Dental Caries Inhibition in Albino Rats by Breynia nivosus Extract
The study investigated the caries inductive capacities
of different sucrose concentrations and the anti-caries activity of Breynia
nivosus extract in experimental albino rats. Different concentrations
(70, 50, 30 and 10%) of sucrose-in-diet, were respectively fed to caries-free
albino rats harboring Streptococcus rattus in their oral
cavity, to determine their caries induction effect. Subsequently, 200
mg mL-1 of Breynia nivosus extract were intra-orally
administered to the teeth surfaces of caries-free and non caries free
rats to ascertain its possible caries curative and/or preventive effects.
Direct observation, probing, microbial count and radiography were used
to monitor the caries status of the rats. There were significant increase
(p<0.05) in the microbial count of dental plaque of rat groups fed
with 70, 50 and 30% sucrose-in-diet. However, milky white spots were only
observed among the rat groups fed with 70 and 50% sucrose-in-diet at the
6th week of observation. The results of this investigation suggests that
Breynia nivosus extract possesses some degree of in vivo
caries preventive and curative effect on the teeth surfaces of albino
rats fed simultaneously with 70% sucrose-in-diet.
Dental caries is characterized by demineralization of the mineral portion of
enamel and dentine, followed by disintegration of their organic material. It
has been attributed to a dieto-bacterial interaction on the tooth-surface which
produces acids such as lactic acid and pyruvic acid. These acids lowers the
pH to below 5 which is enough to dissolve or decalcify the enamel (Talaro
and Talaro, 2002; Ingraham et al., 2001; Johnson
and Beighton, 1992). Although glucose, fructose and other sugars are cariogenic
in varying degrees, sucrose has been described as the main cause of dental caries.
While decreased prevalence of dental caries is reported in some cities in Brazil
(Tagliaferro et al., 2008), it is however on the
increase among adolescents in South Western Nigeria (Umesi-Koleoso
et al., 2007) as well as in Australian children, after marked improvements
in the 1990s (Armfield and Spencer, 2008). Increased
cases among adults and the elderly has also been reported in New Delhi (Patro
et al., 2008).
Dental caries can pose numerous challenges to the sufferers including general
discomfort, inconvenience, expense, masticatory difficulty and alteration of
the facial skeleton and associated musculature (Talaro and
Talaro, 2002; Johnson and Beighton, 1992).
Herbal remedies has been employed since ancient period in the prevention and
treatment of diseases including dental diseases. Although works on the antimicrobial
activities of medicinal plants on dental caries has been widely reported especially
in the western countries (Yamamoto and Ogawa, 2002; Addai
et al., 2002; Sato et al., 2003;
Linke and LeGeros, 2003), not much reports are available on the effect of
locally used plants in Southeastern Nigeria. In this report, we present findings
on the caries inductive capacities of different concentrations of sucrose-in-diet
and the effect of plant extract on the induction of dental caries in experimental
MATERIALS AND METHODS
Selection of Experimental Rats
Plaque samples were collected from the anterior teeth surfaces of eighty 8-10
week old albino rats weighing between 60-120 g. The samples were cultured in crystal
violet blood agar and incubated at 37°C for 24 h (Collins
et al., 1995
). The colonies were sub-cultured by the streak plate technique
to obtain pure culture. The isolates were identified using standard methods as
described by Chessbrough (2002)
. Male rats harboring Streptococcus
were randomly selected from the eighty for use in the experiment.
Collection and Identification of Plant Materials
The fresh leaves and tender stems of Breynia nivosus (ice plant)
were obtained from Enugu, Enugu State, Nigeria and authenticated by a
taxonomist from the Applied Biology Department, Ebonyi State University,
Abakaliki. This study was conducted in 2003.
Extraction of Plant Materials
The plant materials were dried at room temperature and pulverized using
mechanical grinder (Fondicion, Corona). Exactly 40 g of the macerated plant
material was introduced into 200 mL of 95% ethanol and mixed thoroughly. The
mixtures were allowed to stand for 24 h with constant shaking. Whatman`s No.
1 filter paper was used to sieve the extract and later evaporated to dryness
in a water bath at 40Â°C (Ibrahim et al., 1997).
Preparation of Diet
Granulated sugar (sucrose) obtained from Ogbete market, Enugu, Enugu
State, were combined with commercially available rat feed purchased from
Nsukka, Enugu State, Nigeria. The combinations were respectively prepared
to produce 70, 50, 30 and 10% sucrose concentrations in the feed.
Feeding of Rats with Different Sucrose Concentrations in the Diet
Fifteen rats selected as previously described were divided into five
groups (A-E). Prior to the commencement of sucrose-in-diet feeding, the
upper and lower anterior teeth surfaces of each rats were visually observed
and also examined using dental probe and torch. Plaque sample were also
collected from the upper and lower anterior teeth surface of each of the
rats using sterile swab sticks and dental probe. The radiography of the
lower anterior teeth of the rats was also taken. Group A rats were fed
with 70% sucrose-in-diet, group B 50%, group C 30% and group D 10%. Group
E was fed with normal diet without sucrose. The anterior teeth surfaces
of the rats were observed on weekly basis for signs of early dental carries.
At the observation of milky white spots on the anterior teeth surface
of any of the rat groups, dental plaque samples were collected from all
the rats as previously stated, for post microbial count. The radiograph
of the lower anterior teeth of the rats were also taken.
Curative Effects of Breynia nivosus on Experimental Dental
Twelve 8-10 weeks old male albino rats weighing 62-120 g and showing
no signs of dental caries but harboring Streptococcus rattus on
their teeth surfaces were divide into four groups of three each (A-D).
Base-line information was obtained by examining the upper and lower anterior
teeth surface of each rat visually and with dental probe and torch. Plaque
samples from the upper and lower anterior teeth surfaces of the rats were
cultured to establish the pre-microbial count. Also the radiographs of
their lower anterior teeth were obtained. The rat groups A and B were
fed daily with 70% sucrose-in-diet while groups C and D were given normal
diet. On confirmation of early caries signs among the rat groups A and
B, the administration of herbal extract commenced. About 200 mg mL-1
of Breynia nivosus extract was administered via intra-oral
route, on the anterior teeth surfaces of group A rats. However, while
group A were given normal drinking water (containing 0.1 mg L-1 of
fluoride), group B rats were given water containing 0.7 mg L-1
of fluoride. Also commenced was the feeding of group D rats with 70% sucrose-in-diet
to serve as control. Thus, whereas rat groups, A, B and D were given 70%
sucrose-in-diet, those of group C continued with normal diet. On observation
of early signs of caries among rat group D, dental plaque samples from
all the rats in all the groups were collected for post-microbial count.
The teeth surfaces were also examined visually and with dental probe and
torch. The radiograph of the lower anterior teeth of the rats were also
Preventive Effect of Breynia nivosus on Experimental Dental
Fifteen 8-10 weeks old male albino rats weighing 60-120 g without signs
of dental caries but harboring Streptococcus rattus on their anterior
teeth surfaces were divided into 5 groups (A-E). The baseline information
was determined by the examination of the anterior teeth surface of each
rat visually and with dental probe and torch. Plaque samples were also
collected for pre-microbial count as previously stated.
Rat groups A and B were fed with 70% sucrose diet. About 200 mg mL-1
of Breynia nivosus extract were administered intra-orally to the
anterior teeth surfaces of rat group A once daily and group B twice daily.
While group C rats were fed with normal diet without medicinal plant preparation,
group D were fed with 70% sucrose-in-diet and water containing 0.7 mg
L-1 of fluoride. Rat group E however, were given 70% sucrose-in-diet
only, without plant extract treatment or fluoride. Upon observation of
early signs of dental caries in rat group E, plaque samples from all the
rats in all the groups were collected for post-microbial count. Their
teeth were also observed visually and with dental probe and torch. The
lower anterior teeth of the rats were also subjected to radiographic examination.
Detection of Dental Caries
The early signs of dental caries were determined using direct observation, probing,
microbial count and radiograph (Talaro and Talaro, 2002;
Ingraham et al., 2001). In the direct observation,
the colour and texture of the surface of the upper and lower anterior teeth
were examined visually and by using dental probe and torch. The translucencies
were also determined with the aid of dental torch and mirror. The pre and post
microbial plaque counts were determined using dental plaque samples collected
from the anterior teeth of the rats prior to the commencement of the experiment
and after, respectively. The plate count technique as described by Ingraham
et al. (2001) was used to quantify the microbial content of the plaque
The radiographic examination of the lower anterior teeth of the rats
was conducted at the Radiography Department, Ebonyi State University Teaching
The t-distribution was used to analyze the microbial counts obtained
in this study.
Effect of Different Sucrose Concentrations on the Anterior Teeth Surface
of Experimental Rats
The appearance of the upper anterior teeth surface of the rats given
70, 50, 30 and 10% sucrose-in-diet were not different from that of the
control group given normal diet without sucrose. However, the lower anterior
teeth surface of the rats given 70 and 50% sucrose in diet showed presence
of milky white spots and were sticky on probing by the sixth week of observation.
The translucency and stickiness of the lower anterior teeth surface of
the rats reduced with decrease in sucrose concentration. Similarly, the
roughness of the teeth surface (texture) also reduced in the rats with
decrease in the sucrose concentration in diet.
There was a significant increase (p<0.05) in the microbial count in
the dental plaque of rat groups fed 70, 50 and 30% sucrose-in-diet. The
microbial count of the rat groups fed with 10% sucrose-in-diet and those
of the control group did not increase significantly (p<0.05) (Table
Furthermore, the radiograph of the rats fed with 70% sucrose-in-diet
suggested areas of radiolucency compared with those that were given 50,
30 and 10% sucrose-in-diet respectively. There was no evidence of radiolucency
observed for the control rat group.
Curative Effects of Breynia nivosus on the Anterior Teeth of
The appearance and texture of the upper anterior teeth surface of the
rat groups showing signs of early dental caries and given Breynia nivosus
extract and simultaneously fed with 70% sucrose in diet (group A) were
not different from the control group given normal diet. However, while
the appearances of lower anterior teeth surface of the same rat group
were not different from the control group given normal diet, on probing,
they were generally slightly sticky. Similarly, the translucency and texture
of the lower anterior teeth surface of the same rat group were slightly
dull and slightly rough respectively. However, the lower anterior teeth
of the rat group given only 70% sucrose in diet were sticky on probing
and had milky white spots.
There was no significant difference (p<0.05) between the initial microbial
count and the count at the end of the experiment for rat given Breynia
nivosus and fluoride as well as the control group (Table
2). No sign of radiolucency was observed among the rat group showing
early caries signal and given Breynia nivosus and 70% sucrose-in-diet
as well as those given fluoride and the control group. However, the radiograph
of those given 70% sucrose diet only, suggested possible area of radiolucency.
||Effect of different sucrose concentrations on the microbial
count of albino rats
|abDifferent alphabetical superscripts in
a row indicates that the means are significantly different at the
probability level p<0.05 but similar superscripts in a row indicate
a lack of significant difference between the means (p<0.05)
||Effect of plant extracts and fluoride on the microbial
counts of albino rats showing early signs of caries
|abDifferent alphabetic superscripts in a
row indicate that the means are significantly different at the probability
level p<0.05, but similar superscripts in a row indicate a lack
of significant difference between the means (p<0.05)
||Effects of plant extracts and fluoride on the microbial
counts of caries-free albino rats
|abDifferent alphabetical superscripts in
a row indicate that the means are significantly different at the probability
level p<0.05, but similar superscripts in a row indicate a lack
of significant difference between the means (p<0.05)
Preventive Effect of Breynia nivosus on the Anterior Teeth
of Experimental Rats
The appearance, translucency and texture on probing of the upper and
lower anterior teeth surface of caries-free rat groups given simultaneously
70% sucrose-in-diet and Breynia nivosus once and twice daily were
not different from those of the control group given normal diet and no
medicinal plant preparation. The upper and lower anterior teeth surface
of the rat group given fluoride were grayish-white in colour. However,
those of the rat group given only 70% sucrose-in-diet were sticky on probing
and had milky white spots.
There was no significant difference between the pre and post microbial
count (p<0.05) of the caries-free rats given 200 mg mL-1
Breynia nivosus once and twice daily. Similarly, no significant
difference was observed among the caries-free rats given simultaneously
70% sucrose-in-diet and fluoride as well as the control group given normal
diet (Table 3).
Furthermore, there was no sign of radiolucency observed for the caries-free
rat group given Breynia nivosus once and twice daily as well as
those given fluoride and normal diet. However, the radiograph of the rats
given 70% sucrose-in-diet only, suggested area of radiolucency.
The observation of milky-white spots on the lower anterior teeth surfaces of
rat group fed 70% and 50% sucrose-in-diet for six weeks as well as their dull
light reflection and rough texture in this work is noteworthy. Ooshima
et al. (1983) reported the development of severe dental caries when
specific pathogen-free rats infected with Streptococcus mutans was fed
with a diet containing 56% sucrose. Further, Folayan et
al. (2008) stated that children population who consumed sugar once a
day were at risk of developing caries. Several studies have implicated sugared
beverage and drinks (Levy et al., 2003; Declerk
et al., 2008) in the presence of poor oral hygiene to contribute
substantially to caries. Sugared free gum has been reported to significantly
slow progression of approximal caries (Morgan et al.,
That the level of stickiness on probing decreased with decrease in the concentration
of sucrose in the diet was not unexpected since the more the sucrose, the more
the possibility of acid production which eventually demineralizes the tooth
surface. Stickiness has been described to be an indicator of caries development.
The discovery of a significant increase in Streptococcus rattus content
of the plaque samples collected from the rats fed respectively with 70, 50 and
30% sucrose-in-diet in this study indicate possible development of dental caries.
Previous studies has shown that S. mutans regularly increased
at the sites that subsequently becomes carious especially in the presence of
sucrose-in-diet (Loesche et al., 1984; Staat
et al., 1975; Linossier et al., 2003).
It is not surprising however, that the rat group fed with 30% sucrose-in-diet
in this study, though had significant increase in their S. rattus content,
did not produce visible early caries signs after six weeks. This is probably
because, caries is a multifactorial disease and thus other factors such as sugar
source, flow rate and composition of saliva and oral hygiene may come into play
for caries initiation (Ingraham et al., 2001; Johnson
and Beighton, 1992).
The observation in this study of the absence of white spot lesion and presence
of slightly sticky and rough lower anterior teeth of rat group given simultaneously
extract of Breynia nivosus and 70% sucrose-in-diet is notable. Although
may not be suggestive of absolute curative potential of the plant material,
the fact that white spots lesions were absent at the end of the 6 weeks may
indicate some level of early caries curative property. This was further corroborated
by the fact that there was a drastic reduction in the microbial count compared
with the rat group given only 70% sucrose-in-diet. A previous work reported
that the feeding of Streptococcus mutans infected rats with polyphenol
compound (designated Sunphenon) isolated from the leaf of Camellia sinensis
reduced caries incidence (Saito, 1990). Also, fluoride
containing dentifrices tested in another study reduced caries in rats (Guggenheim
et al., 1997). Further research in the area of isolation, identification
and characterization of the active principles in the plant material used in
this study is hereby advocated. The in vitro activity of Breynia nivosus
on dental caries organism has been previously reported (Amadi
et al., 2007). Vanka et al. (2001)
also reported the inhibitory effect of Azadirachta indica in S. mutans
and its incipient caries reversing potential.
Furthermore, that the group of caries-free rats given Breynia nivosus
extract generally did not show observable white spot lesions, reflected light
brightly, had smooth texture and non-sticky anterior teeth surfaces is of significance
given that the rats were all fed simultaneously with 70% sucrose-in- diet daily.
This possibility of in vivo preventive effect of Breynia nivosus
extract was further presented by the radiography of the rats given the plant
extract which showed no signs of radiolucency compared with the control rat
group given only 70% sucrose-in-diet. Addai et al.
(2002) reported that brief chewing of Garcinia manii chewing stick
confers a caries preventive/control benefit by reducing the salivary pH after
glucose rinse. Also, Linke and LeGeros (2003) reported
that frequent intake of black tea can significantly decrease caries formation,
even in the presence of sugars in the diet. The result of the preventive study
in the present work indicates that, this plant material, whether given once
or twice daily, could exert some caries preventive effect on rats simultaneously
fed with 70% sucrose-in-diet.
While the result of this study justifies the local use of this plant
in the South-Eastern Nigeria in the treatment of dental diseases, it does
appear that Breynia nivosus possesses more preventive effect than
The assistance of Dr. Okeke Clement of the Department of Botany, Nnamdi
Azikiwe University Awka and the association for the identification, conservation
and utilization of medicinal plants of Nigeria (ASICUMPON) in the recommendation
and identification of the medicinal plant used in this study, is hereby
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