In vitro anti-biofilm activity of macelignan isolated from Myristica fragrans Houtt. against oral primary colonizer bacteria

Copyright © 2007 John Wiley & Sons, Ltd. Phytother. Res. 22, 308–312 (2008)DOI: 10.1002/ptr

308 YANTI ET AL.

Copyright © 2007 John Wiley & Sons, Ltd.

PHYTOTHERAPY RESEARCHPhytother. Res. 22, 308–312 (2008)Published online 9 October 2007 in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/ptr.2312

Received 2 March 2007Revised 27 June 2007

Accepted 10 July 2007

In Vitro Anti-biofilm Activity of Macelignanisolated from Myristica fragrans Houtt.against Oral Primary Colonizer Bacteria

Yanti1,2, Yaya Rukayadi1,3, Kyu-Hoi Kim1 and Jae-Kwan Hwang1*1Department of Biotechnology, Yonsei University, Seoul 120-749, Korea2Faculty of Biotechnology, Atma Jaya Catholic University, Jakarta 12930, Indonesia3Research Center for Bioresources and Biotechnology, Bogor Agricultural University, Bogor 16002, Indonesia

In early dental plaque formation, oral primary colonizers such as Streptococcus mutans, Streptococcus sanguisand Actinomyces viscosus are initially attached to the pellicle-coated tooth surface to form a biofilm. Thestudy aimed to determine the efficacy of macelignan, isolated from nutmeg (Myristica fragrans Houtt.),in removing each single oral primary biofilm in vitro on a polystyrene 96-well microtiter plate. Four biofilmgrowth phases (4, 12, 20 and 24 h) were evaluated in this study after treatment with macelignan at variousconcentrations (0.2, 2 and 10 μμμμμg/mL) and exposure times (5, 10 and 30 min). Anti-biofilm activity of macelignanwas measured as the percentage of the remaining biofilm absorbance after macelignan treatment in compari-son with the untreated control. At 24 h of biofilm growth, S. mutans, A. viscosus and S. sanguis biofilms werereduced by up to 30%, 30% and 38%, respectively, after treatment with 10 μμμμμg/mL macelignan for 5 min.Increasing the treatment time to 30 min resulted in a reduction of more than 50% of each of the single primarybiofilms. The results indicate that macelignan is a potent natural anti-biofilm agent against oral primarycolonizers. Copyright © 2007 John Wiley & Sons, Ltd.

Keywords: anti-biofilm activity; macelignan; Myristica fragrans Houtt.; oral bacteria.

INTRODUCTION

Within the human oral cavity, the biofilm on toothsurfaces that results in dental caries can contain over700 bacterial species, among which Streptococcus mutans,Lactobacillus spp. and Actinomyces spp. are the mostimportant species (Aas et al., 2005; Pennisi, 2005). Dentalplaque is a complex multi-species biofilm communityand is a direct precursor of caries and periodontal dis-eases (Rosan and Lamont, 2000; Socransky and Haffajee,2005). The general principles of microbial successionfor plaque development involve colonization by pioneerGram-positive bacteria, well known as primary colo-nizers including S. mutans, S. sanguis and A. viscosus,that attach to the tooth surfaces.

Dental caries can lead to tooth dysfunction and loss.The best way to control these negative impacts is toremove biofilm associated diseases prior to the devel-opment of dental caries (Wilson, 1996). Several studieson the effect of intraorally applied antimicrobial agentsto inhibit oral biofilm growth have been conducted.Antimicrobial agents such as chlorhexidine gluconate,cetylpiridinium chloride, amine fluoride/stannousfluoride, triclosan, parabens and phenolic agents wereshown to be effective for inhibiting biofilm develop-ment (Wilson, 1996; Doron et al., 2001; Auschill et al.,2005; Wilson et al., 1998; Marsh and Bradshaw, 1993;Sreenivasan et al., 2004). However, the excessive useof these chemical agents can result in rearrangement of

the oral and intestinal flora and cause undesirable sideeffects such as microorganism susceptibility, vomiting,diarrhea and tooth staining (Chen et al., 1989). Theseproblems implied the importance of further researchfor natural antiplaque agents with safety for humansand specificity for oral pathogens.

Recently, xanthorrhizol isolated from Curcumaxanthorrhiza Roxb. showed in vitro antibacterial activ-ity against both S. mutans biofilm and planktoniccells (Rukayadi and Hwang, 2006; Hwang et al., 2000).However, exploration of natural anti-biofilm agentsfrom medicinal plants to combat oral pathogen bacte-ria is still limited. Nutmeg (Myristica fragrans Houtt.),an endemic plant to the Maluku Province of Indonesia,has been used traditionally for both medicinal andculinary purposes (Van Gils and Cox, 1994). Macelignan(Fig. 1), one of the bioactive compounds from nutmeg,

Figure 1. Structure of macelignan.

* Correspondence to: Jae-Kwan Hwang, Department of Biotechnology,Yonsei University, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-749, Korea.E-mail: [email protected]

ANTI-BIOFILM ACTIVITY OF MACELIGNAN 309


has potent anticariogenic activity against S. mutansplanktonic cells compared with other commercial anti-bacterial agents (Chung et al., 2006). In the search fornatural anti-plaque agents, macelignan was investigatedas a natural antiplaque agent against single oral pri-mary biofilms brought about by S. mutans, S. sanguisand A. viscosus.

MATERIALS AND METHODS

Microorganisms and growth conditions. Three oralprimary strains used in this study were S. mutansATCC 25175, S. sanguis ATCC 10556 (American TypeCulture Collection, Rockville, MD, USA) and A. viscosusKCCM 12074 (Korean Culture Center of Microorgan-isms, Seoul, Korea). All strains were cultured in brainheart infusion (BHI) broth or BHIA (BHI supple-mented with 1.5% (w/v) of bacteriological agar, Difco,Detroit, MI, USA) at 37 °C for 24 h aerobically.

Preparation of anti-biofilm agents and artificial saliva.Macelignan and chlorhexidine gluconate were usedas anti-biofilm agents. Macelignan, a single bioactivecompound with molecular weight of 328, was isolatedand purified from the methanol extract of nutmeg(M. fragrans Houtt.) according to the method of Chunget al. (2006). Macelignan was dissolved in 100% (v/v)dimethyl sulfoxide (DMSO) and was diluted to finalconcentrations of 1, 10 and 50 μg/mL using 10% (v/v)DMSO. Commercial antimicrobial agent, chlorhexidinegluconate (1,1′-hexamethylenebis[5-p-chlorphenylbigua-nide]) (Sigma-Aldrich, St Louis, MO, USA) was dilutedto the same concentration with macelignan in steriledistilled water.

The artificial saliva, 1% (v/v) mucin from porcinestomach type III (Sigma-Aldrich, St Louis, MO, USA),was diluted with adherence buffer (10 mM KPO4, 50 mM

KCl, 1 mM CaCl2, 0.1 mM MgCl2, pH 7.0) and wasautoclaved at 121 °C for 15 min.

Preparation of bacterial inoculum cells. All bac-terial cells were cultured at 37 °C for 24 h in BHI brothwith shaking agitation of 200 rpm. Each 1 mL culturewas centrifuged at 2000 × g and 4 °C for 5 min, fol-lowed by resuspending the pellet twice with 1 mL ofphosphate-buffered saline (PBS) pH 7.2 as a washingstep to remove debris-free cells. The pellets of eachstrain were re-suspended in 1 mL of adherence bufferto give a final concentration of 2 × 105 CFU/mL. Astandard curve of turbidity against colony-forming unitwas used to obtain the number of cells.

In vitro biofilm formation. S. mutans, S. sanguis andA. viscosus biofilms were prepared in commerciallyavailable presterilized, polystyrene, flatbottom 96-wellmicrotiter plate (Microtest™ 96, Becton Dickinson Co.,Franklin Lakes, NJ, USA) by the method of Stepanovicet al. (2000) with slight modification. Briefly, the platewas conditioned with 200 μL mucin as artificial saliva,incubated and shaken gently at room temperature for3 h. Then, excess mucin was removed and the plate wasair-dried for overnight. Cell biofilms were initially grownusing BHI broth supplemented with 3% (w/v) sucrose(BHIS). For testing the positive control (chlorhexidine

gluconate) and treatment (macelignan), 20 μL inoculumcells (S. mutans, S. sanguis and A. viscosus with anapproximate cell density of 2 × 105 CFU/mL) was addedto each well and 180 μL BHIS media to give 10 folddilution. Thus, the final cell density of each culturewas calculated to be about 2 × 104 CFU/mL. For thenegative control (untreated), the steps were taken onlyusing BHIS broth (200 μL) without macelignan andinoculum cell addition. The plate was incubated at 37 °Cfor different growth times of biofilm, i.e. adherencephase (4 h), active accumulated phase (12 h), beginningplateau accumulated phase (20 h) and plateau accumu-lated phase (24 h).

Biofilm treatment and quantification. Cell biofilmsat different phases of biofilm growth were treatedwith 50 μL macelignan solution (1, 10 and 50 μg/mL asprepared above), resulting in final concentrations of0.2, 2 and 10 μg/mL, respectively. Macelignan startedshowing significant anti-biofilm effects at 0.2 μg/mLand its activity increased up to 10 μg/mL in a dose-dependent manner. The macelignan exposure timeswere 5, 10 and 30 min. Biofilm cell was quantified byusing a crystal violet assay as described by Djordjevicet al. (2002). After biofilm formation and sampletreatment, the plate was shaken gently to remove allunbound bacteria. The remaining adherent bacteriawere washed twice with 200 μL of 50 mM PBS (pH 7.2)and air-dried for 1 h. Each well was stained with 110 μLof 0.4% (v/v) crystal violet solution for 1 h and thenwas washed twice using 300 μL sterile distilled waterand immediately destained with 200 μL ethanol abso-lute. Destaining solution (100 μL) was transferredto the new well and the amount of remaining crystalviolet stain in the destaining solution was measuredwith a tunable microplate reader (Versamax, Sunnyvale,CA, USA) at 596 nm. Macelignan activity was definedas the percentage of the absorbance of biofilm remain-ing after macelignan treatment in comparison with theuntreated control (Djordjevic et al., 2002). All experi-ments were performed in triplicates with two repeats.The plate treated with chlorhexidine gluconate as apositive control at final concentrations of 0.2, 2 and10 μg/mL was also included in each trial.

Statistical analysis. Data were expressed as mean (n =3 × 2) and standard deviation (SD) by computationalanalysis (SPSS 11.0 for Windows). Fisher’s exact testswere applied to contingency tables (2 × 2) to compareS. mutans, S. sanguis and A. viscosus biofilms treatedwith macelignan between 2 and 10 μg/mL. A value ofp < 0.05 was taken as statistically significant.

RESULTS

Table 1 shows the effect of macelignan at different con-centrations (0.2, 2 and 10 μg/mL) and exposure times(5, 10 and 30 min) on removing biofilm cells. Therewere four phases of biofilm ages, including adherencephase (4 h), active accumulated phase (12 h), beginningplateau accumulated phase (20 h) and plateau accumu-lated phase (24 h). At the adherence phase (4 h), earlybiofilm cells were formed and all ranges of macelignan(0.2, 2 and 10 μg/mL) could completely remove single


310 YANTI ET AL.

Table 1. Effect of macelignan with different concentrations and exposure times on oral primary biofilms (S. mutans, S. sanguis andA. viscosus) at different phases of growth

Percentage of remaining cells of biofilm after macelignan treatment (% ± SD)a

ExposureS. mutans biofilm growth (h) S. sanguis biofilm growth (h) A. viscosus biofilm growth (h)

time (min) 4 12 20 24 4 12 20 24 4 12 20 24

Macelignan 0.2 μg/mL5 0 82 ± 6 91 ± 4 94 ± 2b 0 83 ± 3 86 ± 1 97 ± 3b 0 85 ± 1 92 ± 3b 98 ± 2b

10 0 78 ± 2 87 ± 2 88 ± 3 0 71 ± 1 81 ± 4 90 ± 7 0 81 ± 4 87 ± 1 92 ± 6b

30 0 73 ± 5 79 ± 7 83 ± 9 0 67 ± 4 75 ± 7 83 ± 2 0 77 ± 3 81 ± 4 85 ± 1

Macelignan 2 μg/mL5 0 65 ± 1 77 ± 3 81 ± 4 0 67 ± 5 69 ± 1 77 ± 1 0 72 ± 2 79 ± 4 80 ± 410 0 60 ± 3 66 ± 3 75 ± 3 0 49 ± 8 64 ± 2 70 ± 7 0 68 ± 1 71 ± 1 74 ± 330 0 51 ± 1 57 ± 5 61 ± 4 0 32 ± 5 59 ± 7 63 ± 4 0 63 ± 3 65 ± 6 69 ± 2

Macelignan 10 μg/mL5 0 46 ± 1 59 ± 7 70 ± 2 0 52 ± 5 59 ± 4 62 ± 7 0 53 ± 3 68 ± 1 70 ± 410 0 38 ± 2 55 ± 4 51 ± 4 0 25 ± 7 51 ± 2 53 ± 3 0 48 ± 1 60 ± 2 59 ± 530 0 23 ± 2 34 ± 9 45 ± 3 0 12 ± 5 35 ± 3 41 ± 4 0 21 ± 8 30 ± 3 49 ± 5

a Values are expressed as the percentage of absorbance (596 nm) of cells in treated wells compared with that in untreated wells(considered to be 100%), and SD is standard deviation of the mean percentage of absorbance (596 nm) of cells derived from threetimes of experiment and two replicates (wells) per experiment.b Not significantly different from the control treatment. All entries in the table are significantly different (p < 0.05) from the controlsunless otherwise indicated.

Table 2. Contingency table for comparing the effectiveness of macelignan treatment on S. mutans, S. sanguis and A. viscosus biofilm growth

Percentage of remaining cells of biofilm after macelignan treatment (% ± SD)

S. mutans biofilm S. sanguis biofilm A. viscosus biofilm

ExposureMacelignan (μg/mL) Macelignan (μg/mL) Macelignan (μg/mL)

time (min) 2 10 pa 2 10 pa 2 10 pa

Active accumulated phase (12 h)5 65 ± 1 46 ± 1 0.419 67 ± 5 52 ± 5 0.474 72 ± 2 53 ± 3 0.441

10 60 ± 3 38 ± 2 0.246 49 ± 8 25 ± 7 0.142 68 ± 1 48 ± 1 0.31530 51 ± 1 23 ± 2 0.020b 32 ± 5 12 ± 5 0.016* 63 ± 3 21 ± 8 0.002b

Beginning plateau accumulated phase (20 h)5 77 ± 3 59 ± 7 0.465 69 ± 1 59 ± 4 0.533 79 ± 4 68 ± 1 0.536

10 66 ± 3 55 ± 4 0.444 64 ± 2 51 ± 2 0.409 71 ± 1 60 ± 2 0.45530 57 ± 5 34 ± 9 0.086 59 ± 7 35 ± 3 0.081 65 ± 6 30 ± 3 0.017b

Plateau accumulated phase (24 h)5 81 ± 4 70 ± 2 0.538 77 ± 1 62 ± 7 0.495 80 ± 4 70 ± 4 0.546

10 75 ± 3 51 ± 4 0.286 70 ± 7 53 ± 3 0.367 74 ± 3 59 ± 5 0.40730 61 ± 4 45 ± 3 0.219 63 ± 4 41 ± 4 0.125 69 ± 2 49 ± 5 0.180

a Values of p were calculated using the Fisher’s exact test.b Significantly different (p < 0.05, Fisher’s exact test).

oral primary biofilm cells. During the active accumu-lated phase (12 h), exposure of macelignan at 2 μg/mLfor 5 min showed the remaining S. mutans and S. sanguisbiofilms was 70%. Increasing the 2 μg/mL macelignanexposure for 30 min showed the remaining S. sanguisbiofilm was 32%. At the beginning plateau accumu-lated phase (20 h), macelignan treatment (10 μg/mL) for30 min exposure demonstrated the remaining S. mutansand S. sanguis biofilms were 35% and 30% for theA. viscosus biofilm. At the plateau accumulated phase(24 h), macelignan treatment (10 μg/mL) for 30 minshowed <50% remaining of A. viscosus, S. mutans andS. sanguis biofilms. The results indicate that variance ofmacelignan concentrations, exposure times and biofilmgrowth phases affects the efficacy of macelignan as ananti-biofilm agent against oral primary strains. Similar

results were also observed when the strains wereexposed to commercial antimicrobial agent (chlorhexidinegluconate at final concentrations of 0.2, 2 and 10 μg/mL) in the same trials (data not shown).

Table 2 shows Fisher’s exact tests comparing theeffectiveness of macelignan between 2 and 10 μg/mLin removing each S. mutans, S. sanguis and A. viscosusbiofilm during 12, 20 and 24 h of growth phases.At 12 h of biofilm growth, exposure of macelignan(10 μg/mL) for 30 min was more effective in reducingall single oral biofilms compared with those of 2 μg/mLmacelignan (p < 0.020, p < 0.016, p < 0.002, respectively).At 20 h of biofilm growth, treatment with all macelignanconcentrations for all exposure times to S. mutans andS. sanguis biofilms had similar effectiveness, however,A. viscosus biofilm with 10 μg/mL macelignan for 30 min

ANTI-BIOFILM ACTIVITY OF MACELIGNAN 311


was more effective than treatment with 30 min of 2 μg/mL macelignan (p < 0.017). All single oral primarybiofilms with 24 h of growth phase showed similarreductions in activity after mace-lignan treatment at allconcentrations and exposure times.

DISCUSSION

Dental caries is an infectious and communicable dis-ease that results in destruction of tooth structure byoral biofilm-forming bacteria or dental plaque. Thedisease causes tooth loss if left untreated. One approachto control oral biofilm formation is to remove or re-duce the biofilm mass by using anti-biofilm agent.A majority of reports to date are relevant to antibacte-rial or anticariogenic activity of natural agents againstoral pathogen bacteria, but not specifically focused onremoving the oral biofilm plaque directly. Previouslyin vitro studies on anti-biofilm agents using syntheticantimicrobial agents such as chlorhexidine gluconateand triclosan were reported (Wilson, 1996; Wilsonet al., 1998; Marsh and Bradshaw, 1993). Exposure ofmulti-species biofilms to triclosan (0.07 mmol/L) for 1 hreduced approximately 40% of the bacteria present(Marsh and Bradshaw, 1993), however, chlorhexidinegluconate at 2.23 mmol/L removed approximately 40%of the multispecies bacteria (Wilson et al., 1998). Theseagents seemed very effective in removing dental plaque,but excessive dosage use can result in alteration ofthe oral cavity and also the development of bacterialtolerance. The routine use of chlorhexidine gluconateat high concentrations as part of daily oral hygiene isparticularly unsafe and sensitive against mutansstreptococci (Ernst et al., 1998; Steinberg et al., 2005).

To date, the search for natural anti-plaque agentswith safe efficacy and potent activity has been focusedin order to reduce the use of synthetic antimicrobialagents in daily oral care products such as toothpastes,mouthrinses, dental floss and chewing gums. Xanthor-rhizol, a single bioactive compound with molecularweight of 218 isolated from an edible C. xanthorrhizaRoxb., was first reported as a potent natural anti-biofilmagent against S. mutans biofilm (Rukayadi and Hwang,2006). At 50 μmol/L, xanthorrhizol could remove upto 76% of S. mutans biofilm after exposure time for60 min as well as chlorhexidine gluconate. Exposure oflyophilized American cranberry juice at 0.066–1.33 mg/mL for 1 h against S. sobrinus biofilm formed in vitroin the presence or absence of sucrose also showed anti-biofilm ability (Yamanaka et al., 2004). However, simple

comparisons are difficult to do because of differencesin the biofilm compositions, the antimicrobial agentsand their concentrations used. Since cranberry juice isnot a single bioactive compound like xanthorrhizol andmacelignan, it is probable that its components may re-duce the cell surface hydrophobicity of oral streptococci.The reduction effect was also dependent on the juiceconcentration (Yamanaka et al., 2004).

This study reported anti-biofilm activity of macelignanagainst single oral primary bacteria including S. mutans,S. sanguis and A. viscosus. Macelignan isolated fromnutmeg (M. fragrans Houtt.), an endemic and edibleplant from Indonesia, has been reported to show potentanticariogenic activity against S. mutans bacterium(Chung et al., 2006). Our findings demonstrated thatmacelignan activity at 10 μg/mL for a 30 min exposuretime could remove more than half of each single oralprimary biofilm formed by S. mutans, S. sanguis andA. viscosus at the plateau accumulated phase (24 h). Inthis case, macelignan might kill biofilm cells at the outeredge of the biofilm in direct contact with macelignan.Then, the dead cell could be released from the biofilmas indicated by the reduction of biofilm quantification.Although macelignan did not completely remove thebacteria at the beginning active accumulated phase(20 h) and plateau accumulated phase (24 h), all bacteriawere removed well in the adherence phase (4 h) of biofilmgrowth. The results also confirmed that S. sanguis andA. viscosus biofilms at 12 h of growth phase were removedup to 80% after treatment of 10 μg/mL macelignan withan exposure time of 30 min. This result had similartrends with planktonic S. mutans that was killed bymacelignan at the same concentration (10 μg/mL) aspreviously reported by Chung et al. (2006).

In conclusion, macelignan has an anti-biofilm effectby removing intraoral biofilm-forming bacteria in vitro.However, this study is only a model and the actualactivity of macelignan on dental plaque reduction hasto be evaluated in further clinical tests. In addition,as dental plaque is a biofilm composed of multi-speciesof bacteria and the adhesive ability of these bacteriaseems to be important pathogenic factor, we are alsoconducting macelignan effectiveness on inhibition ofbiofilm formation due to multi-species oral bacteria.

Acknowledgement

This work was supported by the Korea Foundation for InternationalCooperation od Science & Technology (KICOS) through a grantprovided by the Korean Ministry of Science & Technology (MOST)in 2007 (NO. 2006-8-1797) and party by the Yonsei BiomoleculeResearch Initiative of the Brain Korea 21 Project.

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In vitro anti-biofilm activity of macelignan isolated from Myristica fragrans Houtt. against oral primary colonizer bacteria