INTRODUCTION:

The severe type of periodontal disease is defined by the breakdown of the alveolar bone around the periodontium and the loss of periodontal ligaments. As the main contributor to tooth loss, it is regarded as one of the two gravest dangers to oral health. Severe periodontal disease affects 538million people worldwide, 276 million of whom have lost teeth. The numbers are anticipated to increase as the population ages and grows.^1,2

Researchers have identified Porphyromonas gingivalis as one of the main causal agents in the pathogenesis of periodontitis, and they have found a correlation between the prevalence of this pathogen and the severity of periodontal disease. Current research suggests that Porphyromonas gingivalis, even in low abundance, can cause chronic periodontitis by altering the commensal bacterial population to encourage a condition known as dysbiosis, which causes periodontitis to develop.³ Aggregatibacter actinomycetemcomitans is a Gram-negative bacterium that expresses several virulence factors that activate a host response linked to periodontitis pathogenesis and contribute to early and rapidly progressive forms of periodontitis.⁴

The initial steps attempt to reduce or eliminate harmful bacteria, which is usually done by assisting patients with proper oral hygiene and performing mechanical debridement to remove supragingival and subgingival microbial deposits.⁵ In addition to mechanical scaling and root planing, mouthwashes, irrigations, and devices for prolonged drug release are frequently employed for therapeutic agent administration in the treatment of periodontal disorders. It has been proven that contemporary chemotherapeutic agents are effective in the management of periodontal disease. However, they do have some undesirable side effects, including as altered taste, yellowing of the teeth and tongue, germ resistance, and rising costs.⁶

Because of their promising results and uncommon side effects, medicinal herbs have recently received a lot of attention worldwide for their use in the treatment of various diseases. Among the evidence-based herbal medicines, Nigella sativa is highly regarded as a "miracle herb."⁷ Nigella sativa has been used in traditional medicine for generations to cure various illnesses. It offers several health advantages due to its high nutritional content and active components.⁸ Several bioactive chemicals derived from the seed of Nigella sativa have been documented in the literature, with thymoquinone being the most significant. Thymoquinone (TQ) shows the major pharmacological actions of Nigella sativa, such as significant anti-inflammatory, antioxidant, antibacterial, analgesic, histamine release inhibitory, hypoglycaemic, anticarcinogenic, antihypertensive, immune-activating, hepatoprotective effects, and anti-diabetic characteristics.^9,10 The antibacterial action of Nigella sativa and TQ on various bacterial strains and species has been studied in several reports.¹¹

Based on prior research, this study will evaluate the antibacterial activity of Nigella sativa extract in the form of a mouth rinse to a low dosage of Doxycycline 0.1% against two of the most frequent bacteria in periodontitis, Porphyromonas gingivalis and Agregatibacter actinomycetemcomitans.

MATERIALS AND METHODS:

Nigella Sativa Mouth Rinse:

The mouth rinse product containing Habbatussauda (Nigella sativa) 3% (SATIVA Mouth rinse, PT. Miswak Utama, Pasuruan, East Java, Indonesia) is being used in this study.

Doxycycline 0,1%:

Doxycycline is a broad-spectrum antibiotic drug of the tetracycyline group. In this study, the concentration of doxycycline used was 0.1%.

Preparation of Porphyromonas gingivalis and Agregatibacter actinomycetemcomitans Bacterial Culture:

The bacterium sample preparation was conducted in Laboratory Microbiology Research Centre, Faculty of Dental Medicine, Universitas Airlangga, Surabaya, Indonesia. Porphyromonas gingivalis ATCC 33277 and Agregatibacter actinomycetemcomitans ATCC 43718 bacteria sample is taken from the stocks using an inoculating needle and transferred into the test tube filled with Brain Infusion Broth (BHIB). These bacterium is grown and cultured for 48hours at 37°C in an anaerobic environment. Then, each bacterial culture were diluted in broth medium to produce a McFarland standard 0.5 suspension (equal to 1,5 x 108 CFU/mL).

Evaluation of the Inhibition Zone Diameter Using the Agar Disc Diffusion Method:

The antibacterial activity evaluation of these antimicrobial agent and antibiotics to inhibit the growth of bacteria in vitro is measured by determining the inhibition zone diameter using the Agar Disc Diffusion Method. Each of bacterial culture was spread on each Mueller-Hinton Agar (MHA) media plate using the spreading technique. There are three experimental groups, and each group contains 6 plates of Porphyromonas gingivalis ATCC 33277 bacteria culture and 6 plates of Agregatibacter actinomycetemcomitans ATCC 43718 bacteria culture. Each group will be given a different treatment. The first group is a negative control group, the second group will be given Doxycycline 0,1%, and as for the third group will be given Nigella sativa mouth rinse with 3% concentration. These plates were then transferred to an anaerobic jar and incubated for 48hours. The diameter of the inhibitory zone generated was measured once the incubation time was completed.

Statistical Analysis:

All statistical tests were performed using IBM SPSS v23.0. The Kolmogorov-Smirnov test was used to determine the data's normality. The Levene test was used to determine if the data was homogeneous. To assess if there were significant changes between the treatment groups, One way ANOVA test was used to examine differences between groups. If a significant difference exists, the post hoc Tukey HSD test to was done determine which group has a significant difference.

RESULTS:

In this study the following results were obtained. In the doxycycline 0,1% treatment group against Porphyromonas gingivalis bacteria, the mean diameter of the inhibition zone was 19.6833mm. Meanwhile, in the Nigella sativa treatment group against Porphyromonas gingivalis bacteria, the average diameter of the inhibition zone was 25.141mm (Figure. 1, Table.1). In the Doxycycline 0,1% treatment group against Aggregatibacter actinomycetemcomitans bacteria, we observed that the mean diameter of the inhibition zone was 19.475mm. Meanwhile, the mean of the inhibition zone for Nigella sativa against Aggregatibacter actinomycetemcomitans was 25.1mm (Figure. 2, Table 2).

Figure 1. Results for inhibition zone diameter of Doxycycline 0,1% and Nigella sativa Mouth rinse 3% on Porphyromonas gingivalis bacteria

Table 1: Mean inhibition zone diameter (mm) of the control group, Doxycycline 0,1% group, and Nigella sativa mouth rinse 3% group on Porphyromonas gingivalis bacteria

	Replication	Mean	Std. Deviation
Negative Control	6	0,000	0,000
Doxycycline 0,1%	6	19.68333	0,19149
Nigella Sativa	6	25,1417	0,23112
	18	14,9417	11,11203

Figure 2. Results for inhibition zone diameter of Doxycycline 0,1% and Nigella sativa Mouth rinse 3% on Aggregatibacter actinomycetemcomitans bacteria.

Table 2: Mean inhibition zone diameter (mm) of the control group, Doxycycline 0,1% group, and Nigella sativa mouth rinse 3% group on Aggregatibacter actinomycetemcomitans bacteria

	Replication	Mean	Std. Deviation
Negative Control	6	0,000	0,000
Doxycycline 0,1%	6	19.4750	0,76010
Nigella Sativa	6	25,9333	0,10801
	18	14,8028	11,01984

Moreover, a statistical test is carried out to see the significance of each group's diameter of inhibition zone. Based on the results of the post-hoc Tukey test in Table 3 and 4, it can be seen that the total significance value is 0.000 so that it can be concluded that there are significant differences in all groups.

Table 3: Results of post-hoc Tukey test for the control group, Doxycycline 0,1% group, and Nigella sativa mouth rinse 3% group on Porphyromonas gingivalis bacteria

(I) Treatment	(J) Treatment	Mean Difference (I-J)	Std. Error	Sig.	95% Confidence Interval
(I) Treatment	(J) Treatment	Mean Difference (I-J)	Std. Error	Sig.	Lower Bound	Upper Bound
Negative Control 1	Doxycycline 0,1%	-19.68333^*	0.10005	0.000	-19.9432	-19.4235
Negative Control 1	Nigella sativa	-25.14167^*	0.10005	0.000	-25.4015	-24.8818
Doxycycline 0,1%	Negative Control1	19.68333^*	0.10005	0.000	19.4235	19.9432
Doxycycline 0,1%	Nigella sativa	-5.45833^*	0.10005	0.000	-5.7182	-5.1985
*Nigella* *sativa*	Negative Control1	25.14167^*	0.10005	0.000	24.8818	25.4015
*Nigella* *sativa*	Doxycycline 0,1%	5.45833^*	0.10005	0.000	5.1985	5.7182

*. The mean difference is significant at the 0.05 level.

Table 4. Results of post-hoc Tukey test for the control group, Doxycycline 0,1% group, and Nigella sativa mouth rinse 3% group on Porphyromonas gingivalis bacteria

(I) Treatment 2	(J) Treatment 2	Mean Difference (I-J)	Std. Error	Sig.	95% Confidence Interval
(I) Treatment 2	(J) Treatment 2	Mean Difference (I-J)	Std. Error	Sig.	Lower Bound	Upper Bound
Negative Control (2)	Doxycycline 0,1% (2)	-19.47500^*	0.25591	0.000	-20.1397	-18.8103
Negative Control (2)	Nigella sativa (2)	-24.93333^*	0.25591	0.000	-25.5981	-24.2686
Doxycycline 0,1%(2)	Negative Control (2)	19.47500^*	0.25591	0.000	18.8103	20.1397
Doxycycline 0,1%(2)	Nigella sativa (2)	-5.45833^*	0.25591	0.000	-6.1231	-4.7936
*Nigella* *sativa* (2)	Negative Control (2)	24.93333^*	0.25591	0.000	24.2686	25.5981
*Nigella* *sativa* (2)	Doxycycline 0,1% (2)	5.45833^*	0.25591	0.000	4.7936	6.1231

*. The mean difference is significant at the 0.05 level.

DISCUSSION:

The main goal of periodontal treatment is to eliminate all the pathogenic bacteria that can cause infection on periodontal tissues.^12,13 Scaling and Root Planing is the gold standard in periodontal treatment to eliminate bacteria causing periodontal diseases.¹⁴ There are a lot of bacteria that could contribute to the pathogenesis of periodontal diseases, such as Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Tannerella forsythia, Treponema denticola, Prevotella intermedia, and Campylobacter spp. Among those bacteria mentioned, Porphyromonas gingivalis is the most common bacteria to be associated with periodontitis. Many studies found that Porphyromonas gingivalis could deepen the periodontal pocket, increase host inflammatory response, and cause damage to the alveolar bone.¹⁵ In addition to that, Aggregatibacter actinomycetemcomitans are also believed to have an essential role in developing periodontal diseases. Aggregatibacter actinomycetemcomitans is thought to be the most dominant bacteria in aggressive periodontitis.¹⁶ Aggregatibacter actinomycetemcomitans could cause tooth loss by disrupting normal periodontal tissue homeostasis.¹⁷

Unfortunately, periodontopathogenic bacteria, which have a role in the development of periodontitis, are able to reside in the periodontal tissues or deep pockets, thus could limit scaling and root planning and not completely eradicate the pathogenic bacteria.¹⁸ Remaining bacteria that are not eradicated may cause recurrent periodontitis and the destruction of alveolar bone and supporting connective tissue.¹⁹ Due to the limitations of scaling and root planning, adjunctive treatment is necessary to entirely remove all periodontopathogenic bacteria.

According to the World Health Organisation, antibiotics could be used to eliminate bacteria colonies.²⁰ Tetracycline is frequently used as adjunctive therapy to scaling and root planning. Tetracycline is also frequently prescribed to treat chronic periodontitis and aggressive periodontitis patients.²¹ A member of the tetracycline family, Doxycycline, could work well against gram-negative and gram-positive bacteria as well as periodontal pathogen-specific bacteria.²² However, frequent use of antibiotics could lead to some unwanted adverse effects and bacterial resistance. Sub-antimicrobial Dose Doxycycline (SDD) (formerly known as Low-dose Doxycycline (LDD)) is also frequently used as an additional therapy to scaling and root planing. But, SDD cannot be used as a stand-alone therapy.²³ The use of SDD, like Doxycycline 0.1%, is believed to be able to minimize unwanted adverse effects and bacterial resistance.²⁰

Like any other tetracycline, Doxycycline works against bacteria by entering through the porin protein located on the outer membrane of the bacteria by passive diffusion. After passing through the protein porin, active transport occurs towards the inner cytoplasmic membrane. Then, Doxycycline blocks the 30s subunit of the ribosome. By blocking the 30s subunit, the binding of aminoacyl-tRNA (transfer RNA) to the mRNA-ribosomes will not occur. This prevents bacteria from growing and replicating. This process is known as bacteriostatic.²⁴ Doxycycline also has an anti-inflammatory effect by decreasing the Matrix Metalloproteinase (MMP) enzyme.^20,23

This study used Doxycycline 0.1%, which was given to Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis bacteria. According to the previous study conducted by Setiawatie et al., the use of Doxycycline 0.1% did not cause side effects and had significant bacterial inhibitory activity.¹³

The zone of inhibition is a circular area around the spot of the antibiotic in which bacteria colonies do not grow. It can be used to measure the susceptibility of the bacteria towards the experimental medicine. From this study we can observed that, in the doxycycline 0,1% treatment group against Porphyromonas gingivalis bacteria, the mean diameter of the inhibition zone was 19.6833 mm. Meanwhile, in the Doxycycline 0,1% treatment group against Aggregatibacter actinomycetemcomitans bacteria, we observed that the mean diameter of the inhibition zone was 19.475mm. From these two results, we observed that Doxycycline 0,1% has the inhibitory microbial activity against Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis bacteria. However, we observed a significantly higher result from the Nigella sativa treatment group. In the Nigella sativa treatment group against Porphyromonas gingivalis bacteria, the average diameter of the inhibition zone was 25.141mm. Meanwhile, in the Nigella sativa treatment group against Aggregatibacter actinomycetemcomitans, the mean of the inhibition zone was 25.1mm.

A natural supplement called Nigella sativa can be used instead of Doxycycline. Many minerals and phytoconstituents, such as thymoquinone, alkaloids, flavonoids, saponins, sterols, and essential oils, are present in Nigella sativa. Nigella sativa contains 30-48 percent thymoquinone. Analgesic, antihypertensive, anti-histamine release, hypoglycemic, anticarcinogenic, immune-activating, and hepatoprotective activities are all exhibited by the thymoquinone found in Nigella sativa. Several studies have demonstrated that Thymoquinone, an ingredient in Nigella sativa, has bactericidal characteristics that may be effective against both gram-negative and gram-positive bacteria. It also plays a part in preventing the development of bacterial biofilms. Reactive oxygen species (ROS), which are produced by thymoquinone and have the ability to harm bacterial cell walls, can also protect host tissues from oxidative damage by acting as an antioxidant and scavenger. Furthermore, thymoquinone can harm bacterial membranes by inhibiting pump efflux.²⁵

According to the results of this investigation, Nigella sativa exhibits greater inhibitory action against the microorganisms Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis than the control group and the Doxycycline 0,1% group.

CONCLUSION:

Nigella sativa has better antibacterial activity and many benefits for the host tissues than Doxycycline 0,1%. As a result, Nigella sativa has a susceptible bacterial inhibitory action, and it has the potential to be used as an alternate therapy to Doxycycline 0.1%.

ACKNOWLEDGEMENT:

The authors would like to thank Prof. Dr. Ernie Maduratna S., Drg., M. Kes., Sp. Perio (K) as the chairman of the Periodontology Department in the Faculty of Dental Medicine, Universitas Airlangga, for providing us with a good environment and facilities to complete this project.

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Received on 19.11.2021 Modified on 27.06.2022

Research J. Pharm. and Tech 2023; 16(9):4336-4340.

DOI: 10.52711/0974-360X.2023.00710