Clinical Oral Investigations (2025) 29:182

https://doi.org/10.1007/s00784-025-06267-8

RESEARCH

Anti-inflammatory and antimicrobial efficacy of coconut oil for

periodontal pathogens: a triple-blind randomized clinical trial
Simón Pardiñas López1,2,3,6 · Mónica E. García-Caro4 · Juan A. Vallejo4 · Pablo Aja-Macaya4 · Kelly Conde-Pérez4 ·
Paula Nión-Cabeza4 · Ismael Khouly6,7 · Germán Bou4 · Ana Isabel Rodríguez Cendal2,3 · Silvia Díaz-Prado2,3 ·
Margarita Poza4,5

Received: 19 January 2025 / Accepted: 6 March 2025 / Published online: 14 March 2025
© The Author(s) 2025

Abstract
Objectives To evaluate the effect of coconut oil on the oral bacteriome and inflammatory response in patients with periodon-
titis by integrating next-generation sequencing analyses of pathogenic bacterial shifts and quantification of inflammatory
markers, thereby assessing its potential as a natural adjunct to standard nonsurgical periodontal therapy.
Materials and methods A triple-blind clinical trial was conducted with 30 participants diagnosed with periodontitis, ran-
domized into 3 groups: (1) coconut oil, (2) chlorhexidine and (3) placebo. Saliva and gingival crevicular fluid (GCF) samples
were collected before treatment, one month after treatment, and one month post-non-surgical periodontal therapy. Bacterial
DNA was extracted, and the V3-V4 region of the 16 S rRNA gene was PCR-amplified and sequenced using Illumina MiSeq
technologies. Inflammatory biomarkers, including Interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), were quan-
tified from GCF samples.
Results Coconut oil treatment significantly reduced pathogenic bacterial families such as Spirochaetaceae and Tannerella-
ceae while promoting beneficial bacteria such as Streptococcaceae. At the genus and species levels, coconut oil reduced patho-
gens such as Tannerella forsythia and Treponema denticola along with increase in beneficial bacteria such as Streptococcus.

Simón Pardiñas López and Mónica E. García-Caro contributed Ana Isabel Rodríguez Cendal, Silvia Díaz-Prado and Margarita Poza
equally as first authors contributed equally as last authors

Simón Pardiñas López and Juan A. Vallejo contributed equally as

corresponding authors

4
Simón Pardiñas López Grupo de Investigación en Microbiología, Servicio de
[email protected] Microbiología, Instituto de Investigación Biomédica de
A Coruña (INIBIC)- Hospital Universitario de A Coruña
Juan A. Vallejo
(CHUAC)-Universidade da Coruña (UDC)-CIBER de
[email protected]
Enfermedades Infecciosas (CIBERINFEC, ISCIII), Hospital
1 Universitario, Coruña 15006 A, Spain
Periodontology and Oral Surgery, Clínica Médico Dental
5
Pardiñas, Real 66, 3, A Coruña 15003, Spain Grupo Microbioma y Salud, Facultad de Ciencias- Centro
2 Interdisciplinar de Química y Biología (CICA), Universidade
Grupo de Terapia Celular y Medicina Regenerativa, Instituto
da Coruña, A Coruña 15071, Spain
de Investigación Biomédica de A Coruña (INIBIC), Servizo
6
Galego de Saúde (SERGAS), Complexo Hospitalario Department of Oral and Maxillofacial Surgery, College of
Universitario de A Coruña (CHUAC), A Coruña Dentistry, New York University, New York, NY 10010, USA
15003, Spain 7
Multidisciplinary Implant and Aesthetic Miami Institute
3
Grupo de Terapia Celular y Medicina Regenerativa, (M.I.A.M.I.), Miami, FL 33137, USA
Departamento de Fisioterapia, Medicina y Ciencias
Biomédicas, Facultad de Ciencias de la Salud-Centro
Interdisciplinar de Química y Biología (CICA), Universidade
da Coruña, A Coruña 15701, Spain

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The subgingival microbial dysbiosis index improved significantly in both coconut oil and chlorhexidine groups. Further-
more, the coconut oil demonstrated a reduction in IL-6 and TNF-α levels, indicating decreased local inflammation.
Conclusions Coconut oil treatment significantly modulated the oral microbiome and reduced inflammatory markers in
patients with periodontitis, suggesting its potential as a natural and effective adjunct in periodontal therapy.
Clinical relevance This study highlights coconut oil’s potential as a natural adjunct in periodontal therapy, effectively reduc-
ing pathogenic bacteria and inflammatory markers (IL-6, TNF-α). It offers a safe alternative to chlorhexidine, promoting
microbiome balance and improved periodontal health.

Graphical Abstract

Keywords Periodontitis · Oral Microbiome · Coconut oil · 16S rRNA sequencing · Inflammation · Chlorhexidine

Introduction In the mouth, bacteria aggregate into biofilms within dis-

tinct niches, each supporting specific microbial populations
Periodontitis, a chronic inflammatory disease resulting from shaped by unique environmental conditions. This dynamic
microbiome dysbiosis that affects the supporting structures nature of the oral microbiome makes defining a general
of the teeth is the sixth most prevalent chronic disease composition difficult [5–8].
worldwide [1] and remains a significant global oral health In healthy conditions, the bacterial species that compose
issue [2–4]. the biofilm are primarily aerobic organisms, recognized by
Its pathophysiology involves key molecular pathways the host’s immune system as harmless. They protect the host
that activate host-derived proteinases, causing the loss of from pathogens and maintain a balance that allows symbi-
marginal periodontal ligament fibers, the downward migra- otic relationships, known as microbial homeostasis or eubi-
tion of the junctional epithelium, and the apical spread of osis [7, 8].
bacterial biofilm along the root surface [2].

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Clinical Oral Investigations (2025) 29:182 Page 3 of 20 182

However, poor oral hygiene and factors such as a poor disrupt the membrane, leading to increased permeability,
diet or smoking disrupt this balance, leading to an overp- leakage of cell contents, and ultimately cell death [16, 17].
roliferation of pathogenic species known as dysbiosis that In Gram-positive bacteria, lauric acid can interact with the
promotes the development of oral diseases [7, 8]. This oral enzyme that forms peptidoglycan bonds, leading to cell
microbiome’s complexity, influenced by individual health, lysis [18, 19]thereby reducing dental plaque and inflamma-
limits classical bacterial culture methods, especially for tion [18, 20–22].
uncultivable bacteria in dysbiotic states like periodontitis Beyond its direct antimicrobial activities, lauric acid can
[9] Next-generation sequencing (NGS), particularly 16 S be converted in the body to monolaurin (glycerol monolau-
rRNA metabarcoding, has advanced microbiome research rate), a compound similarly reported to have strong inhibi-
by identifying uncultivable microorganisms and reducing tory effects on various pathogenic organisms [17, 20, 23].
costs [10, 11]. This technique uses 16 S rRNA gene frag- CO has been found to be a cost-effective and easy-to-
ments to distinguish bacteria associated with health or dys- obtain option for maintaining good dental hygiene [24].
biosis and assess treatment effects. While in vitro studies show CO impacts biofilms, sequenc-
Furthermore, inflammation is a hallmark of periodontitis, ing data on its oral health effects remain limited [25].
with several pro-inflammatory cytokines playing a crucial To our knowledge, this study is the first triple-blind, ran-
role in their pathogenesis. Notably, IL-6 and TNF-α are two domized controlled clinical trial evaluating the oral micro-
key cytokines that significantly contribute to the inflamma- biological and inflammatory response of CO rinse as adjunct
tory process associated with periodontitis. IL-6 is involved periodontal therapy.
in the regulation of immune responses and acts as a media-
tor of inflammation by promoting the differentiation of B
cells and the activation of T cells. Elevated levels of IL-6 in Materials and methods
periodontal tissues are strongly associated with the progres-
sion of periodontitis and the destruction of the periodontal Study design
attachment [12].
TNF-α, on the other hand, plays a pivotal role in orches- This single-center, triple-blinded randomized controlled
trating the local inflammatory response and the breakdown trial evaluated the clinical efficacy of CO, 0.12% CHX and
of connective tissue and bone. By stimulating the production placebo mouthwashes as adjunctive therapies in periodontal
of matrix metalloproteinases, TNF-α facilitates the degrada- treatment. The study adhered to the CONSORT 2010 guide-
tion of extracellular matrix components, contributing to the lines for reporting randomized clinical trials [26] as well as
apical migration of the junctional epithelium and alveolar the recommendations of the European Federation of Peri-
bone loss [13]. These cytokines also enhance the expression odontology [27] and Cochrane’s risk of bias tools [28].
of other pro-inflammatory mediators, perpetuating a cycle
of inflammation and tissue destruction that characterizes Ethical approval
periodontitis [14].
Adjuvant rinses with antiseptic properties, including The study protocol, approved by the Comité de Ética de
chlorhexidine (CHX), essential oils, and cetylpyridinium la Investigación con Medicamentos de Galicia (CEIm-G)
chloride, have been suggested as effective methods to con- under protocol number 2017/247, was registered at Clini-
trol the progression of periodontitis by targeting dental calTrials.gov (NCT06049589). Conducted in accordance
plaque and reducing inflammation [15]. with the Declaration of Helsinki and Good Clinical Practice
Among these, CHX is widely used for managing oral guidelines, all participants provided written informed con-
pathologies due to its broad-spectrum antimicrobial activity sent prior to enrollment. The study was conducted at Fun-
[15] However, prolonged CHX use is linked to side effects dación Clínica Pardiñas, INIBIC, and Hospital Universitario
such as tooth and tissue staining, taste alteration, burning de A Coruña between November 2022 and December 2023,
sensations, and type 1 hypersensitivity reactions [5]. prioritizing patient safety, validity, and reproducibility.
On the other hand, natural options like coconut oil (CO)
have been gaining more attention for their potential advan- Eligibility criteria for participants
tages in maintaining oral hygiene. CO is largely composed
of medium-chain fatty acids such as lauric acid and capric Inclusion criteria
acid that have antimicrobial properties against a wide range
of microorganisms. In the case of Gram-negative bacteria, Human patient with over 18 years diagnosed with periodon-
the amphipathic nature of these compounds allows them to tal disease stages II and III (grades B and C) based on the
penetrate the bacterial membrane and form micelles that 2017 World Workshop on the Classification of Periodontal

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and Peri-Implant Diseases and Conditions [29], possessing Standardization / training

at least 16 natural teeth, and capable of understanding and
signing informed consent and follow study instructions. Prior to the study, the dental hygienists involved in the study
were standardized and trained by SPL, emphasizing stan-
Exclusion criteria dardization of techniques, precision in instrumentation, and
strict adherence to study protocols to ensure consistency
Individuals treated with antibiotics in the preceding 4 weeks and reliability across all procedures. Patients were provided
or currently undergoing antibiotic therapy, regular consum- with oral health instructions and instructed to discontinue
ers of xylitol, coconut, or coconut derivatives or CHX, the use of the mouthwash.
patients who had received dental prophylaxis within the last
6 months, pregnant and breastfeeding individuals, patients Primary study outcomes assessed
with allergies to coconut, coconut-derived products and
CHX, those with uncontrolled systemic diseases or current Oral Microbiome from saliva and GCF
use of medications such as phenytoin, cyclosporine, immu-
nosuppressants or anticoagulants, and those with active sys- One 8 mL tube of non-stimulated saliva was collected for
temic diseases (e.g., cancer or infectious diseases other than each patient at T1, T2 and T3, and were immediately stored
periodontitis) or history of chemotherapy or radiotherapy to at -80ºC for later analysis.
the head and neck area. GCF was obtained at T1, T2 and T3 using sterile #30
absorbent paper points (Henry Schein, NY, USA) that were
Intervention protocols inserted in the gingival sulcus of 3 different teeth that pre-
sented between 4 and 6 mm PPD for 30 s and then sub-
merged in 1 mL of RNA later (Qiagen, Venlo, Netherlands).
● Baseline (T1): Diagnostic procedures for determining Eight paper points were used for each patient. Samples were
the presence of periodontal disease based on the 2017 stored at -80ºC for later analysis.
World Workshop on the Classification of Periodontal
and Peri-Implant Diseases and Conditions [27] were Bacterial DNA extraction
performed by a blinded specialist in periodontics (SPL).
Saliva and crevicular fluid sample collection was col- Saliva samples were thawed at room temperature, trans-
lected by the same periodontist. Each patient received ferred to 50 mL tubes, and centrifuged at 13,000 rpm for
oral health instructions, including the Modified Bass 10 min at 4 °C. The supernatant was discarded, and the
brushing technique [30, 31] to be performed three times pellet was resuspended in 100 µL of nuclease-free water
daily and flossing after night brushing, to ensure consis- (Thermo Fisher Scientific, USA) and transferred to a 2 mL
tency across participants and groups. They were also in- Eppendorf tube. For bacterial lysis, 5 µL of 20 mg/mL lyso-
structed to begin rinsing with their assigned mouthwash. zyme, 1.25 KU/mL lysostaphin, and 3 KU/mL mutanolysin
● One month after baseline (T2): Saliva and crevicular (Sigma-Aldrich, USA) was added and incubated at 37 °C
fluid samples collected prior to treatment. At this point, with shaking for 1 h. DNA extraction was performed using
non-surgical periodontal therapy was performed by the MasterPure™ Complete DNA & RNA Purification Kit
three trained dental hygienists following the European (Epicentre, USA), and the extracted DNA was resuspended
Federation of Periodontology (EFP) clinical guidelines in 35 µL of TE buffer (10 mM Tris-Cl, 1 mM EDTA, pH
[32]. Step 1 involved professional mechanical plaque 8.0) and stored at -20 °C.
removal and control of plaque-retentive factors, while For GCF samples, vortexing for 4 min detached bacteria
Step 2 included subgingival periodontal instrumentation from paper points that were squeezed against the tube walls
using hand and powered ultrasonic instruments [32]. and removed. The tubes were centrifuged at 13,000 rpm for
● One Month after periodontal therapy (T3): saliva and 30 min at 4 °C. The precipitates were treated with 30 µL of
crevicular fluid samples were collected. Each patient enzyme cocktail and incubated at 37 °C for 1 h. After add-
was instructed not to eat, wash, smoke or rinse their ing 2 µL of proteinase K, the samples were placed on ice
teeth at least one hour before saliva sampling at each for 10 min, followed by centrifugation for 5 min at 4000 g
visit. at 4 °C. DNA extraction was carried out using the AllPrep
DNA/RNA Kit (Qiagen), and the DNA was eluted in 30 µL
of EB buffer (10 mM Tris-Cl, pH 8.5) and stored at -20 °C.
An extraction control was included for each series.

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Clinical Oral Investigations (2025) 29:182 Page 5 of 20 182

Library Preparation instructions, using the Human IL-6 DY206 ELISA kit from
DuoSet (Minneapolis, USA).
The DNA concentration of each sample was determined To determine the levels of TNF-α, the samples from
using the Qubit dsDNA HS Assay Kit (Invitrogen, USA) to the patients were concentrated using the Savant SpeedVac
prepare a 5 ng/µL dilution for library preparation. Two PCR SPD121P from Thermo Fisher to facilitate the detection of
reactions were required for 16 S rRNA metabarcoding. the protein. Quantification was carried out with the Human
The first PCR amplified the V3-V4 region of 16 S rRNA TNF-alpha DY210 ELISA kit from DuoSet (Minneapolis,
using the primers: USA). All measurements were performed on the Tecan Infi-
Forward: 5’TCGTCGGCAGCGTCAGATGTGTATA- nite® 200 PRO NanoQuant at 450 nm with correction at
AGAGACAGCCTACGGGNGGCWGCAG’3. 570 nm in duplicate. In both cases the protein content was
Reverse: 5’GTGACTGGAGTTCAGACGTGT- expressed in pg/ml.
GCTCTTCCGATCTGACTACHVGGGTATCTAATCC’3.
For each sample, a mixture of 1.25 µL of each primer (10 Statistical analysis
µM), 12.5 µL NZYTECH polymerase, 5 µL DNA (5 ng/
µL), and 5 µL nuclease-free water were prepared, including After sequencing the 16 S rRNA gene, FASTQ files were
a negative control. The amplification program was 95 °C for generated, and their quality was verified using FastQC.
5 min, 25 cycles of 95 °C for 30 s, 50 °C for 45 s, and 75 °C Quality filtering and analysis were performed using
for 45 s, with a final step at 72 °C for 5 min. PCR products QIIME2, where DADA2 was applied to remove primers,
were checked by electrophoresis (550 bp) and purified using adapters, chimeras, and taxonomic groups found in con-
the AMPure XP system (Beckman Coulter, USA). After two trols. Amplicon sequence variants (ASVs) and taxonomic
ethanol washes, DNA was eluted in 50 µL of EB buffer. assignments were generated, and rarefaction curves were
For library preparation, the Nextera XT Index Kit (Illu- produced to assess sample diversity coverage.
mina, USA) was used. A second PCR added the indexes for Taxonomic assignments were made using the SILVA
sequencing, with a mixture like the previous reaction but 138.1 reference database, grouping ASVs and calculating
replacing primers with 2.5 µL of indexes. The conditions relative abundances for each sample at various taxonomic
were 95 °C for 3 min, 5 cycles of 95 °C for 30 s, 60 °C levels. Bar charts were created using Phyloseq and ggplot2
for 45 s, and 72 °C for 45 s, followed by 5 min at 72 °C. in R, with unclassified ASVs labeled by their last known
The products were verified via electrophoresis on a 1% taxon and “NA.” ASVs with an abundance of less than
agarose gel and purified again using AMPure XP. Libraries 0.01% or present in fewer than 30–50% of samples were
were quantified using the dsDNA HS Assay Kit (Invitrogen, filtered out for relative abundance diagrams.
USA). Alpha diversity was assessed using QIIME2 with ACE
(Abundance-based Coverage Estimator), Fisher, Shannon,
Sequencing and Simpson indices. The Wilcoxon rank-sum test was used
to compare data across different time points and treatments.
For each sample, a specific volume was taken based on the Beta diversity was also analyzed using QIIME2 with Bray-
sample with the lowest concentration to obtain an equimolar Curtis, Jaccard, Jensen-Shannon, and Weighted UniFrac
pool in which all samples were equally represented. The final indices to study sample composition similarities.
pool was successively diluted to reach a final concentration A normalized CLR (Centered Log-Ratio) abundance
of 12 pM. Finally, 80% of the pool was mixed with 20% of analysis was performed at family, genus, and species levels
Phix 12 pM (Illumina, USA). The samples were sequenced using ggplot2 in R, with values transformed to a logarithmic
using the Illumina MiSeq v3 2 × 300 paired-end kit (Illu- scale for group comparisons. Box plots were created, and
mina, USA) and the MiSeq platform (Illumina USA). the Wilcoxon rank-sum test was applied. Lastly, the subgin-
gival microbial dysbiosis index (SMDI) was calculated at
Interleukin-6 and TNF-α from saliva the genus level based on the Chen et al. study [33].
A Brunner-Langer model for longitudinal data was
One 8 mL tube of non-stimulated saliva was collected for employed to evaluate and compare changes in Interleukin
each patient at T1, T2 and T3, and were immediately stored levels across follow-up periods between groups, using the
at -80ºC for later analysis. ATS statistics to determine main effects and interactions.
To determine the levels of IL-6 the samples were cen- For specific time-point comparisons, the Mann-Whitney
trifuged at 13,000 g for 5 min at room temperature. The test with Bonferroni correction was applied, while the Wil-
supernatant was measured following the manufacturer’s coxon test with Bonferroni correction was used for within-
group comparisons over time. Baseline group homogeneity

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was assessed using the Kruskal-Wallis test. A significance Randomization and group allocation
level of 5% (α = 0.05) was applied to all analyses.
Participants were randomly assigned to one of three groups
Preparation of the rinses in a 1:1:1 ratio using block randomization (block size of
3) performed by (AD) to ensure balanced sample sizes.
For the CO rinses, four commercial coconut oils were ana- Opaque 250 ml marked containers for measuring rinse vol-
lyzed using gas chromatography to assess their composi- ume, standardized toothbrushes, and toothpaste were dis-
tion. The coconut oil with the highest lauric acid content tributed to participants by (IFM), who was unaware of the
was selected, resulting in the use of pure virgin coconut oil contents and distinct from the person collecting samples.
with 47.92% C12:0 for the study. (Superalimentos Mundo- Each participant received standardized toothbrushes
Arcoiris, Girona, Spain) (Gum Classic, SUNSTAR Suisse, Switzerland) toothpaste
For the CHX rinses, a commercial 0.12% CHX solution and dental floss (Gum, SUNSTAR Suisse, Switzerland) and
(Lacer, 08290, Barcelona, Spain) was chosen, with three dental floss, and was instructed not to use any additional
drops of concentrated coconut flavoring (Nature’s Flavour, dental products. Participants were directed to rinse vigor-
Alphapower Food, Gauting, Germany) added to provide ously with 5 ml of the allocated mouthwash after brushing
coconut flavor. at night as follows:
For the placebo rinses, water was used as the base, with
the same coconut flavoring added as in the CHX group to ● Group 1 (CO Group): Coconut oil for 10 min, as de-
maintain consistency. scribed in similar studies [25, 34–37].
● Group 2 (CHX Group): 0.12% CHX solution with co-
Sample size calculation conut flavor for 1 min.
● Group 3 (Placebo Group): Coconut-flavored water for
The sample size was calculated based on similar studies 1 min.
evaluating the effects of essential-oil mouthrinse on sub-
gingival periodontopathogens [34]. The calculations carried Participants, clinicians, study personnel, and the statistician
out indicate that a minimum of 5 patients per group for a t were all blinded to group assignments.
test to reach 80% power at 95% confidence level were nec-
essary to ensure statistical reliability and avoid overlooking
significant results. Since this study includes a third group, Results
adjustments for multiple comparisons were necessary using
the Bonferroni criterion. Additionally, anticipating a drop- A total of 30 patients were enrolled and completed all study
out rate of 20%, the sample size was increased to 10 patients visits. The cohort included 15 females and 15 males, with
per group. an age range of 33 to 72 years (median age: 53.5 years).
(Table 1) This balanced sex distribution and relatively nar-
row age spread facilitate comparison across groups while
minimizing demographic biases.

Bacterial alpha diversity of the samples

Table 1 Demographics. Coconut oil (CO), chlorhexidine (CHX), SD
(Standard deviation) In the GCF samples, the CO group showed relatively stable
CO CHX Placebo Total diversity across time in all indices. The Shannon and Simp-
(N = 10) (N = 10) (N = 10) (N = 30)
son indices indicated that diversity remained stable over
Age (years)
Mean 53.3 51.2 53.8 52.8
time in all treatments. (Fig. 1).
SD 9.57 13.09 9.82 10.35 In the saliva samples, all treatments showed similar alpha
Median 54 50 57 53.5 diversity values (Fig. 1). In the CO group, a notable reduc-
Minimum 41 33 37 33 tion, although not statistically significant, in diversity was
Maximum 67 72 65 72 observed over time in the ACE, Fisher, and Shannon indi-
Gender (N [%]) ces. However, in the CHX treatment, a statistically signifi-
Male 5 (50) 5 (50) 5 (50) 15 (50) cant decrease in diversity was seen in the ACE, Shannon,
Female 5 (50) 5 (50) 5 (50) 15 (50) and Simpson indices over time.
Race (N [%])
White 10 (100) 10 (100) 10 (100) 30
(100)

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Clinical Oral Investigations (2025) 29:182 Page 7 of 20 182

Fig. 1 Alpha-diversity of gingival crevicular fluid (FCG) and saliva to a treatment type: A) Red color: treatment with placebo, C) green
samples grouped according to treatment type and sample collection color: treatment with coconut oil; and CHX) blue color, treatment with
time using ACE (Abundance-based Coverage Estimator) (I), Fisher Chlorhexidine. T1, T2 and T3 represent the time of sampling. Wil-
(II), Shannon (III), and Simpson (IV) indices. Each color corresponds coxon rank-sum statistical test was used: * p < 0.05, ** p < 0.01

Bacterial Beta diversity of the samples and to a lesser extent, Porphyromonadaceae and Prevotel-
laceae (Fig. 3A).
A clear separation between GCF and saliva samples was The relative abundance of the bacteriome at the genus
observed, indicating that the microbial composition of each level showed an abundance of Fusobacterium, Porphyromo-
of them is different (Fig. 2A). When separately studying nas, and Treponema in GCF samples, while Streptococcus
saliva and GCF samples (Fig. 2B), similarity in bacterial predominated in saliva samples, and to a lesser extent, Por-
diversity between treatments was observed. phyromonas and Prevotella. (Fig. 3B)
In the GCF samples, at the family level, bacteria belong-
Analysis of the oral Microbiome ing to Defluviitaleaceae, Spirochaetaceae, Synergistaceae,
and Tannerellaceae families had a significant descending
In GCF samples the relative abundance of bacterial families correlation in the samples of patients treated with CO over
Spirochaetaceae and Fusobacteriaceae predominated, and time (Fig. 4). Significant differences were obtained between
to a lesser extent, Porphyromonadaceae and Prevotellaceae, T1 and T3 for the Spirochaeraceae and Tannerellaceae gen-
while in saliva samples, Streptococcaceae predominated, era. Regarding Actinomycetaceae, Gemellaceae, Pasteurel-
laceae, and Streptococcaceae families, a significant positive

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Fig. 2 Beta-diversity of samples. A) Beta-diversity of each of the samples separated according to the type of sample (GCF and saliva).
samples analyzed. Each color belongs to a type of sample. Gingival Each color belongs to a type of treatment. A) Red color: treatment with
crevicular fluid (GCF): Red color. Saliva: blue color. The treatments placebo, C) green color: treatment with coconut oil; and CHX) blue
were differentiated using symbols. Treatment A: placebo, Treatment C: color, treatment with Chlorhexidine. In both cases, the Bray-Curtis,
Coconut oil, Treatment CHX: chlorhexidine. B) Beta-diversity of the Jaccard, Jensen-Shannon and Weighted unifrac indices were used

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Clinical Oral Investigations (2025) 29:182 Page 9 of 20 182

Fig. 3 Bacterioma of the pooled samples obtained from gingival cre- sampling A) at the family level and B) at the genus level. The samples
vicular fluid (GCF) and saliva samples from patients with periodon- analyzed were based on a treatment with placebo (A), Coconut oil (C)
titis determined by 16 S rRNA metabarcoding. They were grouped and (CHX) chlorhexidine, obtaining the samples at different times: T1,
according to the type of sample, the type of treatment and the time of T2 and T3

correlation was found in CO samples, with significant dif- T2 and T3 for the latter. Only significant positive correlation
ferences between T1 and T3 and between T2 and T3 in over time was observed for Veillonella NA.
Streptococcaceae. In saliva samples (Fig. 7), CO had a decreasing effect
In the GCF samples at the genus level (Fig. 5), the on Bacteroidales incertae sedis, Carnobacteriaceae, and
abundance of Defluviitaleaceae UCG-011, Fretibacterium, Spirochaetaceae families, with a significant descending cor-
Olsenella, Tannerella, Treponema, and the Eubacterium relation over time, with significant differences between T1
group had a significant descending correlation over time in and T3 for Spirochaetaceae. An increase in abundance was
CO. Additionally, Tannerella and Treponema showed sig- observed for Lachnospiraceae over time, with significant
nificant abundance reduction between T1 and T3. Signifi- ascending correlation and differences between T1 and T3.
cant ascending correlation was observed for Actinomyces, In the analysis of the effect of CO on the oral microbi-
Gemella, Parvimonas, Streptococcus, and Veillonella genera ome at the genus level in saliva samples (Fig. 8), significant
over time, with significant differences between T1 and T3 descending correlation over time was observed for Granu-
for Parvimonas and between T2 and T3 for Streptococcus. licatella, Phocaeicola, Prevotella, and Treponema, with
In the GCF samples at the species level (Fig. 6), the significant differences between T1 and T3. A significant
species Fretibacterium NA, Olsenella uli, Olsenella NA, correlation over time was observed for Oribacterium, with
Tannerella forsythia, Treponema denticola, Treponema significant differences between T1 and T3 and between T2
maltophilum, Treponema NA, and Treponema socranskii and T3.
showed a significant descending correlation over time in In the analysis of bacterial species in saliva samples
CO, with significant differences between T1 and T3 for (Fig. 9), significant descending correlation was observed in
Tannerella forsythia. Significant ascending correlation was the CO group for Granulicatella NA, Phocaeicola absces-
found over time for Parvimonas NA and Streptococcus NA, sus, Prevotella melaninogenica, Tannerella forsythia, and
with significant increases between T1 and T3 and between Treponema NA. Significant ascending correlation over time
was observed for Oribacterium NA and Tannerella NA,

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Fig. 4 Selected bacterial families present in the oral microbiome of boxes: A) Red color: treatment with placebo, C) green color: treatment
gingival crevicular fluid (GCF) samples from patients with periodonti- with coconut oil; and CHX) blue color, treatment with Chlorhexidine.
tis. The box plot represents the normalized Centered Log-Ratio (CLR) Samples from the same patient were connected by dotted lines. The
abundance of each family according to the type of treatment A: pla- gray color is assigned when no significant correlation was obtained
cebo; C: Coconut oil; CHX; and time of sampling (T1, T2 and T3). in the treatment. The Wilcoxon rank-sum statistical test was used: *
Each color belongs to a treatment in which there was a significant p < 0.05, ** p < 0.01
correlation over time, represented by a line that passes through the

with significant differences between T1 and T3 and between between T1 and T3 when comparing CO and CHX, favoring
T2 and T3 for Oribacterium NA. CO (p = 0.045). (Table 2) (Fig. 11).
A significant decrease in SMDI between T1 and T3 was
observed for CO and CXH, with a pronounced decrease
between T2 and T3, demonstrating a significant shift toward Discussion
a more balanced microbial profile, while in the placebo
group significant differences were only observed between To the best of our knowledge, this study represents the first
T2 and T3. (Fig. 10) randomized clinical trial to investigate the effects of coconut
oil on the oral microbiome and inflammatory response in
Interleukin-6 and TNF-α patients with periodontitis. CO is composed of fatty acids
such as lauric acid and monolaurin, which have antibacte-
The CO group showed a significant decrease in IL-6 levels rial activity. Although fatty acids antimicrobial effects on
over the entire period (p = 0.021) and between T2 and T3 various bacteria are well documented [17, 23, 38–42], most
when comparing CO with placebo (p = 0.027). systematic reviews and meta-analyses evaluating oil pull-
Similarly, a significant reduction in TNF-α was observed ing focus on clinical outcomes rather than its direct impact
only in the CO group between T1 and T3 (p = 0.021) and on the oral microbiome [25, 43, 44]. The findings on the
present study demonstrate that CO treatment significantly

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Clinical Oral Investigations (2025) 29:182 Page 11 of 20 182

Fig. 5 Selected bacterial genera present in the oral microbiome of gin- A) Red color: treatment with placebo, C) green color: treatment with
gival crevicular fluid (GCF) samples from patients with periodontitis. coconut oil; and CHX) blue color, treatment with Chlorhexidine. The
The box plot represents the Centered Log-Ratio (CLR) abundance of gray color is assigned when no significant correlation was obtained in
each genus according to the type of treatment (A: placebo; C: coconut the treatment. Samples from the same patient were connected by dot-
oil; and CHX: Chlorhexidine) and time of sampling (T1, T2 and T3). ted lines. The Wilcoxon rank-sum statistical test was used: * p < 0.05,
Each color belongs to a treatment in which there was a significant cor- ** p < 0.01
relation over time, represented by a line that passes through the boxes:

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Fig. 6 Selected bacterial species present in the oral microbiome of gin- A) Red color: treatment with placebo, C) green color: treatment with
gival crevicular fluid (GCF) samples from patients with periodontitis. coconut oil; and CHX) blue color, treatment with Chlorhexidine. The
The box plot represents the Centered Log-Ratio (CLR) abundance of gray color is assigned when no significant correlation was obtained in
each species according to the type of treatment (A: placebo, C: coco- the treatment. Samples from the same patient were connected by dot-
nut oil and CHX: chlorhexidine) and time of sampling (T1, T2 and T3). ted lines. The Wilcoxon rank-sum statistical test was used: * p < 0.05,
Each color belongs to a treatment in which there was a significant cor- ** p < 0.01
relation over time, represented by a line that passes through the boxes:

modulates the oral microbiome, promoting a shift toward a complex: Porphyromonas gingivalis, Tannerella forsythia,
healthier microbial profile, while also reducing key inflam- and Treponema denticola [45].
matory markers. However, the main limitation of this study The most effective treatments for combating the dis-
is its small sample size, indicating the need for larger-scale ease are mechanical, both surgical and non-surgical, and
research to obtain more conclusive results. In patients suf- these can be complemented with antiseptic compounds to
fering from periodontitis, there is a significant dysbiosis of control the bacterial load before, during, and after treat-
the oral microbiota, marked by an increase in certain bac- ment to enhance its effectiveness [15, 46]. One of the most
terial species, particularly in the subgingival sulcus. This used options is CHX, a synthetic chemical compound with
includes the most pathogenic bacteria of Socransky’s red potent antibacterial effects. However, it has been reported

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Clinical Oral Investigations (2025) 29:182 Page 13 of 20 182

Fig. 7 Selected bacterial families are present in the oral microbiome time, represented by a line that passes through the boxes: A) Red color:
of saliva samples from patients with periodontitis. The box plot repre- treatment with placebo, C) green color: treatment with coconut oil;
sents the Centered Log-Ratio (CLR) abundance of each family accord- and CHX: blue color, treatment with Chlorhexidine. The gray color
ing to the type of treatment (A: placebo, C: coconut oil and CHX: is assigned when no significant correlation was obtained in the treat-
chlorhexidine) and time of sampling (T1, T2 and T3). Each color ment. Samples from the same patient were connected by dotted lines.
belongs to a treatment in which there was a significant correlation over The Wilcoxon rank-sum statistical test was used: * p < 0.05, ** p < 0.01

Fig. 8 Selected bacterial genera present in the oral microbiome of time, represented by a line that passes through the boxes: A) Red color:
saliva samples from patients with periodontitis. The box plot repre- treatment with placebo, C) green color: treatment with coconut oil;
sents the Centered Log-Ratio (CLR) abundance of each genus accord- and CHX) blue color, treatment with Chlorhexidine. The gray color is
ing to the type of treatment (A: placebo, C: coconut oil and CHX: assigned when no significant correlation was obtained in the treatment.
chlorhexidine) and time of sampling (T1, T2 and T3). Each color Samples from the same patient were connected by dotted lines. The
belongs to a treatment in which there was a significant correlation over Wilcoxon rank-sum statistical test was used: * p < 0.05

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Fig. 9 Selected bacterial species present in the oral microbiome of resented by a line passing through the boxes: A) Red color: treatment
saliva samples from patients with periodontitis. The box plot represents with placebo, C) green color: treatment with coconut oil; and CHX)
the Centered Log-Ratio (CLR) abundance of each species according to blue color, treatment with Chlorhexidine. The gray color is assigned
the type of treatment (A: placebo, C: coconut oil and CHX: chlorhexi- when no significant correlation was obtained in the treatment. Samples
dine) and time of sampling (T1, T2 and T3). Each color belongs to a from the same patient were connected by dotted lines. The Wilcoxon
treatment in which there was a significant correlation over time, rep- rank-sum statistical test was used: * p < 0.05

to have side effects such as dental staining and taste altera- in bacterial load with a shorter rinsing time, the results from
tion, thereby limiting its suitability for prolonged use [15]. the present study indicate that CO may confer additional
Rinsing with essential oils such as CO, also known as oil anti-inflammatory benefits, likely related to the direct action
pulling, has shown to be an alternative to CHX due to its of lauric acid on proinflammatory cytokines. Although some
antibacterial and anti-inflammatory properties with minimal studies also report that CHX reduces gingival inflammation,
side effects [47]. this effect may predominantly stem from decreasing bac-
Unlike conventional oral rinses designed for 1–2 min of terial load rather than directly targeting cytokines [51–53].
use, oil pulling requires a longer duration to maximize its This study, based on next-generation sequencing technolo-
emulsifying and saponifying actions, which are essential gies, allowed for a detailed analysis of whether there is a
for reducing plaque adhesion and bacterial coaggregation significant decrease in pathogenic species and an increase in
[48]. The extended rinsing time for CO compared to CHX primary colonizers following different treatments, particu-
and placebo used in the present study aligns with findings larly in GCF samples.
from other studies demonstrating the enhanced effective- When studying alpha diversity, a measure of the com-
ness of oil pulling when performed over prolonged periods positional complexity within a specific site or community
[47, 49, 50]. While CHX can yield comparable reductions [54], it was observed that at the start of the treatment, the

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Clinical Oral Investigations (2025) 29:182 Page 15 of 20 182

Fig. 10 Subgingival dysbiosis index (SMDI) at gender level. The box cant correlation over time, represented by a line that passes through the
plot represents the SDMI based on a group of genera associated with boxes: A) Red color: treatment with placebo, C) green color: treatment
subgingival dysbiosis according to the type of treatment (A: placebo, with coconut oil; and CHX) blue color, treatment with Chlorhexidine.
C: coconut oil and CHX: chlorhexidine) and time of sample (T1, T2 Samples from the same patient were connected by dotted lines. The
and T3). Each color belongs to a treatment in which there was a signifi- Wilcoxon rank-sum statistical test was used: * p < 0.05

Table 2 Coconut oil (CO), chlorhexidine (CHX), SD (Standard deviation)

Outcomes CO CHX Placebo CO vs. CHX CO vs. Placebo CHX vs. Placebo
IL6 T1 vs. T2 Mean -47,6 -5,93 12,5 -41,7 -60 -18,4
SD 77,2 80,3 32,8 35,2 26,5 27,4
P value 0,222 1 1 1 0,189 0,945
T1 vs. T3 Mean -72,4 8,72 0,61 -81,1 -73 8,11
SD 67,7 161,3 24,5 55,3 22,8 51,6
P value 0,021* 1 1 0,741 1 1
T2 vs. T3 Mean -24,8 14,7 -11,8 -39,5 -13 26,5
SD 42,2 141,9 38,4 46,8 18 46,5
P value 0,141 1 1 1 0.027* 1
TNA T1 vs. T2 Mean -0,05 0,02 0,01 -0,07 -0,06 0,01
SD 0,07 0,09 0,08 0,03 0,03 0,04
P value 0,177 1 1 0,189 0,369 1
T1 vs. T3 Mean -0,06 0,03 -0,01 -0,07 -0,05 0,03
SD 0,06 0,08 0,04 0,03 0,02 0,03
P value 0,021* 1 1 0.045* 1 1
T2 vs. T3 Mean 0 0,01 -0,02 -0,01 0,01 0,02
SD 0,05 0,09 0,07 0,03 0,03 0,03
P value 1 1 1 1 0,156 1
*p<0,05

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Fig. 11 The graph displays the levels of IL-6 (1) and TNF-α (2) according to the type of treatment (A: placebo, C: coconut oil and CHX: chlorhexi-
dine) and time of sampling (T1, T2 and T3)

bacterial diversity values were similar in both saliva and proteins of the gingival cellular tissue during disease pro-
GCF samples, contrary to what was observed by Kim et al. gression [59].
[55]. For the CO treatment, there was a decrease in saliva However, no studies were found showing a significant
samples, but not a significant variation over time. This effect of CO on T. forsythia, although a significant decrease
suggests that there may be a general decrease in bacterial in this species was observed when using essential oils, espe-
quantity, reducing dental plaque, while the proportion of cially in periodontal pockets [60], and further investigation
organisms that make up the oral microbiota remains similar. into the effects of CO on these two pathogens in cellular
This was observed in greater detail when studying the bac- models is needed.
teriome at different taxonomic levels. Other bacteria showing a decrease in abundance when
Regarding beta diversity, used to describe taxonomical treated with CO include Porphyromonas gingivalis, Eubac-
differences between samples [54], there was a clear dif- terium nodatum, Treponema socranskii, and Treponema
ference between the oral microbiome composition in GCF maltophilum, all associated with periodontitis as reported in
and saliva samples, consistent with the findings of Kim et the literature [60, 61].
al. [55]. However, the oral microbiome during the differ- In our study, a decrease in Olsenella uli and T. maltophi-
ent treatments did not show relevant differences, although lum, present in periodontal pockets [62], was also observed.
it would be interesting to observe the effects of treatments However, there is no existing literature demonstrating the
over different time periods. effect of CO on these bacteria, making the findings of this
In the GCF samples, a decrease was observed in two study particularly interesting, especially for T. maltophilum,
bacterial species belonging to Socransky’s red complex, since CHX treatment did not yield significant differences for
Tannerella forsythia and Treponema denticola, a group of this bacterium.
bacteria central to driving the dysbiotic process underlying For the genus Fretibacterium and the species Defluviita-
periodontitis [56], which was also reported in another study leaceae UCG-011, a decrease in abundance was observed
[57]. with CO treatment. Studies have shown an abundance of
Treponema denticola is recognized as one of the primary these bacteria in patients with periodontitis, making their
etiological agents of periodontitis, owing to its numerous reduction with coconut oil treatment noteworthy [63].
virulence factors, including high motility and chemotaxis, The subgingival microbiota in a healthy state is com-
synergistic interactions with other periodontal pathogens, posed of bacteria from the genera Streptococcus, Actino-
production of cytotoxic metabolites, robust biofilm forma- myces, Gemella, and Veillonella [64, 65]. When periodontal
tion, and cell wall proteins that disrupt host defenses [58]. disease occurs, members of these genera are displaced by
In contrast, the role of Tanerella forsythia in periodonti- pathogenic species. Treatment with CO showed an increase
tis has been more recently elucidated through the identifica- in these beneficial bacteria. This is significant because it
tion of six KLIKK proteases, which actively degrade the suggests that CO not only directly reduces some pathogenic

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Clinical Oral Investigations (2025) 29:182 Page 17 of 20 182

species but also promotes the growth of bacteria associated or normobiotic according to the SMDI criteria, though
with oral health, indicating a return to a more balanced, less species-level analysis is limited by the V3–V4 sequencing
pathogenic state. For instance, some Streptococcus species technology.
can be linked to periodontitis, while others can exhibit activ- The SMDI values obtained suggest that non-surgical
ity capable of displacing bacteria from Socransky’s orange periodontal therapy significantly reduces subgingival dys-
or red complex, such as T. denticola or T. forsythia [5, 66]. biosis. However, the combined action of a mouthwash as
On the other hand, CO had an unexpected effect on the an adjunct therapy, whether CHX or CO, appears to signifi-
genus Parvimonas. P. micra is known to inhabit the sub- cantly reduce periodontitis-associated pathogens before and
gingival cavity and act as a pathogen in periodontitis, being after the treatment.
one of the most predominant species [67]. Thus, an increase CO may reduce dysbiosis predominantly through the
in these bacteria with CO treatment may not be beneficial. selective inhibition of pathogenic taxa without negatively
Regarding the effect of CO on the oral microbiota in impacting beneficial bacteria. Nonetheless, future studies
saliva samples, a decrease in Phocaeicola, Prevotella, and with additional experimental arms or more refined taxo-
Treponema was observed, with the specific species being nomic methods would be valuable to further isolate and
P. abscessus, P. melaninogenica, and T. forsythia. These confirm the specific effects of CO on individual bacterial
genera have been identified as some of the most abundant species within these genera. The role of inflammation in
in the saliva of patients with periodontitis [68], although periodontitis is well-documented, with cytokines such as
the presence of T. forsythia. P. melaninogenica is typically IL-6 and TNF-α playing central roles in disease progression.
classified as an oral commensal, some studies found a high This study’s findings align with previous research that high-
abundance of this species in subgingival areas of periodon- lights the significance of these pro-inflammatory mediators
titis patients [69]. While P. abscessus has been isolated from in the breakdown of periodontal tissues [12]. Elevated lev-
brain abscesses [70], it has been linked to periodontal dis- els of IL-6 in periodontal tissues contribute to the ampli-
ease [71]. Therefore, the observed decrease in these species fication of the inflammatory response, exacerbating tissue
with CO use is significant. breakdown and bone resorption.
For the genera Granulicatella and Oribacterium, an Similarly, TNF-α is critical in orchestrating the local
increase in abundance was observed during CO treatment. inflammatory response and promoting the degradation of
Granulicatella is typically associated with good oral health the extracellular matrix and bone loss through the stimula-
in the subgingival region [72]. However, Oribacterium is tion of matrix metalloproteinases (MMPs) [13]. By enhanc-
a pathobiont present in both supragingival and subgingival ing the production of other inflammatory mediators, TNF-α
microbiomes of periodontitis patients [73]. Therefore, the further amplifies tissue damage, which underscores its
increase in these bacteria due to CO treatment could be less importance as a therapeutic target [14].
beneficial, though these results should be confirmed with a The anti-inflammatory properties of CO, specifically
larger study. its ability to modulate cytokine activity, suggest it may
Non-surgical periodontal therapy performed may also play a role in dampening the chronic inflammation seen in
influence these results, as significant differences were found periodontitis. Previous studies have shown that CO’s key
between the start of the treatment (T1) and one month after components, such as lauric acid, can inhibit the produc-
dental cleaning (T3). Dental cleanings in healthy patients tion of pro-inflammatory cytokines, potentially reducing
have been shown to reduce bacterial quantity while main- the inflammatory burden in periodontal tissues [22]. This
taining the proportional balance of different bacterial spe- suggests that CO could mitigate the destructive effects of
cies, which may explain the alpha diversity results in this IL-6 and TNF-α, providing a novel approach to controlling
study. Unlike previous studies that collected samples imme- inflammation in periodontitis.
diately after cleaning, this study collected samples one While our findings demonstrate a significant reduction in
month later, allowing dental plaque to reestablish. It has IL-6 and TNF-α in the CO group, these two markers rep-
been reported that dental plaque development after cleaning resent just a fraction of the inflammatory processes under-
exceeds initial values by the second day [74]. The observed lying periodontitis. Other cytokines such as IL-1β, IL-8,
effects of CO one month later, showing a decrease in patho- prostaglandin E2, and matrix metalloproteinases also play
genic species and an increase in early colonizers, could indi- essential roles in tissue destruction and disease progres-
cate a return to a healthier oral microbiota. sion [75–77]. Although incorporating a broader panel of
In the present study, the effect of CO on periodontal dys- biomarkers would have provided deeper insights into the
biosis was evaluated using the SMDI, which is calculated exact immunological pathways affected by CO, our primary
based on the median values of each genus over time for objective was to evaluate two well-established mediators in
each treatment. Each genus is classified as either dysbiotic periodontitis. Future research should expand on these results

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182 Page 18 of 20 Clinical Oral Investigations (2025) 29:182

Funding Open Access funding provided thanks to the CRUE-CSIC

by examining additional inflammatory markers and explor- agreement with Springer Nature.
ing how they interact with the oral microbiota to influence The present study received funding from the Instituto de Salud Carlos
longer-term periodontal outcomes. III (ISCIII), Spain, through the projects PI20/00413 and PI23/00696,
Future research should investigate whether CO’s anti- co-funded by the European Union (EU), to M. P and by CIBER de
Enfermedades Infecciosas CIBERINFEC, ISCIII (CB21/13/00055) to
inflammatory effects can effectively alter cytokine levels G. B and M. P.
in clinical settings, offering a new avenue for periodontitis Grupos con Potencial de Crecemento from Xunta de Galicia, Galicia,
treatment. Spain (ED431B 2023/58 and ED431B 2020/55) and Fundación Públi-
ca Galega de Investigación Biomédica (FINIBIC).
Funding for open access charge: Universidade da Coruña/CISUG.

Conclusions Data availability No datasets were generated or analysed during the

current study.
CO significantly reduces bacterial load in both subgingival
and supragingival areas, targeting pathogenic bacteria while Declarations
promoting beneficial species for a healthier oral environ-
ment. Additionally, CO reduces IL-6 and TNF-alpha levels, Competing interests The authors declare no competing interests.
showcasing its anti-inflammatory properties.
Open Access This article is licensed under a Creative Commons
These results highlight the potential clinical relevance of Attribution 4.0 International License, which permits use, sharing,
CO as a natural and effective adjunct in periodontal ther- adaptation, distribution and reproduction in any medium or format,
apy, offering a promising alternative to CHX for managing as long as you give appropriate credit to the original author(s) and the
periodontitis. source, provide a link to the Creative Commons licence, and indicate
if changes were made. The images or other third party material in this
However, larger and long-term clinical studies are needed article are included in the article’s Creative Commons licence, unless
to assess CO’s extended effects on the oral microbiome indicated otherwise in a credit line to the material. If material is not
and periodontal health. Investigating its antimicrobial and included in the article’s Creative Commons licence and your intended
anti-inflammatory mechanisms could lead to more targeted use is not permitted by statutory regulation or exceeds the permitted
use, you will need to obtain permission directly from the copyright
treatments. holder. To view a copy of this licence, visit ​h​t​t​p​:​​​/​​/​c​r​e​a​t​i​​v​e​c​​o​m​m​o​​n​​s​.​​o​
r​​​g​/​l​i​c​e​n​s​​e​s​/​​b​​y​/​4​.​0​/.

Annexes I-VII
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