Natural Product Research

Formerly Natural Product Letters

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Remineralization and antibacterial/

antibiofilm effects of toothpaste containing
nanohydroxyapatite and Curcuma aeruginosa
extract

Yessie W. Sari, Nur A. Nuzulia, Wulan T. Wahyuni, Abdurrahman Bahtiar,

Angga Saputra, M. Hafif A. Subroto, Yessy Ariesanti, Utami Syafitri & Indra
Bachtiar

To cite this article: Yessie W. Sari, Nur A. Nuzulia, Wulan T. Wahyuni, Abdurrahman
Bahtiar, Angga Saputra, M. Hafif A. Subroto, Yessy Ariesanti, Utami Syafitri & Indra Bachtiar
(2021): Remineralization and antibacterial/antibiofilm effects of toothpaste containing
nanohydroxyapatite and Curcuma�aeruginosa extract, Natural Product Research, DOI:
10.1080/14786419.2021.1981316

To link to this article: https://doi.org/10.1080/14786419.2021.1981316

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Published online: 27 Sep 2021.

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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2021.1981316

SHORT COMMUNICATION

Remineralization and antibacterial/antibiofilm effects of

toothpaste containing nanohydroxyapatite and Curcuma
aeruginosa extract
Yessie W. Saria,b, Nur A. Nuzuliaa, Wulan T. Wahyunib,c, Abdurrahman Bahtiara,
Angga Saputraa, M. Hafif A. Subrotod, Yessy Ariesantie, Utami Syafitrif and
Indra Bachtiarg
a
Department of Physics, Faculty of Mathematics and Natural Sciences, IPB University, Bogor,
Indonesia; bTrophical Biopharmaca Research Center, IPB University, Bogor, Indonesia; cDepartment of
Chemistry, Faculty of Mathematics and Natural Sciences, IPB University, Bogor, Indonesia; dMenteng
Dental Clinic, Jakarta, Indonesia; eDepartment of Oral and Maxillofacial Surgery, Faculty of Dentistry,
Trisakti University, Jakarta, Indonesia; fDepartment of Statistics, Faculty of Mathematics and Natural
Sciences, IPB University, Bogor, Indonesia; gStem Cell and Cancer Institute, Kalbe, Jakarta, Indonesia

ABSTRACT ARTICLE HISTORY

The aim of this study was to evaluate the effect of nanohydroxyapa- Received 27 June 2021
tite (nanoHAP) and Curcuma aeruginosa (C. aeruginosa) toothpastes Accepted 11 September 2021
on tooth remineralization and antibacterial/antibiofilm activity.
Remineralization was evaluated by the morphological changes in KEYWORDS
extracted human premolar teeth following toothpaste application. Bioactive; calcium
phosphate; caries; essential
The antibacterial and antibiofilm activities were evaluated by agar dif- oil; toothpaste
fusion and microdilution methods, respectively, against S. mutans.
Statistical approach was utilized to formulate 20 toothpastes with dif-
ferent concentration of nanoHAP and C. aeruginosa. We observed
that the interaction among toothpaste ingredients determined the
remineralization and antibacterial/antibiofilm activities. The optimum
toothpaste formula (OF1) suggested by the prediction model was
shown to induce remineralization and have comparable antibacterial
activity to that of the control (chlorhexidine gluconate). Furthermore,
the antibiofilm activity of this formula was higher to that of the con-
trol. The result obtained indicate that these novel toothpastes have
potential in decreasing caries prevalence.

CONTACT Yessie W. Sari yessie.sari@apps.ipb.ac.id

Supplemental data for this article can be accessed online at https://doi.org/10.1080/14786419.2021.1981316.
ß 2021 Informa UK Limited, trading as Taylor & Francis Group
2 Y. W. SARI ET AL.

1. Introduction
Strategies for caries prevention have included the use of hydroxyapatite for remineral-
ization of dentin and tooth enamel (Jeong et al. 2006; Besinis et al. 2012). Nano-sized
hydroxyapatite (nanoHAP) has also shown potential as a remineralizing agent for initial
enamel lesions (Huang et al. 2011).
Caries, tooth mineral degradation due to microbial activities, is often prevented by
adding chemicals with antimicrobial properties to the toothpaste. These compounds
include triclosan, stannous fluoride, cetylpyridinium chloride, and chlorhexidine
(Gunsolley 2006). Currently, the application of natural ingredients to oral care prod-
ucts, including toothpaste, is gaining increasing attention, and several studies have
identified plant extracts that have the ability to prevent bacterial adhesion, particularly
of cariogenic bacteria such as Streptococcus mutans.
Recently identified plant material that inhibits the growth of S. mutans and enhan-
ces biofilm degradation is Curcuma aeruginosa, a member of the Zingiberaceae family
(Wahyuni et al. 2017). The rhizome of this plant is widely used as a medicinal material
in several countries in Asia and contains high levels of monoterpenes and sesquiter-
penes. Curcuma essential oil contains 1.8-cineol, curzerenone, camphor, and germa-
crone as major constituents and are reported to impart antibacterial activity against a
variety of gram-positive and gram-negative bacteria (Kamazeri et al. 2012;
Theanphong et al. 2015; Akarchariya et al. 2017).
The aim of the present study was to evaluate the effect of combining nanoHAP as
a remineralizing agent with C. aeruginosa essential oil as an antibacterial and antibio-
film agent in toothpastes. A statistical approach was taken to obtain an optimal for-
mulation for the toothpaste that considered the weight percentage of the nanoHAP,
C. aeruginosa essential oil, and toothpaste base materials as the input parameters
(Tables S1 and S2). The percentage of remineralization, the size of antibacterial clear
zones, and the IC50 for biofilm suppression were used as the responses indicating the
degree of remineralization, the antibacterial activity, and the antibiofilm activity,
respectively.
 NATURAL PRODUCT RESEARCH 3

2. Results and discussion

2.1. Nanohap synthesis
The XRD pattern of the nanoHAP and confirms the good agreement with the XRD
ICDD data file No. 00-024-0033 (Figure S1). The crystallite size at the (002) Miller’s
plane was 12.62 ± 0.17 nm. Figure S2a shows a scanning electron micrograph of the
nanoHAP and indicates a spherical network. The nanoHAP particles had a size of
58.29 ± 8.76 nm, (Figure S2b).

2.2. Remineralization effect

SEM micrographs (Figure S3a–e) show the morphological changes in each tooth sam-
ple under three conditions: baseline, demineralized, and remineralized. Porous surface
was observed at demineralized samples. All 20 toothpastes, having different formulas,
were able to induce remineralization as indicated by the disappearance of porous sur-
face. The changes in the image gray levels further quantify the efficacy of each tooth-
paste (Figure S4). Based on this, the minimum remineralization occurred on samples
F4, F5, and F6. While the maximum remineralization occurred on samples F11, F12,
F14, F15, and F16. F4 had high nanoHAp content (2% wt), while F16 had low
nanoHAP content (0.2% wt). Thus, the high content of nanoHAP does not ensure to
enhance remineralization, yet the interaction between components in tooth-
paste matters.

2.3. Antibacterial activity

The antibacterial activity of toothpaste formulas was evaluated against S. mutans, a
gram-positive bacterium responsible for prevalent oral cavity diseases. Most toothpaste
formulas showed antibacterial activity (indicated by the formation of a clear zone)
with a diameter that varied from 11.84 to 23.23 mm (Figure S5).

2.4. Antibiofilm effect

Some toothpaste formulas showed comparable biofilm degradation to that achieved
with chlorhexidine gluconate (CG), as control (Figure S6). The IC50 value (representing
the concentration that causes a 50% degradation of biofilm) of toothpaste F16 and
F17 were much lower than the IC50 of CG, indicating that these toothpaste formulas
have potential for application as antibiofilm agents. Interestingly, the F16 and F17 had
comparable IC50 value although contained differ amount of C. aeruginosa oil, 0% wt
and 0.2% wt, respectively. In the absence of C. aeruginosa oil, the antibiofilm activity
at F16 may be contributed by the high peppermint content. Peppermint has been
showed to have this activity (Shayegh et al. 2008). Peppermint content in F16 and F17
is 2.9% wt and 0.1% wt, respectively. Toothpaste with very small amount of C. aerugi-
nosa could give comparable IC50 value with toothpaste containing much higher
peppermint, indicating that C. aeruginosa oil is promising source of bioactive com-
pound that may contribute to the prevent oral disease.
4 Y. W. SARI ET AL.

2.5. Formula optimization

Data on remineralization, antibacterial and antibiofilm were further used as the
response for formula optimization, using the I-optimal design. The toothpaste optimal
conditions, predicted by the model, are shown in Table S3.

2.6. Formula validation

Figure S7 shows the SEM images obtained after demineralization and remineralization of
tooth samples treated with the two optimum formulas (OF1 and OF2). The tooth morph-
ology changes were similar to those observed with the initial 20 formulas (Figure S3) and
with the 5 commercial toothpastes used as controls. However, the SEM images indicated
that OF1 and OF2 had more positive effects than were observed with the 5 commercial
toothpastes, as reflected by the higher changes of gray level for OF1 and OF2 (Figure S8).
The antibacterial diffusion and microdilution tests against S. mutans showed that the
OF1 formula produced a comparable inhibition zone to that achieved with CG (Table S4),
while the microdilution tests showed that OF1 had a minimum inhibitory concentration
(MIC) and minimum bactericidal concentration (MBC) greater than 10 mg/mL, which was
higher than the values for CG or the other commercial toothpaste. One explanation may
be that the antibiofilm activity of OF1 did not arise by killing the bacteria embedded in
the biofilm but instead arose by detachment of bacteria from the substrate surface. The
degradation of established biofilms might occur either by killing the embedded bacteria
or by the detachment of living bacteria from the substrate (Segev-Zarko et al. 2015).
Pure hydroxyapatite naturally presents with no effective antibacterial activity (Qiu
et al. 2020). The MIC and MBC data (Table S4) indicated that both formulas (OF1 and
OF2) had antibacterial activities, which may be due to the presence of C. aeruginosa
oil. The coupling of nanoHAP and C. aeruginosa oil in OF1 did not reveal a competitive
effect on the antibacterial and antibiofilm activities. This result was supported by a
previous study that showed a coupling of hydroxyapatite with aminopropyltrimethoxy-
silane improved the antibacterial activity (Shalini and Ruban Kumar 2019).
The antibiofilm activity of OF1 was stronger than that of OF2 and CG, the commer-
cial toothpaste fortified with antibacterial agent, the commercial toothpaste fortified
with antibiofilm agent, and the commercial standard toothpaste. From this point of
view, OF1 can be assumed to have the highest potential for development as an anti-
plaque or antibiofilm toothpaste.

3. Experimental
See supplementary materials.

4. Conclusion
In this work, nanoHAP was combined with C. aeruginosa oil as toothpaste fortifiers. A
statistical approach was conducted to formulate the toothpaste composition. The pre-
diction model suggested two toothpaste formulations for validation as optimum
toothpastes. These formulations have slightly different contents of both nanoHAP and
 NATURAL PRODUCT RESEARCH 5

C. aeruginosa oil. The validation of remineralization activity showed that both formulas
had higher remineralization activity compared to the five commercial toothpastes.
Both formulas also showed comparable antibacterial activities to that of CG, an anti-
septic that is widely used in oral care. Furthermore, the antibiofilm activity showed
that OF1 had a much higher antibiofilm activity than either OF2 or CG. The OF1 for-
mula indicated that nanoHAP can be coupled with C. aeruginosa oil as toothpaste for-
tifiers for remineralization and promotion of antibacterial and antibiofilm activities.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This study was funded by the Indonesia Endowment Fund for Education (Lembaga Pengelola
Dana Pendidikan) RISPRO Program (PRJ-31/LPDP/2019). We thank Prof. Dr. Irmanida Batubara,
and Prof. Dr. Y. Aris Purwanto for insightful comments.

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