Evaluation the Effects of Dentifrice Containing Propolis Extract in the Control of Plaque and Gingivitis with Measuring il-1β and il-6 Salivary Cytokines levels, (clinical and immunological trial)

Leenah Zuheer Hassan^* and Maha Abdul Aziz Ahmed

^*Correspondence: Leenah Zuheer Hassan, Department of Periodontics, College of Dentistry, Iraq, Email:

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Introduction

Dental plaque is viewed as the key factor related to both inflammation of the gingiva and dental caries . Selfperformed mechanical plaque removal such as using of tooth brush which is quite possibly the most acknowledged strategies for controlling plaque and gingivitis [1]. A wide types of toothpastes are present commercially and interest in natural items has expanded recently. Propolis herbal active agent one of these products. In the world markets propolis available in various structures as tincture, capsules, lozenges, creams and added to the mouth washes and toothpastes. Depending on published reports showing that propolis resin is a product with anti-inflammatory and bactericidal activities [2,3]. A clinical study regarding propolis-based herbal toothpaste revealed that Propolis was safe and efficient in decreasing accumulation of plaque when compared with Miswak and Colgate toothpaste [4]. Cytokines have been characterized as soluble components overlap or merge, building a complex immune-regulatory network in the immune system that is perturbed frequently in a disease. Many types of the cytokines such as interleukins ( IL-6, IL-1), and tumor necrosis factor alpha (TNFα) are elevated in most, if not all, inflammatory states and have been documented as goals of therapeutic intermediation [5]. The IL-6 although mostly regarded as a pro-inflammatory cytokine, IL-6 also has many regenerative or anti-inflammatory activities while IL-1β is the first cytokine released after microbial stimulation and play a major role in the cell migration to the inflamed sites [6].

Saliva has been considered as a diagnostic liquid that could be utilized in the systemic and oral diseases diagnosis. The salivary markers levels, like cytokines, might actually be utilized to differentiate individuals with healthy periodontium from patients with periodontal disease [7,8]. Demonstrated that the IL-1β and IL-6 concentrations in patients with gingival disease were higher than in the control group, but with a non-significant difference. Studies that have been directed to find the propolis effects on oral and periodontal health conditions were few. Thus, the current study has been conducted to evaluate and compare the Propolis containing tooth paste efficacy with Colgate total dentifrice in controlling plaque and inflammation of gingiva with measurement of salivary IL1β and IL-6 levels.

Materials and Methods

The study design was approved by the Ethics Committee of the College of Dentistry, University of Baghdad and all patients were given detailed information relating to the study aim and an informed consent representing the patient's approval to participate in this study. Inclusion criteria were patients aged 20-35 years, apparently good general health, had generalized dental biofilm induced gingivitis with intact periodontium [9], 24 teeth as a minimum must be present and consented to stop for 24 h the measures of oral hygiene. Criteria for exclusion were persons with chronic disease, immunocompromised patients, pregnant, on contraceptive pills and lactating women, currently using any mouthwash, on antibiotic therapy and anti-inflammatory medications during the study and at the last 2 weeks before the study, having a background for hypersensitivity to any item utilized in the current study, with a new tooth extraction, having probing pocket depth (PPD) ≤ 4 or attachment loss, smoker or alcoholism, with a broad untreated dental caries, soft and hard palate lesions and wearing orthodontic apparatuses, removable dentures, implant, crown and bridge or presenting with abnormal salivary flow. Colgate total fresh mint strip tooth paste Manufactured by Colgate Palmolive Co with Active Ingredient: Stannous fluoride 0.454% (0.15% w/v fluoride ion) and Ecodenta triple force Manufactured by BIOK Laboratory UAB Vilnius with active ingredients White clay+Propolis+Teavigo™ were used in this study. All toothpaste tubes had a plain black covering to confirm appropriate hiding of the item from the examiner and the patients this done by subject not included in this study.

Clinical periodontal parameters examination (performed by the university of Michigan O probe with Williams markings) include: assessment of bleeding tendency by a modified sulcus bleeding index (mSBI) [10], and assessment of plaque accumulation using the modified Quigley Hein plaque index (mQHPI) and fluorescein disclosing tablets [11].

Study design

This study is a cross over double blinded randomized clinical trial had been conducted on 20 males and females. During the initial preparation visit, the patient received motivation and oral hygiene instructions (OHI), recording the bleeding on probing BOP, PPD and clinical attachment level CAL [12] then eliminate plaque, calculus, and stain by supra and sub gingival scaling and polishing. After prophylaxis, patients were gotten guidance about the clinical strides of the investigation. Next visit after one-week from initial visit: subjects must be with clinically healthy gingiva to continue in the study through clinical examination for ( BOP, PPD and CAL) ,then the patient instructed to refrain from any oral hygiene measures for 24 hours. First visit (baseline) after 24 h of refrain from oral hygiene measures: Saliva sample collection was done. After that measure the (mSBI and mQHPI), the patients were given the Ecodenta or Colgate total toothpastes and instructed to use for 7 days twice daily at the morning after breakfast and at the evening before bedtime also take study tooth brush medium (Oral B) and use the modified bass technique tooth brushing method [13].

Second visit (after 7 days from first visit): Saliva sample collection then measuring (mSBI and mQHPI). After that scaling and polishing were done and the patient instructed about the washout period (7 days) instruction. After one week from second visit: Examination of (BOP, PPD and CAL) to ensure presence of clinically healthy gingiva, after that the subjects instructed to refrain from any oral hygiene measures for 24 h. First recall visit (baseline) after 24 h from refrain of oral hygiene measures: Saliva samples collection was done then measurement for (mSBI and mQHPI) after that the patient was given (Colgate) or (Ecodenta) toothpastes use it twice a day for 7 days. Second recall visit (after 7 days from first recall visit): Saliva sample collection then measurement for (mSBI and mQHPI).

Instruction for patients to use adequate amounts of tooth paste and brushing for 2 minutes.

Patients asked during test period to refrain from all other unassigned forms of oral hygiene measures, including non-study toothbrushes or toothpastes, interdental aids, chewing gum, or oral rinses. Patients motivated and instructed during 7 days’ washout period to maintain oral hygiene measures by using whatever toothpaste, tooth brush and interdental aids. Patients instructed to brush their teeth before 2 hours prior to each visit.

Salivary samples collection

Unstipulated whole saliva had been collected prior to clinical periodontal evaluation according to a modification of the previous method by [14]. Subject expectorated at least 1 mL of saliva in a test tube.

After centrifuging 15 minutes 3000 RPM, aliquots had been prepared and samples had been frozen at -20°C in a freeze until analysis by enzyme-linked immunosorbent assay (ELIZA) kit for assessment of IL-6 and IL -1B levels [15,16].

Statistical analysis

Statistical Package for Social Sciences (SPSS) variant 25 was utilized to analyse the data which presented as mean, standard deviation (SD) and ranges with using of Independent t-test and Paired t test. A degree of probability of value p-value ≤ 0.5 represent the significant (S) also > 0.05 is non-significant (NS) while highly significant (HS) as P < 0.001.

Results

Age and gender

Patients' age was from 21 to 33 years with a mean of 25.8 years. The higher percentage at age ≥ 25 years was 65% (13 patients). While the age <25 years was 35% (7 patients) revealed lower percentage. Concerning gender, percentage of males was higher than females (65% against 35%) respectively; hence the numbers were females 7 and males 13.

Baseline 1st visits before dentifrice usage

Non-significant differences were revealed between groups in means of mQHPI and mSBI, while significant differences were detected in means of IL-1β and IL-6, (Table 1).

Table 1: Descriptive statistics and comparison for mQHPI, mSBI, IL 1β ng/L and IL-6 ng/L between study groups at baseline 1st visit.

After dentifrice usage for one week (2nd visits)

Table 2 revealed decreases in means of all parameters at 2nd visits hence, there were significant differences detected between visits at both groups in means of mQHPI and mSBI , while non-significant differences were demonstrated at both groups regarding means of both interleukins except ,the significant difference at Ecodenta group for IL-1beta.

Table 2: Descriptive statistics and comparison of mQHPI, mSBI, IL1β and IL-6 between 1st and 2nd visits at each study group.

There were significant differences between groups regarding mean percentage of change for clinical and immunological parameters at 2nd visit compared to 1st visit hence Ecodenta group registered higher values for mean percentage of change than Colgate group at all parameters, as shown in Table 3.

Table 3: Statistical analysis for mean percentage of change between study groups in mQHPI, mSBI, IL-1β and IL-6 at 2nd visit compared to that at baseline1st visit.

Discussion

Conclusion

Prosthetic stomatitis, when associated with angular cheilitis, may have a multifactorial etiology, despite recent scientific evidence suggesting that most cases are linked to fungal infections, namely by Candida albicans. Further studies and an increase in the sample of institutionalized elderly are needed to analyse other risk factors associated with the disease. To prevent the extension of lesions, prosthetic patients should be called regularly for dental appointments and a clinical examination of the oral cavity and removable prosthesis. Preventive measures should be developed to avoid the colonization of Candida albicans on the palatal mucosa and prosthesis, such as improvement of oral and prosthetic hygiene and prosthetic removable at night during sleep.

References

1. Janakiram C, Varghese N, Ramanarayanan Venkitachalam JJ, et al. Comparison of modified bass, fones and normal tooth brushing technique for the efficacy of plaque control in young adults-A randomized clinical trial. J Clin Exp Dent 2020; 12:e123.
2. Brumfitt W, Hamilton-Miller JM, Franklin I. Antibiotic activity of natural products. Propolis. Microbios 1990; 62:19-22.
3. Hegde KS, Bhat SS, Rao A, et al. Effect of propolis on Streptococcus mutans counts: an in vivo study. Int J Clin pediatr Dent 2013; 6:22.
4. Bhat N, Bapat S, Asawa K, et al. The antiplaque efficacy of propolis-based herbal toothpaste: A crossover clinical study. J Nat Sci Biol Med 2015; 6:364.
5. Scheller J, Chalaris A, Schmidt-Arras D, et al. The pro-and anti-inflammatory properties of the cytokine interleukin-6. Mol Cell Res 2011; 1813:878-888.
6. Hasturk H, Kantarci A, Van Dyke TE. Oral inflammatory diseases and systemic inflammation: role of the macrophage. Front Immunol 2012; 3:118.
7. Teles RP, Likhari V, Socransky SS, et al. Salivary cytokine levels in subjects with chronic periodontitis and in periodontally healthy individuals: A cross�sectional study. J periodont Res 2009; 44:411-417.
8. Becerik S, Öztürk VÖ, Atmaca H, et al. Gingival crevicular fluid and plasma acuteâ?phase cytokine levels in different periodontal diseases. J Periodontol 2012; 83:1304-1313.
9. Caton JG, Armitage G, Berglundh T, et al. A new classification scheme for periodontal and periâ?implant diseases and conditions–Introduction and key changes from the 1999 classification.
10. Newbrun E. Indices to measure gingival bleeding. J Periodontol 1996; 67:555-561.
11. Escribano M, Figuero E, Martin C, et al. Efficacy of adjunctive anti�plaque chemical agents: A systematic review and network meta�analyses of the Turesky modification of the quigley and hein plaque index. J Clin Periodontol 2016; 43:1059-1073.
12. Eickholz P. Clinical periodontal diagnosis: Probing pocket depth, vertical attachment level and bleeding on probing. Perio 2004; 1:75-80.
13. Janakiram C, Venkitachalam R, Fontelo P, et al. Effectiveness of herbal oral care products in reducing dental plaque & gingivitis–a systematic review and meta-analysis. BMC Complement Med Ther 2020; 20:1-2.
14. Syndergaard B, Al�Sabbagh M, Kryscio RJ, et al. Salivary biomarkers associated with gingivitis and response to therapy. J Periodontol 2014; 85:e295-303.
15. Belstrøm D, Constancias F, Liu Y, et al. Metagenomic and metatranscriptomic analysis of saliva reveals disease-associated microbiota in patients with periodontitis and dental caries. NPJ Biofilms Microbiomes 2017; 3:1-8.
16. Papagerakis P, Zheng L, Kim D, et al. Saliva and gingival crevicular fluid (GCF) collection for biomarker screening. InOdontogenesis Hum Press 2019; 549-562.
17. Shahdad SA, Taylor C, Barclay SC, et al. A double�blind, crossover study of biotene oralbalance and bioxtra systems as salivary substitutes in patients with post�radiotherapy xerostomia. Eur J cancer care 2005; 14:319-326.
18. Piekarz T, Mertas A, Wiatrak K, et al. The influence of toothpaste containing Australian Melaleuca alternifolia oil and ethanolic extract of polish propolis on oral hygiene and microbiome in patients requiring conservative procedures. Mol 2017; 22:1957.
19. Dehghani M, Abtahi M, Hasanzadeh N, et al. Effect of Propolis mouthwash on plaque and gingival indices over fixed orthodontic patients. J Clin Exp Dent 2019; 11:e244.
20. Dommisch H, Staufenbiel I, Schulze K, et al. Expression of antimicrobial peptides and interleukin�8 during early stages of inflammation: An experimental gingivitis study. J Periodont Res 2015; 50:836-845.
21. Schincaglia GP, Hong BY, Rosania A, et al. Clinical, immune, and microbiome traits of gingivitis and peri-implant mucositis. J Dent Res 2017; 96:47-55.
22. Meyer S, Giannopoulou C, Cancela J, et al. Experimental mucositis/gingivitis in persons aged 70 or over: microbiological findings and prediction of clinical outcome. Clin Oral Investig 2019; 23:3855-3863.
23. Lee A, Ghaname CB, Braun TM, et al. Bacterial and salivary biomarkers predict the gingival inflammatory profile. J Periodontol 2012; 83:79-89.
24. Ebersole JL, Nagarajan R, Akers D, et al. Targeted salivary biomarkers for discrimination of periodontal health and disease (s). Front Cell Infect Microbiol 2015; 5:62.
25. Giannopoulou C, Kamma JJ, Mombelli A. Effect of inflammation, smoking and stress on gingival crevicular fluid cytokine level. J Clin Periodontol 2003; 30:145-153.
26. Koo H, Gomes BP, Rosalen PL, et al. In vitro antimicrobial activity of propolis and Arnica montana against oral pathogens. Arch Oral Biol 2000; 45:141-148.
27. Orsi RO, Sforcin JM, Rall VL, et al. Susceptibility profile of Salmonella against the antibacterial activity of propolis produced in two regions of Brazil. J Venom Anim Toxins Incl Trop Dis 2005; 11:109-116.
28. Velazquez C, Navarro M, Acosta A, et al. Antibacterial and free�radical scavenging activities of Sonoran propolis. J Appl Microbiol 2007; 103:1747-1756.
29. Morawiec T, Dziedzic A, Niedzielska I, et al. The biological activity of propolis-containing toothpaste on oral health environment in patients who underwent implant-supported prosthodontic rehabilitation. Evidence Based Complem Alternative Med 2013; 2013:704947.
30. Park YK, Koo MH, Abreu JA, et al. Antimicrobial activity of propolis on oral microorganisms. Curr Microbiol 1998; 36:24-28.
31. Ozan F, Sümer Z, Polat ZA, et al. Effect of mouthrinse containing propolis on oral microorganisms and human gingival fibroblasts. Eur J Dent 2007; 1:195-201.
32. Barrientos L, Herrera CL, Montenegro G, et al. Chemical and botanical characterization of Chilean propolis and biological activity on cariogenic bacteria Streptococcus mutans and Streptococcus sobrinus. Braz J Microbiol 2013; 44:577-585.
33. Seidel V, Peyfoon E, Watson DG, et al. Comparative study of the antibacterial activity of propolis from different geographical and climatic zones. Phytother Res 2008; 22:1256-1263.
34. Gonzales JR, Herrmann JM, Boedeker RH, et al. Concentration of interleukinâ?1β and neutrophil elastase activity in gingival crevicular fluid during experimental gingivitis. J Clin Periodontol 2001; 28:544-549.
35. Woo KJ, Jeong YJ, Inoue H, et al. Chrysin suppresses lipopolysaccharide-induced cyclooxygenase-2 expression through the inhibition of nuclear factor for IL-6 (NF-IL6) DNA-binding activity. FEBS Lett 2005; 579:705-711.
36. Wang LC, Lin YL, Liang YC, et al. The effect of caffeic acid phenethyl ester on the functions of human monocyte-derived dendritic cells. BMC Immunol 2009; 10:1-3.
37. Machado JL, Assunçao AK, da Silva MC, et al. Brazilian green propolis: anti-inflammatory property by an immunomodulatory activity. Evid Based Complement Alternat Med 2012; 2012.