Scanning Electron Microscopic study of Piper betle L. leaves extract effect against Streptococcus mutans ATCC 25175

7/27/2019 Scanning Electron Microscopic study of Piper betle L. leaves extract effect against Streptococcus mutans ATCC 251

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J Appl Oral ci. 37

www.scielo.br/jaos

ABTACT

canning lectron Microscopic study ofPiper betle. leaves extract effect against Streptococcusmutans ACC 2515

Zubaidah aji Abdul AIM, Nalina TAIAJA2

1- Bc ons, h, epartment of ral Biology, ental chool, niversity of Malaya, uala umpur, Malaysia.

2- Bc ons, Mc, h, C niversity, ental chool, niversity of Malaya, uala umpur, Malaysia.

Corresponding address: ubaidah j Abd ahim (h - epartment of ral Biology, aculty of entistry - niversity of Malaya, 50603 - uala umpur -

Malaysia - hone: 603-964851 - ax: 603-964536 - e-mail address: [email protected]

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ntroduction: revious studies have shown that Piper betle . leaves extract inhibitsthe adherence ofStreptococcus mutans to glass surface, suggesting its potential rolein controlling dental plaque development. bjectives: n this study, the effect of thePiper betle . extract towards S. mutans with/without sucrose) using scanning electronPLFURVFRS\ 6(0 DQGRQSDUWLDOO\SXULHG FHOODVVRFLDWHGJOXFRV\OWUDQVIHUDVHDFWLYLW\were determined. Material and Methods: S. mutans were allowed to adhere to glass beadssuspended in different Brain eart nfusion broths without sucrose; with sucrose; withoutsucrose containing the extract 2 mg m-1 and 4 mg m-1); with sucrose containing theextract 2 mg m-1 and 4 mg m-1)]. ositive control was .12% chlorhexidine. he glassbeads were later processed for M viewing. Cell surface area and appearance and, cellpopulation ofS. mutans adhering to the glass beads were determined upon viewing usingthe M. he glucosyltransferase activity with/without extract) was also determined.ne- and two-way ANA were used accordingly. esults: t was found that sucroseincreased adherence and cell surface area ofS. mutans p


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J Appl Oral ci. 38

ther plant extracts like that of Syzygium

aromat icum21 , Polygonum cusp idatum 2,

Andrographis paniculata, Cassia alata, Chinese

black tea, uava, Harrisonia perforata1, ropolis

ype 3 and cacao bean18 also have an antiadherence

effect. he adhering property could contribute to

the virulence activity of the bacteria11,12. he ability

to adhere to oral surfaces so successfully is one of

the factors that afford these bacteria a critical role

in the formation of dental plaque which, eventually

lead to oral diseases such as caries, gingivitis and

periodontitis1,13,15. n previous studies, it has been

reported that the crude aqueous extract of Piper

betle . leaves exhibit antivirulence and antibacterial

activities towards S. mutans1. t was suggested

that the antimicrobial activity is attributed to the

hydroxychavicol component of the extract25.

Streptococcus mutans has been implicated

as one of the primary causative agents of dentalcaries in human and experimental animals,19.

he bacteria after the initial colonization of the

pellicle will attach strongly to the tooth surface

by the extracellular polysaccharides ). hese

extracellular polysaccharides are synthesized by

the S. mutans in the presence of sucrose via the

enzymatic action of one or more glucosyltransferases

s). activity may represent the virulence

factor of mutans Streptococciand can be used as

an index for determining the antivirulence activity

of the extract. he presence of -isoforms B, C

and as seen under electron microscopy within alllayers of the in situ formed pelliclePD\DIUPWKHLU

VLJQLFDQWUROHLQSODTXHGHYHORSPHQW7KHUHVXOWV

obtained from previous work1 have indicated the

Piper betle . extract affects the adhering capacity

ofS. mutans via inhibition of the activity and

hence the extracellular polysaccharide formation.

his conclusion was based on biochemical and

microbiological studies. he objectives of this study

were to use scanning electron microscopy M)

to demonstrate visually the effect of the aqueous

extract of Piper betle . leaves in the presence

and absence of sucrose on the cell adherence, cell

growth and extracellular polysaccharide formation

ofS. mutans and to relate the effect of the extract

on the activity with bacterial adherence.

MATEIA AN MET

Preparation of crude aqueous extract of the

leaves ofPiper betle

he leaves ofPiper betle . were obtained from

one source in Mentakab, ahang. Crude aqueous

extract of the leaves was prepared according to

Nalina and ahim1 2). Before use the dried

pellets were weighed, dissolved and diluted to therequired concentrations using deionized distilled

ZDWHU7KLVLVIROORZHGE\OWUDWLRQXVLQJm

Q\ORQV\ULQJHOWHUV0LOLSRUHLOOHULFD0$6$

which is for sterilization process2.

Preparation of bacterial suspension

Streptococcus mutans ACC 2515 American

ype Culture Collection ACC), Manassas, A

2188, A] suspension was prepared according

to Nalina and ahim1 2). he bacterial stock

which was kept frozen in glycerol at -C before

use was thawed at room temperature to revive the

bacteria. he thawed stock was then dispersed in

3 m Brain eart nfusion B) broth xoid,

ampshire, ngland) before incubating it at 3C

for 18-2 h. he number of bacterial cells in the

suspension used in the study was standardized by

adjusting the absorbance of the bacterial suspension

spectrophotometrically 55 nm

) to .144. his

absorbance is equivalent to 1 cells m-1 23. o

ensure that only pure culture of the stock are usedin the study, the revived bacteria were consistently

tested for purity by culturing them on B plates

containing 5% blood.

EM analysis on the adherence capacity and

growth ofS. mutans

M analysis was done to determine a) the effect

of sucrose on the growth and adherence capacity

of S. mutans to glass surface and b) the effect

ofPiper betle . leaves extract on the adherence

capacity and growth of S. mutans on the glasssurface in the presence and absence of sucrose.

he adherence capacity was determined from the

bacterial cell population adhering to the surface

of glass beads and the growth from the size and

dividing appearance of the bacterial cells. he

extracellular surface appearance of bacterial cells

as viewed by M was also noted.

a) ffect of sucrose on the growth and adherence

capacity ofS. mutans to glass surface

7KHDVVD\ZDVEDVHGRQDPRGLFDWLRQRIWKH

method developed by oshima, et al.18 2).

3 m of Brain eart nfusion B) broth xoid)

containing 1% w/v) sucrose B) was pipetted

into a vial containing sterile glass beads diameter:

3 mm) Merck, armstadt, ermany). his was

followed by the addition of bacterial cell suspension

containing S. mutans equivalent to x1 cells m-

1). he mixture was then incubated at 3C in an

inclined position at an angle of 3 degrees for 18 h.

his time duration allows the bacterial cells to be in

the stationary phase. After the 18 h incubation, the

glass beads were then washed with sterile deionized

water and transferred into 1 m of glutaraldehyde

igma Aldrich Co., t. ouis, M, A) 4%) to[WKHFHOOVDQGOHIWRYHUQLJKWDW&IRUWKH6(0

analysis. M was used to view and record the cell

canning lectron Microscopic study ofPiper betle . leaves extract effect against Streptococcus mutans ATCC 27

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J Appl Oral ci. 39

population, cell growth and extracellular surface

appearance ofS. mutans. he assay was carried

out in triplicate.

or blank control, the assay was repeated using

B without bacterial inoculation. he blank control

was to validate that the assay is devoid of bacterial

contamination. or negative control, the experiment

was repeated using B broth without sucrose.

b) ffect ofPiper betle . leaves extract on the

adherence capacity and growth ofS. mutans on the

glass surface in the i) absence and ii) presence of

1% sucrose

he assay procedure in a) was repeated with the

addition of the Piper betle extract at the respective

concentrations 2 mg. m-1 and 4 mg. m-1) to the

inoculated B broth. n the determination of i) the

effect of plant extract in the absence of sucrose, the

B broth devoid of sucrose was used and ii) the

effect of plant extract in the presence of sucrose,the B broth containing 1% sucrose was used.

terile deionized distilled water and .12%

chlorhexidine were used in place of the plant extract

for negative and positive controls respectively and

the assays were also carried out using the B

broth with or without sucrose. he beads from the

experiments were also processed for the M study.

All of the above assays were carried out in triplicates.

here were 9 beads to be viewed under M for each

experiment with the respective broths.

Preparation of the glass beads for EManalysis

7KHJODVVEHDGVWKDWKDGEHHQ[HGRYHUQLJKWLQ

glutaraldehyde were then processed for M analysis

according to the following steps. irst, the glass

beads were washed with sodium cacodylate igma

$OGULFK&REXIIHU0DWS+DQGWKHQ[HG

in 2% w/v) osmium tetroxide Merck) in the buffer

solution overnight. he next day, the beads were

gently washed in distilled water twice for 15 min and

then dehydrated in an ascending series of ethanol

B aboratories upplies; nternational,

utterworth, eicestershire, ) concentrations

1, 2, 3, 4, 5, , , 8, 9 and 95%) for

15 min for each step. ollowing this, the beads were

further dehydrated in 1% ethanol twice for 15

min and subsequently in ethanol:acetone mixture

DVIROORZVUVWO\HWKDQRODFHWRQHIRUPLQ

then ethanol:acetone 1:1) for 15 min and followed

by ethanol:acetone B) 1:3) for 15 min. inally,

the beads were treated 3 times with pure acetone

for 15 min.

he beads were then processed for critical

point drying for about 2 h. nce the beads were

completely dry, they were mounted onto metal

stubs with double sided tape and then coated with

gold. hree beads from each assay were then used

for viewing using a scanning electron microscope

hillips, indhoven, Netherlands).

etermination of the number of bacterial

cells adhering to the glass surface

he number of cells adhering to the glass surfaceon a 1 m2 area of the glass bead viewed at a

PDJQLFDWLRQRI[ZDVFDOFXODWHGDQGWKHWRWDO

number of cells was determined from an area of 1

mm2 of the glass surface. he area with high density

of bacteria cells was used in the calculation of the

number of cells igure 1). n total, 9 beads from

3 different assays per experiment were processed

and analyzed.

etermination of bacteria cell surface area

7KH EHDGVZHUHYLHZHGDWD PDJQLFDWLRQ RI

[$WWKLVPDJQLFDWLRQWKHVLHRIWKHFHOOLVPDJQLHGDQGWKXVPHDVXUHPHQWLVHDVLO\GRQH

he size of the individual adhered bacterial cell was

measured according its length ) and width ) in

PDQGH[SUHVVHGDVWKHPHDQVXUIDFHDUHD>6'

[6'@LQP2 of the total number of adhered cells

from 9 beads used in the determinations.

etermination of the appearance of the

extracellular surface

his was carried out by comparison with the

appearance of the extracellular surface ofS. mutans

adhering to hydroxyapatite beads28.

igure iagram showing the calculation for the number of bacterial cells on 100 Pm2 area in a total area of 1 mm2 of

the glass surface. The area with high density of bacteria cells was used in the calculation of the number of cells in Table 1

AIM ZA, TAIAJA N

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Preparation of partially purified cell-

associated T ofS. mutans

Crude was isolated from S. mutans ACC

2515 strain in the research laboratory, ral

Biology epartment, niversity of Malaya using the

method described by oshima, et al.18 2) with

VOLJKWPRGLFDWLRQ1,21. he crude enzyme was then

VXEHFWHG WRSDUWLDOSXULFDWLRQE\XOWUDOWUDWLRQ

Macrosep mega membrane - mw cut off=3 kd;

all ife cience, xton, A, A). he crude enzyme

ZDV WKHQ FHQWULIXJHGXVLQJ WKHFHQWULIXJDOOWHU

Macrosep mega membrane; all ife cience) at

5, g for 2 min to remove residual cells and the

supernatant collected was then referred as partially

SXULHGFUXGHHQ\PH7KHSDUWLDOO\SXULHGFUXGH

enzyme was kept in the freezer -2C) to be used

for further analyses.

etermination the effect of the extractWRZDUGV SDUWLDOO\ SXULHG FHOODVVRFLDWHG

T (CA) activities

he reaction mixture used in the determination

RIWKHSDUWLDOO\SXULHGFHOODVVRFLDWHG*7DFWLYLW\

was adapted from the method described by Mukasa,

et al.14DQGPRGLHGWRVXLWWKHH[SHULPHQW

carried out in this study. he reaction mixture

consists of .1% of thimerosal igma Aldrich Co.),

1.4 g of sucrose, 34.3 mg of exogenous dextran

CN Biomedicals, Aurora, , A) and mixed in

1 m of 5 mM sodium phosphate igma Chemical

Co.) buffer p .5). 1 m of the partiallySXULHGFHOODVVRFLDWHG*7ZDVSLSHWWHGLQWRWKH

UHDFWLRQPL[WXUHWR JLYHDQDOYROXPHRIP

he mixture was then incubated at 3C. After 18

h to allow the enzyme to catalyze the formation

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the reaction was terminated by placing the tube

in a boiling water bath for 5 min. After 5 min, the

mixture was centrifuged igma 32, sterode,

ermany) at 2, g for 1 min. he pellet obtained

after centrifugation was collected and used in the

determination of water-insoluble ) extracellular

polysaccharide content while the supernatant was

discarded.

he pellet was washed 3 times each with 5 m

of distilled water and then uniformly suspended in

1 m of distilled water by agitation. 5 m of the

extracellular polysaccharide suspension was

then pipetted into a clean test tube and assayed

for glucose content.

he experiment was repeated by adding the plant

extract at the respective concentrations 4. mg

m-1) to the enzyme reaction mixtures. Comparison

ZDVPDGHEHWZHHQWKHDFWLYLWLHVRISDUWLDOO\SXULHG

CA treated with plant extract and that without

the treatment. he activity of the enzyme in thepresence of .12% chlorhexidine igma Aldrich

Co.) was also determined using the same procedure.

etermination of glucose content of the

I glucan

he determination of glucose was carried out

according to the method by ubois, et al.4 195).

o 5 m of the glucan suspension, 5 m of

deionized distilled water was added and this was

followed by the addition of 5 m of phenol igma

Aldrich Co.) 8% w/v). he mixture was vortexed

and subsequently 2 m of concentrated sulfuric

acid M Chemicals, ssex, ) was added to

the mixture. his mixture was then allowed to

stand for 1 min at room temperature before its

optical density at 49 nm was measured. or the

standards, a range of volumes 1 m) of the

stock glucose igma Aldrich Co.) solution 1 mg m-

1ZDVSUHSDUHGWRJLYHDUDQJHRIQDOFRQFHQWUDWLRQ

from to 1 mg m-1. or the blank control, 1 m

of deionized distilled water was used instead. he

determinations were carried out in triplicates.he glucose content determined corresponds to

the amount of extracellular polysaccharide formed

E\WKHSDUWLDOO\SXULHG*7ZLWKLQWKHVSHFLFWLPH

which was then used in the calculation of the

activity. he enzyme activity is expressed as mol

glucose in the extracellular polysaccharide produced

per min mole glucose min-1)21.

tatistical analysis

Normality tests for all the data obtained were

carried out using the hapiro-ilks analysis. he

data for the comparison of the cell populationand cell growth in the respective experiments

were analyzed using a two-way ANA. or the

FRPSDULVRQRIWKHSDUWLDOO\SXULHG&$*DFWLYLWLHV

between the different experiments, a one-way

ANA was used.

ET

Effect of sucrose on the adherence capacity

and growth ofS. mutans on glass surface

igure 2 shows M micrographs 1,x

magnification) for blank control B without

bacterial inoculation), negative control inoculated

B without sucrose) and test inoculated B

with sucrose) based on M viewing at. hese

images provide qualitative observations of the

cell population which was used to determine the

adherence capacity ofS. mutans to glass surface.

t was shown that there were no cells adhering

to the glass surface in blank control B without

bacterial inoculation) igure 2A). his indicates

that the experimental procedures are assured of the

exclusion of bacterial contamination. he number of

cells adhering to the glass surface when grown in the

absence of sucrose was less igure 2B) comparedto that in the presence of 1% sucrose as shown in

igure 2C. t was observed more cells were adhering


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J Appl Oral ci. 4

to the glass surface and they are in aggregates. he

extracellular surface of the cells appeared to have

DOD\HURIXII\DSSHDUDQFHVSUHDGLQJDFURVVWKH

surface of the glass bead igure 2C).

he quantitative measurement of the bacterial

cell population was carried out during the M

YLHZLQJDW[PDJQLFDWLRQWZDVIRXQGWKDW

the number of cells adhering to the glass surface

increases in the presence of sucrose and the

increase was -fold higher than that in the absence

p


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J Appl Oral ci. 42

Effect of the Piper betle . leaves extract on

the growth ofS. mutans with and without

sucrose

he cell surface area of the bacterial cells grown

in the presence of the extract, with and without

sucrose based on the M viewing at 1,x

PDJQLFDWLRQ LVVKRZQLQ7DEOH W ZDV IRXQG

that the bacterial cells grown in the presence of 2

mg m-1 of the extract without sucrose exhibited

FHOOVXUIDFHDUHDWKDWLVQRWVLJQLFDQWO\GLIIHUHQW

Cell surface area (m2)

xperiments Absence of sucrose resence of sucrose

Negative control (without the CA 0.2090.018a,b 0.280.026a

2 mg m-1 CA 0.2160.016b,x 0.2440.018d,x

4 mg m-1 CA 0.1460.010c,y 0.1450.009e,y

ositive control (0.12% chlorhexidine nd nd

The number of cells adhered to the glass beads 100 m 2 was expressed as the mean standard deviation from nine

determinations

a p


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J Appl Oral ci. 43

from those grown in the absence of the extract.

ncreasing the concentration of the extract to 4

mg m-1 decreases the cell surface area by 3%

p


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J Appl Oral ci. 44

was expressed as mol glucose content of the

extracellular polysaccharide perminute and was

found to decrease in the presence of the extract.

he inhibitory effect was found to be concentration

GHSHQGHQWDQGZDV PRUH VLJQLFDQWDW KLJKHU

concentrations of the extract. At .5 mg m-1 of

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reduced by 29% p


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t was thought that the reduced adherence is

attributed to extracellular polysaccharide not being

IRUPHGHIFLHQWO\ZKLFK LVGXH WRUHGXFHG*7

activity in the presence of the extract.

n this study, it was shown that there are

bacteria though few in number adhering to the

glass surface despite the undetectable activity

in the presence of 4 mg m-1 of the extract. n the

presence of .12% chlorhexidine there were no

bacterial cells adhering to the glass surface and at

that concentration, the activity corresponds

to that of 2.5 mg m-1 of the extract and, cell

adherence was observed in the presence of the later.

his suggests that is not involved and that other

factors such as hydrophobic and ionic interactions

maybe involved in the adherence or attachment of

bacteria to the tooth surfaces which, is in agreement

with what had been reported in previous work11,12.

owever, it has been shown that the cell surfacehydrophobicity of S. mutans is reduced in the

presence of the plant extract1. he crude Piper

betle OHDYHVH[WUDFWPD\ KDYH LQXHQFHG WKH

adhesion between the cell surface of the bacteria

and the host surface via ionic interaction which

could be responsible for the adherence effect

demonstrated at 4 mg m-1 extract concentration.

t was also shown that the Piper betle . leaves

H[WUDFWKDVDQLQXHQFHRQWKHFHOOVXUIDFHDUHD

size) of the bacterial cells. he presence of sucrose

appears not to have effect on the cell surface area

of bacterial cells in the presence of the 4 mg m-1

extract as with or without sucrose, the bacterial

cells appear to attain similar cell surface area. his

indicates that bacteria grown in the presence of

the extract could experience environmental stress,

ZKLFKFRXOG LQXHQFH LWVDELOLW\ WRXVHQXWULHQWV

HIFLHQWO\DQGWKHUHE\VORZVGRZQLWVJURZWK5,29. t

has been reported that the aqueous Piper betle .

extract exhibited similar effect towards other oral

bacteria for example, S. sanguinis, S. mitis and

Actinomyces sp.5.

CNCN

he M analysis provided visual qualitative)

and quantitative evidence that the Piper betle

. leaves extract has a reducing effect on cell

adhesion, cell growth and extracellular appearance

ofS. mutans. he effect of the Piper betle . leaves

extract on the activity and adherence capacity

ofS. mutans suggests the involvement of other

factors in the adherence of bacterial cells to the

glass surface.

ACNEEMENT

his project was supported by research grants

ote and A) provided by the Malaysian

government. he authors would like to acknowledge

Ms ubaidah Abu assan of the lectron Microscopy

nit, Medicine chool, niversity of Malaya for their

assistance in the electron microscopy procedure.

EEENCE

ODF&$OODQRUG6LOVRQ00FDELROPVSHFLF

surface properties and protein expression in oral Streptococcus

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2;9(2):3746


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J Appl Oral ci. 46

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VXFURVHLQWDHDQGRZUDWHRIVDOLYD&DULHVHV

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HQ=7DHU+(XUQH$QXHQFHRIUS$RQFULWLFDO

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Documents

Scanning Electron Microscopic study of Piper betle L. leaves extract effect against Streptococcus mutans ATCC 25175