Indonesian Dental Association Journal of Indonesian Dental Association http://jurnal.
id/index.
php/jida ISSN: 2621-6183 (Prin.
ISSN: 2621-6175 (Onlin.
The Effect Ant-Nest Plant (Myrmecodia pendan.
Extract on Streptococcus sanguinis and Treponema denticola Biofilms Nely Soviati1.
Armelia Sari Widyarman2A.
Ciptadhi Tri Oka Binatha2 1 Undergraduate Student.
Faculty of Dentistry.
Trisakti University.
Indonesia 2 Department of Microbiology.
Faculty of Dentistry.
Trisakti University.
Indonesia Received date: August 21, 2019.
Accepted date: November 16, 2019.
Published date: February 20, 2020.
KEYWORDS
Myrmecodia pendans.
Streptococcus sanguinis.
Treponema denticola.
ABSTRACT
Introduction: Ant-nest plant (Myrmecodia pendan.
contains triterpenoid, flavonoid, saponin and tannin that acts as antibacterial substance.
Objectives: This studyAos aim was to analyze the effect of Myrmecodia pendans extract effect towards biofilm of Streptococcus sanguinis and Treponema denticola.
Methods: Ant-nest plant was extracted using maceration technique.
The obtained extract was diluted into five different concentrations using phosphate buffer saline (PBS).
sanguinis and T.
denticola ATCC 35405 were cultured in Brain Heart Infusion (BHI) broth, 48h, 37AC, anaerobic atmosphere and distributed into four 96 well-plate for 24h to form Subsequently, the extract was distributed into the well that contained the mono-species and multispecies biofilm and then incubated for 1h, 3h, 6h and 24h.
Chlorhexidine .
2%) was used as positive control and BHI broth was used as negative control.
The biofilm was stained using crystal-violet and measured by microplate reader with a wavelength of 490 nm.
Data were statistically analyze using one-way ANOVA test and Post Hoc LSD test which p < 0,05 was set as significant difference.
Result: The most effective concentrations to reduce monospecies biofilm were 100% in 1h incubation period for S.
sanguinis (OD = 1,403 A 0,.
and 24h for T.
denticola (OD = 1,012 A 0,.
and multispecies biofilm (OD = 0,984 A 0,.
compared to negative control.
Statistical analysis showed that ant-nest plant extract significantly reduced S.
denticola and multispecies biofilm mass compared to negative control .
< 0,.
Conclusion: The ant-nest plant extract has inhibitory effects against S.
sanguinis and T.
denticola biofilm and it may be used as alternative for dental Future studies are needed to evaluate the potential of ant-nest plant extract in multispecies composed of other oral bacteria.
A Corresponding Author E-mail address: armeliasari@trisakti.
id (Widyarman AS) DOI: 10.
32793/jida.
Copyright: A2020 Soviati N.
Widyarman AS.
Binarta CTO.
This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium provided the original author and sources are credited.
Journal of Indonesian Dental Association 2020 3.
, 11-15 Soviati N, et al.
INTRODUCTION
14,15 However.
pendans extract has not been known to decrease biofilm formation of periodontal pathogens such as S.
sanguinis and T.
Two common dental and oral health diseases are dental cariesAialso known as cavitiesAiand periodontal There are two main types of periodontal tissue inflammation gingivitis and periodontitis.
1 The inducer of these inflammation are bacterial species in sub-gingival Gingivitis, which is an inflammation on gingiva can be prevented by plaque removal, such as by brushing your teeth regularly.
Periodontitis is marked by periodontal tissue damage and the loss of connective tissue attachments, alveolar bone loss, and pathological pockets surrounding the tooth.
2 One of the causes of this periodontal infection is biofilm.
3 Biofilm is a thin layer of microorganisms adhering to a solid surface and covered by a matrix of organic substances .
olysaccharides, extracellular DNA .
DNA), and protein.
4 The production of this extracellular matrix protects the bacteria from the surrounding area, including from chemotherapeutic agents.
MATERIALS AND METHODS
This study is an in vitro experimental laboratory with post-test only control design.
This study was conducted in Microbiology Centre of Research and Education (MiCORE) laboratory.
Faculty of Dentistry.
Trisakti University.
Jakarta.
Indonesia.
The plant used in this study was M.
pendans which obtained from local society in Papua island which is located in eastern Indonesia.
Myrmecodia pendans Extraction Dried M.
pendans plant was washed in running water until clean and rinsed with aquadest to remove sticky soil on the plantAos surface.
The dried sample was blended into a powder and sieved.
Extracts were obtained from as much as 50 g of powder using a maceration technique, wherein the sample was soaked in 500 mL of 70% ethanol for 5 days.
The sample was then left for 24h and filtered with filter paper.
Filtrate was evaporated using a rotary evaporator at 40AC.
The 100% .
extract was diluted in brain heart infusion (BHI) broth (Thermo Scientific.
Waltham.
MA.
USA) to 50%, 25%, 12.
5% and 25% .
Streptococcus sanguinis is an anaerobic, facultative.
Gram-positive bacterium found in the oral cavity that is involved in biofilm development.
6 S.
sanguinis has a role in development of periodontal disease.
7 Treponema denticola is a common spirochete found in sub-gingival plaque and has a role in the development of chronic periodontitis, acute necrotizing ulcerative gingivitis (ANUG), infected root canal, and acute abscess.
8 These bacteria have some common virulence factors such as (MSP.
, lipopolysaccharides (LPS.
, dentilisin, peptidoglycan, cystalysin, peptidases, and phosphatases, which can cause inflammation and induce the host cell to produce chemical mediators, all of which involved in damaging periodontal connective tissues.
Bacterial culture and Biofilm Assay test Brain heart infusion (BHI) broth was used to grow S.
sanguinis and T.
denticola ATCC 35405 and the bacteria were incubated at 37AC in an anaerobic atmosphere for 24h using GasPak jar system (Becton Dickinson.
Franklin Lakes.
NJ.
USA).
The bacterium culture in broth medium was measured at 600nm and diluted to obtain a suspension of McFarland standard 0.
quivalent to 1,5x108 CFU/mL).
After homogenizing, the bacterium suspension was distributed into a 96-well plate, and incubated at 37AC for 24h in an anaerobic atmosphere to formed biofilm.
The supernatant was removed and the biofilm attached on the bottom of well plate was rinsed with phosphate buffer saline (PBS).
uL M.
pendans extracts with different concentrations .
%, 50%, 25%, 12.
5% and 6.
25%) were distributed into the well plate.
Chlorhexidine .
2%) was used for the positive control and biofilm wells without herbal tested was used for the negative control.
Inhibitory effects were observed after incubation at 37AC for 1h, 3h 6h, and 24h in an anaerobic atmosphere.
Crystal violet dye .
5% w/.
was distributed into the well plate and incubated for 15 min.
The extraction of the remaining crystal violet dye in the well plate was measured after the addition of 200 AAL 90% ethanol and the absorbance was measured using a microplate reader at 595 nm.
About 70-80% of the worldAos population uses herbal and natural ingredients for health care, prophylaxis, and oral lesion treatment.
The most populated countries, such as China and India, have been using natural remedies for over 2000 years to cure oral diseases, including periodontal disease.
10 For example, pineapple stem extracts contain high levels of bromelain, an enzyme which acts as an antibacterial agent against Enterococcus 11 Strawberry extracts have been proven to delay biofilm formation of E.
faecalis and Porphyromonas gingivalis in vitro.
12 Pomegranates contain significant amounts of ellagic acid, which displays antibacterial activity against Candida albicans.
13 Myrmecodia pendans .
nt nest plan.
is a plant from Papua in eastern Indonesia, where it is well known as herbal remedy.
pendans is commonly used in western Papua as a traditional medicine for many diseases.
14 Previous studies showed that ant nest plant extracts display antimicrobial activity towards Streptococcus mutans and E.
Journal of Indonesian Dental Association 2020 3.
, 11-15 Soviati N, et al.
Statistical Analysis Streptococcus sanguinis Biofilm Biofilm (Optical densit.
The data was statistically analyzed using a one-way ANOVA test and a Post Hoc LSD test in which p<0.
was set as the significant difference.
Statistical calculations were performed with SPSS Statistics for Windows software (IBM.
Armonk.
USA).
RESULTS
Negative kontrol Control
6,25%
12,50%
Positive Control Ant-nest plant extract concentration Results showed that there were significant reductions of mono-species S.
sanguinis biofilm mass (Optical density/OD C mea.
after treatment with ant nest plant extract for all incubation periods .
< 0.
The most effective concentration to reduce S.
sanguinis biofilm was 100% with a 24-hour incubation time-period (OD = 403 A 0.
compared to the negative control (OD = 354 A 0.
2% of biofilm reduction (Fig .
Figure 1.
Streptococcus sanguinis biofilm reduction .
ptical densit.
after treatment with ant nest plant extract with 100%, 50%, 25%, 12,5%, 6,25% concentration in different incubation time .
h, 3h, 6h, 24.
Chlorhexidine .
,2%) was used as positive control and BHI broth was used as negative control.
Treponema denticola Biofilm Biofilm (Optical densit.
There were significant reductions of mono-species biofilm of T.
denticola biofilm mass after treatment with ant nest plant extract for all incubation periods .
< 0.
The most effective concentration to reduce T.
biofilm was 100% with a 24 hour incubation period (OD = 1.
012 A 0.
compared to negative control (OD = 327 A 0.
6 % of biofilm reduction (Fig .
Negative kontrol - 6,25% Control
12,50%
Positive Control 100% kontrol Ant-nest plant extract concentration Results also showed that there were significant reductions of mixed-species biofilm mass after treatment with ant nest plant extract for all incubation periods .
< The most effective concentration was 100% with 24 hour incubation period (OD = 0.
984 A 0.
compared to the negative control (OD = 3.
383 A 0.
9% of biofilm reduction (Fig.
Furthermore, there were no significant difference of monospecies biofilm of S.
denticola and mixed biofilm mass reduction after treatment with ant nest plant extract .
%) compared to positive control .
> 0.
Thus, this plant extract has the ability to inhibit biofilm formation as effective as chlorhexidine in this study.
Figure 2.
Treponema denticola biofilm reduction .
ptical densit.
after treatment with ant nest plant extract with 100%, 50%, 25%, 12,5%, 6,25% concentration in different incubation time .
h, 3h, 6h, 24.
Chlorhexidine .
,2%) was used as positive control and BHI broth was used as negative control.
Biofilm (Optical densit.
Multispecies Biofilms DISCUSSION In this study, ant nest plant (M.
extract was tested for its effectiveness against S.
sanguinis and T.
denticola biofilm formation for both mono- and mixedspecies biofilm.
Study report the analysis of gene expression profiles in T.
denticola triggered by the contact with other relevant oral bacterial species including two representatives each for the red (P.
gingivalis and T.
, orange (F.
nucleatum and P.
, and yellow (S.
gordonii and S.
The extent of observed transcriptional responses in T.
denticola appears to reflect previously established disease-related associations, interspecies interactions and synergistic relationships.
16 The results Negative kontrol - 6,25% Control
12,50%
Positive 100% kontrol Control Ant-nest plant extract concentration Figure 3.
Multispecies biofilm reduction .
ptical densit.
after treatment with ant nest plant extract with 100%, 50%, 25%, 12,5%, 6,25% concentration in different incubation time .
h, 3h, 6h, 24.
Chlorhexidine .
,2%) was used as positive control and BHI broth was used as negative control.
showed an inhibitory effect for all incubation periods against the periodontal pathogens as mono and mixed species, with the most effective concentration being The lowest optical density in this study is on mixed species biofilm, since both strains have a Journal of Indonesian Dental Association 2020 3.
, 11-15 Soviati N, et al.
many microorganisms.
25 Most plant phenolic compounds also have antibiofilm effects because they interfere with quorum sensing, which can affect the growth of many synergistic relationship, this ant nest plant extract may be have the ability to interfere the synergistic relationship in both strain and inhibit the biofilm mass.
A maceration technique was used to acquire the ant nest plant extract.
It is an active substance retrieval performed by soaking a simplified powder in a solvent for some days at room temperature and protected from the light.
The solvent will penetrate into the cell wall of the simplified powder and infiltrate the cell cavity which contains the active substances.
These active substances will be dissolved due to the different concentrations of solvent inside and outside the cell, causing the concentrated solvent to be pushed out.
This process was repeated until a balanced concentration of the solvent was achieved inside and outside the cell.
The advantage of the maceration technique is that it uses simple tools and it does not destroy heat-sensitive active substances.
CONCLUSION
Based on this study, it can be concluded that Myrmecodia pendans extract has an antibiofilm effect on sanguinis and T.
denticola biofilm as mono and Ant nest plant (M.
extract may be used as an alternative antibacterial agent to inhibit bacterial activities in the oral cavity.
Future studies are needed to evaluate the potential of ant nest plant extract.
CONFLICT OF INTEREST
The authors declare that they have no conflicts of Previous studies that were done by Roslizawaty et , in 2013 showed that M.
pendans decreased C.
albicans growth.
From the saturation level in test tubes of fungus and M.
pendans, it was proven that there was an increasing inhibitory effect of ant nest extract on C.
albicans at higher concentrations.
18 M.
pendans belongs to the Rubiaceae family.
It contains many active phytochemicals, mostly phenolic compound groups such as flavonoids, tannins, saponins, phenolic acids, alkaloids, triterpenoids, and glycosides.
19 M.
displays two antibacterial mechanisms, which are cytotoxicity and apoptosis.
Cytotoxicity is a characteristic that can slow and prevent microbial cell growth, which has potential to harm healthy host cells.
14,20
REFERENCES