Antibacterial Efficacy of Hubballi Propolis against Aggregatibacter Actinomycetemcomitans One of the Major Causative Organisms of Perimplantitis: An In vitro Study.

Background: Peri-implantitis can be attributed to many underlying causes, one of the chief ones being due to infection caused by oral micro flora and particularly Aggregatibacter actinomycetemcomitans. Antibiotics are administered along with mechanical debridement to control the infection. The side effect of conventional antibiotic therapy and drug resistance has led to the necessity for alternate approaches to handle infections. Natural products are being investigated because of their multi-target activity and structurally different from the normal antibiotics. Propolis a product by Apis Mellifera bees as a wound healing and bone regenerating effect along with antimicrobial effect. One of the important features of Propolis is the chemical properties of Propolis changes with the different locations of procurement. Antimicrobial activity of Hubballi propolis against Aggregatibacter actinomycetemcomitans is not been reported in the literature. Aim: The aim of this study is to evaluate the antimicrobial effect of the Hubballi Propolis against Aggregatibacter actinomycetemcomitans.
Methods: The two solvents used for the study were water and 70% Aq ethanol. Minimum inhibitory concentration (MIC), total phenolic contents (TPC), and total flavonoid content (TFC) were tested.
Results: Hubballi Propolis sample showed antimicrobial effect against Aggregatibacter actinomycetemcomitans with MIC range from 0.1 mg/ml to 0.25 mg/ml.
Conclusion: Hubballi Propolis is effective against Aggregatibacter actinomycetemcomitans infection thus may help in treating peri-implantitis. Propolis extracted with water as solvent showed better MIC, higher TPC and TFC than the propolis extracted using alcohol as solvent. This feature is noteworthy as the formulations produced using water extract is favorable than alcohol extract of propolis which irritates the mucosa and hence difficult for its application in dentistry. Copyright:

INTRODUCTION

Implant placement in the oral cavity is a procedure for rehabilitation of edentulous area. The implant body is surgically positioned into the edentulous area. The prosthetic part is placed after a period of 3–4 months of surgical placement of implant. The design and topography of implant surface are known to influence colonization of different bacteria within 30 min of its insertion into the oral cavity.[1] These oral commensal bacterial under unfavorable pH, systemic illness, and in infectious condition lead to peri-implantitis.[2]

Peri-implantitis is an inflammatory process affecting dental implants.[3] Aggregatibacter actinomycetemcomitans, according to Cosyn et al. is present in high numbers in the normal peri-implant sulcus.[4] It is one of the major causative microorganism of peri implantitis. For such condition, patients may not respond to conventional therapy (mechanical debridement, antiseptics and ultrasonic and laser treatments) hence as an adjuvant antimicrobial agent are prescribed before surgical treatment plan. However, most of the microorganisms are resistance to synthetic antimicrobial agent due to inappropriate systemic usage and this is the major clinical problem in treating the infection. Hence, in the field of research, there is high demand for new solutions for these drug resistance situations or at least to offer alternatives such as natural product extracts. Natural products exhibit broad biologically activities and there is no evidence of bacterial resistance occurrence due to these natural products.[5]

Propolis is one such natural product, produced by Apis melifera bees.[6] This species of bees collect the resin from the plants and mix with their saliva and produces the propolis which is a protective product to the hive as well as to the bees in the hive.[7] This product is rich in antioxidant properties. It has antimicrobial, anti-viral, anticancerous properties also. Literature has reported that the propolis when used as a drug has very low or no toxicity. The studies on propolis have been widely carried out and reported that flavonoid content of propolis is known for almost all its medicinal properties.[891011] However, the main drawback of propolis as reported is the constituent of propolis varies with sources of its procurement.[7] Hence, there is a need to evaluate the composition and the potency of the material procured from different source against the Aggregatibacter actinomycetemcomitans to provide more effective products for peri implantitis before its use as therapeutic agent. In this endeavor as an initial step this in vitro study was carried to know minimal inhibitory concentration (MIC), total phenolic and flavonoid content of propolis procured from Hubballi, Karnataka. Novelty of this study lies in presenting an alternative natural therapeutic method for treating peri-implantitis caused by Aggregatibacter actinomycetemcomitans.

Methods

This research assessed antimicrobial property (MIC) against Aggregatibacter actinomycetemcomitans and chemical constituents (total phenolic and total flavonoid content [TFC]) of Hubballi propolis.

Type of study

in vitro, observational study.

Duration of study

One year.

The chemical composition of propolis is important in antimicrobial activity. Extraction of pure form of propolis is must than using raw propolis. Hence, maceration method followed by refluxing was done. These techniques are very simple, affordable and the chemical structures of propolis are not altered. Commonly used solvents water wired equivalent privacy (WEP) and 70% ethanol American Electric Power (AEP) were used.

Preparation of propolis extract

Instruments used: Hot water bath and Rotary vacuum evaporator

Materials required: Ethanol, distilled water, raw propolis

Raw propolis: Hubballi propolis was procured for the research because the sample was collected in the spring season, in a certified single apiary by manual scraping technique from the wooden hive, thereby it was the best standardized quality raw propolis.[6]

Preparation of wired equivalent privacy

Maceration of Hubballi propolis sample was carried out by dissolving the sample in distilled water for the WEP sample of Propolis for 24 h at the room temperature [Figure 1]. Next, the samples were refluxed [Figure 2]. Whatmanno filter paper no. 41 was used to filter the obtained solutions. Later the filtrate was transferred on to the petridish subjected over a hot water bath furnace [Figure 3]. This resulted in the evaporation of the solvent and dry concentrated sample/extract. The resultant samples was cooled which resulted in sticky and semisolid form [Figure 4].

Figure 1

Measured labelled samples placed for maceration

Figure 2

Refluxing the samples

Figure 3

Samples placed on the petridish and solvent is evaporated

Figure 4

Dry concentrated extract

Preparation of American electric power

The procedure for 70% ethanol extract of Propolis is similar to water extract.

Propolis phytochemical constituent analysis

Estimation of total phenolic content was done by Folin-Ciocalteau Colorimetric[12] method and estimation of TFC by aluminum chloride colorimertic method as reported by Woisky and Salatino.[13]

Determination of antimicrobial activity of propolis

The microorganisms evaluated were Aggregatibacter actinomycetemcomitans (American Type Culture Collection: No 43718). Minimum inhibitory concentration test was determined.[14] This test determines the minimum quantity of test material required to inhibit the bacterial growth. In this research, MIC was determined using the serial tube dilution technique from 0.001 to 0.5 mg/ml concentrations.

Statistical analysis: Each in vitro assay was performed at least thrice, and the data were expressed as mean ± SD (n = 3).

RESULTS

The MIC and total phenolic and flavonoid content of both water extract and alcohol extract of

Hubballi propolis is shown in following tables.

The MIC values for the propolis samples are shown in Tables 1 and 2. Both the propolis samples evaluated in the present study showed the antibacterial effect against Aggregatibacter actinomycetemcomitans with MIC ranging from 0.12 mg/mL to 0.25 mg/mL.

Table 1

Minimum inhibitory concentration of propolis (water-extracted propolis) from Hubballi

Microorganisms	Concentration of extract mg/ml (0.001-0.5)
	0.5	0.25	0.12	0.06	0.031	0.01	0.008	0.004	0.002	0.001
Aa	Sensitive	Sensitive	Sensitive	Resistance	Resistance	Resistance	Resistance	Resistance	Resistance	Resistance

Table 2

Minimum inhibitory concentration of propolis (aqueous extract propolis) from Hubballi

Microorganisms	Concentration of extract (mg/ml)
	0.5	0.25	0.12	0.06	0.031	0.01	0.008	0.004	0.002	0.001
Aa	Sensitive	Sensitive	Resistance	Resistance	Resistance	Resistance	Resistance	Resistance	Resistance	Resistance

Total phenolic contents (TPC) and TFC ranged from 175.4 ± 5.7–192.2 ± 3.3 and 31.73 ± 8.5–33.08 ± 10 mg/ml, respectively.

DISCUSSION

Gram-negative coccobacillus and Aggregatibacter actinomycetemcomitans cause tissue destruction. It is frequently associated with peri-implantitis. Aggregatibacter actinomycetemcomitans stimulates bone resorption by lipopolysaccharide-mediated mechanisms and proteolysis sensitive factor in microvesicles.[15] Conventional mechanical debridement, antibiotics, and surgical treatment are the different treatment regimens followed during the peri-implantitis. When peri-implantitis is not controlled with mechanical debridement, antibiotic therapy is administered before surgical treatment. Synthetic antibiotics have basic disadvantages of drug-induced side effects and drug resistance over the natural products from plant extracts or modified plant extract. Among these natural products, propolis comes ahead due to its antimicrobial activity effect on an extensive pathogenic microorganism. Propolis is reported to have wound healing and also bone regeneration properties with least or no toxicity.[616] The previous studies reported that mechanism of action (MOA) of propolis on bacterial could be inhibition of protein synthesis or partial bacteriolysis or by disintegration of the cytoplasm, cytoplasmic membrane, and cell wall.[816] This literature evidence shows that propolis MOA is different and has multi-target activity against each microorganism.

In addition, it was reported that the factors such as extraction of propolis solvents, pH, and acidic solutions of propolis might alter the effectiveness on bacteria.[15] However, if minimal inhibitory concentration (MIC) of a particular plant extract is <100 μg/ml then, its antimicrobial efficacy is considered to be good. If the MIC ranged from 100 to 500 μg/ml, it is regarded to be moderate while MIC more than 500 μg/ml is considered poor.[17] Hubballi Propolis (WEP and AEP) were moderately active against the Aggregatibacter actinomycetemcomitans with the MIC range from 0.1 to 0.25 mg/ml (100–250 μg/ml) Tables 1 and 2. This result is in accordance with the research done by Gebara et al.[18] and Agarwal et al.[19] The results are notable exception as many studies have reported that Propolis is less active or inactive against Gram-negative organism. Further, recently, Khodijah et al. proved that propolis candies were effective in inhibiting the growth of Aggregatibacter actinomycetemcomitans.[20]

Apart from the MIC results, the examined propolis samples possess considerable total phenolic and flavonoid contents as compared to studies carried on the other parts of the world such as Algeria, Brazilian, Chinese, Morocco, Poland Korean, and Turkey which are using propolis commercially.[212223242526272829] TPC and TFC of the present study ranged from 175.4 ± 5.7 to 192.2 ± 3.3 and 31.73 ± 8.5 to 33.08 ± 10 mg/ml [Tables 3 and 4], respectively. The results of our studies clearly indicate that tested propolis samples procured from Hubballi with comparable high phenolic (TP) and flavonoid (TF) contents can be selected for treating the infection related to Aggregatibacter actinomycetemcomitans and also for other commercial propolis products. Another interesting point of this study is that the WEP showed better results than alcohol extract propolis [Graph 1]. The water extract propolis results in the study were in agreement with a study by Nagai et al.[30] This is the major advantageous result to overcome the drawback of alcohol propolis which irritates the site of application.

Table 3

Total phenolic content and total flavonoid content of propolis (water-extracted propolis) from Hubballi

TPC	TFC
192.2±0.33 mg of gallic acid equivalent value/g	33.08±1 mg of quercentine equivalent value/g

TPC: Total phenolic content, TFC: Total flavonoid content

Table 4

Total phenolic content and total flavonoid content of propolis (aqueous extract propolis) from Hubballi

TPC	TFC
175.4±0.57 mg of gallic acid equivalent value/g	31.73±0.85 mg of quercentine equivalent value/g

TPC: Total phenolic content, TFC: Total flavonoid content

Graph 1

Representing MIC of Hubballi propolis (wired equivalent privacy and American Electric Power). Wired equivalent privacy inhibits Aggregatibacter actinomycetemcomitans at a lesser concentration American Electric Power

Hence, with our study results and the evidence from previous studies, it may be considered that sanitation of the peri-implant site regularly with Hubballi Propolis may prevent adhesion and colonization of the Aggregatibacter actinomycetemcomitans thus increasing the longevity of implant treatment and improving the quality of living of the patient.

CONCLUSION

Considering literature evidences and present research results, it may be concluded that the administration of Hubballi propolis at appropriate concentrations is effective on Aggregatibacter actinomycetemcomitans hence may help in the treatment of peri-implantitis. Hubballi Propolis may serve as an alternative natural and reliable antimicrobial agent to avoid the side effects of synthetic antibiotics. However, clinical evidence is required to know and document the mechanism of propolis and its appropriate administration dose on bacteria.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.