Propolis effects in periodontal disease seem to affect coronavirus disease: a meta-analysis

Abstract This meta-analysis aimed to investigate the effects of propolis on the severity of coronavirus disease symptoms by reducing periodontal disease. PubMed, EMBASE, SciELO, Web of Science, and SCOPUS databases were systematically searched. Studies have been conducted analyzing propolis’s effects on COVID-19 and periodontitis. The study was conducted according to the PRISMA statement and registered in PROSPERO. Risk of Bias (RoB) assessment and meta-analysis of clinical studies were performed (Review Manager 5, Cochrane). The certainty of the evidence was assessed using GradePro (GDT). Studies have shown propolis flavonoids inhibit viral replication in several DNA and RNA viruses, including coronaviruses. Propolis components have an aminopeptidase inhibitor activity that can inhibit the main proteases of SARS viruses and seem to inhibit protein spikes, which are sites of most mutations in SARS-CoV strains. The meta-analysis showed favorable results with the use of propolis on probing depth (95%CI: 0.92; p < 0.001), clinical attachment level (95%CI: 1.48; p < 0.001), gingival index (95%CI: 0.14; p = 0.03), plaque index (95%CI: 0.11; p = 0.23), and blending on probing (95%CI: 0.39; p < 0.001). The antibacterial activity of propolis could be mediated through its direct action on microorganisms or the stimulation of the immune system, activating natural defenses. Thus, propolis inhibits the replication of SARS-CoV-2 as well as its bacterial activity. Treatment with propolis improves general health and facilitates the activation of the immune system against coronavirus.


Introduction
Coronavirus disease (COVID- 19) is an infectious disease caused by a novel coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which affects the respiratory and hematological systems.SARS-CoV-2 is a single-stranded RNA virus with an envelope similar to that of coronaviruses, causing severe acute respiratory syndrome and Middle East respiratory syndrome, which mainly causes respiratory and enteric diseases.The primary clinical symptoms of COVID-19 are fever, dry cough, sore throat, body pain, diarrhea, anosmia, and ageusia. 1 Declaration of Interests: The authors certify that they have no commercial or associative interest that represents a conflict of interest in connection with the manuscript.
In the absence of specific drugs to treat COVID-19, there is an urgent need to find alternative approaches to prevent and control the spread of the virus.Bioactive substances, such as propolis, may play an essential role in combating COVID-19.Studies have shown that the pathogenesis of COVID-19 can be minimized by propolis components that act on the functional activity of the main proteases of SARS-CoV-2. 2,3ropolis is a balsamic and resinous product containing different plant parts and molecules secreted by bees. 4,5It is composed of a mixture of 50% vegetable resins, 30% wax, 10% essential and aromatic oils, 5% pollen, and 5% other organic substances, including polyphenols, flavonoids, amino acids, minerals, ethanol, vitamin A, vitamin B complexes, and vitamin E. The chemical composition, aroma, and color of propolis vary according to geographic region and can be found in several types.The most common are green, red, and brown ones. 4,5he biological activity of propolis is attributed to various chemical constituents that block or reduce the chance of viral entry into the host cells. 4,5However, the precise mechanism underlying this antiviral activity remains unknown.Among the proposed hypotheses, the one that stands out suggests that propolis destroys the external envelope of the virus, thereby inhibiting its entry into cells and interrupting viral replication. 6ropolis has anti-inflammatory, antiviral, antioxidant, and immunoregulatory effects.Its components have an inhibitory effect on angiotensin-converting enzyme 2 (ACE2), transmembrane protease, serine 2 (TMPRSS2), main protease (M PRO ), and serine/ threonine-protein kinase PAK 1 (PAK1) signaling pathways, all of which could contribute to reducing the pathophysiological consequences of COVID-19. 7,8 recent study showed an association between the severity of COVID-19 and periodontal inflammation since the latter causes an increase in markers such as D-dimer, white blood cell count, and C-reactive protein level, which are also related to the severity of COVID-19. 9][12][13] Periodontitis is one of the most prevalent oral inflammatory diseases. 14It is characterized as a multifactorial chronic inflammatory disease associated with biofilm dysbiosis and the progressive destruction of dental insertion. 15,16urrently, there are no antiviral therapeutic drugs available for COVID-19 in humans.Thus, in the absence of specific antiviral agents against COVID-19, the reuse of drugs previously used to reduce viral infectivity and replication and improve the immune system has become an alternative that should be investigated.Thus, the present study aimed to highlight the role of propolis in reducing the severity of COVID-19 symptoms by reducing periodontal disease.

Methodology
This systematic review was reported according to Cochrane, the National Health Service Centre for Reviews and Dissemination, 17 and the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA statement 2020) guidelines. 18The study protocol was registered with the International Prospective Register of Systematic Reviews (PROSPERO-CRD42021233759). The eligibility criteria for the participants, interventions, comparisons, outcomes, and study design were defined in accordance with the PRISMA statement.The following questions were raised in this systematic review: a) Does propolis affect the treatment of periodontal diseases?b) Does propolis exert an antiviral action against SARS-CoV-2?c) Can treating periodontal disease with propolis positively influence COVID-19 patients?
This study presented a systematic review of the effect of propolis on periodontitis and considered a comprehensive review of the impact of propolis on coronavirus.These two searches were necessary because of the lack of studies that addressed the direct relationship between periodontal disease and COVID-19, using propolis as a therapeutic agent.

Search strategy and selection criteria
PubMed, EMBASE, Web of Science, SciELO, and SCOPUS databases were systematically searched using the following critical medical subject headings terms using a two-search strategy: ("Propolis" AND ["Periodontal disease" OR "Periodontitis"]) and ("Propolis" AND ["COVID-19" OR "SARS-CoV-2"]).Furthermore, a manual search was performed, and reference lists and previous systematic reviews were reviewed to identify other potentially relevant studies.The final search was conducted on September 28, 2021.
According to our selection criteria, we included only studies published in peer-reviewed journals.The studies were divided into two domains: the first considered the relationship between periodontal disease and propolis, and the second considered the relationship between COVID-19 and propolis.In the first domain, we included case reports, case series, and clinical studies that investigated the treatment of periodontal disease using propolis.For the second domain, we included all studies that investigated the relationship between COVID-19 and propolis because of the small number of published studies on this subject.Duplicate publications and articles that did not meet the objectives of the systematic review were excluded.Figures 1 and 2 outline the procedure used in the literature search.

Data extraction
Two investigators participated in each phase of the review and independently screened the titles and

Quality appraisal and risk of bias
The selected randomized and non-randomized clinical trials were appraised using appropriate tools for quality and risk of bias assessments for each study design.Non-randomized studies were assessed using the Methodological Index for Non-Randomized Studies (MINORS), 19 and randomized clinical trials were evaluated using the Jadad scale. 20he Jadad quality scale considers five items, each of which may vary in score from 0 to 5. Studies with scores ≤ 2 points were considered to be of poor methodological quality, whereas those with a score of ≥ 2 were considered good quality.The MINORS consider eight items for non-comparative studies and 12 for comparative studies.These items can be scored from 0 to 2 points each, with maximum total scores of 16 and 24 points for the comparative and non-comparative studies, respectively.
The Revised Cochrane risk-of-bias tool for randomized trials (RoB-2) 21 and the risk of bias in non-randomized studies 22 were used to assess the risk of bias (ROB) in randomized and non-randomized studies, respectively.The Review Manager (RevMan) software (Cochrane Collaboration) was used to create the ROB graph and summary.The results were categorized as: a) low risk of bias, b) unclear risk of bias, and c) high risk of bias.The certainty of the evidence was performed on GradePro (GDT) based on the study design, risk of bias, inconsistency, indirectness, imprecision, other covariates (confounding and publication bias), and the results of the study (number of patients and Confidence Interval, CI).Certainty was classified as high, moderate, low, or very low.

Summary measures
Periodontal disease × propolis studies were categorized based on sample characterization, exposu re, compa rators, a nd outcomes.For COVID-19 studies, the highlights in each study were selected, grouped according to similarity, and presented in the form of a table.The data on clinical periodontal parameters were analyzed and grouped by two researchers and tabulated by one researcher.Another researcher reviewed the tabulated data, thereby decreasing the likelihood of disagreement.

Data synthesis and meta-analysis
The meta-analysis was performed using the Cochrane Collaboration software Review Manager (RevMan) version 5.2.The effects of propolis use in patients with periodontitis were estimated using the mean differences and 95% confidence intervals (CIs) for each clinical parameter.Statistical heterogeneity was assessed using the chi-squared test (p < 0.1) and the calculation of the I2 statistic.We considered an I2 value > 75% to indicate significant heterogeneity across the studies. 23A randomeffects model was adopted for all meta-analyses to reduce potential heterogeneity because the studies had different characteristics, sample sizes, and group data.A funnel plot was used for all the analyses.

Database search
The process used for selecting studies in the systematic review is outlined in Figures 1 and 2. We identified 61 articles related to ("Propolis" AND ["COVID-19" OR "SARS-CoV-2"]) using this strategy to search all the scientific databases.After removing unrelated and duplicate articles, 18 cases were assessed for eligibility, and 13 articles were identified as eligible for qualitative analysis.Similarly, we identified 123 studies related to ("Propolis" AND ["Periodontal disease" OR "Periodontitis"]) and 12 from other sources.After removing duplicates and unrelated articles, 22 cases were assessed for eligibility, and 10 studies were identified as eligible for qualitative analysis.Eight of the ten final studies were included in the quantitative analysis.

Sample characteristics
Clinical studies assessing the effect of propolis on periodontitis and studies reporting its impact on COVID-19 symptoms were considered for analysis.Nine randomized clinical trials and one non-randomized clinical trial (n = 328 patients) demonstrated propolis's effectiveness in treating periodontitis.Thirteen studies reported the mechanism of action of propolis against coronaviruses.The characteristics of the studies are presented in Tables 1 and 2.

Propolis components, properties, and mechanism of action
The properties of the propolis components and their mechanisms of action against SARS-CoV-2 and COVID-19 are summarized in Table 2.

Clinical outcomes of periodontal treatment with propolis
The periodontal outcomes assessed after propolis treatment are shown in Table 3.Five clinical parameters presented in the articles were grouped and analyzed for meta-analysis (Figure 3a-e).Probing pocket depth (PPD) data were grouped from eight studies, Clinical Attachment Level (CAL) data were grouped from seven studies, Gingival Index (GI) data were grouped from five studies, and Plaque Index (PI) and Blending on Probing (BOP) data were grouped from four studies.None of the other parameters could be grouped for quantitative analysis by the authors.Clinical parameters were reported to improve following propolis therapy in all studies included in our review.

Quality assessment and risk of bias
All clinical studies were analyzed using specific scales for quality and ROB assessments.One randomized clinical trial scored 2 (poor quality) on the Jadad scale.All other studies scored between 3 and 5.A summary of the ROB in each study is presented in Figure 4.The studies obtained an overall bias percentage for the five domains evaluated, as shown in Figure 5.
The non-randomized clinical trial scored 19 on the MINORS scale to evaluate comparative studies (maximum score = 24).The results of the ROB assessment are shown in Figure 6.
RCT studies included in the meta-analysis were considered and evaluated for the certainty of evidence.The certainty grade was evaluated as "High" for the BOP parameter and "Moderate" for PPD, GI, PI, and CAL (Figure 7).

Discussion
This systematic review and meta-analysis aimed to study the relationship between propolis treatment and periodontal disease and its possible systemic effects, leading to a decrease in the risk of COVID-19 severity.According to the results of studies on propolis, there is evidence that it may be a possible herbal agent against coronavirus.The effect of propolis on periodontitis also affects systemic health, supporting the improvement of the patient's immunity and reducing inflammation, thus reducing the possibility of the severity of COVID-19.
COVID-19 is associated with increased levels of activated pro-inflammatory chemokines and cytokines that lead to the development of atypical pneumonia with rapid respiratory impairment and lung failure. 44COVID-19 is characterized by a connection between viral peak protein S (spike) and angiotensin-converting enzyme 2 (ACE2).ACE2 is a protein in the human body that facilitates virus entry into cells and is an essential receptor for SARS-CoV-2.Activation of the spike protein is mediated by proteases, such as TMPRSS2, which are critical in viral infection. 7,34,45After entry and subsequent endocytosis, coronavirus infection causes the positive regulation of PAK1.The main protease of SARS-CoV-2 coronavirus, M PRO (cysteine enzyme), is essential for the processing of polyproteins by the coronavirus and its life cycle.Therefore, inhibition of the active site of this enzyme is of great relevance for the discovery of effective drugs.The inhibition of ACE2, TMPRSS2, PAK1, and M PRO are important targets for treating COVID-19. 7,8urthermore, propolis can be beneficial and efficient against new strains of COVID-19.Mutations in SARS-CoV strains usually occur in the spike protein. 46,47The omicron variant presents many mutations in this virus protein, causing genetic changes that affect virus characteristics such as transmissibility, disease severity, immune escape, and diagnostic and therapeutic escape. 46.Thus, treatment with propolis can improve the prognosis of individuals infected with this and other strains.
Propolis can be used as a complementary treatment for patients with COVID-19.It has a potential impact on the replication of SARS-CoV-2, either by direct antiviral effects, anti-inflammatory effects promoting immunoregulation of pro-inflammatory cytokines, or indirect effects owing to its immunomodulatory effect on the host immune system and interference with the host inflammatory response.The antiviral effects of these compounds are mediated by inhibition of viral transmission to other cells, inhibition of viral propagation, and destruction of the external envelope of the virus. 7,34,45he biological activity of propolis is attributed to various chemical constituents, such as caffeic acid phenethyl ester (CAPE), flavonoids, quercetin, kaempferol, artepillin C, phenolic acid ester, chrysin, galangin, apigenin, pinobanksin 5-methyl ether, pinobanksin. 5APE is a bioactive component of propolis that may inhibit the functional activities of M PRO and PAK1. 7,8Artepillin C, a prenylated derivative of p-coumaric acid, is one of the main phenolic compounds found in Brazilian green propolis.It inhibits PAK1 activity, has anti-inflammatory properties, and can increase immunity against COVID-19 by inhibiting PAK1, which is responsible for suppressing the host immune system. 36,43ropolis components such as CAPE, rutin, chrysin, and myricetin affect ACE2 receptors. 7,37Rutin and CAPE affect the protease COVID-3CL and spike protein. 41Proteases are potential targets for inhibition of COVID-19 replication.Kaempferol is involved in the inhibition of TMPRSS2. 7,37Quercetin and vitamin C have shown aminopeptidase inhibitory activities that can interrupt the main proteases of SARS-CoV-2. 35,36ropolis produced by the bee Tetragonula sapiens inhibits the conversion of ACE2 (the SARS-CoV-2 receptor in the human body).Its compounds, sulabiroin A, isorhamnetin, (2S) -5,7-dihydroxy-4-methoxy-8prenylflavanone, and in particular, glyasperin A and broussoflavonol F, exhibit the ability to inhibit SARS-CoV-2 main protease activity. 38,42ome of the propolis extracts' flavonoid and caffeic acid components can act on the microbial membrane or cell wall, causing functional and structural damage to the microorganisms. 48In addition, they can effectively modulate cytokines and inflammatory mediators, thereby inhibiting the production of prostaglandins and transforming growth factor-ß. 49hese components have also been shown to be effective in treating periodontitis.Previous Propolis effects in periodontal disease seem to affect coronavirus disease: a meta-analysis studies have reported a decrease in the levels of Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum in patients with periodontitis treated with propolis. 26,31In addition to its antimicrobial effects against P. gingivalis, artepillin-C, a component of propolis, also exerts an inflammatory effect. 50revious systematic reviews concluded that treating periodontitis with propolis is safe and combined with conventional therapy may enhance the clinical parameters compared with non-surgical treatment alone or with placebo. 51,52Assunção et al. 51 excluded studies comprising patients with systemic disease.This reduces the power of propolis analysis in patients' systemic health.
This systematic review and meta-analysis analyzed studies that clinically evaluated patients with periodontitis.Eight studies classified patients according to the chronicity of the disease, [25][26][27][28][29][30]32,33 whereas two 24,31 classified patients according to the severity of the disease (moderate or severe). This y have led to a bias in evaluating the studies included in our review. Despie these possible biases, the certainty of evidence in our analyses showed moderate certainty for PPD, GI, PI, and CAL and high certainty for BOP.These results are related to the type of parameter used for evaluation, which is directly linked to the reduction in inflammation in periodontal tissues.For the RCTs RoB, the selected studies had more than 50% on the "measurement of the outcome" domain and more than 75% low risk of bias in the other four evaluated domains.For the non-RCT study, only one domain (missing data) was categorized as high RoB, and the others (measurement of outcomes) were categorized as unclear RoB.In this way, we can consider that, overall, the selected studies were evaluated as having low RoB or unclear RoB.The controlled study design positively influenced the quality of the results.However, all the analyzed studies showed favorable results for using propolis compared with placebo or conventional treatments as control groups.The meta-analysis showed statistical significance for PPD (95%CI: 0.92; p < 0.001), CAL (95%CI: 1.48; p < 0.001), GI (95%CI: 0.14; p = 0.03), and BOP (95%CI: 0.39; p < 0.001).The PI results (95%CI :0.11; p = 0.23) did not present statistically significant differences, but they similarly favored treatment with propolis.These data support the hypothesis that this treatment is an effective option for patients with periodontitis.
Individuals with diabetes and periodontitis may have positive regulation of ACE2 expression, making it a favorable environment for SARS-CoV-2 entry into the cell through ACE2 receptors. 53Treatment of periodontal disease has been shown to positively influence glycemia in patients with diabetes. 3The control of glycemic levels throughout periodontal treatment is of great relevance, as diabetes is one of the most prevalent comorbidities in patients with severe COVID-19.
Propolis has shown positive results as an adjunctive treatment for periodontitis compared with traditional therapy.Likewise, various components of propolis have shown antiviral effects against SARS-CoV-2.The antiviral effect of propolis, in combination with a reduction in the levels of periodontal inflammation, can lead to an improvement in general health and, consequently, a decrease in the risk of severe COVID-19.

Conclusions
The results of our meta-analysis suggest that propolis is a low-cost treatment option that affects SARS-CoV-2 protease through basic treatments in patients with periodontitis.Several components of propolis have anti-inflammatory and immunoregulatory activities, including the inhibition of PAK1, ACE2, M PRO , and TMPRSS2.In addition to this direct relationship, the reduction of comorbidities such as hypertension and diabetes by periodontal treatment is a major factor for improvement in general health and, thus, a reduction in the severity of COVID-19.

Figure 3 .
Figure 3. Forest plots for clinical parameters with and without propolis treatment.a) Pocket probing depth; b) Clinical attachment level; c) Gingival index; d) Plaque index and; e) Blending on probing.

Figure 4 .
Figure 4. Summary Risk of bias of Randomized Clinical Trial studies.

Figure 5 .
Figure 5. Risk of Bias graph of Randomized Clinical Trials.

Figure 6 .Figure 7 .
Figure 6.Summary Risk of bias of non-randomized clinical study.
Prisma 2020 Flow Diagram for Propolis and periodontitis search.

Table 1 .
Sample and intervention characterization for clinical studies assessing propolis effects in periodontitis treatment.

Table 3 .
Assessment methods and outcomes of intervention with propolis in patients with periodontal disease.
Plaque control record (PCR); Tooth mobility (TM); Probing pocket depth (PPD); Clinical attachment level (CAL); Bleeding on probing (BOP).CAL: improved in the propolis and minocycline groups, as compared to the placebo, but with no statistically significant difference; PPD: statistically significant improvement in the propolis group as compared to the placebo; Bacterial number: decrease in the propolis group for P. gingivalis, T. forsythia and P. intermedia.Plaque index (PI); Gingival index (GI); Pocket probing depth (PPD); Bleeding upon probing (BOP); Clinical attachment level (CAL) Viable counts of anaerobic bacteria and P. gingivalis: significant decrease in propolis group compared with others; PPD: significant decrease was observed in propolis group compared with others; GI, PI and CAL: no significant difference between groups.