness of ethanolic extract of propolis (EEP) compared to chlorhexidine gluconate (CHX) on planktonic Streptococcus mutans, Streptococcus sobrinus, Lactobacillus acidophilus, Lactobacillus salivarius subsp. salivarius, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Porphyromonas gingivalis, Staphylococcus aureus, Enterococcus faecalis, Actinomyces israelii, Candida albicans, and their single-species biofilms by agar dilution and broth microdilution test methods was made for [3] . The authors demonstrated both agents inhibited the growth of all planktonic species. On the other hand, CHX exhibited lower minimum bactericidal concentrations than EEP against biofilms of A. actinomycetemcomitans, S. aureus, and E. faecalis whereas EEP yielded a better result against Lactobacilli and P. intermedia. The bactericidal and fungicidal concentrations of both agents were found to be equal against biofilms of Streptecocci, P. gingivalis, A. israelii, and C. albicans. The results of this study revealed that propolis was more effective in inhibiting Gram-positive bacteria than the Gramnegative bacteria in their planktonic state and it was suggested that EEP could be as effective as CHX on oral microorganisms in their biofilm state [3] .
The presence of the subgingival microbiota generates an inflammatory response involving direct and indirect aggression with the involving neutrophils, macrophages y endothelial cells and chemokine activation of integrins and production of Tumor Necrosis Factor Receptor (TNF-R), Heparan Sulfate proteoglycan (HSP), L-selectin, Integrin Adhesion Molecule-intra-Citoplasm (Integrin -ICAM), PSGL-1 and P-Selectin [2,4] .
The periodontal disease has a multifactorial etiology involving besides the microbiota, the oral care of patients, genetic, environmental predisposing factors such as smoking and alcohol, social cultural factors and financial. Although there are several brands on the market of mouthwashes and toothpastes, these products act preemptively acting on the control of dental plaque. However, there is, to date, no product which acts treating inflammation and stimulating the formation of alveolar bone lost during the progression of the disease. Treat the inflammatory process and stimulate new formation of bone, concurrently, continue to be a challenge for scholars and dentists [4] .
Propolis has been used for centuries by world population due to its pharmacological properties such as anti-inflammatory, healing, antimicrobial and antioxidant [4,5] . In dentistry, propolis has been used to control the oral microbiota [ 6,3] . The antibacterial activity of propolis is reported due to flavonoids, aromatic acids, and esters present in resins. Galangin, pinocembrin, and pinostrobin are known as the most effective flavonoids agents against bacteria. Ferulic acid and caffeic acid also contribute to the bactericidal and fungicidal action of propolis. Propolis has plenty of biological and pharmacological properties and its mechanisms of action have been widely investigated in the last years, using different experimental models in vitro and in vivo [7] .
Red propolis, found in Northeast mangrove areas of Brazillian Northeast, is originated from Dalbergia ecastophyllum and it is composed for pterocarpans, isoflavonoids, chalcones, prenylated benzophenones and phenylpropanoids [8] . The red propolis therapeutical effects are imputed to fenolic compounds, in association with fenolic acids, esters, fenolic aldeids [9] . Table 1 shown the chemical markers of red propolis. It act sinergicly and researchers shown that the pool of compounds activity have a more effect than one isolated compound [10] . Studies related clinical uses and biological properties of red propolis as antibacterial, antioxidant, antifungi, anti-inflammatory, healing wounds and that is the reason to investigate propolis role against caries, candidiasis, gengivitis and other oral pathologies [11] . Propolis has an important activity against microorganisms. In vitro studies demonstrated its abilities against virus like influenza, herpex virus type 1 and 2 and against a large range of bacteria, including Staphylococcus aureus and Salmonella enteritis, even resistant strains. It is related the potential of red propolis alone or in synergic combination with certain antibiotics and antifungals [12,13] . Previous reports related the antibacterial activities of isolated compounds of red propolis against S. aureus, Bacillus subtilis and Pseudomonas aeruginosa, evaluated by measuring the Minimum Inhibitory Concentrations (MIC), demonstrated that the main antimicrobial phenolic compounds were found to be vestitol, neovestitol and medicarpin. In particular, medicarpin exhibited the most potent bacterial activity against elected bacteria, indicating that it is the most active antibacterial compound in Brazilian red propolis extracts [7] . There are few studies about the antimicrobial activity of Brazilian red propolis against oral pathogens microorganisms and periodontitis. The therapeutical properties and its low toxicity in low concentrations may enable propolis for many interesting in dentistry use. The development of a gel controlled release system that generates a bioadherence (preventing saliva action) that can act against some of the chronical oral pathologies can have a great value. Table 1 shown the chemical markers of red propolis. So, this study developed a mucoadhesive gel containing Brazilian propolis which was tested in vitro against periodontitis envolved microorganisms and in vivo animal model of periodontitis.

Material and Methods Citoxicity assays using ethanolic extract of red propolis
Murine fibroblasts (L929), murine epithelial cells (CHO) and human gingival fibroblasts (FIB) were seeded on 96-well microtiter plates ( n=12) at the density of 1,0x10 4 cells/well and, after 24 hours, exposured to concentrations of ethanolic propolis extract (0,06 to 0,25%). It was incubated at 37 o C in 5% CO2 for 72 hours. The proliferation index was determined by methyl-thiazolyltetrazolium (MTT, Sigma, USA) assay, which is based on the conversion of MTT to formazan crystals by mitochondrial desidrogenases [14,15] . After the exposition, the medium was removed and dye MTT 1 mg/mL was added. Subsequently, the MTT solution was removed and the crystals of formazan was dissolved in dimethylsulfoxide (DMSO, Sigma, USA). Absorbance was measured using a microplate reader (Spectra Max 190, Molecular Devices, USA) at 570 nm. Antimicrobial Assay Bacterial sensitivity or resistance to mucoadhesive gel was detected by the agar diffusion assay [16,17]  /mL were subcultured in the proper agar and supplemented for each microorganism. 6mm diameter wells were punched into the agar in triplicate and filled with: 20µL of gel containing chitosan only and red propolis gel 5 and 10%. Ethanol propolis extract 5% (EPE), chlorhexidine 0,12% (CHL,Sigma, USA), 70% ethanol (E, Synth, Brazil)), and sterile distilled water (DW) were dispensed in sterile blanc discs (Laborclin, Brazil) soaked with 20µL of each substance. The bacteria plates were incubated at 37º C in 5% CO2 for 24 hours. Candida albicans cultured on Sabouraud dextrose agar plates were incubated agar at 37º C for 24 hours under aerobic atmosphere. The inhibition zone diameter around the well and filter paper formed after 24 and 48 hours at 37º C were measured and taken medium and standard deviations (M± SD). Any inhibition zone around the filter paper measuring ≤ 7mm was considered a negative result. Minimal Inhibitory Concentration (MIC) test was carried out using microtiter plates (96 wells) containing 100 L/well BHI. After being transferred to the first well, serial dilutions were performed to obtain concentrations ranging from 75 to 0,1mg/mL. MIC was defined as the lowest concentration of the propolis gel that inhibited microorganism visible growth indicated by resazurin 0.01% (Sigma-Aldrich, St. Louis, MO, USA). To determine minimal bactericidal concentration (MBC), an aliquot of each incubated well with concentrations higher than MIC was subcultured on BHI medium. MBC was defined as the lowest concentration of the propolis gel that allowed no visible growth on the test medium [17] . Ligature induced periodontitis model. [18] The animal experiment was approved by the Ethics Committee on Animal Use (CEUA / UFMG-protocol number 294/2012). Wistar rats (120-140 g, n=9, divised in 03 groups) were aclimmated to the housing conditions for 5 days. They are allocated into plastic cages with water and food at libitum and a 12 hour light/dark were applied. The animals received a intramuscular injection for general anesthesia by association of ketamine (0,4 mL/mg) and xylazine (0,2 mL/mg). The left second molar was selected for the ligadure, using cotton ligadure number 4.0 (Ethicon, J&J). The ligadure was knotted on the buccal side of the tooth resulting in a subgingival position palatal and a supragingival position buccally for 7 days. The right second molar without ligature was used as an internal control of the experiment. Then, the ligadure was removed and the animals were separated in randomized groups and treated with different dosis of propolis gel (2, 5 and 10%) and vehicle chitosan. After 10 days, animals were sacrified. The mandibles were removed and fixed in 10% formalin buffer for 48 hours, then descalcified by EDTA 10% for 10 days and dehydratedin an ascending series of ethanol solution and embedded in paraffin [19] . The 5µm sections were obtained and stained in a hemotoxicilin and eosin (HE). For the histometric evaluation, the lamins were photographed by a microcamera (JVC TK-1270/RGB) coupled on the microscope. The measurement of the alveolar inserction loss were obtained by the difference of cementum-enamel junction/alveolar bone crest distance of the sides with and without ligature [ Figure 1]. The measurement of bone loss were obtained by the difference of dentinoenamel junction/ alveolar bone crest distance. The measurements were expressed in mm using a computer program Image J.1.48. The One-way ANOVA Bonforronipost test was used for histometric comparisons between ligated and unligated groups at significance level of 1% ( p< 0.01) .

Results and Discussion:
About citotoxity / proliferation detected by MTT method, on that assay, it was shown a statistically significant proliferation using the 0,25% concentration for all lines of cells (L929: 0,407±0,080 %v/v; CHO: 1,44±0,37 % v/v; and FIB:1,43±0,37 % v/v) against the control (0,265±0,04; 0,622±0,080; 0,619±0,56; p<0,05, ANOVA, Bonferroni's post test) ( Table 2 and  Table 3). Other studies found a citotoxity activity of red propolis against tumor cells lines in vitro [14,15] and that is the reason why the antitumor activity of propolis has been largely studied. On this report, we found a proliferation of cells caused by red propolis differently of showed on that studies. But the concentrations of propolis used in our assays were lower, comparing to the ones used on that studies cited. Authors found a not citotoxity activity of propolis against osteoblast-like cells, using, as in our report, lower concentrations of propolis [13] . So, the activity of propolis probably is dosagedependant and it can be specific for certain types of cells. On our studies, it was used cells that are strongly associated to healing process in vivo.    This study shows satisfatory results of ethanolic extract and gel for all the microorganisms, showing that propolis is efficient in both formulation. Table 4 and Table 5 show the antimicrobial properties of red propolis. It retains its antimicrobial properties when associated to chitosan microspheres. Against caries bacteria, propolis gel 5% showed an efficient activity against Streptococcus salivarius and Streptococcus sanguinis similar to the findings of da Silva et al [12] . Even using ethanolic extract 5%, the results were positive and larger than control, but the deviation was wide, close to chitosan and chlorexidine. These results are according to Bueno-Silva et al. [7] that shows a neovestitol-vestitol containing propolis ability of control development of caries growth in vitro and in vivo, considering as gold standard a fluoride. Better deviations were found on results for Lactobacillus casei and Porphyromonas gingivalis using propolis gel 10% and Fusobacterium nucleatum using gel 5%. Better media were obtained using ethanolic extract 5% and it may be related with alcohol that may reduce the surface tension and solubilize propolis compounds, causing a large difusion. The consequence is a large inhibition zone and deviation [20] performed a difusion in wells instead of a difusion in discs to avoid the interference of alcohol. Enterococcus faecalis does not shows signifficant results, but it could not be related to propolis antibacterial activity. Brumfitt et al. [21] related technical problems as low rate of propolis compounds in agar. Gram negative may be less sensitive to ethanolic red propolis extract because of its complex wall cell or its higher fat contents. Righi et al. [5] findings are similar, showing a limited activity against Gram negatives and a major MMC (minimum microbicidal concentration) against Kleibsiella pneumoniae, a Gram positive one. Marcucci et al. [22] and Sforcin et al [23] founded low activity of green propolis against Gram negative bacteria. On this report as in De Luca et al. [14], we found significative results against gram positive and gram negative bacteria. These differences found in propolis activity occurs because according to the variaty of the propolis. Other studies observed that low concentrations of propolis reveals bacteriostatic action instead of bactericidal [5] . This study shows satisfatory results of red propolis gel 5% specially against Aggregatibacter actinomycetemcomitans and ethanolic extract 5% specially against Candida albicans. These results are similar to those observed by Sokolonski et al. [24,25] . The large number of phenolic compounds (as flavonoids, phenolic acids and their esters [3,22] , which have been attributed antibacterial activity, with individual pharmacological activity against microorganisms turns propolis an interesting to act against resistant microorganisms. Reports show that ethanolic extract of red propolis could inhibit even resistant bacteria, as S. aureus that is resistant to methicillin. Propolis can act effectively against resistant fungi as C. glabrata, specially when used as an adjuvant of fluconazole.

Table 2-Cytotoxicity test. Number of viable cells after contact with ethanolic extracts of red propolis with human gingival fibroblasts (FIB), murine fibroblasts (L929) and hamster epithelial cells (CHO). Means and Standard Deviations (M ± SD) of three tests.
Other relates shows the antiinflammatory and immunomodulatory properties that can act combined to the antimicrobial activity and improving it Freires et al., [13] . In addiction, propolis is a natural product reduce orange-complex periodontopathogens [25] and have a good population acceptance used for since ancient times [26] . To analyze pharmacological properties in vivo, it was used an animal model of ligature induced periodontitis. Animal models can provide a high quantity of data but it is not possible to determine if it is possible to extrapolate to human form of the disease. The ligature method for periodontitis induction was studied by de Molon et al [18] and Inui et al. [23] and the authors verified a significant increase in the gene expression of pro-inflammatory cytokines, interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), and proteins involved in osteoclastogenesis, receptor activator of nuclear factor-k B ligand (RANKL) and osteoprotegerin (OPG) was observed in the first week of analysis. In the later periods of evaluation (14-21 days), no significant alterations were noted with regard to inflammatory processes, bone resorption, and expression of cytokine genes. The ligature-induced PD model resulted in progressive alveolar bone resorption with two different phases: Acute (0-14 days), characterized by inflammation and rapid bone resorption, and chronic (14-21 days) with no significant progression of bone loss. Furthermore, the gene expressions of IL-6, IL-1β, TNF-α, RANKL, and OPG were highly increased during the progress of PD in the early periods [18] . It generates a formation of a pathogenic biofilm, inflammation and bone loss. Rodents are naturally more resistant to periodontitis [27,28,29] . It is possible to observe a tendency of reduction in groups treated with propolis, presenting minor medias for this groups, but it is not significant. Groups tested with propolis gel 5% and 2% showed similar results. These results are related to the bioactive properties of D. ecastophylum [30,31] . The Figure  1 showed the periodontal insertion and bone loss (vehicle and treated) compared to the control (without propolis gel).
Probably, on that report, it is not shown a significant different between groups because of the low number of animals used (the deviation is high). Other studies with extract ethanolic of green propolis irrigation combinated to periodontal clinic treatment allowed to obtain better results than scaling and root planning by themselves, which results from the assessment of both clinical and microbiological parameters [25,26] . Considering the world's biodiversity, the various types of propolis [27] , that microorganisms may become are more sensitive to red propolis and that a mucoadhesive formulation can promote a more contact time with the product, the mucoadhesive red propolis gel can be very useful in periodontitis. Considering the physical characterists that can facilite the use and the commercialization of the product and the pharmacological activities described, the red propolis gel is an interesting product for oral uses. In spite of the superior microbial activity of ethanolic extract, the gel has the advantage of prolonged controlled liberation. Furthermore, red propolis induced proliferation in vitro of some cells related to healing processes. Our data demonstrate that propolis, as an ethanolic extract or as a mucoadherent gel, is a promising agent against dental bacteria and may be useful on the treatment of various oral diseases as caries and periodontitis. However, other complex studies in vitro and in vivo are necessary to confirm these results.

Conflict of Interests
The authors report no relationships, financial or otherwise, with any entity that may influence the objectivity of this paper.