Comparative evaluation of subgingival irrigation with propolis extract versus chlorhexidine as an adjunct to scaling and root planing for the treatment of chronic periodontitis: A randomized controlled trial.

Background: This study was carried out to investigate the effectiveness of subgingival irrigation with propolis extract compared to chlorhexidine as an adjunct to mechanical debridement to bring out the restoration of periodontal health in patients with chronic periodontitis. Materials and
Methods: Twenty subjects were selected and randomly assigned into two groups of ten subjects each, which received subgingival irrigation with 0.2% chlorhexidine (control group) and propolis extract (test group) after initial scaling and root planing on the 7th day and 15th day as an adjunctive treatment. Plaque index (PI), gingival index (GI), and probing pocket depth (PPD) were assessed at baseline, on the 15th day, and on the 30th day. Microbial analysis for the colony-forming unit (CFU) was done at baseline and on the 30th day.
Results: Statistically significant reduction was observed in PI, GI, PPD, and CFU counts from baseline to 30 days in both the groups. On intergroup comparison, the group received irrigation with chlorhexidine showed slightly better results; however, this difference was statistically nonsignificant compared to the group that received irrigation with propolis. Conclusions: It was concluded that propolis extract is as effective as chlorhexidine as a subgingival irrigant in reducing the subgingival plaque formation and microbiota from periodontal pockets. Copyright:

INTRODUCTION

Periodontitis is an inflammatory disease of supporting tissue of the teeth, caused by groups of microorganisms that accumulate in the form of biofilm and colonize at the tooth surface both above and at the gingival margin resulting in progressive destruction of the gingiva, periodontal ligament, and alveolar bone.[1] Mechanical debridement in the form of scaling and root planing (SRP) is a conventional and initial therapy of nonsurgical treatment usually effective in reducing the bacterial load by removal of plaque and calculus that harbors putative periodontal pathogens.[2] However, the invasion of these organisms within the gingival tissue and in deeper inaccessible areas does not guarantee complete absolution of the disease with this method of debridement alone.[3] Subgingival irrigation helps to gain access to deep and tortuous pockets, disrupt the subgingival biofilm, and eradicate the subgingival microflora.[4] This results in substantial variation in the effectiveness of nonsurgical periodontal therapy.

Chlorhexidine gluconate regarded as a gold standard is a broad-spectrum antibiotic that demonstrates antibacterial activity against Gram-positive and Gram-negative flora.[5] Chlorhexidine has been used as an anti-plaque agent for its substantivity and bacteriostatic activity. However, prolonged use of chlorhexidine exhibits drawbacks such as brownish discoloration of tooth surfaces and restorations, alteration in taste perception, and parotid duct stenosis.[67] Hence, there exists a need to find an alternative approach to avoid the drawbacks of chlorhexidine and wade of this surge in developing antibiotic resistance.[8]

Recent trends have been leaning toward phytotherapeutic extracts obtained from various plant and animal sources for their antimicrobial properties. One such substance is propolis, a mixture of natural resins produced by honeybees (Apis mellifera) from substances collected from different parts of plants, buds, and exudates. It has been proven to possess antibacterial, antioxidant, anti-inflammatory, and immunomodulatory action.[91011] The principle components of propolis such as bioflavonoids, pinocembrin, quercetin, galangin, aromatic acids, diterpenic acids, tannins, and phenolic compounds have been known to possess pharmacological actions, which work in synergy to bring out the biological action of propolis.[12] These compounds act on the bacterial cell walls and cytoplasm leading to the disintegration of the cell wall, cytoplasmic membrane, reduction in the protein content and its synthesis, and thereby bacteriolysis of oral biofilm.[13] Hence, we hypothesize that propolis extract may play a role in the reduction of periodontal inflammation and possess antibacterial effects against periodontal pathogens. In this study, we aimed to compare the effects of subgingival irrigation with 0.2% chlorhexidine and 25% propolis extract as an adjunct to SRP in subjects with chronic periodontitis on clinical parameters such as plaque index (PI), gingival index (GI), and probing pocket depth (PPD) and on microbiological parameters of the periodontal pocket microbes on the basis of aerobic microbial analysis (colony-forming unit [CFU] counts).

MATERIALS AND METHODS

The study was conducted in accordance with the Helsinki Declaration of 1975, as revised in 2000, and was approved by the institutional ethical committee with approval no. MGV/KBHDC/158/19-20. Informed written consent was obtained from all the participants.

The present study was a single-center randomized double-blind clinical trial conducted over a period of 2 months. A total sample size of 34 sites increased to a total of 40 sites considering dropout/attrition rate (i.e. twenty sites per group; two study groups) would yield 80% power to detect significant differences, with an effect size of 1.0 and significance level at 0.05. Based on this calculation, a total of 20 subjects of either of sexes were selected from the pool of 29 subjects from the Outpatient department of periodontology after a thorough periodontal clinical examination and a single-phase full-mouth SRP based on the inclusion criteria. The subjects were then randomly divided into two groups, Group A (control group) and Group B (test group) with ten subjects in each group using a flip coin method. Two teeth were then selected in each subject based on the sites with maximum probing depth adding up to the total of forty teeth (i.e. twenty teeth per group) which was measured using a UNC-15 probe.

Group A (control group) received subgingival irrigation with 0.2% chlorhexidine (Rexidin mouthwash, Indoco remedies Ltd. India)

Group B (test group) received subgingival irrigation with 25% propolis extract (Super Bee Propolis tincture, Hi-Tech Natural Products India Ltd. India).

The inclusion criteria included systemically healthy patients with mild-to-moderate periodontitis in accordance to American Academy of Periodontology (1999),[14] between 18 and 55 years of age, having a PPD of ≥5 mm, no history of any intake of systemic antibiotics in the last 6 months. Subjects on medication, presence of periapical or pulpal infection on qualifying tooth, patients of aggressive periodontitis, pregnant or lactating females, any allergy to chlorhexidine or propolis, prior history of any surgical periodontal therapy, having any habit of tobacco usage were excluded.

The clinical parameters assessed were PI,[15] GI,[16] and probing depth (PPD).[17] One week after the complete SRP, the first recordings for clinical parameters and collection of subgingival plaque samples for assessment of CFUs were done and considered as a baseline followed by on 15th and on 30th day from the baseline day for clinical parameters, and directly on 30th day from baseline for microbiological parameters.

After the recording of all the clinical and microbiological parameters at baseline, the first application of subgingival irrigation was carried out only in the selected teeth using a needle and a disposable 5 mL syringe. The needle tip was inserted into six different areas (mesiofacial, facial, distofacial, mesiolingual, lingual, and distolingual) of the selected periodontal pocket, the solution was deposited with light pressure, and the area was irrigated subgingivally for 30 s for 5 mL of chlorhexidine or propolis extract based on the selected group [Figure 1]. Subgingival irrigation was again repeated on the 7th day and on 15th day from the day of first application.

Figure 1

Method of subgingival irrigation using a syringe

For the collection of subgingival plaque samples, all the teeth included in this study were isolated using sterile cotton rolls, and then a sterile paper point was inserted in the deepest part of the pocket for 30 s. The paper point was then suspended in 2 mL of thioglycollate broth and transported immediately for microbiological analysis. 0.1 mL of this suspension of the sample was inoculated on a blood agar plate using the lawn culture method and then incubated for 24 h under aerobic conditions in a CO2 incubator at 37°C. CFUs were counted after the incubation period was over.

Subgingival irrigation and recording of the parameters were performed by two different operators to avoid bias. All the subjects were advised to brush their teeth twice a day throughout the study. No mouthwash was prescribed to the subjects.

RESULTS

All the recorded data were compiled and analyzed using GRAPH PAD software Inc. 11452 EI Camino Real #215, San Diego 92130, USA. Intragroup comparisons were made by paired t-test and unpaired t-test for intergroup comparisons. For all the tests, P < 0.05 was considered statistically significant. Both the treatment modalities were well tolerated by the patient and all of the twenty patients completed the 1-month follow-up period.

The mean scores for PI, GI, and PPD at each time interval for both the groups are shown in Table 1. On comparison for clinical parameters from baseline to 15 days, the mean PI statistically significantly reduced by 17.03% for Group A and slightly more by 19.57% for Group B, further statistically significant reduction was observed better for Group A by 31.44% and 29.57% for Group B from baseline to 30 days [Table 2]. However, on intergroup comparison, the mean reduction in PI was statistically nonsignificant between both the groups at 15th as well as at 30th day [Table 1].

Table 1

The mean values, standard deviation of plaque index, gingival index, probing pocket depth, and intergroup comparison with level of significance at different time intervals

Group	Mean±SD
	PI			GI			PPD
	Baseline	15 days	30 days	Baseline	15 days	30 days	Baseline	15 days	30 days
Group A	2.29±0.43	1.9±0.42	1.57±0.29	2.81±0.16	1.66±0.26	1.12±0.30	6.5±1.05	4.95±1.09	3.65±1.18
Group B	2.30±0.39	1.85±0.36	1.62±0.34	2.70±0.22	1.81±0.34	1.21±0.44	6.2±1.00	5.05±1.14	4.1±1.16
Mean difference	0.010	−0.045	0.050	−0.105	0.155	0.085	0.300	0.100	0.450
P*	0.930	0.720	0.620	0.097	0.115	0.490	0.362	0.779	0.216

*P<0.05 is statistically significant between two groups. All values expressed in terms of mean±SD. Group A – Subgingival irrigation with 0.2% chlorhexidine; Group B – Subgingival irrigation with 25% propolis extract; SD – Standard deviation; PI – Plaque index; GI – Gingival index; PPD – Probing pocket depth; P – Level of significance

Table 2

The percentage of mean change in plaque index, gingival index, probing pocket depth at different time intervals, and intragroup comparison with level of significance for both the groups

Clinical parameters	Percentage of mean change for Group A (n=20)			Percentage of mean change for Group B (n=20)
	Baseline versus 15 days (P)	Baseline versus 30 days (P)	15 days versus 30 days (P)	Baseline versus 15 days (P)	Baseline versus 30 days (P)	15 days versus 30 days (P)
PI	17.03% (<0.00011*)	31.44% (<0.0001*)	17.36% (0.0005*)	19.57% (<0.0001*)	29.57% (<0.0001*)	12.33% (0.0263*)
GI	40.92% (<0.0001*)	60.14% (<0.0001*)	17.37% (<0.0001*)	32.97% (<0.0001*)	55.19% (<0.0001*)	33.14% (<0.0001*)
PPD	23.84% (<0.0001*)	43.84% (<0.0001*)	26.26% (<0.0001*)	18.54% (<0.0001*)	34.68% (<0.0001*)	18.81% (<0.0001*)

*P<0.05 indicates statistically significant. Result expressed in terms of percentage change. Group A – Subgingival irrigation with 0.2% chlorhexidine; Group B – Subgingival irrigation with 25% propolis extract; n – Number of sites; P – Level of significance; PI – Plaque index; GI – Gingival index; PPD – Probing pocket depth

The mean GI statistically significant reduced more by 40.92% for Group A and 32.97% for Group B from baseline to 15 days, further statistically significant reduction was observed better for Group A by 60.14% and by 55.19% for Group B from baseline to 30 days [Table 2]. On intergroup comparison, the mean reduction in GI was statistically nonsignificant between both the groups at 15th as well as at 30th day [Table 1].

The mean PPD statistically significant reduced more by 23.84% for Group A and 18.54% for Group B from baseline to 15 days, further statistically significant reduction was observed better for Group A by 43.84% and by 34.68% for Group B from baseline to 30 days [Table 2]. On intergroup comparison, the mean reduction in GI was statistically nonsignificant between both the groups at 15th as well as at 30th day [Table 1].

The blood agar plates showed whitish, circular, raised with smooth edges, opaque group of colonies [Figures 2 and 3]. When the microbiological data were compared, a statistically significant reduction in CFU counts was observed for both the groups from baseline to 30 days. Furthermore, the intergroup comparison showed no statistically significant difference between Group A and Group B for the reduction of CFU counts [Table 3].

Figure 2

Bacterial colonies on blood agar plate from subgingival plaque samples (a) at baseline and; (b) from the site treated with subgingival irrigation with chlorhexidine on the 30th-day

Figure 3

Bacterial colonies on blood agar plate from subgingival plaque samples (a) at baseline and; (b) from the site treated with subgingival irrigation with propolis extract on the 30th day

Table 3

The mean and standard deviation of colony-forming units for both the groups and intergroup and intragroup comparison with level of significance

Group	Colony forming units (mean±SD)		Intragroup comparison (P)
	Baseline	30 days
Group A	1.50×103±135.54	7.5×101±14.28	<0.0001*
Group B	1.445×103±137.21	8.25×101±13.59	<0.0001*
Intergroup comparison (P)	0.1799	0.0995	-

*P<0.05 indicates statistically significant. Results expressed in terms of (mean±SD). Group A – Subgingival irrigation with 0.2% chlorhexidine; Group B – Subgingival irrigation with 25% propolis extract; SD – Standard deviation; P – Level of significance

DISCUSSION

The bacteria play an evident role in the initiation and progression of periodontitis. SRP is required for the removal of supragingival and subgingival plaque and calculus which harbors putative periodontal pathogens. However, the contours of the periodontal pocket and the root anatomy are complex enough to provide sufficient bacterial load reduction.[18] There are no direct effects of antiseptics in mouthrinses on the subgingival microbiota because of almost total lack of penetration of oral rinses below the gingival margin (mean 0.2 mm).[19] Hence, subgingival irrigation comes into play as a treatment modality as an adjunct to routinely performed SRP. In this present study, subgingival irrigation as an adjunct to SRP was used since it provides deeper penetration of periodontal pockets as mentioned in a position paper published by the American Academy of Periodontology.[20] Placement of a device 1 mm apical to the gingival margin attains 90% pocket penetration when probing depths were ≤6 mm.[21] The repeated flushing out action of the inflammation-inducing components by the hydrodynamics of subgingival irrigation and change in composition of subgingival plaque due to the antimicrobial effect of the irrigants helps in maintenance of periodontal tissue integrity.[22] A study conducted by Asari et al.[23] has observed the progressive beneficial effect of subgingival irrigation till 84 days even after the episode of irrigation ended by the 28th day from the baseline.

According to Soh et al.[24] after a prior SRP, effective subgingival irrigation requires a minimum of 2 weeks and maximum of 4 weeks to obtain a clinically significant reduction in periodontal parameters. Furthermore, recolonization of bacteria back to the baseline values requires 3–4 weeks after SRP[2526] hence, the time points of baseline, 15th day and 30th day from baseline for evaluation of clinical parameters, and baseline and 30th day from the baseline for evaluation of microbial parameter were selected.

In this study, both the groups showed progressive improvement in all the clinical and microbiological parameters on the evaluated time periods. The results for both test and control groups for PI score reduce maximum on 15th day and remained stable on further evaluation. These observations were in agreement with a study conducted by Koo et al.[27] in which propolis solution rinse significantly decreased the plaque score after 3 days as compared to placebo and also stated that the mechanism of plaque reduction of propolis differs from that of chlorhexidine as the former is an inhibitor of glucosyltransferases enzyme which is required for plaque formation. Tanasiewicz et al.[28] also concluded the effectiveness of plaque inhibition and improvement in periodontium by using propolis extract in their study. Similarly, a study conducted by Soh et al.[24] using chlorhexidine demonstrated effective plaque reduction with results similar to the present study.

The values for the GI and PPD gradually decreased at 15th-day evaluation and further on the 30th day for both the groups. These observed results of this study corroborated with results of the clinical studies by Gebara et al.,[29] Coutinho,[30] Sanghani et al.,[31] and Andrade et al.[32] using propolis in the form of subgingival irrigation/placement. This finding could be possibly due to anti-inflammatory substances found in propolis which causes tissue suppression of leukotrienes and prostaglandins synthesis by macrophages and have inhibitory effects on myeloperoxidase activity.[33] The radical scavenging effect of ethanolic extract of propolis is equal in effectiveness when compared to that of Vitamin C, this removal of radicals by flavonoids in propolis along with its anti-inflammatory response aids in tissue healing and regeneration.[1133] Systematic review by Freires et al.[34] found propolis extract gel to exhibit strong alveolar bone protective effect in ligature-induced periodontitis in rats, another meta-analysis by López-Valverde et al.[35] found propolis to reduce the PPD as compared to placebo.

In the present study, 25% concentration of propolis extract was used which was based on minimum inhibitory concentration of propolis against keystone periodontal pathogens such as Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium sp., Capnocytophaga sp., and Eikenella sp. observed in an in vitro study in vitro study by Gebara et al.[36] A similar in vitro study was done by Akca et al.[37] comparing propolis with chlorhexidine on oral pathogens with results showing equal effectiveness of both against P. gingivalis biofilm. This antibacterial action of propolis was also evident in the present study. The microbial colonies showed a significant decline in the count from baseline values in both the groups and the intergroup comparison was nonsignificant. Pundir et al.[38] also demonstrated reduction of CFUs after one-stage full-mouth disinfection using 20% propolis hydroalcoholic solution, and attributed this effect due to the long-lasting effect of propolis leading to a transformation in the repopulation process of the organisms in the periodontal pocket.

The present study had limitations in terms of sample size and a follow-up period of 1 month. Further studies should be conducted varying the concentrations and frequency of irrigation with propolis.

CONCLUSIONS

From the evidence of the present study, we can conclude that propolis extract has equal effectiveness as compared to chlorhexidine in reducing the subgingival plaque formation and microbiota from periodontal pockets, and is an effective antimicrobial agent. Subgingival irrigation with propolis extract as an adjunct to SRP enhances periodontal health and has a significant role in periodontal therapy. They are inexpensive, noninvasive, and naturally have an added benefit to the patients suffering from periodontitis.

Financial support and sponsorship

Nil.

Conflicts of interest

The authors declare no conflicts of interest.