ISSN 0975-2366

DOI:https://doi.org/10.31838/ijpr/2020.12.03.494
Research Article

Effectiveness of Grapefruit (Citrus Paradisi) And Lime

(Citrus Aurantifolia) Against Pathogenic Root Canal
Biofilms
NURANI HAYATI1*, ARMELIA SARI WIDYARMAN2, BOEDI OETOMO ROESLAN3
1
Department of Endodontic and Conservative Dentistry, Faculty of Dentistry, Prof. Dr. Moestopo (B)
University, Indonesia
2
Department of Microbiology, Faculty of Dentistry, Trisakti University, Indonesia
3
Department of Oral Biology, Faculty of Dentistry, Trisakti University, Indonesia
*Corresponding Author
Email: [email protected]
Received: 22.07.20, Revised: 14.08.20, Accepted: 09.09.20

ABSTRACT
Root canal bacterial biofilms show significant resistance to antimicrobial agents. Citrus as one of the most
common consumed fruits contain various phenolic compounds that have antibacterial properties and proven
to have low toxicity and unharmful to human tissues. The aim of this study was to examine the effectiveness of
grapefruit (Citrus paradisi) and lime (Citrus aurantifolia) in different concentrations in the inhibition and
eradication of Porphyromonas endodontalis and Porphyromonas gingivalis biofilm in vitro and their
cytotoxicity to human gingival fibroblasts. Pure blended and squeezed juices of grapefruit and lime were
diluted in distilled water into concentration 100%, 50% and 25%. The effect of juices toward microbial biofilms
were examined using biofilm assay. Chlorhexidine gluconate (2%) and NaOCl (2.5%) was used as a positive
control. Data were analyzed using two-way analysis of variance and Tukey’s post-hoc significant difference test
with P<0.05 was considered statistically significant. Additionally, MTT cytotoxicity assay was performed to
measuring fibroblast viability. The cytotoxicity assay showed that cell viability with each citrus was significantly
above the recommended toxicity value for gingival fibroblasts (>90%). The biofilm assays indicated that all
grapefruit and lime juice treatments significantly eradicated and inhibited the formation of P. endodontalis and
P. gingivalis biofilms in all concentration after exposure for and 15 min and 48 h, respectively (P<0.05). All
blended and squeezed-juiced extracts of grapefruit and lime have an antibiofilm effect against P. endodontalis
and P. gingivalis. Grapefruit and lime juice may be useful in preventing and eradicating biofilm in root canal.
Keywords: Biofilm, fibroblast, grapefruit, lime, Porphyromonas endodontalis, Porphyromonas gingivalis.

INTRODUCTION requires exposure to a particular antibiotic in up

Anaerobic black-pigmented Gram-negative to 500x minimum inhibitory concentration [11-
bacteria are commonly found in root canal 12].
systems and endodontic abscesses, and their Various types of herbs and fruits have been
presence is often accompanied by symptoms of exhibit some antibacterial and antibiofilm abilities
swelling and pain caused by percussion and against several root canal pathogenic bacteria
palpation in the oral cavity [1-2]. Porphyromonas and also proved many beneficial in dentistry [13-
endodontalis and Porphyromonas gingivalis are 17]. Citrus fruits have been shown to have low
black-pigmented bacteria commonly found in toxicity and safe for human tissues. They contain
root canal systems [3-5]. These species produce various flavonoids that exert antibacterial,
various virulence factors that can induce the antifungal, and antioxidant activities [18]. A study
release of various cytokines, such as interleukin-1 comparing the chemical compounds of
and tumor necrosis factor alpha, in the host [6-8]. tangerines (Citrus tangerina), grapefruits (C.
The main virulence factors of these two bacteria paradisi), lemons (C. limon), and limes (C.
are lipopolysaccharides (LPS) [6-7]. One of the aurantifolia) found that grapefruits have the
targets of LPS in the human body is gingival highest phenol and flavonoid contents [19]. Limes
fibroblasts, which play an important role in were also shown to inhibit the growth of oral
periodontal soft tissue remodeling [8]. Gram-positive and Gram-negative bacteria [7].
Porphyromonas endodontalis and P. gingivalis Root canal irrigation, now commonly used in
biofilms show significant resistance to antibiotic endodontics, poses challenges due to its toxicity to
agents. Complete elimination of such biofilms tissues. Irrigation agents such as sodium

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Pathogenic Root Canal Biofilms

hypochlorite (NaOCl) can cause issues ranging treatment. After incubation for 24h, cell viability
from a burning sensation in the gingiva and oral was analyzed using the MTT assay method. An
mucosa to necrosis of periapical tissue [20]. MTT solution was made by mixing 5 mg of
These issues can be avoided by switching to thiazolyl blue tetrazolium bromide (98%; Sigma-
natural ingredients as the main components of Aldrich, Singapore) with 1 ml of NaCl (0.9%). The
irrigation agents. The preparation method that cells were then added by 50µl MTT solution into
provides optimal antibacterial activity yet also safe each plates, and re-incubated at 37oC for 4h.
to human tissue has not been determined. Then, 14.5 ml of isopropanol solution and 100 μl
Therefore, this study aimed to evaluate the of 3.7% HCl were added, and the solution was
effectiveness of different preparations of C. re-incubated at 37°C for 1 h. The well plates were
paradisi and C. aurantifolia in inhibiting and subsequently inserted in a microplate absorbance
eradicating P. endodontalis and P. gingivalis reader (iMark; Bio-Rad, USA), and the optical
biofilms and their toxicity to human gingival density (OD) was measured at a wavelength of
fibroblast. 595 nm.

METHODOLOGY Quantitative Test for Active Compounds

Citrus Paradisi and C. Aurantifolia Juice Active compound identification using liquid
Extraction Methods chromatography–mass spectrometry (LC-MS) was
This was a post-test only controlled group design, performed on each preparation of C. paradisi
and the protocol of this research was approved and C. aurantifolia after centrifuged at 4,500
and reviewed by the ethics committee of Faculty rpm for 15 min. The supernatants were
of Dentistry Trisakti University, Indonesia (No. transferred into 2-ml vials and placed in the LC-
307/S2/KEPK/FKG/9/2019). This study used ruby MS system. The quantitative test was performed at
grapefruits (Price Look-Up code 6472) from the Agency for the Assessment and Application of
Australia and limes from plantations in West Technology (Badan Pengkajian dan Penerapan
Java. They were confirmed to be C. paradisi Teknologi), Tangerang, Indonesia.
Macf. and C. aurantifolia (Christm.) Swingle,
respectively, by the Plant Taxonomy Laboratory, Bacterial Cultures
Department of Biology, Faculty of Mathematics Freeze-dried P. gingivalis (ATCC 33227) and P.
and Natural Sciences, Padjadjaran University, endodontalis (ATCC 35406D-5) were opened in
Sumedang, Indonesia. an anaerobic room by breaking the vials. Then, 1
Citrus juice was extracted from the fruits using two ml of enriched tryptic soy broth (TSB) with 0.005%
methods. The first method was mixing the entire hemin, 0.05% L-cysteine HCl, 0.5% yeast extract,
contents of citrus that has been peeled include the and 0.001% menadione were added into the vial
seed, using a fruit mixer/blender machine (Philips to rehydrate the contents. The liquids with the
HR2116/00, Netherlands) to obtained blended bacteria were mixed with 5 ml of liquid medium
juice extract. While the second method after the in the tube and inoculated into an enriched blood
rind was peeled off, the flesh was squeezed using agar medium. All procedures were performed in
a fruit press machine (Calypso, Indonesia) and an anaerobic atmosphere. The liquid and blood
then both preparations were filtered to obtained a agar media were incubated at 37°C for 14 days
pulp-free juice liquid. The pure blended and in anaerobic conditions. After 7 and 14 days,
squeezed juices were diluted in distilled water into pure colonies of black-pigmented P. gingivalis
concentrations of 100%, 50%, and 25%. and P. endodontalis in a blood agar medium
were obtained, respectively. All bacterial
Cytotoxicity Assay suspensions were prepared according to the 0.5
Human gingival fibroblasts (HGFs) were cultured McFarland standard, then incubated at 37°C for
and incubated in a 5% CO2 atmosphere in an 24 h. The suspensions of P. gingivalis and P.
incubator (80% O2 and 20% N2) at 37°C. After the endodontalis were then re-diluted in a liquid
HGFs reached a confluent form, they were treated brain heart infusion medium until a concentration
with the prepared blended and hand-squeezed of 1.5 x 108 CFU/ml was reached.
juice extracts. One group was treated with 50 μl
of each concentration. Two groups used as Biofilm Assay
positive controls were treated with 50 μl of 2% P. endodontalis and P. gingivalis biofilms
chlorhexidine and 50 μl of 2.5% NaOCl. Another inhibition assays were performed by culturing
group, used as a negative control, was treated 200uL of 0.5 McFarland of P. endodontalis
with a Dulbecco’s Modified Eagle’s Medium (ATCC 35406D-5) and P. gingivalis (ATCC
(DMEM) (Sigma-Aldrich Pte Ltd, Singapore). The 35406D-5) in flat-bottom polystyrene 96-well
assay was performed in triplicate for each plates along with treatments (blended or

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Nurani Hayati et al / Effectiveness of Grapefruit (Citrus Paradisi) And Lime (Citrus Aurantifolia) Against
Pathogenic Root Canal Biofilms

squeezed juices with different concentrations) wavelength of 595 nm.

together and incubated at 37°C for 48 h. While
for the eradication assay of P. endodontalis and Data Analysis
P. gingivalis biofilms, bacteria were incubated at The data normality distribution was tested using
37°C for 48 h and exposed to the treatments the Saphiro-Wilk normality test. The data were
(blended or squeezed juices with different analyzed using two-way analysis of variance and
concentrations) for 15 min incubation time. The Tukey’s post-hoc significant difference test. A
inhibition and eradication effects of the blendeed value of P < 0.05 was considered statistically
and squeezed juices of the two citrus were significant. All statistic data were analyzed using
analyzed using a crystal violet biofilm assay. SPSS.16 software.
Biofilm cells were harvested by removing the
culture medium and rinsing each well three times RESULTS AND DISCUSSION
with 0.2 ml of phosphate-buffered saline (PBS) to Cytotoxicity of the Treatments
remove non-adhesive bacteria and Crystal violet The viability measurements based on the
(0.5% w/v) was added into all 96-well plate and absorbance value of living cells showed that the
incubated for 15 min and then removed. To each living HGFs treated with blended and squeezed
of the well plates, 200 µL of absolute ethanol was juices of C. paradisi and C. aurantifolia exhibited
added, and well plate was measured as biofilm viability above 90%, which was significantly
mass. Chlorhexidine gluconate (0.2%) and higher than the viability of those treated with
NaOCl (2.5%) was used as a positive control and 2.5% sodium hypochlorite (24.92%) and 2%
wells without treatment as a negative control. The chlorhexidine gluconate (76.1%; P < 0.05; Figure
OD values were determined using microplate- 1).
spectrophotometer (SAFAS MP96, Monaco) at a

Fig. 1: Human Gingival Fibroblast (HGF) Viability after Exposure to All Juice Treatments, 2%
Chlorhexidine, and 2.5% NAOCl for 24 H. All Citrus Paradisi and Citrus Aurantifolia Treatments
Resulted in HGF Viability of >90%, Whereas the Viability Values for The 2% Chlorhexidine and
2.5% NAOCl Treatments Were 76.1% and 24.92%, Respectively

Quantitative Phytochemical Test Result of the (99.47%), and quercetin (44.65%; Table 1). The
Extracted Juices on Citrus Paradisi same main flavonoids were identified in 50 ml of
Phytochemical analysis of 50 ml squeezed juice blended juice: hidrosmin (48.82%), hesperidin
of C. paradisi identified several flavonoids, (80.13%), naringenin (59.87%), naringin
including hidrosmin (57.73%), hesperidin (99.94%), and quercetin (42.65%) (Table 2).
(63.57%), naringenin (59.87%), naringin

Table 1: Flavonoids Identified in C. Paradisi Squeezed Juice

No. RT (%) Name MF MW (g/mol)
1. 4.35 57.73 Hidrosmin C30H36O16 652,6
2. 0.48 63.57 Hesperidin C28H34O15 610,1898
3. 2.22 59.87 Naringenin C15H12O5 272.25
4. 7.51 99.47 Naringin C27H32O14 580,5
5. 7.76 44,65 Quercetin C15H10O7 302,236
* RT=Retention time, MF=Molecular formula, MW=Molecular weight

Table 2: Flavonoids Identified in C. Paradisi Blended Juice

No. RT (%) Name MF MW (g/mol)

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Pathogenic Root Canal Biofilms

1. 4.36 48.82 Hidrosmin C30H36O16 652,6

2. 7.67 80.13 Hesperidin C28H34O15 610,1898
3. 0.48 59.87 Naringenin C15H12O5 272.25
4. 2.21 99.84 Naringin C27H32O14 580,5
5. 3.41 42.65 Quercetin C15H10O7 302,236
* RT=Retention time, MF=Molecular formula, MW=Molecular weight

Quantitative Phytochemical Test Result of the (68.77%; Table 3). The same main flavonoid was
Extracted Juices on Citrus Aurantifolia also identified in 50 ml of blended juice:
Phytochemical analysis of 50 ml squeezed juice hesperidin (99.79%), naringin (83.01%),
of C. aurantifolia also identified several eriocitrin (80.95%), and diosmin (68.02%) (Table
flavonoids: hesperidin (99.88%), naringin 4).
(82.97%), eriocitrin (81.13%), and diosmin

Table 3: Flavonoids Identified in C. Aurantifolia Squeezed Juice

No. RT (%) Name MF MW (g/mol)
1. 2.99 81.13 Eriocitrin C30H36O16 596.5
2. 3.41 99.88 Hesperidin C28H34O15 610.1898
3. 2.93 68.77 Diosmin C15H12O5 608.5
4. 2.79 82.97 Naringin C27H32O14 580,5
5. 2.99 81.13 Eriocitrin C30H36O16 596.5
* RT=Retention time, MF=Molecular formula, MW=Molecular weight

Table 4: Flavonoids Identified in C. Aurantifolia Blended Juice

No. RT (%) Name MF MW (g/mol)
1. 3.65 80.95 Eriocitrin C30H36O16 596.5
2. 6.55 99.79 Hesperidin C28H34O15 610.1898
3. 3.64 68.02 Diosmin C15H12O5 608.5
4. 4.69 83.01 Naringin C27H32O14 580,5
5. 3.65 80.95 Eriocitrin C30H36O16 596.5
* RT=Retention time, MF=Molecular formula, MW=Molecular weight

Biofilm Assay Result of Porphyromonas values after exposure for 48 h are shown in
Endodontalis Figure 2. The lowest value was obtained from the
The inhibition test revealed that the P. pure (100%) blended juice extract of C. paradisi
endodontalis biofilm’s OD values after exposure (0.003±0.001), and most of the treatments had
to all C. paradisi and C. aurantifolia juice significantly lower value than chlorhexidine as
treatments were significantly lower than that of positive control (0,352±0,111).
the negative control (P < 0.05). The inhibition

Fig. 2: Concentration-Response Graphs P. Endodontalis after Treatment with Blended and

Squeezed C. Paradisi and C. Aurantifolia for 48 H (Inhibition Test). The Vertical Axis of Panels
Indicates the P. Endodontalis Biofilm Mass Optical Density (OD). the Horizontal Axis Indicates the
Concentration of C. Paradisi and C. Aurantifolia (100%, 50% And 25%). Chlorhexidine Gluconate

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Nurani Hayati et al / Effectiveness of Grapefruit (Citrus Paradisi) And Lime (Citrus Aurantifolia) Against
Pathogenic Root Canal Biofilms

(2%) and Naocl (2.5%) Were Used as a Positive Control and Wells Without Treatment as a
Negative Control. All Treatments Were Done in Triplicate.
*p < 0.05 **p < 0.01

The eradication test after exposure for 15 minutes chlorhexidine positive control (P < 0.05). While
revealed that the OD values after exposure to all the lowest value obtained from NaOCl solution
treatments were also significantly lower than as positive control (0,0556±0,00251) (Figure 3).
those of the negative control and the

Fig. 3: Concentration-Response Graphs P. Endodontalis after Treatment with Blended and

Squeezed C. Paradisi and C. Aurantifolia for 15 Min (Eradication Test). The Vertical Axis of Panels
Indicates the P. Endodontalis Biofilm Mass Optical Density (OD). The Horizontal Axis Indicates the
Concentration of C. Paradisi and C. Aurantifolia (100%, 50% And 25%). Chlorhexidine Gluconate
(2%) and Naocl (2.5%) Were Used as a Positive Control and Wells Without Treatment as a
Negative Control. All Treatments Were Done in Triplicate.
*p < 0.05 **p < 0.01

Biofilm Assay Result of Porphyromonas at a 50% concentration (0.025±0.032). Most

Gingivalis juice treatments had significantly lower OD values
The inhibition test revealed that the OD values of than the chlorhexidine positive control
P. gingivalis biofilms after exposure to all C. (0,391±0,161) at P < 0.05, except for the C.
paradisi and C. aurantifolia juice treatments were aurantifolia blended juices at a 50%
also significantly lower than that of the negative concentration (0,227±0,089) and C. paradisi
control (P < 0.05). The OD values after exposure squeezed juice at a 25% concentration
for 48 h are shown in Figure 4. The lowest value (0,221±0,053).

was obtained from the C. paradisi blended juices

Fig. 4: Concentration-Response Graphs P. Gingivalis after Treatment with Blended and Squeezed C.
Paradisi and C. Aurantifolia for 48 H (Inhibition Test). The Vertical Axis of Panels Indicates the P.
Gingivalis Biofilm Mass Optical Density (OD). The Horizontal Axis Indicates the Concentration of C.

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Nurani Hayati et al / Effectiveness of Grapefruit (Citrus Paradisi) And Lime (Citrus Aurantifolia) Against
Pathogenic Root Canal Biofilms

Paradisi and C. Aurantifolia (100%, 50% And 25%). Chlorhexidine Gluconate (2%) and Naocl
(2.5%) Were Used as a Positive Control and Wells Without Treatment as a Negative Control. All
Treatments Were Done in Triplicate.
*p < 0.05 **p < 0.01
C. paradisi and C. aurantifolia juice treatments squeezed juice at a 25% concentration (0.077 ±
were also significantly lower than that of the 0.01538). However, no juice treatment’s OD
negative control (P < 0.05). The OD values after value was significantly different from those of the
exposure for 15 min are shown in Figure 5. The chlorhexidine and NaOCl positive controls.
lowest value was obtained from C. paradisi

Fig. 5: Concentration-Response Graphs P. Gingivalis after Treatment with Blended and Squeezed C.
Paradisi and C. Aurantifolia for 15 Min (Eradication Test). The Vertical Axis of Panels Indicates the
P. Gingivalis Biofilm Mass Optical Density (OD). The Horizontal Axis Indicates the Concentration of
C. Paradisi and C. Aurantifolia (100%, 50% And 25%). Chlorhexidine Gluconate (2%) and Naocl
(2.5%) Were Used as a Positive Control and Wells Without Treatment as a Negative Control. All
Treatments Were Done in Triplicate.
*p < 0.05 **p < 0.01

Citrus fruits have low toxicity, are safe for human juice, include hydrosmin, hesperidin, naringenin,
tissues, and exert various therapeutic activities naringin and quercetin. Meanwhile, four highest
[21]. Thus, they show potential as components of flavanone glycosides were also analyzed in
alternative irrigation solutions [8]. This study blended and squeezed C. aurantifolia juice, they
evaluated the cytotoxicity of different were hesperidin, naringin, eriocitrin, and diosmin.
concentrations of the juices of two citrus fruits, C. This result is consistent to the study conducted by
paradisi and C. aurantifolia, to HGFs by Kanaze et al., who carried out a quantitative test
calculating HGF viability through measuring the of flavonoid glycosides on C. paradisi using hand
optical density of the living cells. All the citrus squeezed preparations. In their research they
groups in this study showed no toxicity towards analyzed three types of flavonoid glycosides,
human gingival fibroblast cells, which is the most namely naringin, hesperidin and diosmin,
important cells in periodontal area [8]. This was whereas naringin had the largest concentration of
showed by the average viability value of each 584.7 µg/ml; and diosmin has the smallest
preparation has higher value than NaOCl and concentration of 1.9 µg/ml [24]. However,
chlorhexidine as positive controls which were diosmin was not detected in this study. These
considered to be more toxic against gingival discrepancies between studies may be due to
fibroblast compared to both citrus since it has geographic differences of the plant sources,
viability value less than 90%. According to storage time, and method of sample preparation.
Konjhodzic-Prcic et al., a substance is considered In other hand, this result of the study also
nontoxic when cell viability is 90% or higher, consistent with previous study by Ghafar et.al,
slightly toxic when viability is 60–90%, moderately who analyzed hesperidine as the highest
toxic when viability is 30–59%, and very toxic flavanoid in C. aurantifolia [25].
when viability is less than 30% [22]. Several In this study, C. paradisi blended juice
phytochemical components, such as glucose preparations with concentrations of 100% and
molecules from tannins or flavonoids, are 50% had the lowest OD value in the P.
associated with high viability [23]. endodontalis and P. gingivalis biofilm inhibition
In this study, five flavonoid compounds were test and all C. paradisi preparations were
identified in blended and squeezed C. paradisi significantly different with negative control (P

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Pathogenic Root Canal Biofilms

<0.005) and control positive chlorhexidine 75%, and 100% inhibits the growth of S. aureus
solution 2%, but not significantly different from biofilms [30]. Another study conducted by Lee
NaOCl solution which also acted as a positive et.al., have also found evidence of the
control (P >0.05). These indicate that C. paradisi antibacterial and antifungal properties of
juice preparations at concentrations of 100% and compounds contained in C. aurantifolia,
50% have significantly higher inhibitory ability including flavonoids [31].
than chlorhexidine solution but not significantly Gram-negative black-pigmented anaerobic
different from the NaOCl solution 2.5% against P. bacteria are predominant microorganisms in the
endodontalis and P. gingivalis biofilm. root canal with pulp necrosis and periapical
Meanwhile, the results of the C. paradisi lesions. P. endodontalis and P. gingivalis are
eradication test against P. gingivalis biofilm black-pigmented bacteria species commonly
showed that the blended juice preparation with found in root canals [3]. The growth of bacteria as
concentration of 100% had the lowest OD value biofilms shows a significant reduction in sensitivity
after squeezed juice preparation with to antibiotics and antimicrobial agents, including
concentration of 100% but did not differ those found in toothpaste and mouthwash
significantly from the two positive control, 2% [11,32]. Root canal irrigation, which commonly
chlorhexidine solution and 2.5% NaOCl (P used in dentistry, is still having issues with its
>0.05). This suggests that all C. paradisi toxicity such as NaOCl irrigation which may
preparations have the same level of effectiveness cause a burning reaction on gingiva and mucosa
as 2% chlorhexidine and 2.5% NaOCl solutions to necrosis of the periapical tissue [20,33-34]
in eradicating P. gingivalis biofilm. Flavonoids exert their antibacterial activity by
In biofilm eradication study, P. endodontalis and inhibiting nucleic acid synthesis, destroying the
P. gingivalis biofilms were exposed to C. paradisi cytoplasmic membrane by perforation and
and C. aurantifolia blended and squeezed juices reduction of membrane fluids, and reduction of
for 15 min, according to Kim et al. who found metabolic energy [35-36]. Flavonoid compounds
that antimicrobial agents required at least 10 in citrus fruits act against bacteria by denaturing
minutes to attach to bacterial cell walls and their proteins and destroying their cytoplasmic
destroy biofilms [26]. Our results regarding the membranes, thereby disrupting their selective
inhibition and eradication of P. endodontalis and permeability, active transport functions, and
P. gingivalis biofilms are consistent with another control of cell protein composition [37]. The
study conducted by Okungbowa et al, which disruption of cytoplasmic integrity causes the
stated that C. paradisi acts as antibacterial agent removal of molecules and ions from the cells,
against several bacteria, such as Salmonella spp., which consequently lose their shape, resulting in
Streptococcus mutans, and Staphylococcus aureus lysis [30]. Flavonoids can also prevent biofilm
[27]. Heggers et al. similarly reported that formation by inhibiting early bacterial adhesion to
grapefruit seed extracts exert significant activity the root canal surfaces, thus preventing the
against Gram-positive and Gram-negative formation of extracellular polymeric substances,
bacteria after exposure for 15 min by destroying which protect biofilms against antimicrobial
the cytoplasmic membrane of bacterial cells via agents, and inhibiting the diffusion and chemical
inhibition of their enzymatic activity [28]. This may interactions of polysaccharides and extracellular
explain the higher antibacterial efficacy of the C. proteins of microorganisms [35,38]. According to
paradisi blended juice compared to the squeezed Vikram et al., flavonoids contained in citrus fruits,
juice preparations, as the mixture of blended juice such as naringin and hesperidin, act as quorum-
also contained the seeds. Peterson et al. reported sensing inhibitors by preventing interactions
that C. paradisi contains high amounts of between acyl-homoserine lactones (the signaling
flavones, flavonols, and flavanones (especially molecules in Gram-negative bacteria) and their
naringin) and other compounds, such as receptors, thereby inhibiting biofilm formation
monoterpenes, sesquiterpenes, carotenoids, [39]. Quorum sensing is a phase of biofilm
pectin, vitamins B1 and C, and mineral salts [29]. growth when bacteria release signaling molecules
The activities of these components include in their local environment, inducing responses
destroying bacterial membrane cells, inhibiting such as changes in the expression of specific
amino acid synthesis and respiration processes, genes once they reach a maximum concentration
and causing leakage of cytoplasmic components [40-41]. It modulates gene expression for
[18]. antibiotic resistance, facilitates the growth of
This study also supports the findings of previous species which beneficial to biofilms, and inhibits
studies on the antibacterial activity of C. the growth of competitors [42-43]. Quercetin is
aurantifolia. Razak and Djamal found that C. also known to inhibit biofilm formation by
aurantifolia juice at concentrations of 25%, 50%, reducing the formation of glucans and the

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Pathogenic Root Canal Biofilms

expression of several virulent genes in S. mutans, treatment. Interbational Journal of Applied

thus inhibiting the formation of glucosyl Dental Sciences, 2019; 5(3): 366-369.
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The limitations of this study is the in vitro design Moeschberger ML, Leys EJ. New bacterial species
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compare the results of both studies. Also, future
root canal infection in apical periodontitis and its
studies should isolate and compare with other
relationship with clinical symptomatology. Brazil
active compounds of C. aurantifolia and C.
Oral Research. 2018; 32: 82-110.
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period, and additional studies tested on animals CD14-mediated signal pathway of
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in actual oral conditions. Immunity, 1996; 64(11): 4488-4494.
9. Ferraz CCR, Diogenez A, Henry MA, Hargreaves
CONCLUSION KM. LPS from Porphyromonas gingivalis
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all C. paradisi and C. aurantifolia blended and Journal of Endodontic, 2011; 37(1): 45-48.
squeezed juice concentrations were nontoxic to 10. Ma N, Yang D, Okamura H, et al. Involvement of
HGF, as indicated by viability values of over 90%. interleukin-23 induced by Porphyromonas
This study demonstrates the promising antibiofilm endodontalis lipopolysaccharide in
activity of C. paradisi and C. aurantifolia against osteoclastogenesis. Molecular Medicine Reports,
P. endodontalis and P. gingivalis. This antibiofilm 2017; 15(2): 559-566.
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