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: apirak.s@rsu.ac.th

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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