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Article

Frankincense (Boswellia serrata) Extract Effects on Growth and Biofilm Formation of Porphyromonas gingivalis, and Its Intracellular Infection in Human Gingival Epithelial Cells

by
David Vang
1,
Aline Cristina Abreu Moreira-Souza
1,
Nicholas Zusman
2,
German Moncada
1,
Harmony Matshik Dakafay
1,
Homer Asadi
1,
David M. Ojcius
1 and
Cassio Luiz Coutinho Almeida-da-Silva
1,*
1
Department of Biomedical Sciences, Arthur A. Dugoni School of Dentistry, University of the Pacific, San Francisco, CA 94103, USA
2
Dental Surgery Program, Arthur A. Dugoni School of Dentistry, University of the Pacific, San Francisco, CA 94103, USA
*
Author to whom correspondence should be addressed.
Curr. Issues Mol. Biol. 2024, 46(4), 2991-3004; https://doi.org/10.3390/cimb46040187
Submission received: 1 February 2024 / Revised: 20 March 2024 / Accepted: 28 March 2024 / Published: 30 March 2024
(This article belongs to the Special Issue Frankincense (Heaven’s Gift)—Chemical and Biological Diversity)
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Abstract

:
Frankincense is produced by Boswellia trees, which can be found throughout the Middle East and parts of Africa and Asia. Boswellia serrata extract has been shown to have anti-cancer, anti-inflammatory, and antimicrobial effects. Periodontitis is an oral chronic inflammatory disease that affects nearly half of the US population. We investigated the antimicrobial effects of B. serrata extract on two oral pathogens associated with periodontitis. Using the minimum inhibitory concentration and crystal violet staining methods, we demonstrated that Porphyromonas gingivalis growth and biofilm formation were impaired by treatment with B. serrata extracts. However, the effects on Fusobacterium nucleatum growth and biofilm formation were not significant. Using quantification of colony-forming units and microscopy techniques, we also showed that concentrations of B. serrata that were not toxic for host cells decreased intracellular P. gingivalis infection in human gingival epithelial cells. Our results show antimicrobial activity of a natural product extracted from Boswellia trees (B. serrata) against periodontopathogens. Thus, B. serrata has the potential for preventing and/or treating periodontal diseases. Future studies will identify the molecular components of B. serrata extracts responsible for the beneficial effects.

1. Introduction

There is growing awareness that over-prescription and misuse of antibiotics by healthcare providers are major contributors to the antibiotic resistance of human pathogens. This leads to an increasing demand for novel antimicrobials to treat infections caused by different pathogens. The synthesis of new synthetic antibiotics is expensive and complex, and the average time from discovery to market for a general broad-spectrum antibiotic is around 14 years [1]. The need for sustainable, safe, and cost-effective therapeutics is increasing. In this context, natural products can reduce the likelihood of antibiotic resistance and, having been used for centuries by humans, are usually considered to be less toxic. Some natural products are believed to enhance the body’s immune response, aiding in the natural defense against infections [2].
Frankincense is a gum resin that is a byproduct of incisions made to the trunks of the Boswellia tree [3,4]. The Boswellia genus comprises around thirty different species and is found in arid regions of Africa, the Arabian Peninsula, and South Asia [4]. Frankincense is primarily obtained from B. frereana, B. sacra, B. papyrifera, and B. serrata. These specific species are indigenous to Somalia, Yemen, Oman, and parts of India and Pakistan [4,5]. For centuries, frankincense has been used as a traditional medicine for constipation and inflammatory diseases as well as incense for religious rituals and funeral ceremonies [6]. Frankincense has been attracting more attention due to its potential effects against inflammation, cancer, diabetes, and microbial infection [4,7,8].
Periodontitis is a chronic inflammatory disease affecting the supporting structures around teeth (gingiva, periodontal ligament, and alveolar bone), and can ultimately lead to uncontrolled bone resorption and irreversible tooth loss [9]. Periodontitis affects approximately 20–50% of the worldwide population [10], and has been linked with several systemic diseases such as cardiovascular disease, diabetes, respiratory tract infections, cancer, and neurodegenerative disorders [11]. Two oral pathogens that have been directly linked to periodontitis include the Gram-negative anaerobic bacteria Porphyromonas gingivalis and Fusobacterium nucleatum [9,12,13]. Interestingly, P. gingivalis and F. nucleatum use different mechanisms to modulate the host immune response and contribute to biofilm formation and dissemination [13,14,15,16]. P. gingivalis and F. nucleatum are intracellular pathogens and have been described to evade the host immune response via several virulence factors [9,14,16,17,18,19,20,21,22].
Given the rise in the antibiotic resistance of human pathogens, it is important to develop effective antibiotic-independent treatments to eliminate periodontopathogens in the oral cavity. The use of natural products can provide a safe alternative to antibiotics to treat chronic inflammation without compromising the host.
As we previously reviewed, different frankincense extracts and compounds have been reported to have antimicrobial effects against different pathogens [4]. Essential oils from B. carterii exhibited antimicrobial activity against several Gram-positive and Gram-negative bacteria, such as Bacillus subtilis, B. circulans, Streptococcus faecalis, and Escherichia coli, as well as against two fungal pathogens, Candida albicans and Saccharomyces cerevisiae [23]. Other studies explored the in vitro [24,25] and in vivo [24] antimicrobial effects of B. sacra against multi-resistant strains of Staphylococcus aureus and Pseudomonas aeruginosa. So far, only two studies investigated the effects of frankincense against oral microbes [26,27]. Raja et al. screened several oral pathogens but focused their in vitro experiments on S. mutans [26]. Attalah et al. described the antibacterial effects of B. sacra on the growth and survival of planktonic bacteria and on the formation of biofilm using P. gingivalis clinical isolates [27]. However, no studies have explored the effects of B. serrata as an antimicrobial that can inhibit biofilm formation and intracellular infection of oral cells by P. gingivalis and F. nucleatum.
In this study, we explored the potential effects of B. serrata extracts against P. gingivalis and F. nucleatum growth in vitro, and infection in human epithelial oral cells. We describe, for the first time, the antimicrobial effects of B. serrata on biofilm formation, biofilm reduction and intracellular infection by oral pathogens. We found that frankincense can decrease P. gingivalis growth and biofilm formation and inhibit intracellular infection by P. gingivalis in human oral cells.

2. Materials and Methods

2.1. Frankincense (B. Serrata Extract)

B. serrata extract USP reference standard (cat# 1076250, Sigma-Aldrich—St. Louis, MO, USA) was isolated from B. serrata trees from India. The obtained extract was resuspended in dimethyl sulfoxide (DMSO; Sigma-Aldrich—St. Louis, MO, USA) at 128 mg/mL and kept at 4 °C until use.

2.2. Bacterial Strains, Human Oral Cells, and Growth Conditions

P. gingivalis (ATCC® 33277) and F. nucleatum (ATCC® 25586) were purchased from American Type Culture Collection (ATTC—Manassas, Virginia, Washington, DC, USA) and grown as previously described [28]. Briefly, P. gingivalis and F. nucleatum were separately grown anaerobically at 37 °C for approximately 7 days in Brucella agar plates (Anaerobe systems, cat# AS-141—Morgan Hill, CA, USA). Isolated and pure colonies were collected from agar plates containing either P. gingivalis or F. nucleatum to be inoculated in supplemented BHI broth at 37 °C for approximately 48 h under anaerobic conditions. P. gingivalis and F. nucleatum broth were prepared as we previously described [28]. Freshly grown bacteria were used for experiments after being collected at the log phase and quantified by optical density at 600 nm in the SpectraMax iD3 microplate reader (Molecular Devices—Ramsey, MN, USA).
Immortalized human gingival keratinocytes (HPV-16GM), referred to as gingival epithelial cells (GECs), were obtained from Applied Biological Materials (ABM, cat#T0717—Richmond, CA, USA) and maintained as we previously described [29]. Briefly, GECs were grown and maintained in keratinocyte-serum-free medium supplemented with 30 µg/mL of bovine pituitary extract, 0.2 ng/mL of human recombinant epidermal growth factor, 100 U/mL of penicillin, and 100 µg/mL of streptomycin (Gibco—Gaithersburg, MO, USA). The cells were grown in a humidified incubator, at 37 °C, 5% CO2, and quantified using trypan blue (Sigma-Aldrich—St. Louis, MO, USA) exclusion before each experiment.

2.3. Minimum Inhibitory Concentration (MIC)

P. gingivalis and F. nucleatum were grown in broth at 37 °C for approximately 48 h prior to the experiment. In a 96-well tissue culture plate (Costar, Corning—Glendale, AZ, USA), a serial dilution was performed from the highest B. serrata extract concentration of 512 μg/mL to the lowest concentration of 0.25 μg/mL, using bacterial broth in duplicates. Freshly grown P. gingivalis or F. nucleatum were added to the wells at a final concentration of 5 × 105 CFU/mL. The plates were incubated under anaerobic conditions at 37 °C. After 48 h, the plates were examined for growth and turbidity under the different concentrations of frankincense extract by measuring the optical density at 600 nm in the SpectraMax iD3 microplate reader.

2.4. Biofilm Formation Assay

P. gingivalis or F. nucleatum biofilm formation was determined using the crystal violet staining assay [30], and adapted to our experiments from previous studies [31]. Biofilm formation assays were performed by adding a serial dilution of B. serrata extract at different final concentrations (128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 μg/mL) in duplicates in 24-well plates (Costar, Corning—Glendale, AZ, USA). Freshly grown P. gingivalis or F. nucleatum were added to all wells at a final concentration of 1 × 107 CFU/mL. Antibiotics (100 U/mL of penicillin and 100 μg/mL of streptomycin, from Gibco - Gaithersburg, MO, USA) were used as positive control for bacterial growth inhibition. Bacterial broth was used as negative control. After 48 h of incubation at 37 °C and under anaerobic conditions, the supernatants were discarded, and non-adherent bacteria were gently rinsed off with sterile phosphate-buffered saline (PBS, Gibco—Gaithersburg, MO, USA). Adhered biofilms were fixed with cold absolute methanol for 15 min at room temperature. Then, the biofilms were gently washed once with PBS to remove the methanol and stained with 500 μL of 0.1% (w/v) crystal violet for 15 min at RT. The excess of crystal violet was removed by washing twice with distilled water. Absolute methanol (300 μL) was added to the wells to dissolve the crystal-violet-stained biofilms. Finally, the methanol was transferred to a 96-well microplate and the optical density (OD) values at a wavelength of 560 nm were recorded by using the SpectraMax iD3 microplate reader (Molecular Devices LLC—San Jose, CA, USA).

2.5. Biofilm Reduction Assay

The effects of B. serrata extract on P. gingivalis or F. nucleatum biofilm reduction were also measured using the crystal violet staining method [32], and adapted to our experiments from previous studies [31]. To form mature biofilms, freshly grown P. gingivalis or F. nucleatum suspensions were seeded in 24-well plates at a final concentration of 1 × 107 CFU/mL for 48 h, at 37 °C, under anaerobic conditions. Then, treatments with or without B. serrata extract at different final concentrations were added to the wells and incubated at 37 °C, under anaerobic conditions, for an additional 24 h. The supernatants were then discarded, and the biomass was determined using crystal violet staining and OD values as we described above.

2.6. Lactate Dehydrogenase Quantification

To measure cell viability, lactate dehydrogenase (LDH) levels were measured spectrophotometrically using the CyQUANT LDH Cytotoxicity Assy Kit (cat# C20300, Thermo Fisher, Waltham, MA, USA), as we previously described [29]. Briefly, GECs (1 × 105 cells/mL) were seeded overnight in 24-well plates (Costar, Corning—Glendale, AZ, USA). Then, the media were discarded, and the cells were treated with or without B. serrata extract at different final concentrations (128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 μg/mL) for 24 h. Lysis buffer or water was added during the last 40 min and served as internal control. After sample collection, supernatants were transferred to clear flat-bottom 96-well plates, and LDH substrate was added to all wells to be incubated for 30 min at RT, protected from light. Prior to measuring the absorbance, the reaction was stopped using the Stop Solution from the kit. Absorbance values were recorded at 490 nm and 680 nm using the SpectraMax iD3 microplate reader (Molecular Devices LLC—San Jose, CA, USA). Cells treated with lysis buffer were used as positive controls and defined as 100% cell death while cells treated with water were used to provide spontaneous cell death, and cells with no treatment were used as negative controls in the experiments.

2.7. Antibiotic Protection Assay

The antibiotic protection assay was performed as we previously described [28] to quantify intracellular bacterial survival after treatments with B. serrata extract. GECs (3 × 105 cells/mL) were seeded in 6-well plates (Costar, Corning—Glendale, AZ, USA) overnight in media without antibiotics. Freshly grown P. gingivalis was added to the cells at an MOI of 100 [28,33] in OptiMEM (Gibco, Gaithersburg, MO, USA), and incubated for 2 h, at 37 °C, 5% CO2. Then, the cells were washed three times with sterile prewarmed PBS and treated with metronidazole (200 μg/mL) and gentamicin (300 μg/mL) in OptiMEM for 1 h, at 37 °C, 5% CO2. After incubation, the antibiotics were removed and the cells were washed three times with sterile prewarmed PBS, followed by an incubation with or without B. serrata extract in OptiMEM medium at different final concentrations (16, 2, 0.25 μg/mL) for an additional 21 h, at 37 °C, 5% CO2. Then, the supernatants were discarded, and the cells were washed three times with sterile prewarmed PBS. Sterile distilled water was added into wells and incubated at RT for 20 min. A cell scraper was used to lyse the cells and 50 μL of each cell lysate was plated onto Brucella Blood Agar Plate (Anaerobe Systems, Morgan Hill, CA, USA). The plates were immediately incubated under anaerobic conditions for 10 days, at 37 °C, before colony forming units (CFU) were quantified.

2.8. Immunostaining for P. gingivalis

GECs (1 × 105 cells) were seeded on 18 mm coverslips in 24-well plates to reach approximately 80% of confluence. The experimental design followed the description above for “antibiotic protection assay”, in which cells were infected for 2 h, treated with antibiotics for an additional 1 h, and then incubated for additional 21 h. At the end of the experiment, infected cells on coverslips were washed three times with PBS, followed by fixation with absolute cold methanol for 10 min at RT. After three washes with PBS, the cells were permeabilized and blocked with a solution of 0.2% Triton X-100 (Sigma-Aldrich—St. Louis, MO, USA) in 5% Goat Serum (Sigma-Aldrich—St. Louis, MO, USA) and 1X PBS (Gibco—Gaithersburg, MO, USA) at 4 °C overnight. The cells were incubated with primary rabbit polyclonal antibody anti-P. gingivalis (cat# ANT0085, Diatheva - Cartoceto Italy) at a concentration of 1:50 prepared in 0.05% Triton X-100 in 5% Goat Serum/PBS at 4 °C overnight. After three washes with PBS, secondary goat anti-rabbit IgG (cat# A11012, Invitrogen—Carlsbad, CA, USA) at a concentration of 1:200 was prepared in 0.05% Triton X-100 in 5% Goat Serum/PBS, and was added into the wells for incubation for 2 h, protected from light, at RT. The coverslips were washed three times, counterstained, and mounted on a slide using Vectashield Hardset Antifade Mounting Medium with DAPI (cat# H-1500, Vector Laboratories—Burlingame, CA, USA). Images were then acquired using a Nikon Eclipse 50i fluorescence microscope with an Infinity 3 camera (Nikon Instruments—Melville, NY, USA) and the Lumenera Infinity Analyze 6.3 software (Teledyne Lumenera—Ottawa, ON, Canada).

2.9. Statistical Analysis

Statistical analysis was performed on a Prism GraphPad (GraphPad Software, Prism 9, version 9.5.1) The results are shown in standard deviation (SD) and were analyzed using one-way Anova followed by Dunnett’s test. Differences resulting in p < 0.05 were considered significant.

3. Results

3.1. B. serrata Extract Differentially Impacts the Growth of P. gingivalis and F. nucleatum

First, the in vitro effects of B. serrata extract were tested on planktonic P. gingivalis and F. nucleatum using the minimum inhibitory concentration assay. The minimum concentration of B. serrata extract needed to inhibit P. gingivalis growth was 32 µg/mL, as shown in Table 1. F. nucleatum growth was detected in the presence of all extract concentrations tested (512 to 0.25 µg/mL). Our data thus imply that P. gingivalis planktonic bacteria are more susceptible to B. serrata extracts than F. nucleatum bacteria.

3.2. B. serrata Extract Inhibits P. gingivalis and F. nucleatum Biofilm Formation

Bacteria grow in the environment and on body surfaces as complex bacterial communities known as biofilms [34,35]. Biofilms provide bacteria with protection, against antibiotics and disinfectants, and a dynamic environment, which facilitates infection and enhances resistance against the immune response [34,35]. Therefore, we examined the effects of B. serrata extract on P. gingivalis or F. nucleatum biofilm formation. To do so, we added either P. gingivalis or F. nucleatum and different concentrations of B. serrata extract at the same time in 24-well plates. After 48 h, the biofilms formed at the bottom of the plate were quantified using the crystal violet staining method, as we described in the Section 2. Figure 1A shows that all concentrations tested (0.25–128 µg/mL) significantly inhibited P. gingivalis biofilm formation in a dose-dependent manner. Concentrations ranging from 4 to 128 µg/mL were as effective in inhibiting P. gingivalis biofilm formation as conventional antibiotics. However, only the higher concentration of 128 µg/mL showed a modest inhibitory effect on F. nucleatum biofilm formation (Figure 1B). Our positive control (penicillin/streptomycin), when added at the same time as bacteria to the wells, completely inhibited both P. gingivalis and F. nucleatum biofilm formation. These data show that B. serrata extract dampens P. gingivalis biofilm formation even at the low concentration of 0.25 µg/mL.

3.3. B. serrata Extract Induces P. gingivalis Biofilm Reduction

To determine whether B. serrata extract could decrease established oral pathogen biofilms, we generated P. gingivalis or F. nucleatum biofilms and then added B. serrata extract at different concentrations (Figure 2). Our results show a significant dose-dependent reduction of P. gingivalis biofilms (Figure 2A). The concentration of 128 µg/mL was the most effective, and concentrations ranging from 8–128 µg/mL significantly decreased P. gingivalis biofilm biomass. Interestingly, B. serrata extract (8–128 µg/mL) caused a more significant decrease in P. gingivalis biofilm biomass compared to the conventional antibiotics, penicillin, and streptomycin. Concentrations less than 1 µg/mL did not reduce P. gingivalis biofilm biomass. Figure 2B shows that B. serrata extract did not affect the F. nucleatum biofilm, suggesting that F. nucleatum biofilms are more resistant to the effects of B. serrata extract compared to P. gingivalis biofilms.

3.4. B. serrata Extract at Low Doses Is Not Toxic to Human Gingival Epithelial Cells

We next examined whether the extract may have cytotoxic effects on human gingival epithelial cells. Figure 3 shows that concentrations ≥ 32 µg/mL are toxic to host cells, but that concentrations ≤ 16 µg/mL did not cause significant oral cell death. Therefore, concentrations lower than 16 µg/mL were safe to human oral cells.

3.5. B. serrata Extract Decreases Intracellular P. gingivalis Infection in Human Gingival Epithelial Cells

Taken together, our data demonstrate that B. serrata extract is effective in decreasing P. gingivalis, but not F. nucleatum, growth and biofilm formation, and reduces biofilm biomass of P. gingivalis. Thus, we focused our examination of intracellular infection only on infections with P. gingivalis. We infected human GECs with P. gingivalis and then treated the cells with different concentrations of B. serrata extract following the antibiotic protection assay described in the Section 2. Extracellular bacteria or uninternalized bacteria attached to the host cell surface were removed during the antibiotic protection assay. This assay allowed us to detect exclusively metabolically active and viable intracellular bacteria by means of CFU quantification in blood agar plates [16,28,36,37,38,39,40]. Figure 4A shows that B. serrata extract significantly reduced the intracellular P. gingivalis load in a dose-dependent manner. The concentrations of 16 µg/mL and 2 µg/mL effectively killed intracellular P. gingivalis in infected human GECs. Our results with CFU counts in Figure 4A were confirmed by immunostaining P. gingivalis after infection of human GECs and treatment with B. serrata at 16 µg/mL (Figure 4B). Figure 4B followed the same experimental design as in Figure 4A but was analyzed by immunofluorescence microscopy to corroborate and illustrate the data in Figure 4A. Figure 4B shows representative images in which P. gingivalis numbers (stained in red) were significantly reduced in the group treated with B. serrata compared to the untreated control.
To exclude the possibility that infection and B. serrata treatments were cytotoxic to infected GECs (which could affect the interpretation of data in Figure 4A,B), we quantified LDH from supernatants of human GECs that were infected with P. gingivalis and exposed them to B. serrata following the same experimental design as in Figure 4A,B. Figure 4C shows that neither infection nor B. serrata treatments induced considerable host cell death. Altogether, these results suggest that B. serrata selectively and significantly kills intracellular P. gingivalis in infected GECs, without compromising host cell integrity.

4. Discussion

We, among others, have reviewed data showing that B. serrata presents anti-cancer, anti-diabetic, and antimicrobial effects [4,10,41,42,43]. In this study, we explored the antimicrobial effects of B. serrata extract on the survival and infection of human GECs with P. gingivalis and F. nucleatum. We demonstrated that B. serrata extract significantly decreased P. gingivalis growth and biofilm formation and reduced P. gingivalis biofilm biomass. We also demonstrated that B. serrata significantly reduced P. gingivalis intracellular infection in human GECs, at concentrations of B. serrata extracts that were not toxic for GECs. Our data show, for the first time, the effects of B. serrata extracts on biofilms.
The overall antimicrobial effects of B. serrata extract on F. nucleatum were lower than for P. gingivalis. These results are consistent with previous findings from our group and others that show that F. nucleatum is more virulent in vitro and in vivo compared to P. gingivalis [18,19,28,44,45]. Additionally, both oral pathogens play different roles during the pathogenesis of periodontitis and the formation of oral biofilms. According to Socransky et al. [12], P. gingivalis is a member of the red complex in the formation of subgingival biofilms, which is directly associated with periodontitis and is considered to be a keystone pathogen for this disease [9]. F. nucleatum, on the other hand, belongs to the orange complex and is believed to serve as a “bridge”, essential for the connection of first colonizers to other oral pathogens of the red complex, such as P. gingivalis [12]. Our experiments, which corroborate previously published data [26], show that B. serrata did not have a direct effect on F. nucleatum and did not impact the growth of F. nucleatum planktonic bacteria or biofilm formation in vitro. Possible mechanisms involved in F. nucleatum resistance to B. serrata could be the presence of drug efflux proteins (such as the ATP-binding cassette transporter system), drug inactivation by molecules secreted by F. nucleatum, or target mimicry, which involves the production of molecules that bind and sequester the agonist and thus limit the effects on F. nucleatum bacteria [46,47]. To the best of our knowledge, no known antimicrobial resistance mechanisms in F. nucleatum against B. serrata have been described. Future studies should be focused on understanding the mechanisms involved in F. nucleatum resistance to the antimicrobial effects induced by B. serrata. Once we understand and can modulate F. nucleatum virulence factors, B. serrata would become an ideal and safe therapeutic target against both P. gingivalis and F. nucleatum during periodontitis.
Previous studies from other groups have investigated the effects of B. serrata and B. sacra on oral pathogens [26,27,48]. Raja et al. [26] screened the antibacterial activity of different boswellic acid molecules against several oral pathogens, including P. gingivalis and F. nucleatum. They showed that P. gingivalis was more susceptible than F. nucleatum to boswellic acids obtained from B. serrata, consistent with our own data in this study. Raja et al., focused on the effects of boswellic acids against biofilms established by S. mutans and Actinomyces viscosus, and showed that boswellic acids reduced biofilm formation by 50% [26]. Here, our study showed for the first time the effects of B. serrata extract on biofilm formation by P. gingivalis and F. nucleatum. Our study contributes to previous studies suggesting that B. serrata molecules may be used in therapies against several oral pathogens involved in different diseases, such as dental caries and periodontitis.
A more recent study evaluated the effects of B. sacra extracts against 12 P. gingivalis clinical isolates [27]. This study observed higher MIC levels (500 or 1000 μg/mL) compared to the MIC found in our study using the type-culture P. gingivalis 33277 strain from ATCC. Similarly to the results found in this study, the work by Attallah et al. demonstrated that frankincense extracts inhibited the biofilm formation of five P. gingivalis clinical isolates [27]. Even though the present study and the study by Attallah et al. [27] used frankincense extract from different sources and different tree species, the data suggest that frankincense extracts from different Boswellia species may have antimicrobial and antibiofilm properties against P. gingivalis.
Our group and others have also characterized intracellular infection by P. gingivalis and F. nucleatum in oral host cells [9,11,14,16,17,28,33,49,50,51,52]. P. gingivalis is an opportunistic intracellular Gram-negative pathogen able to invade several different cell types, such as gingival epithelial cells [51,52,53], periodontal ligament fibroblasts [54], osteoblasts and osteoclasts [55], and immune cells [28,33,56]. Due to P. gingivalis’ ability to infect different cell types and induce inflammation, which can have systemic effects, P. gingivalis infection has also been associated with other non-oral health-related conditions, such as cardiovascular disease, cancer, and Alzheimer’s disease [11]. The oral epithelium represents the first barrier against P. gingivalis and comprises the first cells to be infected before P. gingivalis can spread to other cells in deeper tissues, and therefore our study focused on the effects of B. serrata against P. gingivalis-infected human GECs. Previous studies had not described yet the effects of frankincense extracts against P. gingivalis intracellular infection in host cells. Our data showed for the first time that B. serrata significantly decreased intracellular infection in human GECs.
Other studies described the antimicrobial effects of frankincense extracts against airborne microbes [57], oral pathogens [26], fungi (C. albicans and Malassezia furfur), and different types of bacteria [25]. However, the cellular and molecular mechanisms involved in the decrease of intracellular infection by P. gingivalis in human GECs had not yet been examined. A previous study showed that frankincense essential oil robustly modulated genome-wide gene expression in human dermal fibroblasts [58]. Given that frankincense may affect the expression of several genes in host cells, we hypothesize that B. serrata extracts may modulate the expression of host surface receptors involved in the recognition, uptake, and immune responses against P. gingivalis. Given that P. gingivalis traffics into endoplasmic-reticulum-rich autophagosomes for successful survival in human GECs [17], it is also possible that B. serrata extracts may stimulate the fusion of lysosomes to autophagosomes to contribute to P. gingivalis intracellular elimination in human GECs. Future studies will investigate the molecular mechanisms involved in the decrease of P. gingivalis intracellular infection in human GECs via B. serrata treatments.
Several studies describe clinical trials using frankincense extracts to treat several conditions, such as knee pain [59,60], hepatic inflammation and lipid metabolism [61], and chronic low back pain [62]. Regarding oral health, three publications report the effects of B. serrata extracts in the treatment of periodontitis [63,64,65]. Ardakani et al. showed that patients using a mouthwash consisting of five herbal extracts (including B. serrata extracts) improved their periodontal condition in plaque-induced gingivitis comparable to the effect of 0.2% chlorhexidine mouthwash [63]. However, because the authors used a mix of five natural products, it is not possible to conclude whether B. serrata extracts play a role in the improvement of clinical symptoms. Another study showed that mouthwashes containing natural products, including one with frankincense extracts, improved plaque, gingivitis, and gingival bleeding [64]. These initial studies are encouraging and suggest that clinical trials with larger sample populations and only one natural product (frankincense) are needed to confirm the effects of frankincense against periodontal disease.
We used B. serrata extracts to describe antimicrobial effects against oral pathogens. Boswellic acids in B. serrata have previously been used to explore their antimicrobial effects against oral pathogens. Among β-boswellic acid (BA), 11-keto-β-boswellic acid (KBA), acetyl-β-boswellic acid (ABA), and acetyl-11-keto-β-boswellic acid (AKBA), only BA, KBA, and AKBA showed antimicrobial effects against planktonic P. gingivalis, and none of the boswellic acids were effective against planktonic F. nucleatum [26]. In this manuscript, we described for the first time the effects of B. serrata extracts on biofilm formation and intracellular infection. Future studies will determine whether AKBA inhibits P. gingivalis biofilm formation and intracellular infection in human GECs.
Altogether, our data show that B. serrata extract has antimicrobial and antibiofilm effects against P. gingivalis in vitro. The fact that frankincense extracts have similar effects on a culture-type P. gingivalis strain and clinical isolates [27] reinforces the clinical relevance of studying this natural product as a potential therapeutic for periodontitis in humans. Our study, however, opens several questions that should be addressed in future studies and some limitations of the present study should be considered. The specific bioactive compound with antimicrobial effects in our extract remains to be identified. Frankincense extracts, such as B. serrata extracts, may have hundreds of different bioactive compounds that could be involved in antimicrobial and antibiofilm effects, such as boswellic acids [β-boswellic acid (BA), acetyl-β-boswellic acid (ABA), 3-acetyl-11-keto-β-boswellic acid (AKBA), among others] [4]. The identification of the bioactive compounds could lead to treatment that induces more specific and robust effects at lower concentrations compared to use of the whole extracts. The cellular mechanisms activated by B. serrata during the killing of intracellular bacteria also deserve further study.

5. Conclusions

In this study, we explored the antimicrobial effects of B. serrata extract on the survival and infection of human GECs with P. gingivalis. We demonstrated that B. serrata extract significantly decreased P. gingivalis growth and biofilm formation, and reduced P. gingivalis biofilm biomass. We also demonstrated that B. serrata significantly reduced P. gingivalis intracellular infection of human GECs without damaging the host cell. Our data show, for the first time, the effects of B. serrata extracts on existing biofilms.

Author Contributions

Conceptualization, A.C.A.M.-S., H.A., D.M.O. and C.L.C.A.-d.-S.; methodology, D.V., A.C.A.M.-S., N.Z., G.M., H.M.D. and C.L.C.A.-d.-S.; formal analysis, D.V., A.C.A.M.-S., N.Z. and C.L.C.A.-d.-S.; resources, H.A., D.M.O. and C.L.C.A.-d.-S.; writing—original draft preparation, D.V., A.C.A.M.-S., N.Z. and C.L.C.A.-d.-S.; writing—review and editing, D.M.O. and C.L.C.A.-d.-S.; supervision, H.A, D.M.O. and C.L.C.A.-d.-S.; funding acquisition, H.A., D.M.O. and C.L.C.A.-d.-S. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by intramural funds from the University of the Pacific, Arthur A. Dugoni School of Dentistry to H.A., D.M.O., and C.L.C.A.-d.-S. and Start-Up Funds (D30059—Activity 101) from the University of the Pacific, Arthur A. Dugoni School of Dentistry to C.L.C.A.-d.-S.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available in this article.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. B. serrata extract inhibits P. gingivalis and F. nucleatum biofilm formation in vitro. (A) P. gingivalis or (B) F. nucleatum were plated on 24-well plates (1 × 107 CFU/mL) and incubated with different concentrations of B. serrata extract for 48 h under anaerobic conditions. Non-adherent bacteria were washed with phosphate-buffered saline (PBS), and adherent biofilms were fixed with methanol and then stained with crystal violet. Dissolved biofilms were transferred to 96-well plates and the optical density values at the wavelength of 560 nm were recorded. Graphs show biofilm growth percentage, normalized to the positive control (only bacteria without treatments). Negative control: only media. P/S: penicillin/streptomycin. n = 4. ** p ≤ 0.01, *** p ≤ 0.001.
Figure 1. B. serrata extract inhibits P. gingivalis and F. nucleatum biofilm formation in vitro. (A) P. gingivalis or (B) F. nucleatum were plated on 24-well plates (1 × 107 CFU/mL) and incubated with different concentrations of B. serrata extract for 48 h under anaerobic conditions. Non-adherent bacteria were washed with phosphate-buffered saline (PBS), and adherent biofilms were fixed with methanol and then stained with crystal violet. Dissolved biofilms were transferred to 96-well plates and the optical density values at the wavelength of 560 nm were recorded. Graphs show biofilm growth percentage, normalized to the positive control (only bacteria without treatments). Negative control: only media. P/S: penicillin/streptomycin. n = 4. ** p ≤ 0.01, *** p ≤ 0.001.
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Figure 2. B. serrata extract reduces P. gingivalis, but not F. nucleatum biofilms in vitro. (A) P. gingivalis or (B) F. nucleatum were plated on 24-well plates (1 × 107 CFU/mL) and incubated for 48 h under anaerobic conditions. Different concentrations of B. serrata extracts were added to the biofilms and incubated for an additional 24 h. Using the crystal violet staining method, the optical density of biofilm biomass was measured. Graphs show biofilm percentage, normalized to the positive control (only bacteria with no treatments). Negative control: only media. P/S: penicillin/streptomycin. n = 3. * p ≤ 0.05, **** p ≤ 0.0001.
Figure 2. B. serrata extract reduces P. gingivalis, but not F. nucleatum biofilms in vitro. (A) P. gingivalis or (B) F. nucleatum were plated on 24-well plates (1 × 107 CFU/mL) and incubated for 48 h under anaerobic conditions. Different concentrations of B. serrata extracts were added to the biofilms and incubated for an additional 24 h. Using the crystal violet staining method, the optical density of biofilm biomass was measured. Graphs show biofilm percentage, normalized to the positive control (only bacteria with no treatments). Negative control: only media. P/S: penicillin/streptomycin. n = 3. * p ≤ 0.05, **** p ≤ 0.0001.
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Figure 3. Low, physiologically relevant concentrations of B. serrata extract are not toxic to human gingival epithelial cells. Human gingival epithelial cells were plated on 24-well plates (1 × 105 cells/mL) one day before treatment. Cells were then treated with different concentrations of B. serrata extract for 24 h. Supernatants were collected for LDH quantification. Graph shows cytotoxicity percentage, normalized to the positive control (untreated cell incubated with lysis buffer). n = 3. ** p < 0.001.
Figure 3. Low, physiologically relevant concentrations of B. serrata extract are not toxic to human gingival epithelial cells. Human gingival epithelial cells were plated on 24-well plates (1 × 105 cells/mL) one day before treatment. Cells were then treated with different concentrations of B. serrata extract for 24 h. Supernatants were collected for LDH quantification. Graph shows cytotoxicity percentage, normalized to the positive control (untreated cell incubated with lysis buffer). n = 3. ** p < 0.001.
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Figure 4. B. serrata extract decreases survival of intracellular P. gingivalis in human gingival epithelial cells. Human gingival epithelial cells were plated on 6-well plates and incubated overnight before treatment. Cells were infected, or not, with P. gingivalis for 2 h, followed by incubation with antibiotics (metronidazole and gentamicin) for 1 h to remove extracellular bacteria. Then, the cells were treated with or without different concentrations of B. serrata extract for an additional 21 h. Cells were lysed with sterile distilled water and the bacteria were plated onto blood agar plates and incubated for 10 days for CFU counts (A). After 21 h incubation with B. serrata extract, cells were used for detection of P. gingivalis by fluorescence microscopy (B), and the supernatant was used for LDH quantitation (C). n = 3. Scale bar = 100 µm. **** p ≤ 0.0001.
Figure 4. B. serrata extract decreases survival of intracellular P. gingivalis in human gingival epithelial cells. Human gingival epithelial cells were plated on 6-well plates and incubated overnight before treatment. Cells were infected, or not, with P. gingivalis for 2 h, followed by incubation with antibiotics (metronidazole and gentamicin) for 1 h to remove extracellular bacteria. Then, the cells were treated with or without different concentrations of B. serrata extract for an additional 21 h. Cells were lysed with sterile distilled water and the bacteria were plated onto blood agar plates and incubated for 10 days for CFU counts (A). After 21 h incubation with B. serrata extract, cells were used for detection of P. gingivalis by fluorescence microscopy (B), and the supernatant was used for LDH quantitation (C). n = 3. Scale bar = 100 µm. **** p ≤ 0.0001.
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Table 1. B. serrata extract inhibits the growth of planktonic P. gingivalis, but not F. nucleatum, in vitro. B. serrata extract presents antimicrobial activity against P. gingivalis with a MIC of 32 µg/mL, while there were no antimicrobial effects observed against F. nucleatum at the concentrations tested in our experiments. Results show an average of n = 4 experiments.
Table 1. B. serrata extract inhibits the growth of planktonic P. gingivalis, but not F. nucleatum, in vitro. B. serrata extract presents antimicrobial activity against P. gingivalis with a MIC of 32 µg/mL, while there were no antimicrobial effects observed against F. nucleatum at the concentrations tested in our experiments. Results show an average of n = 4 experiments.
OrganismsMIC (μg/mL)
P. gingivalis ATCC 33277
F. nucleatum ATCC 25586		32
>512
MIC, Minimum inhibitory concentration.
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MDPI and ACS Style

Vang, D.; Moreira-Souza, A.C.A.; Zusman, N.; Moncada, G.; Matshik Dakafay, H.; Asadi, H.; Ojcius, D.M.; Almeida-da-Silva, C.L.C. Frankincense (Boswellia serrata) Extract Effects on Growth and Biofilm Formation of Porphyromonas gingivalis, and Its Intracellular Infection in Human Gingival Epithelial Cells. Curr. Issues Mol. Biol. 2024, 46, 2991-3004. https://doi.org/10.3390/cimb46040187

AMA Style

Vang D, Moreira-Souza ACA, Zusman N, Moncada G, Matshik Dakafay H, Asadi H, Ojcius DM, Almeida-da-Silva CLC. Frankincense (Boswellia serrata) Extract Effects on Growth and Biofilm Formation of Porphyromonas gingivalis, and Its Intracellular Infection in Human Gingival Epithelial Cells. Current Issues in Molecular Biology. 2024; 46(4):2991-3004. https://doi.org/10.3390/cimb46040187

Chicago/Turabian Style

Vang, David, Aline Cristina Abreu Moreira-Souza, Nicholas Zusman, German Moncada, Harmony Matshik Dakafay, Homer Asadi, David M. Ojcius, and Cassio Luiz Coutinho Almeida-da-Silva. 2024. "Frankincense (Boswellia serrata) Extract Effects on Growth and Biofilm Formation of Porphyromonas gingivalis, and Its Intracellular Infection in Human Gingival Epithelial Cells" Current Issues in Molecular Biology 46, no. 4: 2991-3004. https://doi.org/10.3390/cimb46040187

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