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928 Pak J Med Sci 2009 Vol. 25 No. 6 www.pjms.com.pk

Original Article

THE EFFECT OF PSIDIUM GUAJAVA AND

PIPER BETLEEXTRACTS ON THE MORPHOLOGYOF DENTAL PLAQUE BACTERIA

A.R.Fathilah1, M. Yusoff2,Z.H.A. Rahim3

ABSTRACTObject ive: It has been reported that the aqueous extracts ofPsidium guajava and Piper betleleaves showed anti-plaque activities during the early stages of dental plaque formation. The

aim of the study was to elucidate if such anti-plaque activities involve any ultra-structural

changes to the morphology of three early dental plaque bacteria, Streptococcus sanguinis,

Streptococcus mitis andActinomyces sp.

Met hodology: Pure cultures of the bacteria were suspended in BHI medium and treated with

the test herbal extracts at the sub-lethal concentrations. The growth mixtures were incubatedat 37C. At the logarithmic growth phase (t1), aliquots of 1 ml of the growth mixtures were

fixed and used in the preparation of specimens for SEM studies. Ultra-structural alterations to

the morphology of the treated cells noted were compared to those of the cells cultured under

untreated conditions.

Result s: Following exposure of the bacteria to the two test herbal extracts, profound

ultra-structural changes to their morphology were observed. The observed structural or

morphological alterations could attribute to the bacteria being less active in performing

normal physiological metabolic functions and thus rendering them less efficient to multiply.

The changes noted included (i) reduced sizes of the bacteria, and (ii) majority cells at the

non-dividing state as compared to those cultured under controlled conditions.

Conclusions: This study has shown anti-plaque effects of aqueous extract of both P. betle and

P. guajava.

KEY WORDS: Psidium guajava, Piper betle, Bacterial ultra-structural alteration, Anti-plaqueactivity.

Pak J Med Sci October - December 2009 (Part-II) Vol. 25 No. 6 928-933

How t o c i te t h is ar t i c le:

Fathilah AR, Yusoff M, Rahim ZHA. The Effect ofPsidium guajava and Piper betle extracts on

the Morphology of Dental Plaque Bacteria. Pak J Med Sci 2009;25(6):928-933.

Correspondence:

Dr. Fathilah Abdul Razak, PhD,

Associate Professor,

Department of Oral Biology,Faculty of Dentistry,

University of Malaya,

50603 Kuala Lumpur,

Malaysia.

E-mail: [email protected]

* Received for Publication: May 23, 2009

* Revision Received: September 19, 2009

* Accepted: September 23, 2009

INTRODUCTION

Plaque control measures have become thebasic principle in the prevention of plaquere-

lated oral diseases such as caries, gingivitis andperiodontitis.1 Dental plaque becomes thick andunhealthy when its microbial population mul-tiplies and produces extra-cellular polysaccha-rides that contribute to the increase in itsmatrix.2 An effective plaque control strategy isto target at ways to disrupt the accumulation ofplaque on the tooth surface favouring the


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Pak J Med Sci 2009 Vol. 25 No. 6 www.pjms.com.pk 929

Ultrastructural Effect of Plant Extracts On Oral Bacteria

formation of thin and loosely bound plaquewhich are more readily dislodged.

It has been reported that the extracts ofPsidium guajava and Piper betle plants exhibittherapeutic properties and are popularly used

in the formulation of traditional medicines.3-5

Aqueous extract of the leaves of both the plantshave been well studied for their anti-microbial,anti-plaque and anti-caries activities. Their usehas been recommended with possible potentialfor further development and incorporation asactive agents in oral health care products.6-9

Bacteria cells undergo several morphologicalchanges during its growth cycle. The largest cellsize corresponds closely to the period whenrapid division is taking place during the expo-nential growth phase. At the lag and approach-

ing the death phases, cells tend to be smaller insize.10 Attaining optimal cell size is importantfor a bacterium to be able to perform normalmetabolic function to grow and flourish.11 Al-terations in cell morphology may render the cellunable to multiply and hence to remain bacte-riostatic. In this study, the direct effect of P.guajava and P. betle extracts on the ultra-struc-ture of selected plaque bacteria was assessedusing the scanning electron microscope (SEM).Results obtained may help elucidate the mecha-

nism of action of P. guajava and P. betle extractsas anti-plaque agents.

METHODOLOGY

Preparation of plant extracts: Aqueous extracts ofP. betle and P. guajava were prepared by boilingsmall pieces of the fresh leaves of the plants indistilled water for several hours until the finalvolume was one third of the initial volume. Fol-lowing this, the decoction was centrifuged at10,000 rpm to eliminate sediments. The super-natant was then divided, into one ml aliquots,

in micro-fuge tubes. They were then concen-trated using a speed-vacuum concentrator(HETO/HS-1-110, Denmark). The concentratedextracts were weighed into sterile micro-fugevials and prepared into stocks of 20 mg/ml us-ing sterile distilled water as diluent.6

Preparation of bacterial suspensions: Streptococcussanguinis, Streptococcus mitis andActinomyces sp.

were representatives of dental plaque bacteriaused in this investigation. Pure test bacterialcultures were obtained from frozen (-80 C)stocks isolated from dental plaque specimenscollected from volunteers visiting the Dental

Clinic at the Faculty of Dentistry, University ofMalaya. Identification of the bacterial isolateswas done using conventional microbiologicalmethods and the API Identification System(BioMereux, France). Each bacterial species wasrevived in Brain Heart Infusion (BHI, Oxoid)broth at 37 C overnight. Following incubation,bacterial cells were harvested by centrifugationat 10,000 rpm for 10 minutes. The cells were thenre-suspended in BHI broth and the concentra-tion was standardised at 108 cells / ml [opticaldensity (OD) of 0.144] by using a spectropho-

tometer read at 550 nm.Determination of the mid logarithmic phase (t

1): 100

ml of BHI broth in separate sterile conical flaskswere seeded with 100 l of S. sanguinis, S. mitisand Actinomyces sp. suspensions and allowedto grow in a shaking water bath at 37 C. Thechanges in the OD of each bacterial culture wereperiodically monitored and recorded at every15 minutes intervals over a period of 9 hours. Agrowth curve of time versus OD was then plot-ted for each bacteria and the time at which the

mid of the logarithmic growth phase (t1) wasreached were determined.Effect of P. betle and P. guajava extracts on bacte-rial morphology: Similar culturing procedure wasrepeated but the bacteria were instead allowedto grown under three different conditions: (a)with addition of P. betle extract, (b) in additionof P. guajava extract, and (c) with addition ofchlorhexidine (CHX)-containing mouth-rinse.The concentration of extracts in (a) and (b) wasstandardised to a sub-lethal concentration of 4mg / ml to allow for minimal cell growth. The

sub-lethal concentration refers to a concentra-tion that falls between the minimal inhibitionand minimal bactericidal concentrations (MICand MBC) of the extracts. 12 The concentrationof CHX-containing mouth-rinse in the test wasset at 0.12 % which is the concentration of CHXused in many commercially available mouth-rinses such as Oral BTM. This was to represent a


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positive control for the study as CHX is consid-ered the standard anti-microbial agent in thedental and hospital arena.13 All tests were car-ried out in triplicate and repeated three timesfor reproducibility of results.

The culture flasks were placed in a shakingwater bath and the bacteria were allowed togrow at 37 C. When the t

1was reached, the

flasks were removed and 100 l of bacteria cellssuspension were removed and captured onNucleopore membrane discs (pore size 1mm). The membrane was then carefully pickedand transferred into glass vials containing onel of glutaraldehyde (4%). The following morn-ing, the membrane was then washed by displac-ing the fixative using a Pasteur pipette andreplacing it with one ml of sodium cocadylate

buffer (0.1 M). The washed membrane was thenfixed in osmium tetra-oxide (1%) and left over-night in tightly capped vials at 4 C. The osmiumtetra-oxide was then gently displaced withsodium cocadylate buffer and later washedthree times by gently displacing the buffer withfresh buffer each time. The membrane discswere then treated with ascending percentage

(10% to 100%) of ethanol (15 minutes each) todehydrate the cells. Finally the discs weremounted onto aluminium stubs and coated withgold before it was ready for SEM (JOEL, Japan)studies.

RESULTS

Figures-1, 2 and 3 represented SEM micro-graphs of S. sanguinis, S. mitis andActinomycessp., respectively at the mid of their logarithmicgrowth phases. Ultra-structural observations ofbacterial cells were summarised and tabulatedin Table I. In the absence of the extracts, the cellsof S. sanguinis (Fig-1a & b), S. mitis (Fig-2a & b)andActinomyces sp. (Fig-3a & b) were observedto have attained optimum cell sizes of (0.98 x0.80) m to (1.00 x 0.77) m, (0.64 x 0.56) m to(0.76 x 0.72) mm and (2.66 x 0.89) mm to (14.93

x 0.87) m, respectively. Most of these cells werealso shown to be at the actively dividing state.

Comparatively however, in the presence ofthe plant extracts the bacteria cells were ob-served to be smaller in size and many have re-sumed the non-dividing state (Fig-1c & d, 2c &d and 3c & d) (Table-I). In addition to that, thenumber of bacteria cells was observed to be

Fig-1: SEM micrograph of S. sanguinis. (a) Majorityof the cells cultured under the untreated conditionwere at the dividing state (90,000x). (b) Higher mag-nification of dividing cells showing the groove wherethe cell would split into two identical cells (200,000x).(c) Cells cultured following exposure to P. betle ex-tract were of variable sizes and majority existed assingle, non-dividing cells (90,000x). (d) Under the in-fluence of P. guajava, the size of the cells were moreuniform and were clumped together in extracellularmatrices (90,000x).

Fig-2: SEM micrographs of S. mitis. (a) Majority ofthe cells cultured under the untreated growth con-dition were in the dividing state (90,000x). (b) Thedividing cells at higher magnification (200,000x). (c)

Under the influence of P. betle, cell were observed tobe of uniform sizes and existed as single non-divid-ing cells (90,000x). (d) Cells cultured in the presenceof P. guajava were also of uniform sizes & aggregatedtogether in heavy extracellular matrices (90,000x).


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denser in the presence of P. guajava as comparedto cells in the presence of P. betle (Fig-1c & dand 2c & d). Heavy mesh-like matrices wereobserved to form around the cells in the pres-ence of P. guajava that tend to aggregate them

together in large clumps (Fig-1d, 2d & 3d). Thegrowth of S. sanguinis, S. mitis andActinomycessp. was found to be totally inhibited in the pres-ence of CHX and none was captured on thenudeogore membrane. The growth mediumappeared clear, an indication of the bactericidaleffect of the CHX-containing mouth-rinse. Thiswas to be expected as CHX has been acceptedby many researchers to exhibit maximum anti-microbial effect on oral micro-organisms.14

DISCUSSION

For cells to perpetuate themselves they mustgrow, that is increase in mass and size in anorderly fashion before they can reproduce ordivide.15 With respect to the oral environment,once bacteria are attached to the tooth surfacethey will continue to perform their normal bio-logical functions and reproduce by binary fis-sion so that the population will increase geo-metrically.11,16 In addition to biochemical nutri-ents that are needed to provide energy to sup-port cell growth, it is also equally important that

appropriate biophysical environment is pro-vided to ensure that the cells can propagatenormally at a specific growth rate within a spe-cific generation-time. Many factors such as tem-perature, pH and nutrients have been knownto play important role to ensure the completionof a growth cycle.11 Studies have shown that thephysiological growth of a bacterial cell is oftenaccompanied by changes in its morphologicalcharacteristics. Campylobacter jejuni for examplewas shown to exhibit gross morphologicalchanges with time when grown in a liquid me-

dium. The cells were the typical short spirals atthe exponential phase, became twice the lengthat the mid-stationary phase, but eventually wereobserved as coccus at the end of the growthcycle.17 Such changes in cell morphology dur-ing a growth cycle were suggested to be attrib-uted to the differential expression of genes thatcontrol the timing of cell division.

Based on the examination of the micrographs,the treatment of S. sanguinis, S. mitis andActi-nomyces sp. with either P. betle or P. guajava ex-tracts have in a way suppressed the morpho-logical development of the bacteria cells. Theextracts appeared to have slowed down cell

growth as most cells at t1 were small and havenot attained the optimal cell size as observed inthe absence of the herbal extracts (Table-I). Theintroduction of the extracts into the growth en-vironment has created an unfavourable condi-tion for the bacteria to grow. Therefore longertime was required for the bacteria to adapt andsynthesize new inducible enzymes that are nec-essary to metabolise the substrates introducedinto the growth medium. This was an indica-tion of the disruption of the normal physiologi-cal activities of the cells and hence, accounted

for the extension of lag phases of the growthcycles The retarding effect of these plant extractson the growth profiles of dental plaque bacte-ria has been reported.18,19

In the immature condition, the underdevel-oped bacterial cells may not be able to performtheir biological function efficiently and as such,metabolic energy becomes insufficient to allow

Fig-3: SEM micrographs ofActinomyces sp. (a) Cellsresumed rod shape with smooth surface under theuntreated growth condition (90,000x). (b) The vary-ing length resumed by the cells under the untreatedcondition was obvious at lower magnification

(30,000x). (c) Cells cultured under the influence of P.betle display tapering ends and irregular surfaces(90,000x). (d) In the presence of P. guajava extracts,the cells were observed to be covered in extracellu-lar matrices (90,000x).


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for growth activities such as cell division andmultiplication.20 The formation of heavy matrixwith the introduction of P. guajava in the growthenvironment also poses an aggregative effect tothe bacterial cells. In the aggregated form, the

growth performance of bacteria cells mayalso be affected.

With respect to the dental plaque, the suppres-sion of bacterial growth may slow down theprocess of plaque formation and minimize the


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accumulation of plaque on the tooth surface.Thin plaque is healthy as it is loosely bound tothe tooth surface and can easily be dislodgedby tooth brushing.21 Thus, the incorporation ofeither P. guajava or P. betle extract in a mouth-

rinse formulation may be considered for use inoral plaque control. Such attempt however, re-quires further investigation to ensure that theherbal preparations would not incur any ad-verse effect on the mucosal cells when exposedto the oral surfaces. Although preliminary stud-ies have shown that both extracts showed notoxic effect on Artemia salina (unpublished re-sult), investigating the responses of the mucosalcells to the extracts using tissue culture tech-nique is necessary to ensure the safe use of theextracts before it can be developed into com-

mercial oral products. In addition to that, in vivotrials of the products on study groups wouldalso be relevant. This would avoid any biasesthat may arise due to the limitation of in vitrotoxicity testing.

CONCLUSION

The aqueous extracts of P. betle and P. guajavawere found to have profound effect on the ul-trastructure of selected dental plaque bacteria.The extracts appeared to have interfered with

the normal growth cycle and development ofthe bacterial cells rendering them less efficientto divide and multiply. Hence, the developmentof dental plaque may be slowed down and con-trolled. This property indicates the antiplaqueactivity of P. betle and P. guajava extracts.

ACKNOWLEDGEMENT

This research was financially supported by theIRPA 09-02-03-0197-EA197 Grant from theMalaysian Government and Vote F0392/2004Bfrom the University of Malaya.

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