Strong antimicrobial activity of collinin and isocollinin against periodontal and superinfectant pathogens in vitro

Article abstract of DOI:10.1016/j.anaerobe.2020.102163

Periodontitis pathogenesis involves activation of host immune responses triggered by microbial dysbiosis. Therefore, controlling periodontal pathogens in-vivo is a main goal of periodontal therapy. New antimicrobials might help to control periodontal infection and improve treatment outcomes at “the dark times” of increasing antibiotic resistance. Here, we determined the biological activity of collinin and isocollinin against 8 bacterial strains. Antimicrobial activity of collinin and isocollinin, chlorhexidine digluconate (CHX) and sodium hypochlorite (NaClO) was evaluated against clinically relevant periodontal bacteria, like Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia, Dialister pneumosintes strains and superinfectants like Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa strains. A broth microdilution test was carried out to determine the minimum inhibitory concentration of collinin and isocollinin against those strains, and bacterial viability was determined by resazurin assay at diverse concentration and exposure times. P. gingivalis was the most susceptible strain to collinin and isocollinin (MIC 2.1 μg/mL and 4.2 μg/mL respectively). Other periodontal pathogens showed MICs <17 μg/mL for collinin and MICs between 20 and 42 μg/mL for isocollinin, whereas CHX and NaClO showed MICs of 62 and 326 μg/mL, respectively. Collinin and isocollinin also exhibited antimicrobial activity against superinfectant bacteria (MIC < 21 and < 42 μg/mL, respectively). Overall, collinin and isocollinin showed a remarkable antibacterial activity against relevant periodontal and superinfective bacteria, especially against P. gingivalis (MIC 2.1 μg/mL and 4.2 μg/mL respectively) and the highly virulent P. aeruginosa (MIC 5.2 and 20.8 μg/mL, respectively).

Full text of DOI:10.1016/j.anaerobe.2020.102163

Anaerobe 62 (2020) 102163
Contents lists available at ScienceDirect
Anaerobe
journal homepage: www.elsevier.com/locate/anaerobe
Antimicrobial susceptibility of anaerobic bacteria
Strong antimicrobial activity of collinin and isocollinin against
periodontal and superinfectant pathogens in vitro
a
b
a
b, *
~
ꢀ
~
Camilo Pardo-Castano , Daniel Vasquez , Gustavo Bolanos , Adolfo Contreras
a Applied Thermodynamic Research Group, School of Chemical Engineering, Universidad del Valle, 760032, Cali, Colombia
b Periodontal Medicine Research Group, School of Dentistry, Universidad del Valle, 760043, Cali, Colombia
a r t i c l e i n f o
a b s t r a c t
Article history:
Periodontitis pathogenesis involves activation of host immune responses triggered by microbial dys-
biosis. Therefore, controlling periodontal pathogens in-vivo is a main goal of periodontal therapy. New
antimicrobials might help to control periodontal infection and improve treatment outcomes at “the dark
times” of increasing antibiotic resistance. Here, we determined the biological activity of collinin and
isocollinin against 8 bacterial strains. Antimicrobial activity of collinin and isocollinin, chlorhexidine
digluconate (CHX) and sodium hypochlorite (NaClO) was evaluated against clinically relevant peri-
odontal bacteria, like Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Fusobacterium
nucleatum, Prevotella intermedia, Dialister pneumosintes strains and superinfectants like Escherichia coli,
Staphylococcus aureus, and Pseudomonas aeruginosa strains. A broth microdilution test was carried out to
determine the minimum inhibitory concentration of collinin and isocollinin against those strains, and
bacterial viability was determined by resazurin assay at diverse concentration and exposure times.
Received 25 November 2019
Received in revised form
16 January 2020
Accepted 20 January 2020
Available online 21 January 2020
Keywords:
Microbial-sensitivity-test
Periodontal-pathogens
Collinin
Coumarins
Chlorhexidine
P. gingivalis was the most susceptible strain to collinin and isocollinin (MIC 2.1
respectively). Other periodontal pathogens showed MICs <17 g/mL for collinin and MICs between 20
and 42 g/mL for isocollinin, whereas CHX and NaClO showed MICs of 62 and 326 g/mL, respectively.
Collinin and isocollinin also exhibited antimicrobial activity against superinfectant bacteria (MIC < 21
and < 42 g/mL, respectively). Overall, collinin and isocollinin showed a remarkable antibacterial activity
against relevant periodontal and superinfective bacteria, especially against P. gingivalis (MIC 2.1 g/mL
and 4.2 g/mL respectively) and the highly virulent P. aeruginosa (MIC 5.2 and 20.8 g/mL, respectively).
mg/mL and 4.2 mg/mL
m
m
m
m
m
m
m
© 2020 Elsevier Ltd. All rights reserved.
1. Introduction
tooth mobility are typical features of periodontal disease, which are
caused by the activation of immune response, triggered by peri-
odontal pathogens and virulence factors [3,4]. Possibly, these well
described signs and symptoms are result of undetermined genetic
susceptibility, early microbial dysbiosis and immune system dys-
regulation [5].
Significant increase in the proportion of important pathogenic
microorganisms of periodontal lesions have been reported by
cultivation and molecular identification methods [3,6,7]. The most
important periodontal pathogens are Aggregatibacter actino-
mycetemcomitans, Porphyromonas gingivalis, Treponema denticola,
Fusobacterium nucleatum, Tannerella forsythia, Eikenella corrodens,
Dialister pneumosintes and new species such as Filifactor alocis
among other unculturable species as well as opportunistic species
from family Enterobacteriaceae [6]. In 1988 Slots and collaborator’s
first reported enteric rods such as Pseudomonas and Candida species
in severe periodontitis and then, detected Staphylococcus aureus in
periodontitis demonstrating the importance of opportunistic
Worldwide preventable oral diseases, such as caries and peri-
odontitis, affect four billion people, generating eating problems and
pain, reducing quality of life, increasing Disability-Adjusted Life
Year (DALYs) and increasing health systems costs related to disease
treatment [1,2].
These prevalent diseases are mainly produced by microbial
dysbiosis, caused by proliferation of pathogenic microbiota and
concomitant suppression of beneficial species, which could cause
severe damage in oral tissues homeostasis [3]. Gingival inflamma-
tion, increased bleeding on probing, apical migration of junctional
epithelium, detach of gingival tissues from root surfaces, pocket
formation, gingival recession, alveolar bone loss, and increased
* Corresponding author.
E-mail address: adolfo.contreras@correounivalle.edu.co (A. Contreras).
https://doi.org/10.1016/j.anaerobe.2020.102163
1075-9964/© 2020 Elsevier Ltd. All rights reserved.

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C. Pardo-Castano et al. / Anaerobe 62 (2020) 102163
2
pathogens in oral disease [8,9] which carry important virulence
factors and are recalcitrant to elimination/control by mechanical
therapy.
It has been shown that routine mechanical removal of bacterial
biofilm by brushing and flossing should promote oral health and
prevent caries and periodontal disease and is the most valuable
antimicrobial approach when rigorously practiced [7]. However,
most people do not exercise proper and meticulous biofilm control
due to diverse reasons, including poverty, limited oral health care
access, lack of access to oral care technology, lack of conscience and
motivation and poor oral education [10].
Chemical control of oral biofilm is a promising tool to prevent
and to treat oral disease [11,12]. Currently, several chemical com-
pounds are been used to control pathogenic members of the oral
microbiota. However, oral microbes are showing increased resis-
tance against antibiotics and medicated rinses. Thus, new prom-
ising products from natural sources are being tested for more
effective oral biofilm control [12].
A natural coumarin derivative so-called collinin has shown
important activity against a key periodontal pathogen such as
P. gingivalis [13], and has been successfully tested against several
other pathogenic and prevalent diseases [14e20]. However, there is
still scarce evidence to support its antimicrobial activity in peri-
odontal disease and against superinfectant bacteria.
In this work, collinin and isocollinin were synthesized, purified
and characterized following reported methods [21]. The antimi-
crobial activity of both isomers was tested against pathogens of
medical and oral clinical interest, in addition bacterial susceptibil-
ity, cell viability and time-kill assays were evaluated.
Fig. 1. Chemical structures of (a) 7-methoxy 8-geranyloxy coumarin (Isocollinin), and
(b) 7-geranyloxy 8-methoxy coumarin (Collinin).
additional recrystallization procedure (hexane/ethanol 96% v/v)
due to the presence of impurities, including the raw material that
was not consumed. A stock solution of isocollinin and collinin
dissolved in ethanol (100
vials.
m
g/mL both) were stored at 4 ꢀC in amber
Two commonly used biocides, chlorhexidine digluconate (CHX)
(PerioGard, Colgate, DF, Mexico) and sodium hypochlorite (NaClO)
(Blancox, Bogota, Colombia), were tested to compare the perfor-
mance of both collinin isomers. Stock solutions of CHX and NaClO
2. Materials and methods
were prepared at 1,200
sterile water.
mg/mL and 50,000 mg/mL, respectively, in
2.1. Preparations of collinin isomers and reference biocides
Briefly, collinin and isocollinin (Fig. 1) were synthesized by first
producing the chemicals daphnetin and geranyl bromide, which
were obtained by two parallel reactions. First, pyrogallol (Alfa
Aesar, Tewksbury, MA, USA), propiolic acid (Alfa Aesar, Tewksbury,
MA, USA) and sulfuric acid (Merck, Darmstadt, Germany) were
stirred at 125 ꢀC for 50 min. Daphnetin was isolated from the re-
action product as a brown color solid after several purification
steps, which are presented in Ref. [21]. On the other hand, geraniol
(Alfa Aesar, Tewksbury, MA, USA), phosphorus tribromide (Alfa
Aesar, Tewksbury, MA, USA), and diethyl ether (Merck, Darmstadt,
Germany) were stirred at 0 ꢀC for 3 h, in a light-isolated flask under
N2 (Cryogas INDURA group, Cali, Colombia) atmosphere. Geranyl
bromide was obtained as a golden oil once purified from the re-
action crude according to Ref. [21].
Geranyl bromide was then added to daphnetin in acetone (Alfa
Aesar, Tewksbury, MA, USA) and DBU (Merck, Darmstadt, Ger-
many), and stirred at 28 ꢀC for 5 h. The resulting crude was purified
according to Ref. [21]. Thus, three main products were obtained:
one dialkylated compound, which was separated as an oil using
solvent extraction, and two alkylated compounds separated by
using column chromatography, to obtain the chemical precursors of
collinin and isocollinin as white solids. The isocollinin precursor, 7-
hidroxy 8-geranyloxy coumarin, was added to a suspension of so-
dium hydride (Alfa Aesar, Tewksbury, MA, USA) in THF (Merck,
Darmstadt, Germany) at 0 ꢀC, under N2 (Cryogas INDURA group,
Cali, Colombia) atmosphere, and the resulting solution was stirred
at 28 ꢀC for 2 h. Iodomethane was then added and the solution was
continuously stirred until the precursor was totally consumed (ca.
16 h). Isocollinin was obtained as a white solid after several puri-
fication steps described in Ref. [21], A similar procedure was also
performed to obtain Collinin as a white solid. Collinin required an
2.2. Bacterial strains and culture conditions
Reference strains of Escherichia coli ATCC 25922, Staphylococcus
aureus ATCC 6538, Pseudomonas aeruginosa ATCC 27853, and
strains of A. actinomycetemcomitans ATCC 29522, P. gingivalis ATCC
33277, F. nucleatum ATCC 25586, P. intermedia ATCC 25611, and
Dialister pneumosintes ATCC 33048, were cultivated in BHI broth
medium (Sharlau, Barcelona, Spain) with 0.0005% hemin (Sigma
Aldrich, St. Louis, MO, USA). Anaerobic bacteria were cultured in
anaerobic jars (Oxoid, AnaeroJar 2.5L, Hampshire, UK) (1% O2, 9%
CO2) and A. actinomycetemcomitans was grown under micro-
aerophilic conditions (5% CO2), at 37 ꢀC for 48 h, whilst aerobic
strains were cultured at 37 ꢀC during 24 h. The initial inoculum for
the susceptibility assays was prepared in sterile PBS (Thermo Fisher
Scientific, Gibco, Paisley, Scotland, UK) and adjusted to reach optical
density (OD) of 0.08 at 600 nm (Biochrom, Ultrospec-10, Cam-
bridge, UK) equivalent to 0.05 on the McFarland standard
(1.5 ꢁ 108 CFU/mL). This standard inoculum was diluted 1:10 in BHI
broth for all assays.
2.3. Bacterial susceptibility assay
The MIC of each agent was determined following the guidelines
of the Clinical and Laboratory Standards Institute (CLSI) with some
modifications according to the nutritional characteristics of each
specie [22]. Serial two-fold dilutions of biocides (CHX 1,200 to
2.3
mg/mL), (NaClO 50,000e97
mg/mL) and (collinin and isocollinin,
100 to 0.2
mg/mL) were prepared in culture media supplemented
with BHI and mixed in wells of 96-well plates. Positive control wells
were used, containing bacteria but not the biocide and also,

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3
negative control wells with CHX at 1,200
inoculum.
m
g/mL without bacterial
NaClO presented the lowest antimicrobial activity against all bac-
terial strains tested (MIC ꢂ 300 g/mL) (data not shown).
P. gingivalis was the most resistant strain to CHX (MIC 62.9
mL), while it showed significantly more sensitivity to collinin and
isocollinin (MIC 2.1 g/ml and 4.2 g/ml, respectively). In addition,
the other periodontal bacteria showed high sensitivity to collinin
(MIC < 17 g/mL), while it was lower with isocollinin and CHX
(20 < MIC < 42 and MIC ¼ 31.0 g/mL, respectively). Whereas,
superinfectant strains showed a similar sensitivity to collinin and
CHX (MIC < 20 g/mL), while these were less sensitive to isocollinin
(MIC > 20 g/mL).
m
Cultures were incubated at 37 ꢀC for 48 h under conditions
previously described. Bacterial growth was monitored measuring
the OD at 600 nm in a spectrophotometer (NanoDrop One, Thermo
Fisher, Madison, WI, USA). The MIC was taken as the lowest con-
centration of biocide that prevents visible growth and reduces
absorbance up to 0.04 or less.
mg/
m
m
m
m
2.4. Cell viability
m
m
The nonviable cells lose rapidly the metabolic capacity to reduce
resazurin into resorufin (fluorescent dye). An assay based on this
biological activity was used to assess cell viability at different times
(CellTiter-Blue Cell Viability Assay, Promega, Madison, WI, USA).
Briefly, the procedure consisted in adding the single reagent
Fig. 2 presents the antimicrobial effect of collinin isomers and
CHX against periodontopathic and superinfective pathogens.
Overall, the differences between the effect of collinin and CHX were
statistically significant, considering both the effect of those two
biocides on all bacteria strains (p < 0.0001) and between strains
(p < 0.001). In the case of isocollinin and CHX, the differences be-
tween the effect of biocides were not statistically significant
(p ¼ 0.9852), while between strains were significant (p < 0.1). There
were differences in the antimicrobial activity of both isomers.
Collinin showed better antimicrobial activity against peri-
odontal pathogens than CHX, and differences in their performance
were statistically significant (p < 0.04), except against P. intermedia
(p ¼ 0.2683). On the other hand, the effect of collinin and CHX over
superinfectant strains did not showed significant differences.
Moreover, isocollinin showed a similar antimicrobial activity to that
performed by CHX, except against P. gingivalis and S. aureus, which
were higher and statistically significant for isocollinin (p < 0.0001)
and CHX (p < 0.001), respectively. Finally, comparing the perfor-
mance of both isomers, they showed a similar antimicrobial activity
against all tested bacteria, except for D. pneumosintes, A. actino-
mycetemcomitans and S. aureus, which exhibited more suscepti-
bility to collinin (p < 0.001).
directly to cells cultured in 100 ml of BHI broth and placed in a 96-
well plate (Costar, Corning, NY, USA), the intensity of the color
produced is directly proportional to the number of viable cells.
After a 3-h incubation under anaerobic or aerobic conditions
depending on the specie, absorbance was measured using a plate-
reading fluorometer (Stat Fax 2100 Microplate Reader, Palm City, FL.
USA).
Minimal Bactericidal Concentration (MBC) was the lowest
biocide concentration at which the well showed no capacity to
reduce resazurin, that is, no color change was observed. The
absence of bacterial growth was confirmed in new agar cultures
with the optimal growth conditions. At MBC, no bacterial growth
was detected.
2.5. Statistical analyses
All experiments were performed in triplicate. Quantitative data
is presented as means with standard deviations (SD), and qualita-
tive data is presented as proportions or percentages. The means and
SD were compared using a two-way ANOVA followed by Sidak’s
multiple comparison post hoc test using statistics software (Prism,
version 7.0, GraphPad, San Diego, CA, USA). Values with p < 0.05
were considered statistically significant.
3.3. Cell viability assay
The MBC values obtained in the cell viability assay were similar
to the MIC values presented in Table 1. However, these results
showed that collinin isomers were more bactericidal over peri-
odontal pathogens than CHX. By contrast, CHX was more bacteri-
cidal over superinfective strains than the collinin isomers.
Particularly, P. gingivalis was the most susceptible strain against
3. Results
3.1. Synthesis and identification of collinin isomers
both isomers (MBC 5.2 and 10.4
CHX (MBC 62.7 g/mL). While E. coli was more resistant to either
collinin isomers (MBC 33.3 g/mL). E. coli resulted more susceptible
to CHX (MBC 15.7 g/mL) (Fig. 3).
The time-kill assay used E. coli as a reference strain to perform
all tests, given that it showed more resistance against both collinin
isomers. With concentrations of 50 mg/mL of isocollinin, E. coli was
killed even after a 24-h follow-up; and at any lower concentration
of isocollinin, E. coli remained viable (Fig. 4). Collinin showed a
better performance to kill E. coli since 25 mg/mL were enough to
control bacterial viability. At lower concentrations, it was observed
a significant reduction in the bacterial growth; however, after 8-h
follow-up, an exponential rising in bacterial population was
detected, and E. coli was viable, confirmed by the resazurin assay
and agar sub-cultivation.
mg/mL), and the most resistant to
m
From 1 g of pyrogallol, 140 mg of collinin and 550 mg of iso-
collinin were obtained, which correspond to overall molar yields of
2.6 and 13.9%, respectively. Both isomers were characterized by
nuclear magnetic resonance (1H-NMR and 13C-NMR) and differ-
ential scanning calorimetry (DSC). Additional 1D NOESY NMR ex-
periments were performed with the precursors of collinin and
isocollinin, to verify the identity of each isomer. Purities of 98.7 and
98.0 0.2% for isocollinin and collinin were estimated by DSC, ac-
cording to the methodology described in the ASTM E928 standard
(ASTM, 2014) [23]. Further information about the synthesis and
characterization procedures of these compounds are reported
elsewhere [21].
m
m
3.2. Bacterial susceptibility and cell viability assays
4. Discussion
Table 1 presents the antimicrobial activity of different chemical
agents against bacteria of clinical importance. Clorhexidine
digluconate (CHX) and sodium hypochlorite (NaClO) were used to
compare the antimicrobial effect of the collinin isomers. Overall,
and considering the standard deviation, periodontophatogens were
more susceptible to collinin and isocollinin than to CHX and NaClO.
In this study, antimicrobial activity in vitro of collinin and
isocollinin was tested against individual microbial strains, such as
five important periodontal pathogens - ATCC strains and another
three important bacteria of medical interest. Collinin and iso-
collinin demonstrated remarkable antibacterial activity against

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C. Pardo-Castano et al. / Anaerobe 62 (2020) 102163
4
Table 1
Evaluation of the antimicrobial activity of different chemical agents against bacteria of clinical importance after 24-48 h of exposure.
Strains
Isocollinin (
m
g/mL)
Collinin (
m
g/mL)
CHX (
m
g/mL)
NaClO (mg/mL)
MIC (mean)
(SD)
MIC (mean)
(SD)
MIC (mean)
(SD)
MIC (mean)
(SD)
Periodontophatics
A.actinomycetemcomitans
P. gingivalis
F.nucleatum
P.intermedia
D.pneumosintes
Superinfectants
E.coli
S.aureus
P.aeruginosa
41.7
4.2
20.8
33.3
41.7
11.8
1.4
5.9
11.8
11.8
5.2
2.1
10.4
16.7
10.4
1.4
0.8
2.9
5.9
2.9
31.7
62.9
31.7
31.7
31.7
9.0
17.4
9.0
9.0
9.0
325.8
651.0
651.0
651.0
325.8
92.3
183.8
183.8
183.8
92.3
20.8
41.7
20.8
5.9
11.8
5.9
20.8
10.4
5.2
5.9
2.9
1.4
15.7
3.9
15.7
4.7
1.1
4.7
651.0
651.0
1302.0
183.8
183.8
368.6
Fig. 2. Minimum Inhibitory Concentration (MIC) of collinin isomers and CHX against periodontopathic and superinfective strains, in a multivariable analysis. Bars represent the
mean values and standard deviation of the MIC, obtained in the bacterial susceptibility assays, made by triplicate. *: p < 0.1, ***: p < 0.001, ns: not significant.
A. actinomycetemcomitans ATCC 29522, P. gingivalis ATCC 33277,
P. intermedia ATCC 25611, D. pneumosintes ATCC 33048 and
F. nucleatum ATCC 25586. Collinin isomers also presented high
antimicrobial activity against superinfectant ATCC strains, partic-
ularly P. aeruginosa. These results are promising for developing
further research directed to control oral and medical pathogenic
biofilms, where microbes have shown increasing resistance against
antimicrobials [3,6,7].
Caries and periodontal diseases affect a large proportion of the
world population and are considered a public health problem [5]. In
the case of periodontitis, the presence and overgrowth of patho-
genic microorganisms are related to disease progression and dis-
ease aggravation [6,24,25]. A variety of active agents have been
tested during the last 40 years to prevention, treatment and control
of periodontitis.
The keystone pathogen P. gingivalis has been reported as the
most frequent microorganism in patients with chronic and
aggressive periodontitis, with a prevalence of approximately 72% in
Colombia [26], and in Latin America, Missailidis et al. [27] affirm
that on average this microorganism can be found in 89% of patients
with periodontitis, 30% with gingivitis and 8% in periodontally
healthy subjects. Other periodontal pathogens are also frequently
reported in patients with periodontitis at diverse countries in Latin
Fig. 3. Minimum Bactericidal Concentration (MBC) of collinin isomers and CHX against
periodontopathic and superinfective strains. Bars represent the mean values and
standard deviation of the MBC, obtained in the bacterial susceptibility assays and
resarzurin assay, made by triplicate.

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C. Pardo-Castano et al. / Anaerobe 62 (2020) 102163
5
Fig. 4. Time-Kill and Cell Viability assays with 3 critical concentrations of Collinin and Isocollinin in E. coli ATCC 25922. A. Line graphs showing the change in optical density at
600 nm (OD 600 equivalent 1.5 ꢁ 108 CFU/ml) in the different time points of exposition in hours respect to control. B. Example of resarzurin assay with collinin to evaluate cell
viability in different times, the pink dots represent the viable cells, the blue dots represent the non-viable cells, the positive control (Cþ) had no biocide, so the bacteria were viable,
and the negative control (C-) had CHX at high concentration (0.12%), so the bacteria were not viable. C. Example of negative and positive controls used in the viability confirmation
assay by blood agar culture. D. Example of MBC confirmation with blood agar culture, this result corresponds to the blue dots observed in the resarzurin assay (non-viable cell).
America [28]. In Chile for example, found A. actinomycetemcomitans
(19.4%), P. gingivalis (83.4%) and superinfective bacteria (17.6%) in
patients with chronic periodontitis [29]. In Argentina, P. gingivalis,
Treponema denticola, Tannerella forsythia (red-complex) [30,31]. In
Venezuela, P. intermedia, P. gingivalis are the most frequently
recovered microorganism [32]. Finally, in Mexico, reported patients
with periodontitis and rheumatoid arthritis, P. intermedia in 89%
and P. gingivalis in 58% of the subjects [33].
This work showed that both collinin isomers have a remarkable
antimicrobial effect against all periodontal pathogens tested,
especially against P. gingivalis. Previous studies have shown that
collinin significantly inhibits the growth of P. gingivalis at concen-
between (5.2e41.7 mg/mL) and (10.4e20.8 mg/mL), respectively.
Hence, the antimicrobial effect demonstrated herein by both col-
linin isomers against main periodontal pathogens, confirm and
improve previous findings [13] that might result in alternatives for
antimicrobial periodontal therapy.
We also observed that both molecules at the right concentra-
tions are also able to kill the test microbes even up to 24 h after
exposure. This suggests that the antimicrobial activity is persistent,
a very useful feature to control recalcitrant oral biofilms that harbor
in deep periodontal lesions where there is a greater density of
pathogens [28].
On the other hand, superinfectants are considered transient
colonizing pathogens that can impair oral health status in some
patients. A multicenter study conducted in 5 cities of Colombia [26],
showed that 38% periodontitis patients harbored superinfective
bacteria. In this work, compared to CHX, collinin and isocollinin
showed similar antimicrobial activity against the superinfecting
strains. In addition, Betancourt and collaborators [35], also showed
that 36% periodontitis patients harbored superinfectant bacteria,
among them, E. coli and P. aeruginosa were the most frequent
pathogens. These results suggest that pathogens, considered as
superinfectants, are a problem in periodontitis patients mainly in
Latin America and possibly other developing countries.
It was shown that both collinin isomers could have a broad
spectrum of action against other microorganisms of medical
importance that not only participate in periodontal disease, but
also in important nosocomial and life-threatening infections. Col-
linin activity against resistant strains of Mycobacterium tuberculosis
trations ꢂ12.5
m
g/mL [13], whereas we find that even at lower
g/mL of collinin and isocollinin,
concentrations ꢂ 2.1 and ꢂ 4.2
m
respectively, are enough to inhibit P. gingivalis, a result even better
compared with the gold standard chlorhexidine (CHX), the anti-
microbial used most frequently in the clinical practice. Differences
on the MIC values for collinin of that reported by Santos et al., in
2013, can be due to a difference in the purity of the compound,
which is not reported in the publication. Furthermore, the char-
acterization of collinin performed by Santos et al., follow the work
~
done by Curini et al. in Ref. [34]. As Pardo-Castano et al. argued [21],
Curini et al. probably obtained the position isomer isocollinin
instead of collinin, which according to our study exhibited a higher
MIC (4.2 mg/mL) than collinin (2.1 mg/mL).
Collinin and isocollinin also exhibited the capacity to reduce the
growth of other important periodontal pathogens, such as
A. actinomycetemcomitans and F. nucleatum, at concentrations

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6
pp. 154e165, https://doi.org/10.1111/omi.12161.Differential.
[5] K.S. Kornman, Contemporary approaches for identifying individual risk for
periodontitis, Periodontology 2000 78 (2018) 12e29, https://doi.org/10.1111/
prd.12234.
has been reported [18] and a similar inhibition pattern for S. aureus,
E. coli and P. aeruginosa, at concentrations >24 mg/mL, was found in
this work.
[6] S.S. Socransky, A.D. Haffajee, Periodontal microbial ecology, Periodontology
2000 38 (2005) 135e187, https://doi.org/10.1111/j.1600-0757.2005.00107.x.
[7] J. Lindhe, N.P. Lang, T. Karring, Clinical Periodontology and Implant Dentistry,
vol. 2, Wiley, 2008. https://books.google.com.co/books?id¼0yGItwEACAAJ.
[8] J. Slots, T. Rams, M. Listgarten, Yeasts, enteric rods and pseudomonads in the
subgingival flora of severe adult periodontitis, Oral Microbiol. Immunol. 3
(1988) 47e52, https://doi.org/10.1111/j.1399-302x.1988.tb00080.x.
[9] T. Rams, D. Feik, J. Slots, Staphylococci in human periodontal diseases, Oral
Currently, the gold standard for the treatment of periodontal
disease is scaling and root planing and some studies have suggested
that better results are obtained if antibiotic therapy with
amoxicillin
þ
metronidazole is included [36]. However, the
generalized increase in bacterial resistance against many antimi-
crobials suggests that new therapeutic alternatives to control
pathogenic microorganisms are needed. Studies conducted in
Colombia [37,38] found a decrease in the susceptibility of peri-
odontal pathogens to amoxicillin and metronidazole; among them,
P. gingivalis and A. actinomycetemcomitans were highlighted. This
suggests that the development of new antimicrobial agents against
these pathogens could be a good therapeutic alternative. Addi-
tionally, chlorhexidine gluconate is the antimicrobial agent most
frequently used in clinical practice for its accepted effect [39].
However, the collinin isomers showed a greater antimicrobial ef-
fect, which suggests that studies with these compounds need to be
expanded to determine their clinical application in medicine and
dentistry.
Microbiol. Immunol.
302x.1990.tb00222.x.
5
(1990) 29e32, https://doi.org/10.1111/j.1399-
[10] J.T. Newton, K. Asimakopoulou, J.T. Newton, Behavioral Models for Periodontal
Health and Disease, 2018, pp. 201e211, https://doi.org/10.1111/prd.12236.
[11] H.Y. Wang, J.W. Cheng, H.Y. Yu, L. Lin, Y.H. Chih, Y.P. Pan, Efficacy of a novel
antimicrobial peptide against periodontal pathogens in both planktonic and
polymicrobial biofilm states, Acta Biomater. 25 (2015) 150e161, https://
doi.org/10.1016/j.actbio.2015.07.031.
[12] X. Kuang, V. Chen, X. Xu, Novel approaches to the control of oral microbial
biofilms, BioMed Res. Int. 2018 (2018), https://doi.org/10.1155/2018/
6498932.
[13] J. Santos, A. Marquis, F. Epifano, S. Genovese, M. Curini, D. Grenier, Collinin
reduces Porphyromonas gingivalis growth and collagenase activity and inhibits
the lipopolysaccharide-induced macrophage inflammatory response and
osteoclast differentiation and function, J. Periodontol. 84 (2013) 704e711,
https://doi.org/10.1902/jop.2012.120118.
[14] W. Li, Y.N. Sun, X.T. Yan, S.Y. Yang, E.J. Kim, H.K. Kang, Y.H. Kim, Coumarins
and lignans from Zanthoxylum schinifolium and their anticancer activities,
J. Agric. Food Chem. 61 (2013) 10730e10740, https://doi.org/10.1021/
jf403479c.
[15] J.S. Kim, S.T. Lee, C.R. Han, D.Y. Jun, M.H. Woo, Y.H. Kim, Induction of apoptosis
by collinin from Zanthoxylum schinifolium is mediated via mitochondrial
pathway in human Jurkat T cells, Process Biochem. 48 (2013) 945e954,
https://doi.org/10.1016/j.procbio.2013.03.010.
[16] T. Tanaka, Development of an inflammation-associated colorectal cancer
model and its application for research on carcinogenesis and chemopreven-
tion, Int. J. Inflamm. (2012) 1e16, https://doi.org/10.1155/2012/658786, 2012.
[17] H. Kohno, R. Suzuki, M. Curini, F. Epifano, F. Maltese, S.P. Gonzales, T. Tanaka,
Dietary administration with prenyloxycoumarins, auraptene and collinin,
inhibits colitis-related colon carcinogenesis in mice, Int. J. Canc. 118 (2006)
2936e2942, https://doi.org/10.1002/ijc.21719.
[18] S. Kim, H. Seo, H. Al Mahmud, M.I. Islam, B.E. Lee, M.L. Cho, H.Y. Song, In vitro
activity of collinin isolated from the leaves of Zanthoxylum schinifolium
against multidrug- and extensively drug-resistant Mycobacterium tubercu-
losis, Phytomedicine 46 (2018) 104e110, https://doi.org/10.1016/
j.phymed.2018.04.029.
5. Conclusions
Collinin and isocollinin demonstrated a remarkable antibacte-
rial activity against relevant periodontal pathogens, such as
A. actinomycetemcomitans, F. nucleatum, and especially against
P. gingivalis (MIC of 2.1 and 4.2,
chlorhexidine digluconate (CHX) and sodium hypochlorite (NaClO)
showed MIC against P. gingivalis of 62.7 and 651 g/mL, respec-
tively. Both collinin isomers also presented antimicrobial activity
against superinfectant strains, particularly P. aeruginosa (MIC 5.2 of
collinin and MIC 20.8
to inhibit its growth.
mg/mL, respectively). Whereas
m
mg/mL of isocollinin), while CHX 15.7 mg/mL
Funding
[19] M. Curini, F. Epifano, F. Maltese, M.C. Marcotullio, A. Tubaro, G. Altinier,
S.P. Gonzales, J.C. Rodriguez, Synthesis and anti-inflammatory activity of
natural and semisynthetic geranyloxycoumarins, Bioorg. Med. Chem. Lett 14
(2004) 2241e2243, https://doi.org/10.1016/j.bmcl.2004.02.009.
[20] I.L. Tsai, W.Y. Lin, C.M. Teng, T. Ishikawa, S.L. Doong, M.W. Huang, Y.C. Chen,
I.S. Chen, Coumarins and antiplatelet constituents from the root bark of
Zanthoxylum schinifolium, Planta Med. 66 (2000) 618e623, https://doi.org/
10.1055/s-2000-8648.
This work was supported by Administrative Department of
Science, Technology and Innovation of Colombia - Colciencias
(Grant 1106-715-51462).
Declaration of competing interest
~
~
[21] C. Pardo-Castano, A.C. Garcia, P. Benavides, G. Bolanos, Solubility of collinin
and isocollinin in pressurized carbon Dioxide : synthesis , solubility param-
The authors declare no competing financial interest.
eters
, and equilibrium measurements, J. Chem. Eng. Data 64 (2019)
3799e3810, https://doi.org/10.1021/acs.jced.9b00234.
[22] Clinical and Laboratoy Standars Institute, Performance Standards for Anti-
microbial Susceptibility Testing, CLSI Document M100-S25, Wayne,PA, 2015.
[23] ASTM D-E928-08, Standard Test Method for Purity by Differential Scanning
Calorimetry, American Society for Testing and Materials, West Conshohocken,
PA, 2014.
[24] N.J. Kassebaum, E. Bernabe, M. Dahiya, B. Bhandari, C.J.L. Murray,
W. Marcenes, Global burden of severe periodontitis in 1990-2010: a sys-
tematic review and meta-regression, J. Dent. Res. 93 (2014) 1045e1053,
https://doi.org/10.1177/0022034514552491.
Acknowledgments
The authors express their gratitude to the Periodontal Medicine
and the Applied Thermodynamic research groups, Universidad del
Valle, Colombia, for their contributions in this study.
References
[25] J. Slots, Periodontitis: facts, fallacies and the future, Periodontology 2000 75
(2017) 7e23.
ꢀ
[1] N.J. Kassebaum, A.G.C. Smith, E. Bernabe, T.D. Fleming, A.E. Reynolds, T. Vos,
ꢀ
[26] G.I. Lafaurie, A. Contreras, A. Baron, J. Botero, I. Mayorga-Fayad, A. Jaramillo,
C.J.L. Murray, W. Marcenes, Global, regional, and national prevalence, inci-
dence, and disability-adjusted life years for oral conditions for 195 countries,
1990-2015: a systematic analysis for the global burden of diseases, injuries,
and risk factors, J. Dent. Res. 96 (2017) 380e387, https://doi.org/10.1177/
0022034517693566.
ꢀ
ꢀ
A. Giraldo, F. Gonzalez, S. Mantilla, A. Botero, L.H. Archila, A. Díaz, T. Chacon,
D.M. Castillo, M. Betancourt, M. Del Rosario Aya, R. Arce, Demographic, clin-
ical, and microbial aspects of chronic and aggressive periodontitis in
Colombia: a multicenter study, J. Periodontol. 78 (2007) 629e639, https://
doi.org/10.1902/jop.2007.060187.
[2] S. Listl, J. Galloway, P.A. Mossey, W. Marcenes, Global economic impact of
dental diseases, J. Dent. Res. 94 (2015) 1355e1361, https://doi.org/10.1177/
0022034515602879.
[3] R.J. Lamont, H. Koo, G. Hajishengallis, The oral microbiota: dynamic commu-
nities and host interactions, Nat. Rev. Microbiol. 16 (2018) 745e759, https://
doi.org/10.1038/s41579-018-0089-x.
[4] G. Hajishengallis, J.L. Krauss, R. Jotwani, J.D. Lambris, I. Diseases, Differential
Capacity for Complement Receptor-Mediated Immune Evasion by Porphyr-
omonas Gingivalis Depending on the Type of Innate Leukocyte, vol. 32, 2018,
[27] C.G. Missailidis, J.E. Umeda, C. Ota-Tsuzuki, D. Anzai, M.P.A. Mayer, Distribu-
tion of fimA genotypes of Porphyromonas gingivalis in subjects with various
periodontal conditions, Oral Microbiol. Immunol. 19 (2004) 224e229, https://
doi.org/10.1111/j.1399-302X.2004.00140.x.
[28] A. Contreras, S.M. Moreno, A. Jaramillo, M. Pelaez, A. Duque, J.E. Botero, J. Slots,
Periodontal microbiology in Latin America, Periodontology 2000 67 (2015)
58e86, https://doi.org/10.1111/prd.12074.
[29] D. Herrera, A. Contreras, J. Gamonal, A. Oteo, A. Jaramillo, N. Silva, M. Sanz,

~
C. Pardo-Castano et al. / Anaerobe 62 (2020) 102163
7
ꢀ
J.E. Botero, R. Leon, Subgingival microbial profiles in chronic periodontitis
patients from Chile, Colombia and Spain, J. Clin. Periodontol. 35 (2008)
106e113, https://doi.org/10.1111/j.1600-051X.2007.01170.x.
051X.2009.01496.x.
[34] M. Curini, F. Epifano, F. Maltese, M. Marcotullio, S. Gonzales, J. Rodriguez,
Synthesis of collinin, an antiviral coumarin, Aust. J. Chem. 56 (2003) 59e60,
https://doi.org/10.1071/CH02177.
ꢀ
[30] L. Fernandez Canigia, A. Nogueira Moreira, C. Furman, S. Piovano, H. Bianchini,
Microbiological assessment of subgingival plaques from Argentinian patients
[35] M. Betancourth, R.M. Arce, J. Botero, A. Jaramillo, C. Cruz, A. Contreras,
ꢀ
with adult periodontitis, Anaerobe
10.1006/anae.1999.0212.
[31] G. Bazzano, R. Parodi, S. Tabares, A. Sembaj, Evaluacion de la terapia mecanica
5
(2000) 263e265, https://doi.org/
Microorganismos inusuales en surcos y bolsas periodontales, Colomb. Med. 37
(2006) 6e14. http://colombiamedica.univalle.edu.co/index.php/comedica/
article/view/406/1092.
ꢀ
ꢀ
ꢀ
periodontal en bolsas profundas: respuesta clínica
y
bacteriologica TT
-
[36] M. Feres, L.C. Gursky, M. Faveri, C.O. Tsuzuki, L.C. Figueiredo, Clinical and
microbiological benefits of strict supragingival plaque control as part of the
active phase of periodontal therapy, J. Clin. Periodontol. 36 (2009) 857e867,
https://doi.org/10.1111/j.1600-051X.2009.01471.x.
evaluation of periodontal mechanical therapy in deep pockets: clinical and
bacteriological response, Rev. Clin. Periodoncia Implant. Rehabil. Oral 5 (2012)
122e126.
http://www.scielo.cl/scielo.php?script¼sci_arttext&pid¼S0719-
01072012000300004&lng¼es&nrm¼iso&tlng¼es.
[37] A. Jaramillo, M. Betancourth, I. Mayorga-Fayad, D. Castillo, A.C.M.R.,
G. Lafaurie, J. Botero, A. Contreras, Antimicrobial profiles of subgingival bac-
teria from peri- odontititis patients in Colombia, Rev. Clin. Periodoncia
Implant. Rehabil. Oral 1 (2008) 61e65.
[32] A. Brito, L.A. Escalona, M. Correnti, M. Perrone, I.M. Bravo, V. Tovar, Peri-
odontal conditions and distribution of Prevotella intermedia, Porphyromonas
gingivalis and Aggregatibacter actinomycetemcomitans in HIV-infected pa-
tients undergoing anti-retroviral therapy and in an HIV-seronegative group of
the Venezuelan population, Acta Odontol. Latinoam. 21 (2008) 89e96.
[33] R.E. Martinez-Martinez, C. Abud-Mendoza, N. Patino-Marin, J.C. Rizo-Rodri-
guez, J.W. Little, J.P. Loyola-Rodriguez, Detection of periodontal bacterial DNA
in serum and synovial fluid in refractory rheumatoid arthritis patients, J. Clin.
Periodontol. 36 (2009) 1004e1010, https://doi.org/10.1111/j.1600-
ꢀ
[38] C.M. Ardila, M.I. Granada, I.C. Guzman, Antibiotic resistance of subgingival
species in chronic periodontitis patients, J. Periodontal. Res. 45 (2010)
557e563, https://doi.org/10.1111/j.1600-0765.2010.01274.x.
[39] M. Feres, L.C. Figueiredo, G.M.S. Soares, M. Faveri, Systemic antibiotics in the
treatment of periodontitis, Periodontology 2000 67 (2015) 131e186, https://
doi.org/10.1111/prd.12075.