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Table of Contents   
ORIGINAL RESEARCH  
Year : 2012  |  Volume : 23  |  Issue : 2  |  Page : 294
Honeybee propolis extract in periodontal treatment: A clinical and microbiological study of propolis in periodontal treatment


Department of Periodontics, V.K. Institute of Dental Sciences, KLE University, Belgaum, India

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Date of Submission30-Apr-2011
Date of Decision13-Aug-2011
Date of Acceptance08-Oct-2011
Date of Web Publication3-Sep-2012
 

   Abstract 

Objective: This study was conducted to evaluate by clinical and microbiological parameters the effect of subgingival irrigation with propolis extract.
Materials and Methods: Twenty patients diagnosed with chronic periodontitis, each presenting three non-adjacent teeth with deep pockets, were selected. Subgingival plaque sampling and clinical recording (at baseline) and scaling and root planing was performed. Two weeks later the selected periodontal sites were submitted to one of the following treatments: Irrigation with a hydroalcoholic solution of propolis extract twice a week for 2 weeks (group A); irrigation with a placebo twice a week for 2 weeks (group B); or no additional treatment (group C). Clinical and microbiological data was collected at baseline and after 4, 6, and 8 weeks.
Results: A decrease in the total viable counts of anaerobic bacteria (P=.007), an increase in the proportion of sites with low levels (≤10 5 cfu/mL) of Porphyromonas gingivalis (P=.044), and an increase in the number of sites negative for bleeding on probing was observed in group A sites as compared to group B and C sites.
Conclusion: Subgingival irrigation with propolis extract as an adjuvant to periodontal treatment was more effective than scaling and root planing as assessed by clinical and microbiological parameters.

Keywords: Propolis, periodontitis, irrigation, periodontal pocket, placebo, anaerobic bacteria, adjuvant, scaling and root planing, clinical parameters, microbiological parameters

How to cite this article:
Coutinho A. Honeybee propolis extract in periodontal treatment: A clinical and microbiological study of propolis in periodontal treatment. Indian J Dent Res 2012;23:294

How to cite this URL:
Coutinho A. Honeybee propolis extract in periodontal treatment: A clinical and microbiological study of propolis in periodontal treatment. Indian J Dent Res [serial online] 2012 [cited 2019 Mar 25];23:294. Available from: http://www.ijdr.in/text.asp?2012/23/2/294/100449
Propolis, sometimes called bee glue, is a natural resinous substance collected by honey bees (Apis mellifera L.) from plant buds and bark exudates. Mixed with their hypopharyngeal gland secretions, beeswax, and pollen, [1] propolis is used by the bees to fill cracks and crevices in the hive, smooth out the interior surface, and block hive entrances to prevent the entry of robber bees, antagonistic insects, or rodents.

Propolis is a very complex mixture and its chemical constituents vary according to its source. A broad analysis reveals approximately 55% resinous compounds and balsam, 30% beeswax, 10% ethereal and aromatic oils, and 5% bee pollen. Bioflavonols are the key contributors to propolis' properties. According to a research undertaken at the Second Leningrad Scientific Conference on the Application of Apiculture (bee culture) in Medicine, bee propolis was found to be rich in vitamins A (carotene), B 1 , B 2 , and B 3 ; biotin; an array of bioflavonoids (key contributors to propolis' properties); and 14 of the 15 minerals that the human body requires for normal function. It also contains a number of unidentified compounds that work together synergistically to create a balanced, nutritive substance. [2]

Propolis is one of the few treatments, which has drawn the attention over a long period of time. The potential of this beehive product as a natural antibiotic has long attracted interest. The ancient Egyptians used it to embalm their dead. In the Balkan states, propolis remains one of the most frequently used medications even today. It has recently found use in various formulations for dermatology, otorhinolaryngology, gynecology, odontology, and veterinary medicine. [2]

In addition, it results in one of many most sterile substances recognized inside the animal kingdom. Propolis is found to have strong inhibitory effects on at least 21 species of bacteria, 9 species of fungi, 3 species of protozoa, and a wide range of viruses. One study has confirmed that propolis exerts some of its anti-inflammatory and anti-infection properties through the inhibition of dihydrofolate reductase activity, which plays an important role in intermediary metabolism, mainly in rapidly dividing cells such as bacteria or tissues showing uncontrolled growth (e.g., tumors). [3]

The infectious nature of periodontal disease and the inherent limitations of scaling and root planing leads sometimes to the use of antimicrobial agents in order to reduce periodontal pathogens as advocated by the Committee on Research, Science, and Therapy in 1996. [4] Locally delivered antimicrobials are an alternative to systemic antibiotics and may help to arrest periodontal disease progression. [5],[6]

This study aimed to evaluate using clinical and microbiological parameters the additional effects of subgingival irrigation with propolis extract after mechanical therapy on deep periodontal pockets.


   Materials and Methods Top


Propolis samples

Propolis was collected from the beekeeping section of the Institute of Agricultural Sciences, Bangalore, Karnataka, India. Propolis samples were obtained from colonies of Africanized honeybees (Apis mellifera) and collected over a period of one year using plastic nets. The nets were removed and frozen at the end of each month to facilitate propolis removal [Figure 1].
Figure 1: Raw Propolis (unprocessed) collected from Apis mellifera colonies

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The unprocessed propolis was sent to the Department of Biochemistry, Jawarharlal Nehru Medical College, Belgaum, Karnataka, India, for preparation of the study and placebo solutions.

Preparation of 20% propolis hydroalcoholic solution (study solution)

Extracts of the propolis samples were prepared by freezing the samples at -20°C and grinding the frozen material in a precooled mortar and pestle. The ground material was mixed with 99.8% (v/v) ethanol in hermetically-sealed glass vessels at a ratio of 1 g of propolis powder to 3 ml of ethanol. Vessels were then incubated for 1 week at room temperature in darkness, with constant agitation. The resulting ethanol solutions were clarified by centrifugation at 7000 g for 60 s and the supernatants were collected and filtered through Whatman #4 filter paper. Ethanol-soluble components were then collected by evaporation to dryness under vacuum. The extracts were re-dissolved in pure ethanol to obtain 20% (w/v) solutions. The final solutions were stored in hermetically-sealed brown-glass bottles at room temperature. [1],[7] Previous studies have shown that propolis extract is stable for 6 months, maintaining its antimicrobial activity over this period. [8]

Preparation of 14% ethanol solution (placebo)

Fourteen milliliters of ethyl alcohol was mixed in 100 ml of distilled water to obtain a 14% ethanol solution; this was stored till use.

Procedure [Figure 2]

A total of 20 patients (11 females and 9 males, aged 25-57 years) diagnosed with chronic periodontitis, exhibiting at least three single-rooted teeth with periodontal pocket depths ≥5 mm, who reported to the Department of Periodontics, K.L.E University, Belgaum, Karnataka, India, were selected for the study. The Ethical Committee of K.L.E Institute of Dental Sciences, Belgaum, Karnataka, India, approved all procedures. Informed consent was obtained in writing from each patient [Figure 3].
Figure 2: Clinical protocol of the study

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Figure 3: Chronic periodontitis patient

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On their first visit all patients were examined (clinical exam I) in order to register the plaque index, [9] gingival index, [10] pocket probing depth, [11] bleeding upon probing, [11] and clinical attachment level. [11] On the patient's second visit, subgingival plaque was sampled (sample 1) and scaling and root planing of all teeth included in the study was performed using curettes.

Two weeks later the selected teeth were submitted to one of the following treatments:

  • Group A: Irrigation with 3 mL 20% propolis hydroalcoholic solution twice a week for 2 weeks [Figure 4]
  • Figure 4: Group A (study group)

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  • Group B: Irrigation with 3 mL of a placebo (14% ethanol solution) twice a week for 2 weeks
  • Group C: Ultrasonic scaling and root planing.


Subgingival plaque samples were collected with a sterile curette from the deepest portion of the pocket after 2 weeks (sample II), 4 weeks (sample III), 6 weeks (sample IV), and 8 weeks (sample V) after scaling and root planing procedures. The samples were sent for anaerobic culturing to the Department of Microbiology, Jawarharlal Nehru Medical College, K.L.E University, Belgaum, Karnataka, India.


   Results Top


Eighteen of the twenty selected patients were followed till the end of the study. Two patients did not attend the scheduled appointments.

At baseline, there were no differences between the three groups in clinical or microbiological parameters.

The effect of treatment with propolis in sites from group A was compared with clinical and microbiological data obtained from groups B and C by simple correlation analysis (Pearson correlation coefficient). A decrease in the total viable counts of anaerobic bacteria was observed in sites from group A as compared to sites from the other groups (P=.007) [Table 1]; [Figure 5]. An increase in the proportion of sites with low levels of P gingivalis (≤10 5 cfu/mL) was observed in sites from group A as compared to the other groups [Table 2]; [Figure 6].
Figure 5: Proportion of sites with ≥105 cfu of total viable counts/ sample

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Figure 6: Proportion of sites with ≤105 cfu of Porphyromonas gingivalis sample

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Table 1: Proportion of sites with ≥105 cfu of total viable counts/sample

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Table 2: Proportion of sites with ≤105 cfu/sample of Porphyromonas gingivalis

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When clinical parameters were evaluated, the reduction in the proportion of sites positive for bleeding upon probing was significantly greater for group A (P=.001) and group B (P=.002) when compared with group C (P=.026) [Table 3]; [Figure 7]. There was a significant decrease in the proportion of sites positive for bleeding upon probing and in the clinical probing depth even at 6 weeks, when the patients were recalled for follow-up.
Figure 7: Proportion of sites negative to bleeding upon probing

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Table 3: Proportion of sites negative to bleeding upon probing

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Six weeks after the irrigation procedures a significant decrease in clinical probing depth was observed in group A (P=.001) when compared to groups B and C [Table 4] and [Figure 8].
Figure 8: Proportion of sites showing pocket probing depth reduction from 5-8mm to ≤ 3mm

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Table 4: Proportion of sites showing pocket probing depth reduction from 5-8 mm to ≤3 mm

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   Discussion Top


Local-delivery antimicrobial agents (LDAs) are available for use as adjuncts to scaling and root planing (SRP) in the treatment of periodontitis. [12] These products are placed into the periodontal pockets in order to reduce subgingival bacterial flora and the clinical signs of periodontitis. Clinicians may consider the use of LDAs in chronic periodontitis patients as an adjunct to scaling and root planning when localized recurrent and/or residual probing depth of ≥5 mm with inflammation is still present after conventional therapies. [12]

Propolis is highly regarded for its medicinal properties, especially in Eastern Europe, South America, and Asia. The antimicrobial properties of propolis against human pathogens have been known since antiquity. [13] A number of studies have presented evidence that propolis has strong hepatoprotective, antitumor, antioxidative, antimicrobial, and anti-inflammatory properties. [14],[15],[16] Curiously, few studies have examined the antimicrobial properties of propolis against periodontopathogens.

In this study, the effect of propolis irrigation was compared with that of irrigation with placebo (B) and no irrigation (C) in the same patient. This was done in order to avoid differences in response to the treatment by different individuals.

In this study we have expressed the micobiological parameters in terms of total viable count, which is of clinical interest since Wolff et al. (in 1994) have reported that the total viable counts between 10 5 cfu/ml and 10 5 cfu/mL are related to low numbers of periodontopathogenic organisms. [17] In this study, a reduction in the total viable counts of anaerobic bacteria was detected at 2 weeks after the irrigation procedures for group A, and this reduction was maintained at 6 weeks after the irrigation procedures.

At the beginning of the experiment, as well as after scaling and root planing procedures, the majority of the evaluated sites exhibited high levels of P gingivalis (≥10 5 cfu/sample) [Figure 9]. The use of propolis extract in group A sites increased the proportion of sites with low levels (≤10≤ cfu/sample) of P gingivalis from 50% to 80% [Figure 10].
Figure 9: Anaerobic culture plate at baseline showing colonies of Porphyromonas gingivalis

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Figure 10: Anaerobic culture plate 8 weeks after baseline showing colonies of Porphyromonas gingivalis

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Repopulation by periodontopathogenic organisms usually occurs 4-8 weeks after scaling, [18] and may influence the success of the therapy. In this study, there was an increase in the proportion of sites with low levels of P gingivalis for group A sites which received irrigation with propolis; this was observed at 2 weeks, with further increases observed at 4 weeks, 6 weeks, and 8 weeks after propolis irrigation. This data suggests the effect of propolis irrigation might be long lasting, leading to a change in the repopulation process occurring in the periodontal pocket.

Scaling and root planing were efficient in increasing the proportion of sites negative to bleeding upon probing as well as in reducing the pocket probing depth to levels of ≤3 mm for all studied sites. A greater improvement in these clinical parameters was observed 4-8 weeks after the irrigation procedures in group A sites as compared to the other sites. Two studies have advocated that one session of scaling and root planing is not enough to maintain subgingival microbiota compatible with health in patients with pocket probing depth ≥5 mm. [19],[20] The antimicrobial and the anti-inflammatory benefits provided by propolis extract even after 6 weeks of the commencement of the treatment indicates that its use should be considered as an adjuvant to scaling and root planing.


   Conclusion Top


The results of this clinical study demonstrate the benefits provided by propolis extract and indicate that it should be considered for use as an adjuvant to scaling and root planing. Additional studies are needed to show whether an increase in the concentration of the propolis extract used and increase in the frequency of application could bring even better results.

 
   References Top

1.Dolci P, Ozino OI. Study of the in vitro sensitivity to honey bee propolis of Staphylococcus aureus strains characterized by different sensitivity to antibiotics. Ann Microbiol 2003;53:233-43.  Back to cited text no. 1
    
2.Gebaraa EC, Pustiglioni AN, de Lima LA, Mayer MP. Propolis extract as an adjuvant to periodontal treatment. Oral Health Prev Dent 2003;1:29-35.  Back to cited text no. 2
[PUBMED]    
3.Kosalec1 SL, Pepeljnjak S, Bakmaz M. Flavonoid analysis and antimicrobial activity of commercially available propolis products. Acta Pharma 2005;55:423-30.  Back to cited text no. 3
    
4.Committee on Research, Science and Therapy. Systemic antibiotics in Periodontics. J Periodontol 1996;67:831-8.  Back to cited text no. 4
    
5.Greenstein G. Effects of subgingival irrigation on periodontal status. J Periodontol 1987;58:827-36.  Back to cited text no. 5
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6.Rams TE, Slots J. Local delivery of antimicrobials in the periodontal pocket. Perio 2000;1996:139-59.  Back to cited text no. 6
    
7.Almeida EC, Menezes H. Anti-inflammatory activity of propolis extracts: A Review. J Venom Ani Tox Trop Dis 2008;2:191-212.  Back to cited text no. 7
    
8.Silva B Sr. Long-term stability of natural propolis: Antimicrobial and chemical evaluations. IADR 2009.  Back to cited text no. 8
    
9.Loe H. The Gingival Index, the Plaque Index and the Retention Index Systems. J Periodontol 1967;38:610.  Back to cited text no. 9
    
10.Loe H, Silness J. Periodontal disease in pregnancy. I. Prevalence and severity. Acta Odontol Scand 1963;21:533.  Back to cited text no. 10
    
11.Carranza FA, Newman MG, Takei H, Klokkevold RP. Clinical Periodontology. 10th ed. Philadelphia: WB Saunders and Co; 2006. p. 551-3.   Back to cited text no. 11
    
12.American Academy of Periodontology Statement on Local Delivery of Sustained or Controlled Release Antimicrobials as Adjunctive Therapy in the Treatment of Periodontitis. J Periodontol 2006;77:8.  Back to cited text no. 12
    
13.Ghisalberti EL. Propolis: A review. Bee World 1979;60:59-84.  Back to cited text no. 13
    
14.Banskota AH, Tezuka Y, Kadota S. Recent progress in pharmacological research of propolis. Phytother Res 2001;15:561-71.  Back to cited text no. 14
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15.Sforcin JM. Propolis and immune system: A review. J Ethnopharmacol 2007;113:1-14.  Back to cited text no. 15
[PUBMED]    
16.Martos VM, Navajas RY, Fernandez LJ, Alvarez JA. Functional properties of honey, propolis, and royal jelly. JFS 2008;73:117-24.  Back to cited text no. 16
    
17.Wolff L, Dahlen G, Aeppli D. Bacteria as risk markers for periodontitis. J Periodontol 1994;65:498-510.  Back to cited text no. 17
    
18.Magnusson I, Lindhe J, Yoneyama T, Lilijenberg B. Recolonisation of subgingival microbiota following scaling in deep pockets. J Clin Periodontol 1984;25:30-3.  Back to cited text no. 18
    
19.Lembariti BS, Van der Weijden GA, Van Palestein Helderman WH. The effect of a single scaling with or without oral hygiene instruction on gingival bleeding and calculus formation. J Clin Periodontol1998;25:30-3.  Back to cited text no. 19
    
20.Ludovico S, Ramaglia L, Gulletta E, Iacono V. Recolonization of subgingival microflora after scaling and root planning in human periodontitis. J Periodontol 1990;61:579-84.  Back to cited text no. 20
    

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Correspondence Address:
Amita Coutinho
Department of Periodontics, V.K. Institute of Dental Sciences, KLE University, Belgaum
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-9290.100449

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]

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