Next Article in Journal
Mandibular Advancement and Skeletal Anchorage in Class II Malocclusion Patients: A Systematic Review with Meta-Analysis
Previous Article in Journal
Predicting Leukoplakia and Oral Squamous Cell Carcinoma Using Interpretable Machine Learning: A Retrospective Analysis
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Oral
Volume 4
Issue 3
10.3390/oral4030033
oral-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Natural Therapeutic Agents’ Efficacy in Preventive Strategies against the Periodontal Pathogen Aggregatibacter actinomycetemcomitans: An In Vitro Study

by
Sima Hermes
,
Sham Alatwan
,
Anders Johansson
* and
Anna Bogren
Department of Odontology, Umeå University, 901 87 Umeå, Sweden
*
Author to whom correspondence should be addressed.
Oral 2024, 4(3), 405-416; https://doi.org/10.3390/oral4030033
Submission received: 3 July 2024 / Revised: 13 September 2024 / Accepted: 14 September 2024 / Published: 19 September 2024
Browse Figures
Versions Notes

Abstract

:
Adolescent carriers of the Aggregatibacter actinomycetemcomitans JP2 genotype have an increased risk of developing periodontitis, due to the bacterium’s high leukotoxin (LtxA) production. LtxA contributes to marginal bone loss by killing immunity cells, thus activating the proinflammatory interleukin-1β (IL-1β), which, in turn, activates the osteoclasts. A possible strategy to prevent periodontitis might be to neutralize LtxA in JP2-infected individuals. The aim of this study was to investigate whether extracts from Matcha or Guava leaves can prolong the viability of macrophages in cell cultures by neutralizing the highly leukotoxic JP2 genotype bacteria. The A. actinomycetemcomitans JP2 genotype was pretreated with extracts from either Matcha or Guava leaves. Later, the extracts were rinsed off, before JP2 bacteria were exposed to macrophage cell cultures. The experiment was repeated, where JP2 bacteria were persistently treated with the extracts instead, i.e., the extracts were not rinsed off. The macrophage viability after bacterial exposure was analyzed and compared with that of macrophages exposed to untreated JP2 bacteria. IL-1β secretion in the cell culture medium was quantified in all group samples. Pretreatment of the A. actinomycetemcomitans JP2 genotype with Matcha or Guava leaf extracts moderately neutralized LtxA activity, which resulted in prolonged macrophage viability and decreased IL-1β secretion. These effects of prolonged macrophage viability were enhanced when extracts were persistently present during the exposure period. The results indicate that Matcha and Guava leaf extracts have effects on the virulence of the A. actinomycetemcomitans JP2 genotype that may be useful in future treatment strategies to prevent periodontitis in JP2 bacterium carriers.

Graphical Abstract

1. Introduction

Gum disease, also known as gingivitis and periodontitis, is a global health issue according to the World Health Organization [1]. Gingivitis is a plaque-microbiota-induced gingival confined inflammation. If left untreated, gingivitis may progress to periodontitis, where the supporting tissue of the tooth gradually degrades [2].
It has been demonstrated that 2 to 4 days of plaque accumulation results in an “initial” inflammatory lesion (vasculitis) in the gingival tissues. Within 4 to 10 days, an “early” lesion develops, characterized by lymphocytes and collagen break down. With further accumulation of plaque, an “established” inflammatory lesion with a predominance of plasma cells develops within two to three weeks. In some individuals, this stage transitions to an “advanced” lesion with progressive loss of attachment, loss of the alveolar bone and formation of deep periodontal pockets, clinically manifest as periodontitis [3]. Deep periodontal pockets serve as a reservoir for bacterial dysbiosis.
The prevalence of periodontitis in adult populations is relatively high. Cross-sectional studies have shown that about 40% of adults are affected. However, the severity of the disease varies highly, where about 10% of adult populations exhibit a rapidly progressive periodontal disease pattern [3].
During the 1980s, A. actinomycetemcomitans was found in both healthy individuals and those with periodontitis [4]. During the mid-1990s, a clone of A. actinomycetemcomitans, the so-called JP2 clone, was discovered to be highly leukotoxic. The origins of JP2 bacteria can be traced back to North Africa, with its subsequent spread to West Africa and later global dissemination via the slave trade [5]. However, it was not until the 2010s that studies established that carriers of the JP2 genotype face a higher risk of developing rapidly progressive and severe periodontitis compared with non-JP2 carriers [6,7]. More recently, a study revealed that 13% of adolescents in North and West Africa carry the JP2 genotype [8].
The cytotoxicity of JP2 bacteria is due to a protein toxin called leukotoxin (LtxA), which is a virulence factor that lyses blood cells such as PMNs and monocytes [9]. LtxA is a large pore-forming protein with a region that exhibits high specificity for the receptor lymphocyte function-associated antigen 1 (LFA-1), which is present on PMNs and monocytes, and therefore makes bonding inevitable. Upon binding, the intracellular signaling system inside the target cells results in the activation of the intracellular cytokine Caspase-1, subsequently cleaving and activating the proinflammatory cytokine interleukin-1β (IL-1β) [10].
Gram-negative pathogens lead to increased cytokine production, including the production of intracellular IL-1β in an inactive form. However, the LtxA in JP2 bacteria leads to the activation and extracellular secretion of IL-1β by inducing inflammatory cell death [11]. The secretion of IL-1β causes further inflammation and can lead to further osteoclast activity, resulting in severe bone loss [3]. A study concluded that the JP2 genotype is highly cytotoxic, and even a 1:1 bacterium–macrophage ratio is sufficient for inducing the abovementioned process [12]. Recent studies have suggested that a targeted LtxA therapy, which blocks the cytotoxicity of JP2 bacteria, may prevent rapid disease progression [13].
It has been shown that adolescents affected by the JP2 clone exhibit rapidly progressive periodontitis patterns and that most of those adolescents are of African origin from countries with low socioeconomic status, where access to dental care is limited [3,6,7,14]. Therefore, a targeted LtxA treatment could potentially prevent periodontitis [15]. In this study, natural and easily accessible therapeutic agents are evaluated as options for preventing periodontitis in countries with a high prevalence of the JP2 clone.
Recent studies discovered that Matcha (a high-grade green tea powder from the Camellia sinensis tree) can neutralize LtxA [16]. The neutralizing mechanism involves Matcha changing the structure of LtxA and even increasing the bacterial affinity to its own LtxA. This means that LtxA binds to the bacteria rather than to the target cell, preventing the consequences of LtxA [16,17]. Another plant material that has been shown to have anti-inflammatory and antibacterial effects is Guava leaves. The antibacterial effect is achieved by bacterial cell membrane disruption, making it more permeable and thus causing the leakage of essential cell contents, ultimately resulting in bacterial death [18].
Chemical analysis indicated epigallocatechin-3-gallate (EGCg) as the molecule responsible for LtxA neutralization in extracts from both Matcha and Guava leaves [19,20]. A recently conducted in vivo study revealed EGCg’s ability to promote symbiosis among oral bacteria by inhibiting the colonization of periodontal pathogens [21]. Recent in vitro studies reported similar results, where EGCg had a positive effect on both oral and digestive system microbiota by stimulating probiotics and prohibiting bacterial pathogens [17,22]. A unique property of EGCg is its ability to decrease bone loss by obstructing the bond between RANK and RANKL, thus inhibiting osteoclast formation [23]. However, Matcha has been associated with liver toxicity at extremely high doses, i.e., exceeding 338 mg EGCg/day [24]. Conversely, Guava leaves only exhibit cytotoxic effects on cancerous cells, without causing harm to normal cells [25]. In conclusion, the unique properties of these plants give them the potential of being a promising preventative treatment against JP2 bacteria, when applied in safe therapeutic doses [26].
Therefore, the objective of this in vitro study was to estimate the stability of extracts from Matcha tea and Guava leaves as potential therapeutic agents in neutralizing the leukotoxin from A. actinomycetemcomitans JP2 bacteria. The obtained data may provide useful knowledge that contributes to identifying valuable tools for the prevention of periodontitis in countries with low socioeconomic status and/or limited access to dental care.

2. Materials and Methods

2.1. Summary of Experimental Design

In the applied method, the JP2 genotype of A. actinomycetemcomitans was cultured, treated with Matcha or Guava leaf extracts, and subsequently exposed to THP-1 cells with macrophage-like phenotypes. After a period of exposure, the viability of the macrophages was determined. Different concentrations of JP2 bacteria per macrophage (MOI; multiplicity of infection) were analyzed to screen for the lethal dose of JP2 bacteria (LD50) at which 50% of the macrophages were non-viable. In essence, this study aimed to examine the effect of the extract treatments on JP2 cytotoxicity and IL-1β secretion.

2.2. Cultivation of A. actinomycetemcomitans

For cultivation, A. actinomycetemcomitans JP2 bacteria (strain HK921) [27] were spread on a blood agar plate and allowed to grow for an incubation period of 48 h in a 37 °C cabinet with an atmosphere of 5% CO2. Afterward, significant JP2 bacterial cultures from the agar plate were harvested and suspended in peptone yeast glucose broth (PYG; Sigma-Aldrich. St. Louis, MA, USA) [28]. Afterward, the mixture was divided into three test tubes, each containing 12 mL, and measured using a spectrophotometer with an optical density (OD) of 600 nm, where OD 1 corresponds to about 109 bacteria/mL.

2.3. Matcha and Guava Leaf Extract Preparation

2.3.1. Matcha Extract

Matcha tea powder is commercially available and was acquired from Rawpowder AB (Huddinge, Sweden). Matcha powder and H2O were mixed to achieve a concentration of 250 mg/mL. Then, the mixture was autoclaved to obtain both boiling and sterilization effects, pipetted into an Eppendorf-Tube (Hamburg, Germany), and finally centrifuged to obtain a particle-free extract. The procedure for achieving an effective Matcha extract concentration and preparation was previously tested and described in detail by Sasagawa et al., 2021 [29].

2.3.2. Guava Leaf Extract

Dried leaves from Guava trees grown in India were kindly provided by Dr. Kartheyanene Jayaprakashat, Örebro University, Sweden. A weight of 5 g of dried Guava leaves was minced and mixed with the proper volume of H2O, calculated to achieve a concentration of 100 mg/mL. The mixture was autoclaved, pipetted into an Eppendorf Tube®, and then centrifuged. The procedure for Guava leaf extract preparation, with the recommended proper concentration, was previously executed and described in detail by Ennibi et al., 2019 [30].

2.4. Exposure of A. actnomycetemcomitans to the Extracts

To expose A. actinomycetemcomitans to the extracts, Matcha and Guava leaf extracts (360 μL of each) were added to two of the three tubes of bacterial suspensions in 12 mL of PYG broth with a bacterial density of OD 600 nm = 0.254, yielding a final concentration of 2.5% for each extract. An amount of 360 μL of sterile H2O was added to the third tube to demonstrate an untreated JP2 bacterium sample.
To facilitate the interaction of JP2 bacteria with the respective extracts and sterile H2O, the three tubes were mixed for 30 min and centrifuged for 15 min, at 5100 rpm and room temperature (17–23 °C). Each tube was emptied gently, to ensure the bacterial pellet remained at the base. An amount of 10 mL of phosphate-buffered saline (PBS; Sigma-Aldrich. St. Louis, MA, USA) was added to each tube. The three tubes were then centrifuged and emptied again to remove unbound extract components. Lastly, the bacterial pellet in each tube was suspended in 3 mL of RPMI (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich. St. Louis, MA, USA), yielding 109 JP2 bacteria/mL, which was then poured into three aliquots.

2.5. Cultivation of THP-1 Cells

Human monocytic leukemia cells (THP-1; Sigma-Aldrich, St. Louis, MO, USA) were used as target cells in the test assay. To grow the monocytes, phorbol myristate acetate (PMA; Sigma-Aldrich, St. Louis, MO, USA) was added to differentiate them into macrophage-like phenotypes. An amount of 100 μL of the solution of monocytes in PMA was pipetted into each well in a 96-well microtiter plate. Lastly, the plate was incubated, and then the medium in each well, except for two wells that were designated to serve as a reference control for PMA-stimulated THP-1 cell viability and IL-1β secretion, was replaced with 100 μL of RPMI, which is a culture medium that promotes human cell growth in vitro. Afterward, the plate was incubated again and the medium in each well, except the reference control wells, was replaced once again with fresh RPMI. The plate containing macrophages was then ready to be exposed to JP2 bacteria. The procedure was previously described in detail by Höglund Åberg et al., 2014 [7]. Hereafter, PMA-stimulated THP-1 cells will be referred to as macrophages.

2.6. Exposure of Macrophages to A. actinomycetemcomitans

The procedure for exposing macrophages in the plate to JP2 bacteria was achieved using 50 μL from each of the three bacterium aliquots, which were pipetted into duplicate wells on a 96-well culture plate with macrophages. Afterward, serial dilution was performed, resulting in a JP2 bacterium concentration that was three-times lower each time, which, in total, corresponded to an MOI of 0 to 300. The 96-well culture plate was later incubated for 2 h at 37 °C in a 5% CO2 cabinet. The reference control wells were not exposed to JP2 bacteria or test agents.
The same method was applied, wherein the macrophages were exposed to JP2 bacteria with 1% extracts of Matcha and Guava leaves present throughout the entire incubation period, i.e., persistent treatment.

2.7. Cytotoxicity Tests

Neutral red uptake (NRU) was the method used to stain macrophages to determine their viability after exposure to JP2 bacteria. The protocol for neutral red uptake was previously described in detail by Repetto et al., 2008 [31]. Briefly, 100 μL of neutral red solution (Neutral red; Sigma-Aldrich, St. Louis, MO, USA) was added to 10 mL of RPMI, resulting in a 5 mg/mL neutral red solution. Subsequently, the solution was incubated at 37 °C for 1 h. Afterward, the solution was centrifuged for 10 min at room temperature at 3000 rpm.
After exposure of the macrophages to the different bacterial mixtures for 2 h, the medium of the plate with the serial dilution was changed by aspirating and adding 100 μL of neutral red solution to each well. The plate was incubated for 90 min and then examined under a standard microscope to observe possible morphological alterations in the cells. The neutral red solution was removed from each well and the cell monolayer was washed with 150 μL of PBS. In the dry wells, 150 μL of lysis buffer (1% acetic acid in 50% ethanol/water) was applied, as well as in the reference control wells. After 30 min, the quantity of surviving macrophages was examined using a spectrophotometer at 540 nm, and the accumulating red color was quantified in each well. Macrophage viability (%) was estimated in all test samples.

2.8. IL-1β Analysis

IL-1β concentrations were determined with an enzyme-linked immunosorbent assay (ELISA; R&D Systems®, Minneapolis, MN, USA), following the manufacturer’s protocol. Briefly, a primary ELISA antibody was bound to the plastic surface and blocked with 2% FBS, to avoid nonspecific binding. Washing procedures were performed between each procedure with 200 μL of ELISA washing buffer solution in each well. Culture supernatants (samples) and standards at different concentrations (0–250 pg/mL recombinant IL-1β) were then added to the plate and allowed to bind to the primary antibody. After 90 min, the plate was washed three times and the wells were filled with a detection antibody and incubated for 90 min. Afterward, the wells were washed three times, and 100 μL of Streptavidin HRP was added. Next, the plate was incubated for 20 min. Lastly, the wells were washed five times, and then enzyme substrate was added to each well and incubated for another 20 min. The reaction was terminated by adding 50 μL of 1 M H2SO4 to each well. The concentration of IL-1β was determined using a spectrophotometer at 450 nm in relation to the standard curve with a known concentration of recombinant IL-1β. The concentration in samples was calculated with linear regression in relation to standards with known concentrations of recombinant IL-1β.

2.9. Statistical Analyses

Microsoft Excel software (Version 16.67) was used for calculations and graph illustrations of the results. For the statistical analyses of the collected data, IBM SPSS version 28.0.1.0 (142) was used. When the data were determined to be normally distributed and the subject included more than two test samples, an ANOVA test was used to calculate significance values (p ≤ 0.05).

2.10. Ethical Reflection

THP-1 cells are commercially available cancer cells for research purposes, and JP2 bacteria are a reference strain without the possibility of tracing any carrier.
It is our hope that the results of this study will contribute to the following: (i) identifying easily available therapeutic agents with potential effects for primary prevention of periodontitis, and (ii) improving periodontal health in adolescents in countries in Africa and Southeast Asia with low socioeconomic status and/or limited access to dental care.

2.11. Literature Review

A literature search was first conducted on PubMed using the following related MeSH terms: A. actinomycetemcomitans, JP2 clone, periodontal disease, adolescents, interleukin-1β, Matcha, Guava, NRU, and/or EGCg. The title and abstract of the identified articles were reviewed, and those relevant to this study were included. Additional data were obtained from the textbook Clinical Periodontology and Implant Dentistry (2015) and the official website of the World Health Organization. In some cases, when relevant studies were difficult to find, Google Scholar was used to source relevant articles by searching, for example, “Psidium Guajava cytotoxicity”.

3. Results

3.1. Untreated JP2 Bacteria

Dose-Dependent Effect on Macrophage Viability and IL-1β Secretion

Untreated JP2 bacteria were exposed to macrophages for 2 h at different concentrations (MOI 0.03–300) to estimate LD50. As illustrated in Figure 1, a cytotoxic effect of secreted LtxA was observed starting from a bacterium–macrophage ratio of 3:100 (MOI 0.03), and LD50 was observed between MOI 0.03 and 0.1.
As illustrated in Figure 2, IL-1β secretion remained stable until MOI 10, followed by a rapid increase and subsequent gradual decrease.

3.2. Therapeutic Agent: Matcha Extract (Test)

3.2.1. JP2 Bacteria Pretreated with Matcha Extract: Dose-Dependent Effect on Macrophage Viability and IL-1β Release

Compared with untreated JP2 bacteria, pretreatment with Matcha extract increased macrophage viability on exposure to JP2 bacteria at different concentrations for 2 h. The results indicate that the LD50 value increased to approximately MOI 0.3, as illustrated in Figure 1.
IL-1β secretions were generally lower compared with those from untreated JP2 bacteria, but at MOI 3, a rapid increase to a high concentration (190 pg/mL) occurred, as illustrated in Figure 2.

3.2.2. JP2 Bacteria Persistently Treated with Matcha Extract: Dose-Dependent Effect on Macrophage Viability and IL-1β Release

Macrophage viability increased substantially compared with pretreatment alone, with around 65% viable macrophages at MOI 100 after 2 h of exposure. LD50 was found to be greater than MOI 100 but less than MOI 300, as illustrated in Figure 1.
IL-1β secretions were significantly less compared with the untreated and pretreated JP2 bacteria, which was consistently noted across all samples in this test, as illustrated in Figure 2.

3.2.3. Pretreated and Persistently Treated JP2 Bacteria (MOI 1) with Matcha Extract: Effect on Macrophage Viability and IL-1β Release at 1 MOI Concentration

After assessing the LD50 of JP2 bacteria through screening, MOI 1 was chosen for further experiments with Matcha extract. Therefore, replicates of eight wells for each of the following conditions were tested: untreated JP2 bacteria; JP2 bacteria pretreated with Matcha extract; and JP2 bacteria persistently treated with Matcha extract. The macrophage viability was measured after 2 h of exposure.
Both pretreatment and persistent treatment with Matcha extract exhibited significant results in enhancing macrophage viability, as indicated in Table 1. The IL-1β concentrations consistently correlated with macrophage viability, as shown in Table 2. JP2 bacteria triggered IL-1β secretion from the exposed macrophages, with a significant reduction observed when JP2 bacteria were pretreated or persistently treated with Matcha extract.

3.3. Therapeutic Agent: Guava Leaf Extract (Test)

3.3.1. JP2 Bacteria Pretreated with Guava Leaf Extract: Dose-Dependent Effect on Macrophage Viability and IL-1β Release

Guava leaf extract pretreatment increased macrophage viability similarly to the Matcha extract pretreatment. Figure 1 illustrates that both Guava and Matcha extracts in pretreatment resulted in the same LD50 at MOI 0.3.
In general, the IL-1β secretion was slightly elevated compared with untreated JP2 bacteria. Specifically, at MOI values of 1 and 3, IL-1β secretion surpassed 130 pg/mL, as illustrated in Figure 2.

3.3.2. JP2 Bacteria Persistently Treated with Guava Leaf Extract: Dose-Dependent Effect on Macrophage Viability and IL-1β Release

Figure 1 illustrates that at MOI 3, 80% of the macrophages remained viable. However, at MOI 10, the viability decreased to 10%. Half of the macrophages were viable between MOI values of 3 and 10 (the exact concentration is unknown).
The secretion of IL-1β remained consistently negligible across all concentrations (45 pg/mL), as illustrated in Figure 2.

3.3.3. Pretreated and Persistently Treated JP2 Bacteria (MOI 1) with Guava Leaf Extract: Effect on Macrophage Viability and IL-1β Release at MOI 1 Concentration

MOI 1 was also chosen for further experiments with Guava leaf extract. Therefore, replicates of eight wells for each of the following conditions were tested: untreated JP2 bacteria; JP2 bacteria pretreated with Guava leaf extract; and JP2 bacteria persistently treated with Guava leaf extract. Macrophage viability was measured after 2 h of exposure.
Both pretreatment and persistent treatment with Guava leaf extract yielded a significant increase in macrophage viability, as indicated in Table 1.
The results for IL-1β concentration were consistent with macrophage viability, as shown in Table 2. A significant reduction in IL-1β secretion was observed when JP2 bacteria were persistently treated with Guava leaf extract. However, pretreatment with Guava leaf extract did not result in a significant reduction in IL-1β secretion of JP2 bacteria-exposed macrophages.

4. Discussion

The results from this study demonstrate that untreated JP2 bacteria have a dose-dependent impact on macrophage viability. The results also indicate that a ratio of 1:1 (bacterium to macrophage) is sufficient for a leukotoxic effect, as previously demonstrated by Kelk et al., 2008 [12]. As a result of JP2 bacterial cytotoxicity, IL-1β reached its peak at MOI 10. At high JP2 bacteria and IL-1β concentrations, rapid and excessive macrophage cell death occurred. By MOI 30, natural cell death began, leading to a subsequent decrease in the secretion of IL-1β from the macrophages. In summary, macrophage viability and IL-1β concentration influenced each other.
An important property of oral care therapeutic agents is their ability to bind to biofilm. In our study, the pretreatment of the bacteria with Matcha and Guava leaf extracts resulted in a color shift in the bacterial pellet, indicating the polymerization of EGCg and binding to JP2 bacteria [16,32]. The effect observed with the Matcha extract pretreatment proved sufficient to initially increase macrophage viability and inhibit IL-1β secretion. However, this effect was not observed with Guava leaf extract pretreatment or with the untreated group. Therefore, our results suggest that the EGCg in Matcha extract enhanced the viability of the LtxA-exposed macrophages.
The pretreatment of JP2 bacteria with the two extracts had no significant impact on macrophage viability at MOI 1. Regarding IL-1β concentrations, pretreatment with Matcha extract resulted in a significant decrease in IL-1β secretion, whereas pretreatment with Guava leaf extract did not show any statistical significance. In summary, when tested in cell cultures, Matcha extract proved to be more efficient than Guava leaf extract in the pretreatment of JP2 bacteria, presumably due to the higher content of EGCg in Matcha extract.
Moreover, significant inhibitory effects on JP2 bacterial leukotoxicity and macrophage IL-1β secretion were observed with persistent treatment using Matcha extract. The physiological properties of EGCg, particularly its potential for degradation, may elucidate why the presence of the extract during JP2 bacterium exposure to the macrophages enhanced its efficacy. Similar in vitro degradation of EGCg was previously documented by Krupkova et al., 2016 [32].
Persistent treatment with Guava leaf extract had significant effects, although they were less pronounced compared with persistent treatment with Matcha extract. IL-1β secretions decreased to around 45 pg/mL and did not decrease further, despite an increase in macrophage viability. A possible explanation for this is that the Guava leaf extract had a lower EGCg content than Matcha extract, which resulted in a less efficient decrease in proinflammatory response.
The experimental protocols were repeated six months after initiation, yielding a high LtxA neutralizing capacity, suggesting the robust stability and sustainability of the extracts. This can be attributed to the applied extract-preparation method, namely decoction. Decoction not only amplifies the positive and survival properties of the plant extract but also proves to be particularly well suited for heat-tolerant natural plants such as Matcha and Guava leaves [33,34,35].
To obtain high inter-rater reliability and reproducibility of the result in the present study, all steps included in the employed methods were fully reviewed before being carried out. The experiments commenced with the selection and collection of the natural therapeutic agents, followed by the preparation and storage of their extracts according to protocols. Next, THP-1 cells were acquired and grown into macrophages. After that, the A. actinomycetemcomitans JP2 genotype was acquired, cultured in vitro, and treated with the prepared extracts, followed by proper storage according to protocols. Finally, JP2 bacteria were exposed to macrophages, and a definitive quantitative analysis of macrophage viability and IL-1β secretion was conducted. To ensure that reliable results and conclusions were obtained, it was critical to meticulously follow the abovementioned steps.
The limitations of this study are as follows; Firstly, the results were based on the initial efficacy of the use of a single dose of the plant extracts. Secondly, the observation period was relatively short, and potential responses of the JP2 bacterium were not examined. The results were based on a screening that demonstrated initial efficacy; however, further studies are needed using a more periodontitis-like biofilm model, including the JP2 bacterium, to validate the findings.
In summary, our study presented promising results; some clinical trials with Matcha or Guava leaf extracts have shown similar results in patients with periodontal diseases [36,37]. In addition to inhibiting LtxA activity, these herbal plants have shown antimicrobial activity against several periodontitis-associated bacteria [21,38]. Furthermore, Matcha and Guava leaf extracts have shown their ability to decrease bone loss by inhibiting the formation of osteoclasts [23]. All things considered, Matcha and Guava leaf extracts contribute to overall health benefits through a favorable impact on the host and the bacteria, when applied in proper doses.

5. Conclusions

The results of the present study are compatible with previous studies that examined products based on the tested plants [16,17,39]. Recent studies focused on the persistency of the effect of the plant extract when either or both LtxA and JP2 bacteria were pretreated [40,41]. However, there was a gap in the literature concerning the effects of Matcha and Guava leaf extracts on prolonging macrophage viability and decreasing IL-1β secretion. This study aimed to address this gap in the literature. Based on our findings, we present the following findings:
-
Matcha and Guava leaf extracts are effective therapeutic agents against JP2 bacteria in treatments with persistent exposure.
-
This study provides valuable insights for further research; however, the findings are not currently recommended for direct application in patient dental care for the prevention or treatment of periodontitis.
Finally, we suggest that future research investigates therapeutic doses of Matcha and Guava extracts.

Author Contributions

Conceptualization, A.J. and A.B.; methodology, S.A., S.H. and A.J.; software, S.H., A.J. and A.B.; validation, A.J. and A.B.; formal analysis, S.H., A.J., A.B. and S.H.; investigation, S.A., S.H., A.J. and A.B.; resources, A.J. and A.B.; data curation, S.H., S.A., A.J. and A.B.; writing—original draft preparation, S.H., S.A. and A.B.; writing—review and editing, S.H., S.A., A.J. and A.B.; visualization, S.H., S.A., A.J. and A.B.; supervision, A.B. and A.J.; project administration, A.J.; funding acquisition, A.J. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by TUA grants from Region Västerbotten, Sweden (A.J.; 7003193).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data are available upon request to the corresponding author.

Acknowledgments

We would like to express our gratitude to Anders Esberg, who assisted in conducting the statistical analysis.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. World Health Organization. Oral Health. Available online: https://www.who.int/health-topics/oral-health#tab=tab_1 (accessed on 2 July 2024).
  2. Papapanou, P.N.; Sanz, M.; Buduneli, N.; Dietrich, T.; Feres, M.; Fine, D.H.; Flemmig, T.F.; Garcia, R.; Giannobile, W.V.; Graziani, F.; et al. Periodontitis: Consensus report of Workgroup 2 of the 2017 World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions. J. Periodontol. 2018, 89 (Suppl. S1), S173–S182. [Google Scholar] [CrossRef] [PubMed]
  3. Lang, N.P.; Lindhe, J. Clinical Periodontology and Implant Dentistry, 6th ed.; Wiley-Blackwell: Oxford, UK, 2015. [Google Scholar]
  4. Nørskov-Lauritsen, N.; Claesson, R.; Jensen, A.B.; Åberg, C.H.; Haubek, D. Aggregatibacter actinomycetemcomitans: Clinical Significance of a Pathobiont Subjected to Ample Changes in Classification and Nomenclature. Pathogens 2019, 8, 243. [Google Scholar] [CrossRef] [PubMed]
  5. Haubek, D. The highly leukotoxic JP2 clone of Aggregatibacter actinomycetemcomitans: Evolutionary aspects, epidemi-ology and etiological role in aggressive periodontitis. APMIS Suppl. 2010, 118, 1–53. [Google Scholar] [CrossRef] [PubMed]
  6. Haubek, D.; Ennibi, O.K.; Poulsen, K.; Vaeth, M.; Poulsen, S.; Kilian, M. Risk of aggressive periodontitis in adolescent carriers of the JP2 clone of Aggregatibacter (Actinobacillus) actinomycetemcomitans in Morocco: A prospective longitudinal cohort study. Lancet 2008, 371, 237–242. [Google Scholar] [CrossRef]
  7. Höglund Åberg, C.; Haubek, D.; Kwamin, F.; Johansson, A.; Claesson, R. Leukotoxic activity of Aggregatibacter actinomycetemcomitans and periodontal attachment loss. PLoS ONE 2014, 9, e104095. [Google Scholar] [CrossRef]
  8. Haubek, D.; Mulli, T.; Kemoli, A.; Lindholm, M.; Gjørup, H.; Nørregaard, M.-L.M.; Johansson, A. Prevalence of JP2 and Non-JP2 Genotypes of Aggregatibacter actinomycetemcomitans and Oral Hygiene Practice of Kenyan Adolescents in Maasai Mara. Pathogens 2021, 10, 488. [Google Scholar] [CrossRef] [PubMed]
  9. Kachlany, S.C. Aggregatibacter actinomycetemcomitans leukotoxin: From threat to therapy. J. Dent. Res. 2010, 89, 561–570. [Google Scholar] [CrossRef]
  10. Hakmi, M.; Bouricha, E.M.; El Harti, J.; Amzazi, S.; Belyamani, L.; Khanfri, J.E.; Ibrahimi, A. Computational modeling and drug-gability assessment of Aggregatibacter actinomycetemcomitans leukotoxin. Comput. Methods Programs Biomed. 2022, 222, 106952. [Google Scholar] [CrossRef]
  11. Kelk, P.; Moghbel, N.S.; Hirschfeld, J.; Johansson, A. Aggregatibacter actinomycetemcomitans Leukotoxin Activates the NLRP3 Inflammasome and Cell-to-Cell Communication. Pathogens 2022, 11, 159. [Google Scholar] [CrossRef]
  12. Kelk, P.; Claesson, R.; Chen, C.; Sjöstedt, A.; Johansson, A. IL-1beta secretion induced by Aggregatibacter (Actinobacillus) actinomycetemcomitans is mainly caused by the leukotoxin. Int. J. Med. Microbiol. 2008, 298, 529–541. [Google Scholar] [CrossRef]
  13. Krueger, E.; Brown, A.C. Aggregatibacter actinomycetemcomitans leukotoxin: From mechanism to targeted anti-toxin therapeutics. Mol. Oral Microbiol. 2020, 35, 85–105. [Google Scholar] [CrossRef] [PubMed]
  14. Ghafari, M.; Bahadivand-Chegini, S.; Nadi, T.; Doosti-Irani, A. The global prevalence of dental healthcare needs and unmet dental needs among adolescents: A systematic review and meta-analysis. Epidemiol. Health 2019, 41, e2019046. [Google Scholar] [CrossRef] [PubMed]
  15. Bostanci, N.; Belibasakis, G.N. Precision periodontal care: From omics discoveries to chairside diagnostics. Clin. Oral Investig. 2023, 27, 971–978. [Google Scholar] [CrossRef] [PubMed]
  16. Chang, E.H.; Giaquinto, P.; Huang, J.; Balashova, N.V.; Brown, A.C. Epigallocatechin gallate inhibits leukotoxin release by Aggregatibacter actinomycetemcomitans by promoting association with the bacterial membrane. Mol. Oral Microbiol. 2020, 35, 29–39. [Google Scholar] [CrossRef]
  17. Kong, C.; Zhang, H.; Li, L.; Liu, Z. Effects of green tea extract epigallocatechin-3-gallate (EGCG) on oral disease-associated microbes: A review. J. Oral Microbiol. 2022, 14, 2131117. [Google Scholar] [CrossRef]
  18. Biswas, B.; Rogers, K.; McLaughlin, F.; Daniels, D.; Yadav, A. Antimicrobial Activities of Leaf Extracts of Guava (Psidium guajava L.) on Two Gram-Negative and Gram-Positive Bacteria. Int. J. Microbiol. 2013, 2013, 746165. [Google Scholar] [CrossRef]
  19. Kochman, J.; Jakubczyk, K.; Antoniewicz, J.; Mruk, H.; Janda, K. Health Benefits and Chemical Composition of Matcha Green Tea: A Review. Molecules 2020, 26, 85. [Google Scholar] [CrossRef]
  20. Wang, D.; Zhou, L.; Zhou, H.; Hu, H.; Hou, G. Chemical composition and protective effect of guava (Psidium guajava L.) leaf extract on piglet intestines. J. Sci. Food Agric. 2021, 101, 2767–2778. [Google Scholar] [CrossRef]
  21. Nakao, R.; Takatsuka, A.; Mandokoro, K.; Narisawa, N.; Ikeda, T.; Takai, H.; Ogata, Y. Multimodal inhibitory effect of matcha on Porphyromonas gingivalis. Microbiol. Spectr. 2024, 12, e03426-23. [Google Scholar] [CrossRef] [PubMed]
  22. Liu, Z.; de Bruijn, W.J.C.; Bruins, M.E.; Vincken, J.P. Reciprocal interactions between epigallocatechin-3-gallate (EGCG) and human gut microbiota in vitro. J. Agric. Food Chem. 2020, 68, 9804–9815. [Google Scholar] [CrossRef]
  23. Sarkar, J.; Das, M.; Howlader, M.S.; Prateeksha, P.; Barthels, D.; Das, H. Epigallocatechin-3-gallate inhibits osteoclastic differentiation by modulating mitophagy and mitochondrial functions. Cell Death Dis. 2022, 13, 908. [Google Scholar] [CrossRef] [PubMed]
  24. Zhao, T.; Li, C.; Wang, S.; Song, X. Green Tea (Camellia sinensis): A Review of Its Phytochemistry, Pharmacology, and Toxicology. Molecules 2021, 27, 3909. [Google Scholar] [CrossRef] [PubMed]
  25. Lok, B.; Babu, D.; Tabana, Y.; Dahham, S.S.; Adam, M.A.A.; Barakat, K.; Sandai, D. The Anticancer Potential of Psidium guajava (Guava) Extracts. Life 2023, 13, 346. [Google Scholar] [CrossRef] [PubMed]
  26. Pasupuleti, M.K.; Nagate, R.R.; Alqahtani, S.M.; Penmetsa, G.S.; Gottumukkala, S.N.V.S.; Ramesh, K.S.V. Role of Medicinal Herbs in Periodontal Therapy: A Systematic Review. J. Int. Soc. Prev. Community Dent. 2023, 13, 9–16. [Google Scholar] [CrossRef]
  27. Haubek, D.; Dirienzo, J.M.; Tinoco, E.M.; Westergaard, J.; López, N.J.; Chung, C.P.; Poulsen, K.; Kilian, M. Racial tropism of a highly toxic clone of Actinobacillus actinomycetemcomitans associated with juvenile periodontitis. J. Clin. Microbiol. 1997, 35, 3037–3042. [Google Scholar] [CrossRef]
  28. Kalfas, S.; Pour, Z.K.; Claesson, R.; Johansson, A. Leukotoxin A Production and Release by JP2 and Non-JP2 Genotype Aggregatibacter actinomycetemcomitans in Relation to Culture Conditions. Pathogens 2024, 13, 569. [Google Scholar] [CrossRef]
  29. Sasagawa, K.; Domon, H.; Sakagami, R.; Hirayama, S.; Maekawa, T.; Isono, T.; Hiyoshi, T.; Tamura, H.; Takizawa, F.; Fukushima, Y.; et al. Matcha Green Tea Exhibits Bactericidal Activity against Streptococcus pneumoniae and Inhibits Functional Pneumolysin. Antibiotics 2021, 10, 1550. [Google Scholar] [CrossRef]
  30. Ennibi, O.K.; Claesson, R.; Akkaoui, S.; Reddahi, S.; Kwamin, F.; Haubek, D.; Johansson, A. High salivary levels of JP2 genotype of Aggregatibacter actinomycetemcomitans is associated with clinical attachment loss in Moroccan adolescents. Clin. Exp. Dent. Res. 2019, 5, 44–51. [Google Scholar] [CrossRef]
  31. Repetto, G.; del Peso, A.; Zurita, J.L. Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nat. Protoc. 2008, 3, 1125–1131. [Google Scholar] [CrossRef]
  32. Krupkova, O.; Ferguson, S.J.; Wuertz-Kozak, K. Stability of (−)-epigallocatechin gallate and its activity in liquid formulations and delivery systems. J. Nutr. Biochem. 2016, 37, 1–12. [Google Scholar] [CrossRef]
  33. Azwanida, N.N. A Review on the Extraction Methods Use in Medicinal Plants, Principle, Strength and Limitation. Med. Aromat. Plants 2015, 4, 3. [Google Scholar] [CrossRef]
  34. Sharma, A.; Ruiz-Manriquez, L.M.; Serrano-Cano, F.I.; Reyes-Pérez, P.R.; Tovar Alfaro, C.K.; Barrón Andrade, Y.E.; Hernández Aros, A.K.; Srivastava, A.; Paul, S. Identification of microRNAs and Their Expression in Leaf Tissues of Guava (Psidium guajava L.) under Salinity Stress. Agronomy 2020, 10, 1920. [Google Scholar] [CrossRef]
  35. Chen, H.; Song, Y.; Li, H.; Zaman, S.; Fan, K.; Ding, Z.; Wang, Y. Enhancing the Adaptability of Tea Plants (Camellia sinensis L.) to High-Temperature Stress with Small Peptides and Biosurfactants. Plants 2023, 12, 2817. [Google Scholar] [CrossRef] [PubMed]
  36. Abdul-Wahab, H.Y.; Salah, R.; Abdulbaqi, H.R. Salivary levels of catalase, total antioxidant capacity and interleukin-1beta and oral health-related quality of life after matcha and green tea consumption for patients with gingivitis: A randomized clinical trial. Int. J. Dent. Hyg. 2024. online ahead of print. [Google Scholar] [CrossRef] [PubMed]
  37. Nayak, N.; Varghese, J.; Shetty, S.; Bhat, V.; Durgekar, T.; Lobo, R.; Nayak, U.Y.; Vishwanath, U. Evaluation of a mouthrinse containing guava leaf extract as part of comprehensive oral care regimen—A randomized placebo-controlled clinical trial. BMC Complement. Altern. Med. 2019, 19, 327. [Google Scholar] [CrossRef] [PubMed]
  38. Morais-Braga, M.F.; Carneiro, J.N.; Machado, A.J.; Dos Santos, A.T.; Sales, D.L.; Lima, L.F.; Figueredo, F.G.; Coutinho, H.D.J. Psidium guajava L., from ethnobiology to scientific evaluation: Elucidating bioactivity against pathogenic microorganisms. J. Ethnopharmacol. 2016, 194, 1140–1152. [Google Scholar] [CrossRef] [PubMed]
  39. Kwamin, F.; Gref, R.; Haubek, D.; Johansson, A. Interactions of extracts from selected chewing stick sources with Aggregatibacter actinomycetemcomitans. BMC Res. Notes 2012, 5, 203. [Google Scholar] [CrossRef]
  40. Moghbel, N.S.; Mugambe, J. Inflammatory Cell Death Induced by Titanium or Leukotoxin—Effect of Extracts. Master’s Dissertation, Umeå University, Umeå, Sweden, 2021. Available online: https://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-190336 (accessed on 2 July 2024).
  41. Toma, C.; Genet, K. The Interaction between Guava-Extract and Aggregatibacter actinomycetemcomitans Leukotoxin. Master’s Dissertation, Umeå University, Umeå, Sweden, 2014. Available online: https://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-102414 (accessed on 2 July 2024).
Oral 04 00033 g001
Figure 1. Illustration of the screening test results. Cytotoxicity tests for detection of macrophage viability (%) after exposure to JP2 bacteria: (i) without treatment (JP2); (ii) pretreated with Matcha extract (Ma); (iii) pretreated with Guava leaf extract (Gu); (iv) persistently treated with Matcha extract (JP2 + Ma); and (v) persistently treated with Guava leaf extract (JP2 + Gu).
Figure 1. Illustration of the screening test results. Cytotoxicity tests for detection of macrophage viability (%) after exposure to JP2 bacteria: (i) without treatment (JP2); (ii) pretreated with Matcha extract (Ma); (iii) pretreated with Guava leaf extract (Gu); (iv) persistently treated with Matcha extract (JP2 + Ma); and (v) persistently treated with Guava leaf extract (JP2 + Gu).
Oral 04 00033 g001
Oral 04 00033 g002
Figure 2. Illustration of the screening test results. IL-1β secretion after macrophages were exposed to JP2 bacteria: (JP2) JP2 bacteria without treatment; (Ma) JP2 bacteria pretreated with Matcha extract; (Gu) JP2 bacteria pretreated with Guava leaf extract; (JP2 + Ma) JP2 bacteria persistently treated with Matcha extract; and (JP2 + Gu) JP2 bacteria persistently treated with Guava leaf extract. Note that a technical error occurred at the 0.1 concentration; therefore, this concentration should not be taken into consideration.
Figure 2. Illustration of the screening test results. IL-1β secretion after macrophages were exposed to JP2 bacteria: (JP2) JP2 bacteria without treatment; (Ma) JP2 bacteria pretreated with Matcha extract; (Gu) JP2 bacteria pretreated with Guava leaf extract; (JP2 + Ma) JP2 bacteria persistently treated with Matcha extract; and (JP2 + Gu) JP2 bacteria persistently treated with Guava leaf extract. Note that a technical error occurred at the 0.1 concentration; therefore, this concentration should not be taken into consideration.
Oral 04 00033 g002
Table 1. Effect of Matcha and Guava leaf extract on the viability of JP2 bacteria-exposed macrophages (%). Macrophages were exposed to JP2 bacteria for 2 h at MOI 1 under the following conditions: (i) JP2 bacteria without any treatment; (ii) JP2 bacteria pretreated with Matcha/Guava leaf extract; and (iii) JP2 bacteria persistently treated with Matcha/Guava leaf extract. Mean values ± SD, n = 8. A significant decrease in the cytotoxicity of JP2 bacteria via extract treatment is indicated (p-value).
Table 1. Effect of Matcha and Guava leaf extract on the viability of JP2 bacteria-exposed macrophages (%). Macrophages were exposed to JP2 bacteria for 2 h at MOI 1 under the following conditions: (i) JP2 bacteria without any treatment; (ii) JP2 bacteria pretreated with Matcha/Guava leaf extract; and (iii) JP2 bacteria persistently treated with Matcha/Guava leaf extract. Mean values ± SD, n = 8. A significant decrease in the cytotoxicity of JP2 bacteria via extract treatment is indicated (p-value).
Macrophage Viability (%)Standard
Deviation		p-Value
Control *100.0±8.6n.d.
JP231.8±13.7n.d.
JP2 Ma pretreated78.6±33.40.003
JP2 Ma present82.6±15.8<0.001
JP2 Gu pretreated78.6±26.40.024
JP2 Gu present84.2±20.9<0.001
* Macrophages with no JP2 bacteria or extract exposure.
Table 2. Effect of Matcha and Guava leaf extracts on JP2 bacteria-exposed macrophage IL-1β secretion (pg/mL). Macrophages were exposed to JP2 bacteria for 2 h at MOI 1 under the following conditions: (i) JP2 bacteria without any treatment; (ii) JP2 bacteria pretreated with Matcha/Guava leaf extract; and (iii) JP2 bacteria persistently treated with Matcha/Guava leaf extract. Mean values ± SD, n = 7. A significant decrease in the cytotoxicity of JP2 bacteria is indicated (p-value).
Table 2. Effect of Matcha and Guava leaf extracts on JP2 bacteria-exposed macrophage IL-1β secretion (pg/mL). Macrophages were exposed to JP2 bacteria for 2 h at MOI 1 under the following conditions: (i) JP2 bacteria without any treatment; (ii) JP2 bacteria pretreated with Matcha/Guava leaf extract; and (iii) JP2 bacteria persistently treated with Matcha/Guava leaf extract. Mean values ± SD, n = 7. A significant decrease in the cytotoxicity of JP2 bacteria is indicated (p-value).
IL-1β Secretion (pg/mL)Standard
Deviation		p-Value
Control *8.0±21.1n.d.
JP2240.3±41.7n.d.
JP2 Ma pretreated42.2±111.6<0.001
JP2 Ma present0.0±0.0<0.001
JP2 Gu pretreated176.9±42.10.441
JP2 Gu present0.0±0.0<0.001
* Macrophages with no JP2 bacteria or extract exposure.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Hermes, S.; Alatwan, S.; Johansson, A.; Bogren, A. Natural Therapeutic Agents’ Efficacy in Preventive Strategies against the Periodontal Pathogen Aggregatibacter actinomycetemcomitans: An In Vitro Study. Oral 2024, 4, 405-416. https://doi.org/10.3390/oral4030033

AMA Style

Hermes S, Alatwan S, Johansson A, Bogren A. Natural Therapeutic Agents’ Efficacy in Preventive Strategies against the Periodontal Pathogen Aggregatibacter actinomycetemcomitans: An In Vitro Study. Oral. 2024; 4(3):405-416. https://doi.org/10.3390/oral4030033

Chicago/Turabian Style

Hermes, Sima, Sham Alatwan, Anders Johansson, and Anna Bogren. 2024. "Natural Therapeutic Agents’ Efficacy in Preventive Strategies against the Periodontal Pathogen Aggregatibacter actinomycetemcomitans: An In Vitro Study" Oral 4, no. 3: 405-416. https://doi.org/10.3390/oral4030033

APA Style

Hermes, S., Alatwan, S., Johansson, A., & Bogren, A. (2024). Natural Therapeutic Agents’ Efficacy in Preventive Strategies against the Periodontal Pathogen Aggregatibacter actinomycetemcomitans: An In Vitro Study. Oral, 4(3), 405-416. https://doi.org/10.3390/oral4030033

Article Metrics

Back to TopTop