INTRODUCTION:

Periodontitis is a chronic inflammatory condition of the periodontium defined by the deterioration of the periodontal ligament and loss of alveolar bone.¹ It is the most common cause of tooth loss and is regarded as one of the most serious threats to dental and oral health.^2,3

According to the most recent Global Burden of Disease Study, periodontitis ranks as the sixth most prevalent illness worldwide, affecting approximately 20% of the global population.^4,5 It is a type of infection that arises due to various factors and involves the interaction of multiple bacteria species with the host's tissues and cells. This interaction results in the secretion of a variety of inflammatory mediators, cytokines, and chemokines, which can cause damage to the structures of the periodontal region.⁶

The main clinical characteristic that differentiates gingivitis from periodontitis is the deterioration of the supporting periodontal tissue. This is demonstrated clinically through attachment loss and alveolar bone loss, revealed by radiographic assessment. Other signs of periodontitis include gum bleeding and the manifestation of a periodontal pocket, an enlarged area beneath the gum line that provides a protective environment for the microorganisms that contribute to periodontal disease.⁷

Several anaerobic and gram-negative bacterial species that reside in the subgingival region have been linked to the onset of inflammatory reactions, and they possess potent mechanisms that can disrupt the host's defenses. Aggregatibacter actinomycetemcomitans (A.a), Porphyromonas gingivalis (P.g), and Fusobacterium nucleatum (F. nucleatum) were proposed as the keystone pathogens in the development of periodontitis and ineffective periodontal therapy.^8,9

The administration of local antimicrobial therapy offers the advantage of delivering an effective drug concentration to the site of infection with less systemic exposure and reducing the risk of bacterial resistance.^10–12While antibiotics and other antimicrobial agents have been widely investigated as adjuvants in treating periodontitis, concerns about the development of resistance and adverse effects have led researchers to explore safer alternatives with minimal side effects, such as herbal-based antimicrobials. As a result, natural medicine, including apitherapy, has gained significant attention as a potential solution.^8,13,14

Sidr honey, which is derived from the nectar of the Ziziphus spina-Christi plant in Yemen, is widely considered the best and most valuable honey worldwide due to Yemen's ideal environment and climate for honey production. Yemen Sidr honey is monofloralwith a distinctive flavor and paramount therapeutic properties. Ziziphus spina-christi trees are known for their medicinal properties and are used in medication research and development throughout Yemen,the Middle East; and Southeast Asia.^15,16The exceptional quality of Sidr honey can be attributed to its elevated levels of fructose, acidity, vitamins, phenolic acid, and flavonoidsThese components contain powerful properties that can combat inflammation, microbes, oxidation, and bacteria, and these components are effective against both types of bacteria, gram-negative and gram-positive.^9,17

Previously, studies had shown the antibacterial properties of various types of honey, such asManuka honey and Alfalfa honey, in in-vitro experiments. However, there was a lack of research into the impact of Yemeni Sidr honey on the bacteria responsible for causing periodontitis. Therefore, the study aims to investigate the effectiveness of Yemeni honey against the bacteria responsible for causing periodontitis, namely A.a, P.g, and F. nucleatum. This was done with the goal of exploring honey as a potential alternative in optimizing medicinal resources for the treatment of periodontal diseases.

MATERIALS AND METHODS:

Study design:

A true experimental laboratory study with post-test-only control group design. The protocol of the study was approved by ethical clearances No.386/HRECC.FODM/ VIII/2020 (Date approval: 31 August 2020) from the Faculty of Dentistry Research Ethics Commission, Airlangga University, Surabaya, Indonesia.

Bacterial Strains:

A. actinomycetemcomitans (ATCC 43718), P. gingivalis (ATCC33277), F. nucleatum (ATCC22586) obtained from reservoirs in the Research Centre / Faculty of Dental medicine/Universitas Airlangga/ Surabaya/ Indonesia. The tested bacterial colonies were isolated, transferred, and cultured for 24 hours at 37°C in 3ml of BHI broth medium. 0.5 McFarland standard (1.5 x 10⁹ CFU/ml) was used to synchronize the bacterial solution, and then it was taken and dropped using a micropipette onto the surface containing Mueller-Hinton agar media. The bacteria were cultured on MHA media and placed in a 5% CO₂ anaerobic atmosphere for 24hours at 37°C.

Honey samples:

Yemen Sidr Honey was obtained from the city of Hadhramaut-Yemen. In sterile beakers/culture bottles, honey was gathered. The honey samples were filtrated first through sterilized mesh/gauze to eliminate the debris and then stored at 2–8°C until required.¹⁸

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC):

The experimental setup involved test tubes that were filled with 5ml of Brain Heart Infusion (BHI) medium. The honey was serially diluted to achieve the desired honey concentration, i.e., 100%, 50%, 25%, 12.5%, and 6.25% from the first to the fifth tube. The tubes were inoculated with the test organisms at a concentration of 0.5 (1.5 x 10⁹ CFU/ml). The control group included a tube with media with bacteria to serve as a negative control. The positive control tubes contain only 5ml of BHI medium for each of the tested organisms. For 24 h, the tubes were incubated (37°C). Each tube's turbidity was examined visually following incubation. A clear test tube showed the breakpoint. The bacteria that did not exhibit any signs of growing or turbidness during the MIC determination were transferred onto sterile nutrient agar plates using the streak plate technique. The agar plates were subsequently incubated in a 37°C temperature-controlled environment for 24hours. The MBC was determined to be the lowest concentration at which test organisms did not grow.¹⁸

Disc Diffusion Method:

The disk diffusion (Kirby-Bauer) method was used for susceptibility testing following the standards established by CLSI in 2016. The MHA surface-containing plates were evenly seeded with tested microorganisms and then placed on the bench to allow the extra fluid to be absorbed. Disc papers with a diameter of 5mm that had been dripped with 10μl of the honey with a concentration of 25%, 20%, 15%, and 12.5% were placed on the surface of the media. The positive control was equally dripped with Doxycycline disc paper, while aquadest was used as a negative control.The plates were incubated for 2x24 hours at 37°C anaerobically, then the diameter of the inhibition zone was measured in mm using a caliper¹⁸.

Statistical analysis:

The statistical analysis was carried out using SPSS version 25. One-way ANOVA and Post hoc (HSD Tukey) were used to investigate if there was a significant difference, with a significance level of 0.05 (α = 0.05).

RESULT:

This study utilized Yemeni Sidr honey at concentrations of 100%, 50%, 25%, 12.5%, and 6.25%, along with positive and negative controls. The swab test results on Mueller Hinton's media Fig 1. indicated that there was no bacterial colony growth from the concentration of 100% to 25% of Yemeni Sidr honey across all three bacterial strains. However, bacterial growth was observed at the test concentrations of 12.5% and 6.25%.

Figure 1. Growth colonies forming of (a) A. actinomycetemcomitans, (b) P. gingivalis, and (c) F. nucleatum microorganismafter replanting Yemen Sidr honey in MHAmedia petri dishes.

Table 1. presents the mean ± standard deviation results of the study, illustrating the antibacterial power of Yemeni Sidr honey against the tested bacteria. Specifically, A.a exhibited a growth rate of 11.3 CFU/ml, while P. g and F. nucleatum displayed growth rates of 11.0 CFU/ml and 11.6 CFU/ml, respectively. Based on these findings, the MIC Yemeni Sidr honey was determined to be 12.5%, and the MBC of Yemeni Sidr honey against A. a, P. g, and F. nucleatum was found to be at a concentration of 25%.

Regarding the disc diffusion method that was conducted to measure the inhibition zone. Fig 2. showed clear inhibition zones surrounding the honey concentrations at 25% and 20%, ensuring that the honeypossesses antibacterial activity against A.a, P. g, and F. nucleatumbacteria in comparison with the antibiotic group.

Figure 2. Inhibition Zone of Yemen Sidr honey at concentrations 25%, 20%, 15%, and 12.5% against (a) A. actinomycetemcomitans, (b) P. gingivalis, and (c) F. nucleatum bacteria compared with control positive (doxycycline 100mg), and control negative (Aquadest).

Moreover, Graph 1. revealed the result of the diameter of the inhibition zones expressed as mean ± standard deviation of the experimental groups of Yemen Sidr honey at different concentrations against A.a, P. g, and F. nucleatum bacteria compared to control positive and control negative. According to the comparison of the zone diameter test for Yemen Sidr honey concentration, it was found that 12.5% represented the lowest inhibition zone diameter. At the same time, 25% showed the highest zone diameter for all tested bacteria. However, the zone diameter test for A.a, P.g, and F.nucleatum bacteria growth with honey concentrations was compared, and it was found that F. nucleatum bacteria represented the lowermost inhibition zone diameter. At the same time, A. actinomycetemcomitans bacteria showed the maximum zone diameter.

Graph 1. Yemen Sidr honey inhibition zones (mm) against A.a, P g, and F. nucleatum bacteria.

The results of the Post hoc (Tukey HSD) test obtained p-value = 0.000, which means that there are significant differences between all the groups of Yemen Sidr honey (MIC, and MBC) and disk diffusion groups against all of A.a, P.g, and F.nucleatum bacteria.

DISCUSSION:

The primary objective of the experimental laboratory research is to investigate and elucidate the antibacterial efficacy of Yemeni Sidr honey against periodontopathic bacteria, including A.a, P.g, and F. nucleatum. These bacteria are gram-negative, anaerobic, facultative bacteria and are associated with the local development of periodontitis. In this study, Yemen Sidr honey was effective against the three well-known oral pathogens in an in-vitro utilizing MIC/MBC determinations and the agar diffusion technique. The findings of the current investigation come alongside several previous studies that basically prove that honey from different flower species or bee species can be used against A.a¹⁹, P.g^8,20, and F. nucleatum²¹ in an in-vitro set-up.

A broth micro-dilution technique was conducted to determine the MIC and MBC value of Yemeni honey versus the above periodontopathic bacteria. Minimum inhibitory concentration is referred to as the lowermost concentration of an antimicrobic agent that completely inhibits and prevents microorganisms from growing visibly to the naked eye.²²According to that, the result showed that the MIC of Sidr honey against the three tested microorganisms is 12.5%, ensuring that the honey is a strong bacterial growth inhibitor. In a prior study, oral streptococci, one of many of bacteria found in the oral cavity, exhibited MIC values ranging from 12.5% to 25%.²⁰However, the current investigation primarily concentrated on periodontal pathogens such as A.a, P.g, and F. nucleatum, and the results were consistent with the previous study.

Moreover, the MBC is the lowest concentration required to kill or destroy particular microorganisms. In this study, it was observed by counting the bacterial colonies that MBC in the range of 25% of Yemen Sidr honey managed to kill about 100% of the tested bacteria. The outcomes recorded by are in agreement with the current findings.²¹Comparable studies reported the MIC of Sidr honeyversus various gram-negative bacterial species revealed that the tested microorganisms were susceptible to Sidr honey ranges at 10-20% concentrations. Meanwhile the MBC of Sidr honey against the similar bacteria ranges at concentrations between 40 to 80%. However, Sidr honey's antibacterial activity was more potent than that of other Mountain honey samples tested for the same bacteria.¹⁶

The variability in the ability of honey to inhibit the growth of microorganisms studied in this research may be influenced by several factors, including its osmotic activity, hydrogen peroxide content, acidity, and phytochemical components.²³Honey's antibacterial properties either damage the bacteria's cell wall or impede the intracellular metabolic processes. Honey is hygroscopic, drawing moisture from its surroundings. This characteristic dehydrates the bacterium, stripping it of all the water it needs to survive. Additionally, honey's sugar concentration is sufficient to prevent microbial development.^24–26 The pH range of honey, which ranges from 3.4 to 5.5, which is low enough to be inhibitory to several bacterial pathogens, may also contribute to the antibacterial action.²⁰

The third and most significant antibacterial component is hydrogen peroxide, while other researchers suggest that the non-peroxide action is more paramount. The enzyme glucose oxidase creates it during the dilution of honey by sterilized double-distillation water. These enzymes require a high enough level of free water to be effective by a factor between 2500 to 50,000, providing slow-release antiseptics at an antibacterial level. Finally, honey contains several phytochemical components that act as an inhibitor of bacterial growth. Additionally, the potential may be connected to the various ways that each microbe species is susceptible to the antibacterial properties of the honey used, the different floral sources that the bees used, and the geographical conditions, such as temperature and humidity, where the honey was generated.²³

Regarding the disc diffusion method, the inhibition zone diameter surrounding Yemen Sidr honey of 25% and 12.5% against A.a was 18.52 -7.78mm, respectively. The inhibition zone diameter surrounding Yemen Sidr honey of 25% and 12.5% against F. nucleatum was 17.25- 7.45mm, respectively. These outcomes are consistent with previous studies, which revealed that the inhibition zone for dissimilar types of honey against the above bacteria has a convergent result.^19,20Our findings also indicate that an increase in honey concentration was associated with an expansion of the inhibitory zone of the tested microorganisms. This investigation was compared with the doxycycline control group, which reported the highest inhibition zone (20.367-19.367) among the other treatment groups. This was made clear by the statistical analysis, which showed a significant difference in values (P ≤0.05) among the various concentrations of honey as well as with the control group. Our results are supported by other studies showing the effectiveness of Sidr honey against gram-negative pathogens.¹⁷As well as the activity of honey from various kinds against periodontopathic bacteria.⁸

The limitation of this study is that it was conducted on planktonic bacteria. The dental infections that cause periodontitis are mostly polymicrobial, consisting of Tannerella forsythia, Treponema denticola, Provotella, and many more bacteria. They present different properties in biofilm. Further study is required to assess the antimicrobial effect of Yemen Sidr honey on dental biofilm. In addition, most of the time, in-vitro results do not correlate with in-vivo activity. Further, in vivo studies are needed to evaluate the effectiveness of Yemen Sidr honey in a practical setting.

CONCLUSION:

In conclusion, the study demonstrated the efficacy of Yemeni Sidr honey in inhibiting the growth of major periodontopathogens, such as A.actinomycetemcomitans, P. gingivalis, and F. nucleatum, in vitro.

CONFLICT OF INTERESTS:

No conflict of interest.

ACKNOWLEDGMENTS:

Universitas Airlangga and the Directorate of Research, Technology and Community Service, Indonesian Ministry of Education, Culture, Research and Technology support this study.

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Received on 26.12.2023 Revised on 11.04.2024

Accepted on 15.07.2024 Published on 10.04.2025

Available online from April 12, 2025

Research J. Pharmacy and Technology. 2025;18(4):1789-1794.

DOI: 10.52711/0974-360X.2025.00256

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Group	Control Negative	Control Positive	100%	50%	25%	12.5%	6.25%
(CFU/ml)	Control Negative	Control Positive	100%	50%	25%	12.5%	6.25%
*A.a*	184.667±7.024	0	0	0	0	11.333 ±0.577	36.333±3.215
*P. g*	190.667±4.726	0	0	0	0	11±1	37±5.292
*F. nucleatum*	164±9.165	0	0	0	0	11.667±1.528	36± 4