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TJPS Vol.41 (Supplement Issue) 2017
TJPS 2017, 41 (Supplement Issue): 153
Antimicrobial activity of 22 plant extracts against oral bacteria (Streptococcus mutans)
Apirak Sakunpak1 and Lukman Sueree2
1Department of Pharmacognosy, Faculty of Pharmacy, Rangsit University, Pathum Thani, Thailand, 12000 2The Herbal Medicinal Products Research and Development Center (Cooperation between Rangsit University and Harbin Institute of Technology and Heilongjiang University of Chinese Medicine), Faculty of Pharmacy, Rangsit University, Pathum Thani, Thailand, 12000
* Corresponding author: E-mail: [email protected]
Keywords: antimicrobial activity; plant extracts; Streptococcus mutans
Introduction Dental caries is major health problem worldwide1. The main reason of dental caries are a group of Streptococcal
species of which Streptococcus mutans is the most important agents of human dental caries2,3. Antibiotics such
as erythromycin and penicillin are reported as effective drugs to prevent dental caries in animal but these are
never used clinically because of hypersensitivity reactions4. Therefore, as the current therapeutic strategies to
prevent dental diseases are not fully void of side effects5. The development of novel and alternative guidelines
for microbial control should be considered not only advantageous but also necessary. Antimicrobial substances
from natural sources like plants have been investigated to achieve higher levels of food safety6. In addition, the
natural compound isolated from medicines plant could offer effective alternatives to antibiotics and represent a
promising approach to the prevention and treatment of dental caries and other oral infections7. Screening for
medicinal plant effective against oral bacteria is the required first step in the identification of natural compound
that could be used as antimicrobial substances8. In this study, 22 plant extracts were select to evaluate the
possible inhibit the growth of oral bacteria pathogens.
Material and method Chemicals
Ethanol (analytical grade) was obtained from Merck (Darnstadt, Germany). Gentamicin was purchased
from Sigma (St. Louis, MO, USA). Sodium chloride were purchased form Sigma (St. Louis, MO, USA).
Microorganisms and Media
The microorganisms used in this study including; S. mutans ATCC 12175, S. mutan NRPC 801, and S. mutans NRPC 804 were obtained from Department of Microbiology, Faculty of science, Prince of Songkla
University, Songkhla, Thailand, 90110. Brain-Heart infusion (BHI) and agar were purchased from Becton,
Dickinson and Company (MD, USA).
Preparation of extracts
Plants as describe in Table 1 were collected from Pathum Thani Province, Thailand, in June 2013 and
reference voucher specimens were deposited at the Sino-Thai Traditional Medicine Research Center, Faculty
of Pharmacy, Rangsit University, Pathumthani, Thailand. These plants were dried at 50 ºC for 24 h in a hot air
oven and reduced to coarse powders using a grinder. Dried plant powders were submitted to solvent extractions
by sonication method with ethanol at room temperature for 1 hour. The extract was then passed through filter
paper. The marcs were subsequently extracted again under the same conditions. The extracts were combined
and concentrated with a rotary evaporator. Extracts were stored in sterile bottle at 4 °C and dissolved in purified
water before use.
Thai Journal of Pharmaceutical Sciences (TJPS) The JSPS-NRCT Follow-Up Seminar 2017 and
33rd International Annual Meeting in Pharmaceutical Sciences
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TJPS 2017, 41 (Supplement Issue): 154
Table 1 Plant used
Family Botanical names Part used
Leguminosae Senna garrettiana (Craib) H.S.Irwin & Barneby heartwood
Pinaceae Pinus merkusii Jungh et de Vriese heartwood
Dracaenacea Dracaena lourieri Gagnep heartwood
Anacardiaceae Anacardium occidentale L. leaves
Zingiberaceae Curcuma mangga Valeton & van Zijp rhizome
Verbenaceae Clerodendrum serratum L. leaves
Capparaceae Crateva adansonii DC. (Roxb.) Jacobs leaves
Acanthaceae Pseuderanthemum palatiferum (Nees) Radlk. ex Lindau leaves
Euphorbiaceae Croton stellatopilosus Ohba leaves
Euphorbiaceae Glochidion perakense Hook. f. leaves
Annonaceae Annona reticulata L. leaves
Leguminasae Adenanthera pavonina L. seed
Apocynaceae Carissa carandas L. leaves
Labiatae Clerodendrum chinense (Osbeck) Mabb. leaves
Leguminosae Cassia fistula L. leaves
Rubiaceae Paederia foetida L. leaves
Sapotaceae Mimusops elengi L. heartwood
Bignoniaceae Oroxylum indicum (L.) Kurz leaves
Moraceae Maclura cochinchinensis Corner heartwood
Piperaceae Piper pendulispicum C. DC. stem
Rubiaceae Tarenna hoaensis Pitard heartwood
Euphorbiaceae Euphorbia antiquorum L. heartwood
Preparation of inoculums
The microorganisms used in this study including; S. mutans ATCC 12175, S. mutans NRPC 801, and
S. mutans NRPC 804 were grown on Brain Heart Infusion agar plate at 37 °C for 48 h in an anaerobic jar.
Disc diffusion method
The bacterial was suspended in 0.85% normal saline solution and its turbidity was adjusted to be
equivalent 0.5 McFarland standard with approximately 108 CFU/ml. This suspension was then inoculated on
the agar surface of BHI agar plate using sterile swabs. Allow the plate to dry before applying plant extract discs
(2 mg/disc). These plates were incubated at 37°C for 48 h in an anaerobic jar. Zone of inhibition was measured
around the disc. All disc diffusion tests were performed in triplicate and antibacterial activity was expressed as
the mean of inhibition zones (mm). Gentamycin was used as positive control.
Determination of minimum inhibition concentration (MIC)
Minimum inhibition concentration of the extract was tested by broth dilution method. There extract were
diluted in 10% ethanol with the concentration of 100 mg/ml and diluted with media to concentration of 1000-2
µg/ml. The inoculums were prepared and adjusted with 0.85% NaCl to contain 108 CFU/ml by adjusting the
turbidity of saline culture to match the McFarland 0.5 standard. It was then further diluted 1:100 in media to
contain 106 CFU/ml and 50 µl of the adjusted inoculum was added to each bacterium well plate. Ten-microliter
of plant extract solution was added and incubated at 37 °C for 48 h in an anaerobic jar. The MIC was defined
as the lowest concentration of the compound to inhibit the growth of microorganisms.
Determination of minimum bactericidal concentration (MBC)
The MBC was defined as the lowest concentration of the compound to kill microorganism. The
incubation mixtures of bacteria that showed positive result of inhibitory effect were streaked on each bacterium
media plate and incubated at 37 °C for 24 hours in anaerobic condition. The lowest concentration that did not
show any growth was taken as the MBC.
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TJPS 2017, 41 (Supplement Issue): 155
Results Antibacterial activities of 22 ethanolic plant extracts against the tested organisms are shown in Table
2. Only three ethanolic extracts of S. garrettiana, P. merkusii and D. lourieri showed the inhibitory activity against
all bacteria strains. Highest antibacterial activity (S. mutans ATCC 12175, S. mutans NRPC 801 and S. mutans
NRPC 804) was observed with ethanol extract of P. merkusii with MIC values of 16, 32 and 16 µg/ml and MBC
values of 512, 1024 and 1024 µg/ml, respectively (Table 3). Results obtained in the current investigation
revealed that the studied of 22 ethanol plant extracts possess potential antibacterial activity against S. mutans.
Table 2 Inhibition zone of 22 plant extracts against S. mutans ATCC 12175, S. mutans NRPC 801 and S.
mutans NRPC 804.
Sample Inhibition zone (mm)
S. mutans ATCC 12175 S. mutans NRPC 801 S. mutans NRPC 804
S. garrettiana 12.80 0.00 13.80 0.00 14.40 0.09
P. merkusii 18.00 0.00 13.60 0.06 15.00 0.00
D. lourieri 11.00 0.04 - 10.70 0.06
A. occidentale - - -
C. mangga - - -
C. serratum - - -
C. adansonii - - -
P. palatiferum - - -
C. stellatopilosus - - -
G. perakense - - -
A. reticulata - - -
A. pavonina - - -
C. carandas - - -
C. chinense - - -
C. fistula - - -
P. foetida - - -
M. elengi - - -
O. indicum - - -
M. cochinchinensis - - -
P. pendulispicum - - -
T. hoaensis - - -
E. antiquorum - - -
Gentamycin 16.70 0.00 15.20 0.00 14.00 0.00
- ; No activity
Table 3 MIC and MBC of plant extracts against S. mutans ATCC 12175, S. mutans NRPC 801, and S. mutans
NRPC 804.
Samples
S. mutans ATCC 12175 S. mutans NRPC 801 S. mutans NRPC 804
MIC
(µg/ml)
MBC
(µg/ml)
MIC
(µg/ml)
MBC
(µg/ml)
MIC
(µg/ml)
MBC
(µg/ml)
S. garrettiana 256 512 256 >1024 256 1024
P. merkusii 16 512 32 1024 16 1024
D. lourieri 1024 >1024 >1021 >1024 1024 >1024
Gentamycin 16 32 16 32 16 32
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TJPS 2017, 41 (Supplement Issue): 156
Discussion Dental caries is infectious diseases caused by S. mutans. Therefore, controlling the levels of these
causative pathogens is a key step in the prevention and treatment of these diseases. In this study the ethanolic
extracts of 22 plants were evaluated for anti-S. mutans activity. Crude ethanol extracts of S. garrettiana, P.
merkusii and D. lourieri inhibited the growth of S. mutans, showing inhibition zones from 10-20 mm. However,
19 plants extracts including; A. occidentale, C. mangga, C. serratum, C. adansonii, P. palatiferum, C.
stellatopilosus, G. perakense, A. reticulata, A. pavonina, C. carandas, C. chinense, C. fistula, P. foetida, M.
elengi, O. indicum, M. cochinchinensis, P. pendulispicum, T. hoaensis and E. antiquorum did not show
antibacterial activity against S. mutans. These results are in accordance with several previous studies. The
ethanol extracts of P. merkusii exhibited the highest antibacterial activity against S. mutans. It would be related
to their resin acids components which show antibacterial property9, 10. Our findings also suggest that P. merkusii
is medicinal plant that good for protection of S. mutans infection. In addition, these plant extracts could be used
for therapeutic purpose in case of S. mutans dental caries.
Conclusion Twenty two of plants were extracted with ethanol and evaluated for antibacterial activity against S.
mutans. Only three plants extract, S. garrettiana, P. merkusii, and D. lourieri exhibited the growth of S. mutans.
The results indicated that P. merkusii have the potential to be developed into agents that can be used as
preventative or treatment therapies for oral diseases.
Acknowledgments The authors wish to thank the Faculty of Pharmacy and Sino-Thai Traditional Medicine Research Center
(Cooperation between Rangsit University, Harbin Institute of Technology, and Heilongjiang University of
Chinese Medicine), Rangsit University, Pathum Thani, Thailand, for all chemicals and instruments. This
research was partly funded by the Research Institute of Rangsit University, Pathum Thani, Thailand (Grant No.
44/2557).
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