Antimicrobial activity of Ruta angustifolia L. Pers against periodontal pathogen: Porphyromonas gingivalis

Introduction

According to the World Health Organisation (WHO), nearly 3.5 billion people worldwide are affected by oral diseases, with three out of four affected individuals living in middle-income countries. Globally, severe periodontal disease is estimated to affect around 19% of the global adult population, representing more than 1 billion cases (Santhosh et al., 2023). The global oral care market has witnessed a significant rise, primarily due to the increasing prevalence of oral health issues such as periodontal diseases, dental caries, plaque-induced gingivitis, and oral cancer (Amil et al., 2024). Periodontal diseases are intricate, multifactorial conditions characterised by the progressive resorption of the structures that support the teeth. This process involves a variety of microorganisms that trigger inflammation, resulting in damage to the supportive tissues. Consequently, this condition leads to the formation of periodontal pockets, loss of alveolar bone, and ultimately, tooth loss (Lorenzi et al., 2024). It is also associated with several systemic health conditions, such as diabetes mellitus and atherosclerosis (Sanz et al., 2020). However, treating periodontal disease presents several challenges, including difficulties in early diagnosis, the complexity of managing its multifactorial nature, and variability in patient responses to treatment. Additionally, the bacterial biofilm that forms in periodontal pockets can be highly resistant to conventional therapies, often requiring a combination of mechanical, antimicrobial, and sometimes surgical interventions. Furthermore, patient compliance in maintaining long-term oral hygiene practices plays a critical role in the success of periodontal disease management (Chang, Kantrong & Darveau, 2021).

A model of periodontitis pathogenesis has been proposed that emphasises polymicrobial synergy and dysbiosis. Dysbiosis refers to an imbalance within the oral microbial community. In a healthy state, the microbiome is diverse and balanced; however, dysbiosis occurs when harmful bacteria outnumber beneficial ones, leading to inflammation and tissue damage. In the context of periodontal disease, dysbiosis is characterised by an overgrowth of pathogenic bacteria, disrupting microbial homeostasis and promoting chronic inflammation, which ultimately contributes to the destruction of periodontal tissues. According to this model, the disease originates from an imbalance between the host and the oral microbiota, where specific species, such as P. gingivalis, Tannerella forsythia, and Treponema denticola, collaborate to establish an inflammatory microbiota, resulting in periodontal inflammation (Belibasakis et al., 2023).

P. gingivalis is considered a predominant pathogenic bacterium associated with periodontitis due to its capacity to disrupt the balance of the oral microbiota and impair host immune responses. This disruption significantly contributes to the pathogenesis of periodontal disease by driving gum inflammation, breaking down periodontal tissues, causing loss of alveolar bone, and forming periodontal pockets, all of which complicate treatment and accelerate the chronic progression of the disease (Howard, Gonzalez & Garneau-Tsodikova, 2021). The bacterium possesses various virulence factors, including enzymes that degrade host tissues and molecules that modulate immune responses, enabling it to efficiently colonise the subgingival area and evade immune detection. This not only triggers periodontal disease but also promotes oral dysbiosis, a shift in the microbial community that exacerbates disease progression. Its ability to alter the local environment by modulating inflammatory pathways further contributes to the chronic nature of the condition. Indeed, colonisation by P. gingivalis disrupts the normal balance of oral microbial communities, leading to inflammation and subsequent bone loss, which complicates effective treatment and management (Benahmed et al., 2022).

Factors such as poor oral hygiene, tobacco use, and systemic health conditions can contribute to the development of dysbiosis. Consequently, the growing awareness of oral hygiene has prompted individuals to invest in oral care products for themselves and their families. As a result, the oral care market has experienced significant revenue growth, with an estimated cumulative annual growth rate of 8.1%. Among the diverse range of products available, mouthwashes have generated the highest revenue, closely followed by toothpaste (Siripipatthanakul & Sixl-Daniell, 2021). Nonsurgical traditional treatment methods for periodontal diseases, such as scaling and root planing (SRP), are insufficient to fully eliminate oral pathogens or prevent their recurrence. Additionally, the use of systemic antibiotics for treating periodontitis is constrained by the necessity of administering high doses to achieve effective concentrations in gingival fluid. This approach is further complicated by the rapid emergence of antibiotic resistance among bacteria and the potential side effects associated with these medications (Uddin et al., 2021). Moreover, prolonged use of chemical ingredients in oral care products, such as chlorhexidine, can lead to tooth and tissue staining, as well as other adverse effects, including calculus accumulation, transient taste disturbances, and impacts on the oral mucosa (Böhle et al., 2022). Allergic reactions to chlorhexidine may also provoke type IV hypersensitivity responses, which can manifest as a burning sensation in the mouth, erythema of the gingival tissues, and stomatitis. These issues present significant challenges for both healthcare providers and patients (Kotsailidi et al., 2020). Given these challenges, there is increasing interest in exploring natural alternatives, particularly herbs and spices known for their antibacterial and chemopreventive properties (Refaey et al., 2024). Thus, finding a safe and effective therapeutic agent for oral health is deemed necessary.

Historically, plants and their extracts have been utilised for therapeutic purposes across various cultures, with traditional medicine practices highlighting their benefits. The appeal of herbal extracts lies in their perceived safety, cost-effectiveness, and efficacy, as well as their role as alternatives or complements to conventional treatments (Abdalla et al., 2022). Herbal extracts have been employed for centuries to address a variety of health conditions, including oral health issues such as gingivitis, periodontitis, and dental caries. The recent popularity of herbal medicines and products is evident among dental patients, professionals, and a wider range of consumers and healthcare practitioners, due to their potential benefits for oral health and alveolar bone protection (Mosaddad, Hussain & Tebyaniyan, 2023). A previous study has indicated that the systemic administration of curcumin, a compound derived from turmeric, significantly reduced alveolar bone loss in a ligature-induced mice model, suggesting its potential as an alternative treatment for initial periodontal therapy (Wang et al., 2023). Another study revealed that extracts from Cinnamomum zeylanicum and Salvadora persica exhibited antibacterial activity against various periodontal pathobionts such as P. gingivalis, T. denticola, T. forsythia, and Actinobacillus actinomycetemcomitans (Saquib et al., 2019). The Rutaceae family, particularly the genus Ruta, such as Ruta angustifolia L. Pers, commonly referred to as “garuda”. This plant has been traditionally used in Indonesia for treating jaundice and liver disorders. Furthermore, it is cultivated in Java and Vietnam, where its decoction is commonly employed to relieve cramps, bloating, and fever (Noer, Abinawanto & Basith, 2018).

Previous studies reported that various extracts of R. angustifolia exhibit a wide range of biological activities, including strong antiviral effects (Wahyuni et al., 2014) and significant cytotoxic activity against several cancer types, such as lung cancer (A549), colon cancer (HCT-116 and HT29), and cervical cancer (CaSki) (Richardson, Aminudin & Abd Malek, 2017; Suhaimi, Hong & Abdul Malek, 2017). Additionally, R. angustifolia has demonstrated antibacterial properties, attributed to naturally occurring compounds like flavonoids, alkaloids, and coumarins. These compounds have shown various medicinal effects, particularly against microbial infections. For example, chalepin, isolated from the leaves of R. angustifolia, demonstrated modest antiparasitic activity against Trypanosoma cruzi, the pathogen responsible for Chagas disease (Bailly, 2023). Furthermore, essential oil extracted from R. angustifolia has shown significant antifungal activity against Fusarium oxysporum and Botrytis cinerea (Nahar et al., 2021). However, there is a lack of comprehensive research on the antibacterial effects of R. angustifolia extracts against oral bacteria associated with periodontal diseases. Therefore, this study aims to investigate the antimicrobial efficacy of R. angustifolia extracts against P. gingivalis, a prominent periodontal pathogen, by assessing biofilm biomass, cell viability, and morphological characteristics. The hypothesis of this study posited that extracts of R. angustifolia exhibit significant antimicrobial activity against P. gingivalis, with the chloroform fractionated extract demonstrating the highest efficacy in inhibiting bacterial growth, reducing biofilm formation, and inducing cell death, thereby suggesting its potential as a viable alternative treatment for periodontitis.

Materials and Methods

Results

Inhibitory effects of R. angustifolia extracts against P. gingivalis

Based on the well diffusion test presented in Fig. 1, all R. angustifolia extracts effectively suppressed the growth of P. gingivalis in a concentration-dependent manner. Inhibition zone measurements indicated that the highest antimicrobial activity occurred at a concentration of 100 mg/mL for the chloroform fractionated extract of R. angustifolia, as shown in Table 1. Among the three extracts tested, the chloroform fractionated extract exhibited the largest inhibition zone, followed by the crude methanol extract (9.30 ± 0.26 mm) and the hexane fractionated extract (8.19 ± 0.27 mm). These results demonstrate that the chloroform fractionated extract of R. angustifolia most effectively inhibited the growth of P. gingivalis, while the negative control (1% DMSO) showed no inhibition.

Table 1:

The inhibition zone diameters of R. angustifolia extracts against P. gingivalis.

Microorganism		Inhibition zone diameter (mm)
		R. angustifolia extracts
		Crude methanol	Hexane	Chloroform
P. gingivalis ATCC 33277	50 mg/mL	8.12 ± 0.77*	6.98 ± 0.26*	8.32 ± 1.08*
	100 mg/mL	9.30 ± 0.26	8.19 ± 0.27*	11.65 ± 0.62
	0.12% CHX(a)	13.79 ± 0.71	13.45 ± 0.79	13.34 ± 0.73

DOI: 10.7717/peerj.18751/table-1

Notes:

(a) 0.12% CHX served as the positive control, whilst 1% DMSO as the negative control against P. gingivalis.

Experiments were conducted in triplicate and tabulated values are represented as mean ± standard deviation. Statistically significant with Kruskal-Wallis (p-value < 0.05) and Dunn’s post-hoc analysis (*p < 0.05 vs. control according to analysis of variance).

In comparison, the inhibition zone for 0.12% CHX was 13.34 ± 0.73 mm, while the chloroform fractionated extract at 100 mg/mL demonstrated an inhibition zone of 11.65 ± 0.62 mm. Thus, these findings suggest that its efficacy is comparable to that of 0.12% CHX, a widely used antibacterial agent for oral diseases. Although CHX exhibited a slightly larger zone, the difference is not substantial, indicating that R. angustifolia may serve as an effective alternative or adjunct in managing oral infections.

Effect of chloroform fractionated extract of R. angustifolia against P. gingivalis biofilm biomass

As shown in Fig. 2, the overall biomass of P. gingivalis biofilms treated with various concentrations of chloroform fractionated extract of R. angustifolia was performed using crystal violet staining. The extract demonstrated notable antibiofilm effects by suppressing the formation of biofilm in P. gingivalis. A clear, concentration-dependent decrease in absorbance was observed, indicating the efficacy of the extract in reducing the biofilm biomass. Notably, the biofilm biomass of P. gingivalis was remarkably reduced by up to 91% at the highest concentration of chloroform fractionated extract of R. angustifolia. This result was consistent with the MIC determined in Table 2, confirming that the chloroform fractionated extract of R. angustifolia effectively disrupted the formation of P. gingivalis biofilm compared to the control.

Live/dead cell viability assay using fluorescence microscope

LIVE/DEAD^® BacLight™ viability staining was conducted to evaluate the impact of the chloroform fractionated extract of R. angustifolia on P. gingivalis biofilm (Fig. 3). In the absence of the extract, the untreated P. gingivalis biofilm displayed a dense and highly uniform structure after 24 h of incubation. In contrast, increasing concentrations of the extract led to marked sparsity and dispersion of the biofilm, indicating a significant reduction in viable cells. As a result, biofilm formation was substantially inhibited, with a notable decrease in bacterial presence at higher concentrations of the chloroform fractionated extract of R. angustifolia.

Similar results were observed at 48 h, where the untreated P. gingivalis biofilm remained dense and uniform, while higher extract concentrations led to increased biofilm disruption and cell death. Significantly, an abundance of lysed cells and cellular debris was visible at the higher concentration of 6.25 mg/mL. These findings demonstrate that the chloroform fractionated extract of R. angustifolia is capable of disrupting P. gingivalis biofilm formation and viability in a concentration-dependent manner.

The effects of the R. angustifolia extract on P. gingivalis cell morphology via FE-SEM

FE-SEM imaging (Fig. 4) was conducted to examine morphological alterations in P. gingivalis exposed to the chloroform fractionated extract of R. angustifolia. Untreated P. gingivalis exhibited a characteristic of smooth cell surface that appeared as tiny grape-like clusters with intact cell membranes. In contrast, the P. gingivalis biofilm treated with 6.25 mg/mL of the chloroform fractionated extract of R. angustifolia displayed significant changes in cellular morphology. The treated cells displayed an unravelled, crater-like appearance of the cell walls, as along with swollen, elongated, and lysed forms that aggregated into large clusters of cellular debris. Therefore, the FE-SEM analysis conclusively demonstrated that the chloroform fractionated extract of R. angustifolia completely inhibited P. gingivalis cells. At this MIC concentration, the integrity of the P. gingivalis bacterial cell membrane was compromised, resulting in extensive cell lysis and disruption of cellular morphology.

Discussion

The present study reveals that the crude methanol extract, hexane, and chloroform fractionated extracts of R. angustifolia exhibit inhibitory activity against P. gingivalis. Among these, the chloroform fractionated extract of R. angustifolia was found to be the most effective in inhibiting the growth of P. gingivalis, leading to its selection for further investigation. This report is the first to document the antimicrobial susceptibility of R. angustifolia extracts against P. gingivalis. The antimicrobial effect of the extracts was dose-dependent, with higher concentrations of the chloroform fractionated extract of R. angustifolia displaying greater bactericidal activity. A previous report identified twelve components isolated from the chloroform fractionated extract of R. angustifolia (Richardson, Aminudin & Abd Malek, 2017). These compounds were graveoline, kokusaginine, bergapten, moskachan B, moskachan D, chalepensin, rutamarin, arborinine, chalepin and neophytadiene. Most of these compounds belong to the classes of alkaloids, furanocoumarins, and dihydrofuranocoumarins, which possess significant antiviral, anti-inflammatory, antioxidant, and antiproliferative properties (Bailly, 2023). Specifically, rutamarin and chalepin are categorised as furanocoumarins (Richardson et al., 2016). Current research indicates that coumarin derivatives, recognised for their antioxidant, antifungal, and antibacterial properties, hold promise as therapeutic agents across various medical applications. Structural modifications, such as the incorporation of nitrogen atoms and azomethane groups, are thought to enhance antibacterial activity by increasing membrane permeability, thereby contributing to potent bactericidal effects (Wahyuni et al., 2023). These compounds likely benefit from increased lipophilicity, which enhances their ability to penetrate bacterial membranes. This lipophilic characteristic may also interfere with bacterial metabolic processes by inactivating essential enzymes and denaturing cellular proteins, ultimately leading to bacterial cell death (Betti et al., 2024). Additionally, recent studies have revealed that coumarin derivatives exhibit significant cytotoxicity towards bacterial DNA, potentially due to their modification of bacterial membrane components, including lipopolysaccharides (LPS). This modification may activate enzymes such as topoisomerases and helicases, destabilizing the DNA structure and obstructing replication, which culminates in bacterial cell apoptosis (Koszelewski et al., 2023).

Furthermore, graveoline, one of the isolated components identified from the chloroform fractionated extract of R. angustifolia (Richardson et al., 2016), exhibits antibacterial, anticancer, antifungal, and spasmolytic effects. Graveoline is a quinolone alkaloid that interacts with bacterial cell membranes, affecting their function and causing cell lysis. Alkaloids can suppress bacterial growth by interfering with nucleic acid, protein synthesis, and metabolism. Additionally, these compounds may disrupt essential metabolic processes and energy sources for various enzymatic reactions in bacteria, such as adenosine triphosphate (ATP), thereby inhibiting growth. Alkaloids can also promote the generation of reactive oxygen species (ROS), which damage bacterial cells (Yan et al., 2021). Hence, R. angustifolia harbours numerous bioactive compounds, showcasing significant potential for therapeutic applications in various diseases, including periodontal diseases.

The periodontal pathogen P. gingivalis is known as one of the late colonisers of the oral cavity, which possess a range of virulent characteristics and robust biofilm-forming capabilities that facilitate persistent infection at the gingival site (Xu et al., 2020). These late colonisers consist of slower-growing, obligate anaerobes, and Gram-negative species, such as P. gingivalis, along with other members of the red complex, T. forsythia and T. denticola, which are typically found at elevated levels in diseased conditions (Siddiqui et al., 2022).

Gram-positive and Gram-negative bacteria are primarily distinguished by the thickness of their cell walls and the presence of an outer membrane, which is unique to Gram-negative bacteria. The thickness of the cell walls of Gram-positive and Gram-negative bacteria ranges from 20 to 80 nm and 1.5 to 10 nm, respectively (Bismelah et al., 2022). Despite having thicker cell walls, Gram-positive bacteria can absorb foreign chemicals, such as bioactive compounds, making them more susceptible to elimination. In contrast, the effectiveness of some antibiotic treatments against Gram-negative bacteria is impaired by the presence of an outer membrane composed of lipopolysaccharides and phospholipids (Fivenson et al., 2023). This additional membrane layer functions as an effective permeable barrier, preventing antibiotic molecules from entering the bacterial cell.

The presence of virulence factors in various periodontopathic bacteria poses significant challenges in treating infections. For example, P. gingivalis and T. forsythia are characterised by high proteolytic and peptidolytic activities, which can hydrolyse antibiotics, leading to the inactivation of their antimicrobial properties (Bismelah et al., 2022). These factors may explain why higher concentrations of plant extracts are required to effectively inhibit P. gingivalis in susceptibility tests. Therefore, the extract of R. angustifolia could serve as an adjunctive treatment for periodontitis, as it effectively targets the growth of late colonisers, thereby helping to prevent the progression of periodontal infections.

Correspondingly, the chloroform fractionated extract of R. angustifolia showed promising results for MIC and MBC, completely inhibiting P. gingivalis at a concentration of 6.25 mg/mL. Furthermore, the biofilm assay provided evidence that the chloroform fractionated extract of R. angustifolia exhibits inhibitory effects on the formation of P. gingivalis biofilms. This significant discovery suggests that the extract has potential as a viable method for mitigating the metabolic activity of P. gingivalis within biofilms. Biofilms are complex three-dimensional structures composed of microorganisms bound together by an extracellular matrix (Mitchell, 2011). Once microorganisms form biofilms, they become increasingly challenging to control and eradicate. According to Raut & Karuppayil (2016), biofilms are associated with over 80% of microbial illnesses in humans. Since periodontal disease involves the formation of biofilms on gingival tissues, disrupting the biofilm formed by P. gingivalis is critically important (Bustamante et al., 2020). The ability of the chloroform fractionated extract of R. angustifolia to interfere with biofilm formation at varying concentrations is crucial for preventing P. gingivalis from recolonising the periodontal pocket, as this treatment eliminates the protective layer of the subgingival biofilm (Bostanci & Belibasakis, 2012).

Moreover, the outcomes of our investigation closely correlate with a recent study that have demonstrated the ability of R. angustifolia extracts in diminishing the formation of biofilm by S. mutans at concentrations of 20%, 50%, and 100% (Noer, Abinawanto & Bowolaksono, 2021). In general, the antimicrobial activities of plants are attributed to their secondary metabolites. Previous studies has confirmed that R. angustifolia extract contains steroids, flavonoids, tannins, and quinones, suggesting its potential as an antibiofilm agent (Lu et al., 2019). Compounds such as flavonoids in R. angustifolia extract have been extensively studied for their bioactivity in inhibiting both Gram-positive and Gram-negative bacteria through the inactivation of extracellular proteins (Bismelah et al., 2022). Recent studies indicate that the antibacterial activities of plant flavonoids against Gram-negative bacteria are related to their lipophilicity. The cell envelope of Gram-negative bacteria contains an inner lipophilic membrane and an outer hydrophilic membrane, while Gram-positive bacteria possess only a cell membrane. Consequently, it is suggested that plant flavonoids employ multiple mechanisms against Gram-negative bacteria, with the cell membrane serving as a critical site of action, in contrast to Gram-positive bacteria, which are primarily affected at the cell membrane. Furthermore, DNA gyrase is another important target of plant flavonoids against Gram-negative bacteria. Thus, plant flavonoids can damage the cell membrane and inhibit DNA gyrase, representing a dual mechanism of action against Gram-negative bacteria (Yan et al., 2024).

Live/dead cell viability assays demonstrated that the chloroform fractionated extract of R. angustifolia, at both sub-MIC and MIC levels, induced a significant reduction in the viability of P. gingivalis compared to the control group. The biofilm formation of P. gingivalis was markedly inhibited by the chloroform fractionated extract of R. angustifolia, which led to a greater dispersion of biofilm structures and an increase in cell death, particularly at a concentration of 6.25 mg/mL after 48 h of incubation. Moreover, non-viable/dead (red fluorescence) cells were evidently in greater proportion than viable/live (green fluorescence) cells. These findings suggest that the chloroform fractionated extract of R. angustifolia exhibits biocidal effects on the P. gingivalis biofilm, as evidenced by the visualization of dead microorganisms. Similar results were reported from a previous study by He et al. (2020), which investigated the impact of quercetin on P. gingivalis aggregation and biofilm formation.

FE-SEM images revealed morphological changes in the cell structure of P. gingivalis after treated with the chloroform fractionated extract of R. angustifolia. The results indicated that the extract caused damage to the cell membrane, leading to subsequent cell death. Active bioactive compounds, such as graveolinine, flavonoid, chalepin, and rutamarin, have been identified in R. angustifolia extracts and are known to contribute to alterations in cell morphology in cancer cells (Kamal et al., 2021; Richardson, Aminudin & Abd Malek, 2017). Rutamarin and chalepin were reported to exhibit similar mechanisms, resulting in cell shrinkage, the formation of apoptotic bodies, and rounding of cancer cells (Richardson et al., 2016; Suhaimi, Hong & Abdul Malek, 2017). Furthermore, flavonoids have been shown to damage the cytoplasmic membrane, inhibit nucleic acid production, and disrupt energy metabolism of bacterial cells (Shamsudin et al., 2022). These factors may collectively contribute to the cell death of P. gingivalis. In contrast, untreated control cells grew exponentially while maintaining an intact structure. He et al. (2020) observed similar effects, where quercetin-treated P. gingivalis exhibited cell elongation from its coccobacillus shape, followed by cell shrinkage and eventual lysis due to deformed cell walls. Notably, the mechanism of action of the extract resembles that of chlorhexidine, in which low concentrations of chlorhexidine disrupt the integrity of the cell wall, ultimately resulting in damage. Consequently, chlorhexidine penetrates the cell, inducing leakage of the cytoplasm that leads to cell death (He et al., 2020).

The initial findings on the antimicrobial activity of the R. angustifolia extracts against the monospecies bacterium P. gingivalis are promising, indicating potential effectiveness in inhibiting this essential periodontal pathogen. However, since P. gingivalis is only one species within the complex oral microbiome, these results may not fully reflect the impact of the extracts on other oral microorganisms that contribute to oral health and disease. Therefore, to enhance the robustness and generalizability of the findings, future studies should encompass a broader range of oral bacterial species, such as T. denticola, T. forsythia, S. mutans, and Fusobacterium nucleatum. This approach will provide a better understanding of the antimicrobial potential across the diverse microbial community in the oral cavity and enhance the relevance of the findings.

In this present study, 0.12% CHX was used as the positive control. CHX is a widely used antibacterial agent in dentistry, known for its broad-spectrum antibacterial properties against various pathogens, including periodontal bacteria such as P. gingivalis. The concentration of 0.12% CHX is frequently incorporated into mouthwashes and other oral care products, establishing it as a standard concentration commonly used in numerous studies as a positive control in antibacterial tests. Utilizing 0.12% CHX as a positive control provides researchers with a reliable benchmark to compare the antibacterial effectiveness of natural products or extracts against a well-established and clinically validated antibacterial agent. This approach ensures that the findings are relevant to clinical practice.

Furthermore, while the in vitro results are encouraging, it is essential to thoroughly evaluate the biocompatibility of the antimicrobial agents to ensure their safety for human use. This evaluation should include testing for potential cytotoxicity and assessing the effects on normal human oral cells such as gingival and periodontal cells. Additionally, clinical efficacy should be explored through further studies utilizing human cellular models to replicate real-life conditions. Moreover, in addition to in vitro studies, in vivo research using animal models, such as rodents, is crucial for evaluating the effectiveness of the extracts in a living organism. This evaluation should consider factors such as bioavailability, immune response, and long-term safety. Ultimately, these additional studies will offer a more comprehensive understanding of the therapeutic potential of the extracts while ensuring their safety and effectiveness for future application in human oral healthcare.

Conclusions

In conclusion, this in vitro study provides new evidence that R. angustifolia exhibits antimicrobial activity against periodontal pathogens, notably P. gingivalis. These findings suggest that R. angustifolia has the potential to be used as an alternative treatment for periodontal disease and warrants further investigation.

Supplemental Information