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ANTIMICROBIAL AND SYNERGISTIC ACTIVITY OF INGREDIENTS OF BETEL QUID ON ORAL AND ENTERIC PATHOGENS

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In this study, antimicrobial and synergistic activity of ingredients of betel quid i.e. kattha, lime, betel leaf, betel nut, cardamom, clove and fennel seeds was tested against microbial population of oral cavity and four enteric pathogens namely Staphylococcus aureus, Salmonella typhi, Escherichia coli and Shigell flexneri. For this purpose two methods were used. Pour plate method was used for calculating the reduction in microbial population in oral cavity and disk diffusion method was used for detecting the effect of ingredients of betel quid on enteric pathogens. In the first method, microbial population of saliva before and after chewing above ingredients individually and in different combinations was compared. It was found that the each ingredient of betel quid separately and in different combination shows reduction in microbial population by different percentage. The highest percentage of reduction in microbial population was shown by the combination of betel leaf, lime and kattha (Blk) followed by betel leaf, cardamom etc. In the second experiment it was found that the bacteria investigated showed most susceptibility against kattha followed by clove and then combination of betel leaf, lime and kattha. From the study it was concluded that chewing of betel quid after every meal reduces the population of oral micro flora which may be responsible for dental carries and bad breath. It was also seen that enteric pathogens like Staphylococcus aureus, Salmonella typhi, Escherichia coli and Shigell flexneri could be inhibited by ingredients of beetle quid.
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Bioscience Discovery, 3(1):47-51, Jan. 2012 ISSN: 2229-3469 (Print)
ANTIMICROBIAL AND SYNERGISTIC ACTIVITY OF INGREDIENTS OF BETEL QUID ON
ORAL AND ENTERIC PATHOGENS
Niraj A Ghanwate and Prashant Thakare1
Department of Microbiology, Sant Gadge Baba Amravati University, Amravati. 444602
1Department of Biotechnology, Sant Gadge Baba Amravati University, Amravati. 444602
nirajghanwate@gmail.com
ABSTRACT
In this study, antimicrobial and synergistic activity of ingredients of betel quid i.e. kattha,
lime, betel leaf, betel nut, cardamom, clove and fennel seeds was tested against microbial
population of oral cavity and four enteric pathogens namely Staphylococcus aureus, Salmonella
typhi, Escherichia coli and Shigell flexneri. For this purpose two methods were used. Pour plate
method was used for calculating the reduction in microbial population in oral cavity and disk
diffusion method was used for detecting the effect of ingredients of betel quid on enteric
pathogens. In the first method, microbial population of saliva before and after chewing above
ingredients individually and in different combinations was compared. It was found that the each
ingredient of betel quid separately and in different combination shows reduction in microbial
population by different percentage. The highest percentage of reduction in microbial population
was shown by the combination of betel leaf, lime and kattha (Blk) followed by betel leaf, cardamom
etc. In the second experiment it was found that the bacteria investigated showed most susceptibility
against kattha followed by clove and then combination of betel leaf, lime and kattha. From the
study it was concluded that chewing of betel quid after every meal reduces the population of oral
micro flora which may be responsible for dental carries and bad breath. It was also seen that enteric
pathogens like Staphylococcus aureus, Salmonella typhi, Escherichia coli and Shigell flexneri could
be inhibited by ingredients of beetle quid.
Key words: Antimicrobial activity, synergistic activity, betel quid, oral pathogens, enteric pathogens.
INTRODUCTION
The practice of chewing the betel (Piper
betel) for its stimulating qualities is indulged in
between a quarter and a tenth of the world’s
population, which makes it one of the most
popular of all psychoactive substances( Norton
1998). In India, Burma, Nepal, Sri Lanka and other
parts of South Asia, as well as Southeast Asia, the
betel leaves are chewed together in a wrapped
package along with the areca nut and mineral
slaked lime (calcium hydroxide). Catechu, called
"Kattha" in Hindi or “kaath” in Marathi, and other
flavoring substances and spices might be added.
This combination is known as a "betel quid". In
India, betel leaves are used as masticators together
with scraped areca nut, lime, cardamom, clove,
kattha and fennel seeds.
Dental carries is the most common
problem that affects all age groups. Tooth decay
occurs when the teeth are frequently exposed to
food containing carbohydrates. After taking food
there are some bacteria which grow on the teeth
and leads to the formation of dental plaque. It is
the process in which the enamel and the dentine
are demineralized by acids produced by bacterial
fermentation of carbohydrates, which is
responsible for oral dental carries as well as bad
breath. In the prospective of oral health
maintenance, the aqueous extract of Piper betel
has showed positive antiplaque activities that act
on dental plaque bacteria at the early phase of
plaque formation (Fathilah and Rahim, 2003).
Brushing removes some of these microorganisms,
but those that persists starts making the plaque
again. Betel leaves, areca nut, fennel seeds, clove,
cardamom, etc are traditionally chewed as mouth
fresheners. Generally these natural mouth
fresheners are consumed separately or as a betel
quid. So, the present study is an attempt to
understand the effect of traditional habitual
chewing of betel quid and its ingredients,
individually and in different combinations on oral
as well as enteric pathogens. It was determined
whether, owing to their antimicrobial properties,
betel leaves, clove, fennel seeds, cardamom, areca
nut, kattha and lime individually or in different
combinations are able to inhibit the population of
oral microorganisms or not and which combination
is best for that.
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Ghanwate and Thakare
Similarly effect of the different ingredients of betel
quid on enteric pathogens like Staphylococcus
aureus, Salmonella typhi, Escherichia coli and
Shigell flexneri was also determined.
MATERIALS AND METHODS
A) Effect of ingredients of betel leaf on oral
microbial population
Collection of plant materials:
Betel leaves, betel nuts, cardamom, clove,
and fennel seeds, lime, kattha were procured from
the local market.
Collection of saliva sample:
The saliva from individuals before and after
masticating two mature betel leaves was collected
in two sterile containers. In similar manner
samples were collected before and after
masticating betel nuts, cardamom, clove buds,
fennel seeds individually and in following
combinations: Betel leaf, lime, kath(Blk) and betel
nut, (Blk) and cardamom, (Blk) and clove, (Blk) and
fennel seeds, and finally all combined
together(betel quid).
Effect on oral pathogens
All the above collected saliva samples
were diluted five times in sterile normal saline and
0.1 ml of each was added in tubes containing
sterile molten nutrient agar and then poured into
sterile Petri plates. The plate was incubated
overnight at 370C. The number of colonies was
counted and the number of bacteria present was
calculated as:
No. of viable bacteria (Cfu/mL) = No. of bacterial
colonies × dilution.
B) Antibacterial activity against enteric
pathogens:
Preparation of crude aqueous extract
All the plant material was washed carefully
with running tap water followed by sterile distilled
water, air dried and then pounded using a grinder
and stored in air tight bottle. 1 g of each grinded
sample was soaked in 10 ml sterile distilled water
and left undisturbed for 24 h. after 24 h the
mixture was boiled for 30 min. The extract was
allowed to cool. The prepared aqueous extract was
filtered.
Inoculum preparation
Cultures used for study were Escherichia
coli (MTCC 729), Staphylococcus aureus (MTCC 96),
Shigella flexneri (MTCC 1457), and Salmonella typhi
(MTCC 98). For every experiment, freshly prepared
sterile nutrient broth (5 ml) was inoculated from
the slant and incubated at 370C for 24 h. After 24 h
incubation loop full of culture was transferred to
another freshly prepared sterile nutrient broth and
incubated at 370C for near about 2 h so as to get
the density of 0.5 Mac Farland standard i.e. 106
cfu/mL.
Antibacterial assay
Antimicrobial susceptibility test was
carried out by Kirby Bauer disc diffusion method.
All assays were applied in triplicates and the means
were calculated.
Table 1: Reduction in microbial population of oral cavity
RESULTS AND DISCUSSION
In the present study, total 7 ingredients of
betel quid were tested for their antimicrobial and
synergistic activity on oral and enteric pathogens.
The results obtained were as follows.
Ingredients of Betel Quid
Reduction in microbial population in oral cavity (%)
Betel leaf
57.05
Betel nut
21.08
Cardamom
54.50
Clove
44.48
Fennel seeds
50.00
Blk
59.42
(Blk)+Betel nut
52.58
(Blk)+Cardamom
48.57
(Blk)+Clove
51.47
(Blk)+Fennel seeds
37.96
Betel quid
41.97
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Bioscience Discovery, 3(1):47-51, Jan. 2012 ISSN: 2229-3469 (Print)
Antimicrobial and synergistic activity on oral pathogens:
All the ingredients of betel quid showed reduction in microbial population of oral cavity individually
and in different combinations as shown in table-1 and fig.-1.
On the basis of statistical analysis of observation it was found that betel leaf, betel nut, the
combinations of Blk, Blk and clove and also whole betel quid showed significant reduction in microbial
population of mouth cavity.
Antimicrobial and synergistic activity on enteric pathogens:
The aqueous extracts of above ingredients individually and in different combinations were
prepared and tested for its antimicrobial activity against Escherichia coli, Staphylococcus aureus,
Salmonella typhi and Shigella flexneri by disc diffusion method. It was observed that the betel nut, kattha,
and clove show maximum activity against test organisms. The combination of Blk showed moderate
activity while betel leaf, cardamom, fennel seeds, lime and whole betel quid did not show any
antimicrobial activity. (Table-2 and figure-2)
Fig. 1: Reduction in oral microbial population.
Fig. 2: Antimicrobial activity of constituents against test pathogens.
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50
Ghanwate and Thakare
Table 2- Antimicrobial effect against enteric pathogens.
Ingredients of Betel Quid
Zone of inhibition in mm
E. coli
Salmonella typhi
Shigella
flexneri
Betel leaf
0
0
8
Betel nut
8
11
25
Blk
0
9
25
Cardamom
0
0
0
Clove
7
0
7
Fennel seeds
0
0
7
Kattha
8
8
0
Lime
0
0
0
Betel quid
0
0
0
Chloramphenicol
16
20
43
Saini et al., in 2009 stated that the microbial count
of saliva is more than 1 lakh cfu/ml (colony forming
unit). Microbial flora starts decreasing after
chewing betel leaf. They observed that betel nut
also decrease oral flora to great extent. In this
study it was found that the microbial flora of oral
cavity (saliva) was decreased by chewing of natural
mouth fresheners individually and in different
combination in varying degrees. It was found that
there was a significant reduction in total microbial
count of the mouth after masticating the betel
quid. The count was found to be reduced to
59.42% in presence of betel leaf, lime and kattha.
Betel leaf, cardamom, Blk and betel nut and Blk
and clove reduced the count by more than 50%.
Fatilah et al in 2009 in their study also
demonstrated bacteriostatic effect of betel leaf on
dental plaque bacteria. The effect of betel leaves
on oral pathogens was studied by Bissa et al
(2007). Along with leaves of different landraces,
the effect of fruits of cardamom and clove buds
was also tested by them. Different combinations,
viz. betel leaves and cardamom; betel leaves and
clove; betel leaves, cardamom and clove were also
applied. All the tested materials gave good results
against the oral microbes but the most effective
was the combination of betel, cardamom and
clove. Gururaj et al in their study also
demonstrated that cardamom and clove chewing
reduce the microbial flora of oral cavity
significantly.
In our study, the combination of Blk showed
highest reduction in microbial population. As it
contains lime it produces alkaline saliva which may
inhibit the growth of oral microorganisms. The
main constituents of betel leaf are fatty acids,
Hydroxyl fatty acids, esters and hydroxyl chavicol
(Katsura et al., 2001) which are said to exhibit
antimicrobial activity (Pauli, 2002). This
combination also showed very good results against
Salmonella typhi, Staphylococcus aureus and
Shigella flexneri. It was also seen that betel nut
showed highest antimicrobial activity against
enteric pathogens. Kattha which is powder of stem
of Acacia catechu shows surprising inhibition of
E.coli, Staphylococcus aureus and Salmonella typhi.
Adeltrudes and Marina, (2010) found that the Piper
betle oil have significant antibacterial and
antifungal activity against S. aureus, Streptococcus
pyogens, Candida albicans and Trycophyton
mentagrophytes.
The ingredients of betel quid showed
significant reduction in microbial population
individually and in different combination, so these
can be used as an effective mouth fresheners
which can prevents dental carries and bad breath.
Among this betel nut, clove, Blk and Kattha also
exhibited antimicrobial activity against enteric
pathogens. So it can be said that, chewing of betel
quid after every meal is good for oral as well as
enteric health.
LITERATURE CITED
Adeltrudes BC and Marina OO, 2010. Characterization and evaluation of antimicrobial activity of the essential oil
from leaves of Piper betel L. E-International Scientific Research J, 2: 2-13.
Bissa S, Songara D and Bohra A, 2007. Effect of traditional P. betel leaves on oral pathogens. Current Science,
92(1):26-28.
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Fatilah AR and Rahim ZHA, 2003. The anti adherence effect of Piper betel and Psidium gaujava extracts on the
adhesion of early settlers in dental plaque. J Oral Sci, 45: 201-206.
Fatilah AR and Rahim ZHA, Othman Y and Yusoff M, 2009. Bacteriostatic effect of Piper betel and Psidium gujava
extracts on dental plaque bacteria. Pakistan J Bio Sci, 12: 518-521.
Gururaj HB, Giridhar P and Ravishankar GA, 2007. Traditions in oral hygiene: Chewing of betel ( Piper betle L.) leaves.
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Katsura H, Tsukiyama R, Suzuki A and Kobayaship M. 2001. In vitro antimicrobial activities of bakuchiol against oral
microorganisms. Antimicrobial agents and Chemother, 45:3009-3013.
Nalina T and Rahim ZHA, 2007. The crude aqueous extract of Piper betle L. and its antimicrobial effect towards
Strptococcus mutans. Amr. J of Biotech and Biochem, 3: 1015.
Norton SA, 1998. Betel consumption and consequences. J AM Acad Demt, 38: 81-88.
Pauli A, 2002. Antimicrobial Properties of Catechol derivatives, 3rd World Congress on Allelopathy, Tsukuba, Japan, 26-
30.
Saini S, Saini SR, Katiyar R, Bhalerao DS and Munde A, 2009. The use of Tobacco and Betel leaf and its effect on the
normal microbial flora of Oral Cavity. Pravara Med Rev, 4(4): 17-19.
... All concentrations of ethanol extract of Areca cathecu L. nut demonstrated zones of inhibition against mixed-oral flora A, B, C, and D with the smallest diameter measuring 7 mm (30 % in mixed-oral flora B, Figure 1) and the largest measuring 18 mm (50 % and 70 % in mixed-oral flora C, Figure 2 and 3). Minimum inhibitory concentration of Areca catechu L. nut ethanol extract effective against mixed-oral flora isolated from tooth scum was found to be 30 % concentration with 9 mm, 7 mm, 16 (arecoline, arecaidine, guvacine and guvacoline 11,12 ; these alkaloids, tannins and polyphenols possess antihelminthic, antifungal, antibacterial, antiinflammatory and antioxidant activities 13 . In a study by Anjali and Rao (1995) water and methanol extracts of the seeds in various ages presented higher percentage of tannin and total phenols than other parts of the areca tree extracts 13 which makes the nut an abundant source of bioactive components with antimicrobial activity. ...
... Also, betel quid extracts are mutagenic against bacteria; similar studies of aqueous extracts of areca nut produced gene conversion in yeast and arecoline and other arecanut alkaloids gave positive responses in most bacterial mutagenicity assays 15 . A study by Ghanwate and Thakare (2012) demonstrated that all ingredients of betel quid showed reduction in microbial population of oral cavity individually and in different combinations, betel nut decreased oral flora greatly; and showed highest antimicrobial activity against enteric pathogens 16 which confirmed the results of this study as similar inhibitory effects were demonstrated against S. typhi, S. non-typhi, S. flexneri and V. cholerae. Studies by De Miranda et al, (1996) reported that areca nut extract exerts a direct antimicrobial effect against oral bacteria, including S. mutans, S. salivarius, C. albicans, and F. nucleatum 17 which also correlates with the results of this study against mixed-oral flora. ...
... Also, betel quid extracts are mutagenic against bacteria; similar studies of aqueous extracts of areca nut produced gene conversion in yeast and arecoline and other arecanut alkaloids gave positive responses in most bacterial mutagenicity assays 15 . A study by Ghanwate and Thakare (2012) demonstrated that all ingredients of betel quid showed reduction in microbial population of oral cavity individually and in different combinations, betel nut decreased oral flora greatly; and showed highest antimicrobial activity against enteric pathogens 16 which confirmed the results of this study as similar inhibitory effects were demonstrated against S. typhi, S. non-typhi, S. flexneri and V. cholerae. Studies by De Miranda et al, (1996) reported that areca nut extract exerts a direct antimicrobial effect against oral bacteria, including S. mutans, S. salivarius, C. albicans, and F. nucleatum 17 which also correlates with the results of this study against mixed-oral flora. ...
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... Various pharmacological activities of Piper betle such as antifungal (Tewari and Nayak, 2003), gastro protective (Majumder et al., 2003), wound healing (Santhanam and Nagarajan, 1990), hepato protective activity (Saravanan et al., 2002), antioxidant (Santhakumari 2003; Pin et al. 2010), anti inflammatory (Pin et al. 2010), anti-malarial activity (Al-Adhroey et al., 2011) , antiallergic (Wirotesangthong et al., 2008), antidiabetic activity and antifertility on male rats (Rathnasooriya and Premakumara,1997) have been reported. The antibacterial activity against human pathogenic bacteria has been reported by several researchers (Ghanwate and Thakare, 2012; Rajeshbabu et al.,2011; Chakraborty and Bharkha Shah, 2011), but antibacterial activity against plant pathogenic bacteria causing several important diseases in crops, fruits and vegetables has not been studied so far. The literature revealed that not much work has been done regarding P. betle leaf extracts as botanical pesticides to control plant pathogens. ...
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In this study, the antimicrobial influence of crude aqueous extract of Piper betle L. on Streptococcus mutans (S. mutans) was investigated. The focus of the antimicrobial effects includes the ultrastructure and acid producing properties of S. mutans. Transmission electron microscopy (TEM) was used to determine the effect of the extract on the ultrastructure of S. mutans. Analysis of the effect on the acid producing properties was analyzed by pH drop assay. The investigation was further carried out to determine the possible chemical components of the extract using thin layer chromatography (TLC), bioautography and gas chromatography mass spectrometry (GCMS). From the micrographs of the transmission electron, it was found that the crude extract of Piper betle L. leaves causes plasma cell membrane damage and coagulation of the nucleoid. The extract was found to significantly reduce acid producing properties of the bacteria. Chemical analysis of the extract showed that hydroxychavicol, fatty acids (stearic and palmitic) and hydroxy fatty acid esters (stearic, palmitic and myristic) as the main components. It was suggested from the results obtained that the crude extract of Piper betle L. leaves may exert anticariogenic activities that are related to decrease in acid production and changes to the ultrastructure of S. mutans. Further study will be carried out to determine if the effect observed is attributed to the presence of hydroxychavicol, fatty acids and hydroxy fatty acid esters in the extract.
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The use of tobacco, betel nut, betel leaf (pan) and chillies cause certain changes in the oral cavity because of different chemicals they contain. A total of twenty five patients were examined, both male and females,ranging between 10-70 years. Oral examination was carried out and most of them presented with, submucus fibrosis or blanched mucosa. Samples from oral cavity were collected with sterile swabs, swabbing it all round in the oral cavity and carried to the laboratory for both aerobic and anaerobic in Robertson’scooked meat medium(RCM) for further processing. The growth in the culture medium was specifically looked for the normal microbial flora of oral cavity i.e Streptococcus viridans, Coagulase NegativeStaphyloccus, Diptheroides, Neisseria catarrhalis (aerobic flora) and the anaerobic flora. In twenty five patients most of them had no normal microbial flora except five patients who showed presence of 4 to 5 colonies i.e scanty growth of Streptococcus viridans indicating that the normal microbial flora was reduced considerably in these patients. Such patients are more prone to develop precancerous conditions and ultimately the carcinoma of the oral cavity with hundred percent reduction in the normal microbial flora.
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Bakuchiol was isolated from the seeds of Psoralea corylifolia, a tree native to China with various uses in traditional medicine, followed by extraction with ether and column chromatography combined with silica gel and octyldecyl silane. In this study, the antimicrobial activities of bakuchiol against some oral microorganisms were evaluated in vitro. The cell growth ofStreptococcus mutans was inhibited in a bakuchiol concentration-dependent manner, and growth of S. mutanswas completely prevented by 20 μg of bakuchiol per ml. The bactericidal effect of bakuchiol on S. mutans was dependent on temperature and stable under the following conditions: sucrose, 0 to 10% (wt/vol); pH, 3.0 to 7.0; organic acids (3% [wt/vol] citric and malic acids). Bakuchiol showed bactericidal effects against all bacteria tested, includingS. mutans, Streptococcus sanguis,Streptococcus salivarius, Streptococcus sobrinus, Enterococcus faecalis,Enterococcus faecium, Lactobacillus acidophilus, Lactobacillus casei,Lactobacillus plantarum, Actinomyces viscosus, and Porphyromonas gingivalis, with MICs ranging from 1 to 4 μg/ml and the sterilizing concentration for 15 min ranging from 5 to 20 μg/ml. Furthermore, bakuchiol was also effective against adherent cells of S. mutans in water-insoluble glucan in the presence of sucrose and inhibited the reduction of pH in the broth. Thus, bakuchiol would be a useful compound for development of antibacterial agents against oral pathogens and has great potential for use in food additives and mouthwash for preventing and treating dental caries.
The anti adherence effect of Piper betel and Psidium gaujava extracts on the adhesion of early settlers in dental plaque
  • A R Fatilah
  • Zha Rahim
Fatilah AR and Rahim ZHA, 2003. The anti adherence effect of Piper betel and Psidium gaujava extracts on the adhesion of early settlers in dental plaque. J Oral Sci, 45: 201-206.
Antimicrobial Properties of Catechol derivatives
  • A Pauli
Pauli A, 2002. Antimicrobial Properties of Catechol derivatives, 3 rd World Congress on Allelopathy, Tsukuba, Japan, 26-