IntroductionIntroduction

Oral bacteria are major causative agents of pulpal and peri-

apical diseases. The treatment of these is performed through

dental root canal enlargement, cleansing, and disinfection and

the main purpose of root canal therapy is to remove bacteria

present in the root canal and to prevent reinfection [1-3]. The

persistent or secondary infection of the root canal is the main

cause of root canal treatment failure. Staphylococcus aureus

and Enterococcus faecalis are the pathogenic bacteria of en-

dondontic infection [4]. S. aureus is a representative purulent

bacterial species and is also isolated from endodontic infection

lesions, so it has been used as a target bacterium for evalua-

tion of antimicrobial property of dental materials used for vari-

ous root canal treatments [5,6]. E. faecalis has been reported

as a major causative agent of root canal failure [7,8].

Recently, chlorhexidine or Mixture of Tetracycline, an Acid,

and a Detergent (MTAD) has been used as antimicrobial agent

mixed with root canal sealer for root canal treatment [3,9].

However, chlorhexidine has toxicity to oral tissue cells and

Int J Oral Biol 46:45-50, 2021pISSN: 1226-7155 • eISSN: 2287-6618https://doi.org/10.11620/IJOB.2021.46.1.45

The antibacterial effect of Endoseal TCS mixed with water-soluble mangostin derivatives of Garcinia mangostana L. ethanol extract against Enterococcus faecalis and Staphylococcus aureusTae-Young Park1†, Yun Kyong Lim2,3†, Jin-Hee Kim3,4, Dae Sung Lee4*, and Joong-Ki Kook2,3*

1Department of Conservative Dentistry, Chosun University, Gwangju 61452, Republic of Korea 2Korean Collection for Oral Microbiology, Chosun University, Gwangju 61452, Republic of Korea 3Department of Oral Biochemistry, School of Dentistry, Chosun University, Gwangju 61452, Republic of Korea 4Medi Bio Lab Co., Ltd., Seoul 61452, Republic of Korea

This study evaluated the antimicrobial activity of Endoseal TCS, an mineral trioxide aggregate-based root canal sealer, mixed with water-soluble mangostin derivatives (WsMD) of Garcinia mangostana L. (mangosteen) ethanol extract against Enterococcus faecalis and Staphylococcus aureus. The antibacterial activity of Endoseal TCS mixed with WsMD against three strains of E. faecalis and three strains of S. aureus was performed using agar diffusion test. The data showed that Endoseal TCS mixed with 0.115% WsMD had a zone of inhibition of 0.7 ± 0.2–2.4 ± 0.1 mm. The results suggest that Endoseal TCS mixed with WsMD of Garcinia mangostana L. ethanol extract is useful as a root canal sealer with antibacterial activity against E. faecalis and S. aureus.

Keywords: Endoseal TCS, Garcinia mangostana L., Enterococcus faecalis, Staphylococcus aureus, Antibacterial effect

Received January 25, 2021; Accepted March 19, 2021*Correspondence to: Joong-Ki Kook, E-mail: jkkook@chosun.ac.kr https://orcid.org/0000-0003-2628-2870*Correspondence to: Dae Sung Lee, E-mail: dsl2008@naver.com https://orcid.org/0000-0003-2317-7848†Tae-Young Park and Yun Kyong Lim contributed equally to this work.

Copyright © The Korean Academy of Oral BiologyCC This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Original Article IJOBInternational Journal of Oral Biology

Int J Oral Biol Vol. 46, No. 1, March 2021

46 www.kijob.or.kr

MTAD has possibility of inducing new tetracycline-resistant

bacteria or has not effect tetracycline-sensitive bacteria that

are existed in root canal [10,11]. Therefore, they are used as

root canal cleaners rather than as sealers for root canals [10,12].

Therefore, studies are being conducted to find an antimicrobial

substance derived from natural products that can be used in

combination with an endodontic treatment material to over-

come these problems [13]. The previous study, the ethanol

extract of Garcinia mangostana L. (magosteen) had strong an-

timicrobial activity against S. aureus and E. faecalis [14,15].

Since mineral trioxide aggregate (MTA) was introduced by

Torabinejad and Chivian [16], it has been widely used in the

field of endodontic treatment. Recently, MTA-based root seal-

er has been developed and delivered via a syringe. This sealer

was reported to have better biocompatibility than conventional

resin sealers for root canal filling [17]. However, it was reported

that the antimicrobial effect of MTA was very low [18]. There-

fore, in this study, the antimicrobial activity of Endoseal TCS®

(MARUCHI Co., Ltd., Wonju, Korea), an MTA-based root canal

sealer, mixed with the water-soluble mangostin derivatives

(WsMD) of the ethanol extract of magosteen against E. faeca-

lis and S. aureus was evaluated.

Materials and MethodsMaterials and Methods

1. Bacteria and bacterial culture

The E. faecalis (KCOM 1083, KCOM 1161, KCOM 2823) and

S. aureus (KCOM 1335, KCOM 1395, KCOM 1491) strains used

in this study were obtained from the Korean Collection for

Oral Microbiology (Gwangju, Korea). These strains were grown

on brain heart infusion (BHI, BD Difco Laboratories, Franklin

Lakes, NJ, USA) broth or BHI agar medium in an incubator at

37℃ for 24 hours.

2. Preparation of water-soluble mangostin derivative

powder

Mangosteen ethanol extract was purchased from Maypro

Industries® (Purchase, NY, USA). To impart water solubility to

the mangosteen ethanol extract, a certain amount of mango-

steen ethanol extract powder was added to purified water and

stirred at 50℃ and 1 atmosphere (atm) for 1 hour. Thereafter,

L-arginine was added to the solution and stirred at 50℃ and

1 atm for 1 hour. The pH at this time was maintained at 9–10.

L-arginine (1%, w/w) and KOH, an alkaline metal salt used as

a catalyst, were added to the solution and stirred at 70℃ and

1 atm for 2 hours to form WsMD. The pH was maintained at

9–10. After the color of the solution changed from ocher to

black, it was cooled to 4℃ and left at room temperature for 12

hours to increase the stability of the WsMD. The solution was

vacuum-dried at 0.4 atm and 60℃ to prepare WsMD powder

(Fig. 1).

3. Preparation of mixed sample of WsMD powder and

Endoseal TCS®

Endoseal TCS® was mixed with a WsMD at weight propor-

tions of 0.023%, 0.07%, or 0.115% to prepare premixed inject-

able pastes. These were provided by MARUCHI Co., Ltd.

4. Antibacterial test

Agar diffusion tests were performed by a modification of the

method of Hasheminia et al. [19]. Briefly, 10 mL of the steril-

ized BHI agar medium was poured into a sterilized 90-mm

Petri dish and then a paper disk (8 × 0.5 mm) was placed into

the agar medium. The bacterial strain was grown in BHI broth

at 37℃ for 16 hours in an incubator. It was diluted to 106 CFU/

mL with BHI agar solution cooled to 40℃ and then 10 mL was

poured onto the previously solidified BHI agar medium. After

Mangostin

L-arginine

KOH

Stirred at 50

in pH 9 10for 1 hr

Stirred at 70

in pH 9 10for 2 hr

Cool to 40

and left at 25

for 2 hrVacuum-driedat 0.4 atm and

60

OHHO

OCH3

O

CH =CHCH2

CH3OH O CH CH=CCH2 3

CH3

3C

OHHO

OCH3

O

CH =CHCH2

CH3OH O CH CH=CCH2 3

CH3

3C

+NH

C = NH

NH

(CH )

CHNH

COOH

2

2 3

2

NH

C = NH

NH

(CH )

CHNH

COOH

2

2 3

2

NH

C = NH

NH

(CH )

CHNH

COOH

2

2 3

2

Fig. 1. Schematic diagram for preparing water-soluble mangostin derivatives from the ethanol extract of mangosteen.

Tae-Young Park, et al. Antimicrobial effect of Endoseal TCS and mangosteen

www.kijob.or.kr 47

the BHI agar solution mixed with the bacterial strain was so-

lidified, the paper disc was carefully removed. Two mL of En-

doseal TCS® (control), or Endoseal TCS® mixed with 0.023% (w/

w), 0.07% (w/w), or 0.115% (w/w) of WsMD by injecting us-

ing a syringe with a 24G needle. The plates were incubated for

24 hours in an incubator at 37℃ and the zone of inhibition was

measured. The length from the edge of the Endoseal TCS® to

the edge of the inhibition zone was measured and recorded.

This experiment was repeated three times and the antibacte-

rial activity is presented as the mean ± standard error of the

length of the zone of inhibition.

Results Results

The antimicrobial activity of the WsMD mixed with Endoseal

TCS® against E. faecalis and S. aureus strains was evaluated

by agar diffusion tests. The data showed that Endoseal TCS®

mixed with 0.115% WsMD created a zone of inhibition that

ranged from 0.7 ± 0.2 to 2.4 ± 0.1 mm (Fig. 2 and Table 1).

Among the strains used in this study, E. faecalis KCOM 2823

was more resistant to Endoseal TCS® containing the most

WsMD compared to the other strains (Fig. 2 and Table 1). In

contrast, the S. aureus KCOM 1491 strain was the most sus-

ceptible (Fig. 2 and Table 1). In the case of Endoseal TCS®,

which was used as a control, antibacterial activity was not ob-

served (Fig. 2 and Table 1).

DiscussionDiscussion

In this study, WsMD were mixed with Endoseal TCS®, an

MTA-based root canal sealer, to impart antibacterial properties,

which were investigated by agar diffusion tests. The zone of

inhibition was increased by adding higher amounts of WsMD

(Fig. 1 and Table 1). According to previous studies, the mini-

mum bactericidal concentration (MBC) values of the ethanol

extract of mangosteen containing about 40% alpha-mangostin

against E. faecalis (KCOM 1083, KCOM 1161, KCOM 2823) and

S. aureus (KCOM 1335, KCOM 1395, KCOM 1491) were be-

tween 2 and 8 µg/mL [14,15]. To investigate the antimicrobial

activity of the WsMD compared to the mangosteen ethanol

extract, the MBC value of WsMD against S. aureus KCOM

1491 was measured. The MBC value of WsMD against S. au-

1122

33 44

A B C

D E F

1122

33 44

1122

33 44

1122

33 44

1122

33 44

1122

33 44

Fig. 2. Agar diffusion test. The zones of inhibition against (A) Enterococcus faecalis KCOM 1083, (B) E. faecalis KCOM 1161, (C) E. faecalis KCOM 2823, (D) Staphylococcus aureus KCOM 1335, (E) S. aureus KCOM 1395, and (F) S. aureus KCOM 1491 were increased depending upon the amount of water-soluble mangostin derivatives (WsMD) from the etha-nol extract of mangosteen added to Endoseal TCS®. 1, Endoseal TCS® (control); 2, Endoseal TCS® + 0.023% WsMD from the ethanol extract of mangosteen; 3, Endoseal TCS® + 0.07% WsMD from the ethanol extract of mangosteen; 4, Endoseal TCS® + 0.115% WsMD from the ethanol extract of mangosteen.

Table 1. Antimicrobial activity of mineral trioxide aggregate-based root canal sealer (Endoseal TCS®) with water-soluble mangostin derivatives from the ethanol extract of mangosteen against Enterococcus faecalis and Staphylococcus aureus

Species and strains

Zone of inhibition (mm)

Control (Endoseal TCS®)Endoseal TCS® + 0.023%

mangostin derivativesa Endoseal TCS® + 0.07%

mangostin derivatives Endoseal TCS® + 0.115%

mangostin derivatives

E. faecalis KCOM 1083 0 0 0.7 ± 0.2 1.6 ± 0.2

E. faecalis KCOM 1161 0 0 0.4 ± 0.1 0.9 ± 0.2

E. faecalis KCOM 2823 0 0 0 0.7 ± 0.2

S. aureus KCOM 1335 0 0 0.6 ± 0.3 1.5 ± 0.1

S. aureus KCOM 1395 0 0 0.8 ± 0.1 1.4 ± 0.2

S. aureus KCOM 1491 0 0.7 ± 0.1 2.0 ± 0.1 2.4 ± 0.1

Values are presented as mean ± standard deviation.aWater-soluble mangostin derivatives from the ethanol extract of mangosteen.

Int J Oral Biol Vol. 46, No. 1, March 2021

48 www.kijob.or.kr

reus KCOM 1491 was the same, 2 µg/mL, as that of mango-

steen ethanol extract (data not shown). These results indicate

that the antimicrobial activity of the WsMD was maintained

and might be released when mixed with Endoseal TCS®. How-

ever, further study is needed to investigate how the WsMD

were produced and released from the Endoseal TCS® mixture.

Little antimicrobial activity of MTA Fillapex, another MTA-

based root canal sealer developed in Brazil, against E. faecalis

was seen in the agar diffusion tests in previous studies [18,19].

However, the antimicrobial activity of MTA Fillapex that was

cured and pulverized against E. faecalis was excellent [19]. The

antimicrobial mechanisms of MTA are explained in two ways.

The first is that MTA produces reactive oxygen species in the

atmosphere, with antibacterial effects [20]. Second, MTA con-

tains calcium oxide. Therefore, when it comes into contact

with water, calcium hydroxide is formed, which increases the

pH and exhibits an antibacterial effect [21-23]. It is known that

E. faecalis cannot survive at a pH of 11.5 or higher. Therefore,

water is essential for MTA to have an antibacterial effect on

E. faecalis. This means that the MTA-based root canal sealer

loses its antimicrobial activity after curing. Thus, the addition of

a water-soluble antibacterial agent is considered necessary to

impart antibacterial activity to the MTA-based root canal sealer,

as in this study.

As a result of studies comparing the antimicrobial activity of

various types of root canal sealer currently in use, the antibac-

terial effect of the MTA-based sealer against E. faecalis was

lower than that of the other types of root canal sealers [18,24-

26]. When using only MTA, the antibacterial effect was lower

than that of the conventional sealer. Therefore, there have

been attempts to increase the antibacterial effect by adding

chlorhexidine to MTA [9,27]. However, when chlorhexidine was

added, the antibacterial effect was increased, but the physical

properties of the MTA itself were deteriorated [9]. Based on

these results, we intend to conduct future studies on whether

Endoseal TCS® containing the WsMD used in this study has

clinically usable properties.

Another study on the antimicrobial effect of MTA-based

sealers against E. faecalis showed some antimicrobial effects

of MTA Fillapex and Endofill against E. faecalis but did not dis-

play complete inhibition and the high pH of the MTA-based

sealer was increased after seven days [28]. In this study, the

Endoseal TCS® component spread to the inhibitory zone from

the second day and began to become opaque, and from the

third day, the agar medium dried, making it difficult to identify

the antibacterial activity (data not shown). Therefore, animal

experiments will be used to evaluate the duration of the an-

timicrobial activity of Endoseal TCS® containing WsMD in the

future.

According to the above results, the higher the content of

WsMD mixed with Endoseal TCS®, the higher the antibacterial

effect. With more than 0.115% WsMD mixed with Endoseal

TCS®, the color tone of the sealer changed and the flowability

was impaired, resulting in the loss of function as a sealer for

root canal filling. Therefore, the proper concentration of WsMD

for mixing with Endoseal TCS® was 0.115%.

In summary, Endoseal TCS® mixed with the WsMD of the

ethanol extract of Magosteen might be useful as a root canal

sealer with antibacterial activity against E. faecalis and S. au-

reus.

AcknowledgementsAcknowledgements

We thank CEO Sung-Wook of MARUCHI Co., Ltd. for mak-

ing a pre-mixed injectable paste containing the mixture of En-

doseal TCS® and WsMD.

Conflicts of InterestConflicts of Interest

Dae Sung Lee and Jin-Hee Kim are co-applicants on patent

application 10-2020-0152138 entitled “Composition for filling

root canal comprising mangosteen extract.” The other authors

have no conflicts of interest to declare.

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