Bacteriostatic Effect of Piper betle and Psidium guajava Extracts on Dental Plaque Bacteria
In this study, the bacteriostatic effect of Piper betle
and Psidium guajava extracs on selected early dental plaque bacteria
was investigated based on changes in the doubling time (g) and specific
growth rates (μ). Streptococcus sanguinis, Streptococcus mitis
and Actinomyces sp. were cultured in Brain Heart Infusion (BHI) in the
presence and absence of the extracts. The growth of bacteria was monitored periodically
every 15 min over a period of 9 h to allow for a complete growth cycle. Growth
profiles of the bacteria in the presence of the extracts were compared to those
in the absence and deviation in the g and μ were determined and analyzed.
It was found that the g and μ were affected by both extracts. At 4 mg mL-1
of P. betle the g-values for S. sanguinis and S. mitis
were increased by 12.0- and 10.4-fold, respectively (p<0.05). At similar
concentration P. guajava increased the g-value by 1.8- and 2.6 -fold,
respectively (p<0.05). The effect on Actinomyces sp. was observed
at a much lower magnitude. It appears that P. betle and P. guajava
extracts have bacteriostatic effect on the plaque bacteria by creating a stressed
environment that had suppressed the growth and propagation of the cells. Within
the context of the dental plaque, this would ensure the attainment of thin and
healthy plaque. Thus, decoctions of these plants would be suitable if used in
the control of dental plaque.
The extracts of the Piper betle (L.) and Psidium guajava (L.)
plants have been reported to posses many biological activities that have contributed
to their role in the development of therapeutic products (Nair
and Chanda, 2008; Wirotesangthong et al., 2007;
Kamath et al., 2008). Piper betle is popularly
used in traditional medicine as it possesses antioxidant, antibacterial, antifungal,
antidiabetic and radioprotective activities (Wirotesangthong
et al., 2007). Psidium guajava is often used as astringent
for skin diseases (Ponglux et al., 1987) and also
showed antidiarrheal, hepatoprotective, hypoglycemic, lipid lowering, antibacterial
and antioxidant activities (Kamath et al., 2008).
The methanolic extract of P. betle and P. guajava have been shown
to exhibit antimicrobial effects on various Gram positive and negative food
borne pathogens (Francis Parillon, 2006). While the effect of P. betle
was on both the Gram-positive and Gram-negative bacteria, the effect of P.
guajava was specific to the Gram-negatives. Membrane damage that causes
loss of cell viability and leakage of intracellular constituents was suggested
to be the main mechanism of action of these extracts.
In the perspective of oral health maintenance, the aqueous extracts of P.
betle and P. guajava have showed positive antiplaque activities that
act on dental plaque bacteria at the early phase of plaque formation. These
extracts were reported to act by first reducing the adhering capacity of the
acquired pellicle which forms on the tooth surface at the early phase of plaque
formation, to receive and bind the bacteria (Fathilah and
Rahim, 2003) and second by diminishing the cell-surface hydrophobicity of
the bacteria which are required to assist the adherence process (Fathilah
et al., 2006). Study by Nalina and Rahim (2006)
has also shown that the crude extract of P. betle inhibited the activity
of glucosyltransferase (GTF) which is required for glucan synthesis by the cariogenic
bacteria Streptococcus mutans. The Minimal Inhibitory Concentration (MIC)
and Minimal Bactericidal Concentration (MBC) of aqueous extracts of P. betle
and P. guajava were reported within the range of 2.16-4.69 and 5.21-10.42
mg mL-1, respectively (Fathilah and Rahim, 2003).
The objective of this study was to investigate the bacteriostatic effect of
the aqueous crude extracts of P. betle and P. guajava on early
dental plaque bacteria, S. sanguinis, S. mitis and Actinomyces
sp. Bacteriostatic effect will be determined based on deviations in the doubling
time and specific growth rate of the growth profiles produced in the presence
and absence of the extracts.
MATERIALS AND METHODS
Preparation of plant extracts: Fresh leaves of P. betle and P. guajava were obtained from the universitys botanical garden. Aqueous extracts of P. betle and P. guajava were prepared by concentrating decoctions of the fresh leaves of the plants using a speed-vacuum concentrator (HETO/HS-1-110, Denmark). The dried extracts were then kept refrigerated at -80°C (Hetofrig, Denmark) prior to use in the experiment. The dried extracts of P. betle and P. guajava were weighed into sterile microfuge vials and prepared into stocks of 20 mg mL-1 using sterile distilled water as the diluents. The extracts were dissolved by sonicating the microfuge vials in a sonicator (Ultrasonic sonicator, Selecta CE95).
Preparation of bacterial suspensions: Streptococcus sanguinis,
S. mitis and Actinomyces sp. used in the investigation were pure
cultures obtained from frozen (-80°C) stocks isolated from dental plaque
specimens collected from volunteers visiting the Dental Clinic at the Faculty
of Dentistry, University of Malaya (Fathilah and Rahim, 2003).
Each bacteria species was revived in Brain Heart Infusion (BHI, Oxoid) broth
at 37°C overnight. Following incubation the bacteria cells were harvested
by centrifugation at 10,000 rpm for 10 min. The cells were then resuspended
in BHI broth and the concentration was standardized at 106 cells
mL-1 [Optical Density (OD) of 0.014] by using a spectrophotometer
read at 550 nm.
The effect of P. betle and P. guajava aqueous extracts on
bacterial growth profiles: The antibacterial activity of P. betle
and P. guajava was determined using an assay procedure which involved
the changes in the optical absorbance as an indication of changes in the bacterial
growth profile. Metal capped borosilicate glass tubes (13x75 mm) were sterilized
and used as culture tubes in the experiments. The growth of S. sanguinis,
S. mitis and Actinomyces sp. under four different conditions was
monitored by measuring the increased in the OD of the growing cells every 15
min. The four growth conditions were: (a) in 5 mL BHI broth to represent the
control, (b) in 5 mL BHI added with P. betle extract at 4 mg mL-1,
(c) in 5 mL BHI added with P. guajava extract at 4 mg mL-1
and (d) in 5 mL BHI added with chlorhexidine (CHX)-containing mouth rinse at
neat concentration of 0.12 mg mL-1. Each of the test tubes was then
inoculated with 50 μL of the respective bacterial cells suspension. The
concentration of extracts was selected at 4 mg mL-1 to be within
the range of the MIC. This would ensure that the addition of the extract would
not kill the bacteria cells but instead would allow the growth of cells to be
at its minimum. CHX-containing mouth rinse in the test was used to represent
a positive control for the study as CHX is considered the standard antimicrobial
agent in the dental and hospital arena (Jones, 1997;
Kornman, 1986). All tests were carried out in triplicate
and repeated three times for reproducibility of results.
The content of each culture tubes were mixed well using a vortex mixer. The
OD readings of each tube were set to zero with a test tube containing everything
else except for the bacterial cells. This was to accommodate differences in
the OD of the mixture caused by the varying color intensities of the plant extracts
and the CHX-containing mouth rinse. The cultures were then incubated at 37°C
in a shaking water bath and changes in the OD readings of each tube were periodically
monitored and recorded at every 15 min intervals over a period of 9 h. The growth
curves of each bacterium under the four growth conditions were plotted and compared
with the profile of the CHX-containing mouth rinse. The growth rate (μ)
and doubling time (g) of S. sanguinis, S. mitis and Actinomyces
sp. under the different growth conditions were then determined using the following
equations (Gerhardt et al., 1981; Cappuccino
and Sherman, 2005):
μ = [(log10 N-log10 N0)
g = (log10 N - log10 N0)/log10
where, N is No. of cells at log phase, N0 is No. of cells at zero time and t is time to reach, t0 is zero time log phase.
Statistical analysis: The effect of the extracts on the growth of the bacteria was illustrated by comparative analysis of their growth profiles under the various growth conditions. Statistical analysis was carried out using the one way analysis of variance (ANOVA). The MINITAB 13 for Windows statistical program was used to determine the Mean, Standard Deviation and evaluate the significance of the data in the experiments. Results were expressed as Mean±SD from one nine determinations (n = 9) set at a significance level of p<0.05.
RESULTS AND DISCUSSION
Throughout the study, the concentration of P. betle and P. guajava was set within the sub-MIC concentration of 4 mg mL-1 and not exceeding the MBC concentration.
This is important as the aim of a good antiplaque agent is not to kill all
but to allow some of the plaque bacteria especially those of the normal species,
to grow at a minimal rate. Within the sub-MIC range the bacteria exist in a
condition where the biological cell functions are not disrupted. The growth
profiles in Fig. 1a-c strongly suggested that the antimicrobial
activity of P. betle and P. guajava towards S. sanguinis,
S. mitis and Actinomyces sp. was bacteriostatic and may have been
targeted at the early lag phase of the growth cycle. P. betle and P.
guajava extracts seem to have created a stressed environment for the cells
to perform their normal biological functions. This explains for the extended
g-values and reduction in the μ-values in Table 1. The
attainment of minimal population size as the bacteria enters the stationary
phase indicated the bacteriostatic activities of P. betle and P. guajava
towards S. sanguinis, S. mitis and Actinomyces sp. Under
the stressed growth environment the bacteria were unable to perform normal biological
function and eventually ceased to propagate. Such growth inhibiting mechanism
has also been reported when the requirement for nutrient was restricted for
S. sanguinis growth (Fathilah et al., 2007).
The search for new alternative agents to be used as adjuncts in oral health
care products has spurred due to the unfavorable staining effect caused by the
prolonged usage of CHX (Cummins, 1992; Baehni
and Takeuchi, 2003). In this study CHX-containing mouth rinse at 0.12 mg
mL-1 was found to be bactericidal to S. sanguinis,
S. mitis and Actinomyces sp. as no growth profile can be generated
following the 9 h incubation period. This finding confirmed the reputation of
CHX as the most effective antimicrobial agent for the oral microorganisms. Alternative
to CHX, plant based bioactive compounds such as sanguinarine, gallotannins and
catechins have been isolated using solvent extraction procedures from Sanguinaria
canadensis (Kopczyk et al., 1991; Harper
et al., 1990), Melaphis chinensis (Wu-Yuan
et al., 1988) and Japanese green tea (Hirasawa
et al., 2006; Otake et al., 1991),
respectively. Exhibiting significant antiplaque activities, these compounds
have been incorporated as adjuncts in dental dentifrices. In other plants like
Azarachdita indica (Neem) (Wollinsky et al.,
1996), P. betle (Fathilah and Rahim, 2003;
Fathilah et al., 2006; Nalina
and Rahim, 2006, 2007) and P. guajava (Fathilah
and Rahim, 2003; Fathilah et al., 2006), aqueous
extraction procedure were employed and the crude extracts have been used. The
exploration of antimicrobial activities of these plants has been based on their
effective used in folklore medicines which often make use of simple decoction
using water. Aqueous extraction procedure was employed in this study as it is
environment friendly and would also avoid any possibility of side effect that
could arise from the exposure to solvents if these extracts are to be used in
the development of oral health care product.
||The growth profiles of (a) S. sanguinis, (b) S.
mitis and (c) Actinomyces sp. in the absence of extract, presence of
P. betle and presence of P. guajava. Deviation of profiles from
the untreated growth condition indicated the bacteriostatic effect of the
||Changes in the generation times (g) and specific growth rates
(μ) of S. sanguinis, S. mitis and Actinomyces
sp. when cultured in the absence (untreated) and presence (extract treated)
of P. betle and P. guajava were compared
The aqueous extracts of P. betle and P. guajava exhibited bacteriostatic effect on early dental plaque bacteria S. sanguinis, S. mitis and Actinomyces sp. under the stressed growth environment the bacteria appear to be unable to perform normal biological function and eventually ceased to propagate. Such events would ecologically control the development of dental plaque. Thus, both the plant extracts may have potential to be used as an active ingredient in the development of oral health care products.
This research was financially supported by the IRPA 09-02-03-0197-EA197 Grant from the Malaysian Government and Vote F0392/2004B from the University of Malaya.
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