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Ethnobotanical Leaflets 14:
960-75, 2010. Antimicrobial Activity of Syzygium aromaticum and Its Bud Oil Against Dental Cares Causing
Microorganisms Kamal
Rai Aneja and Radhika Joshi Department of Microbiology, Corresponding Author: Email: joshi_radhika31282@yahoo.com; radhikasharma31282@gmail.com Issued: August 01, 2010 Abstract The antimicrobial activity of
clove and clove bud oil were investigated by agar well diffusion method
against five dental caries causing microorganisms namely Streptococcus mutans, Staphylococcus aureus, Lactobacillus
acidophilus (bacteria), Candida
albicans and Saccharomyces
cerevisiae (yeast). The results indicated that clove and clove oil have a
potent antimicrobial activity against the tested dental caries causing
microorganisms. The highest antimicrobial activity of clove was found against
Saccharomyces cerevisiae (25.32mm)
in methanolic extract and an MIC of 50mg/ml and that of clove oil was found
against Streptococcus mutans
(34.32mm) with a MIC value of 3.125mg/ml. This study has shown the importance
of clove and clove oil and indicated that clove and clove bud oil can be used
as an antimicrobial agent to cure dental caries. Key
words: Clove,
clove oil, antimicrobial activity, inhibition zone, dental caries. Introduction Dental caries is a chronic disease
of multifactor etiology and pathogens. The early
stage of dental caries is characterized by a destruction of superficial
dental structures caused by acids which are by-products of carbohydrate
metabolism by Streptococcus mutans,
a cariogenic bacterium (Loesche, 1986). Dental caries are one of the
public health concerns for several reasons. Teeth affected with dental caries
are sources of infection, which can cause an inflammation of dental pulp,
periodonteum and gums. If left untreated, this disease gradually leads to
teeth loss, which causes chewing difficulties and aesthetic problems (Allen,
2003). It remains one of the most
widespread diseases of the mankind. In developing countries, dental caries is
often at epidemic proportions, especially among the poor. Since the 19th century, when
sucrose became a daily used sweetener by many people worldwide, the
increasing prevalence of dental caries had also been noticed (Hamada, 2002). Despite a low mortality rate associated
with dental diseases they have a considerable effect on the self esteem,
eating ability, nutrition and health both in childhood and older age. In the
modern society the most important role of teeth is to enhance facial
appearance. Teeth also play an important role in speech and communication. Dental
decay also leads to tooth loss which reduces the ability to eat a varied diet
(Moynihan et al., 1994; Steele et al., 1998). Tooth loss has also
been associated with loss of enjoyment of food and confidence to socialize
(Steele et al., 1998). It is therefore
clear that dental diseases have detrimental effect on quality of life both in
childhood and older age (Moynihan and Petersen, 2004).
Cloves (Syzygium aromaticum) are dried aromatic
unopened floral buds of an evergreen tree 10-20 m in height, belonging
to the family Myrtaceae, indigenous to Clove oil (Syzygium
aromaticum) is widely used as a perfume and food flavouring (Zheng et
al., 1992; Kalemba and Kunicka, 2003), as a medicine for the treatment of
asthma, rheumatoid
arthritis, acne, warts, scars
and various allergic disorders (Kim et al., 1998), as an analgesic, anti spasmodic, and as a general antiseptic
in medical dental practices (Cai and Wu, 1996). Clove bud oil, has been used
for a long time by dentists as a dressing in dentistry for minor wounds, as
an analgesic in painful and infective diseases of the oral cavity and pharynx
as well as general hygiene. Importantly,
Lee and Shibamoto (2001) reported that clove oil might also be used as an
anti-carcinogenic agent due to its antioxidant properties. Their results also
suggested that clove oil might be of use as a potential chemopreventative
agent (Zheng et al., 2005). Research
has shown that clove oil is an effective mosquito repellent (Trongtokit et
al., 2005). However,
clove oil is toxic to human cells (Prashar et al., 2006). If ingested or injected in sufficient
quantity, it has been shown to cause life threatening complications,
including Acute Respiratory Distress Syndrome, Fulminant Hepatic Failure and
Central Nervous System disorder. The lethal oral dose is 3.752 g/Kg body
weight (Kirsch, 1990; Lane et al.,
1991; Hartnoll et al.,
1993).
Herbs are staging a comeback and herbal ‘renaissance’ is happening all
over the globe. The herbal products today symbolize safety in contrast to the
synthetics that are regarded as unsafe to human and environment. Over
three-quarters of the world population relies mainly on plants and plant
extracts for health care. The demand of plant based therapeutics is
increasing in both developed and developing countries due to the growing
recognition that they are natural products, being non narcotic, having no
side effects, easily available at affordable prices and sometimes the only
source of healthcare available to the poor. The objective of this study was to validate the
observations made by earlier workers and to assess 1) the in vitro antibacterial and antifungal
properties of different extracts of dried clove buds and clove oil against
common dental caries causing microorganisms, 2) determination of minimum
inhibitory concentration (MIC) of each extract and clove oil against each
pathogen with a view of finding the minimum concentration of clove and it’s
oil that can be assigned as a novel remedy for dental caries. Materials
and Methods Clove buds and clove oil
were collected from the local market of Extraction
The samples were carefully
washed under running tap water followed by sterile distilled water. These
were air dried at room temperature (30OC) for two days and
pulverized to a fine powder using a sterilized mixer grinder and stored in
air-tight bottles. Four different solvents namely ethanol, methanol, acetone
and aqueous (hot and cold) were used for extraction. A 10g amount of pulverized buds was separately soaked in 100ml
of acetone, ethanol, methanol, and cold sterile distilled water for 24h. Also
the same amount (i.e. 10g) of pulverized buds were immersed in 100ml of hot
sterile distilled water (100OC) and allowed to stand for 30min on
a waterbath with occasional shaking and kept undisturbed for 24h. Each
preparation was filtered through a sterilized Whatman No.1 filter paper and
the filtered extract was concentrated under vacuum below 40oC
using Heidolph, VE-11 rotaevaporator (Bag et al., 2009; Ogundiya et al.,
2006). The dried extract thus obtained was exposed to UV rays for 24h and
checked for sterility on nutrient agar plates and stored in labeled sterile
bottles in a freezer at 4oC until further use (Nkere and Iroegbu,
2005). Test
Microorganisms
Three dental caries causing
bacteria Streptococcus mutans
(MTCC*497), Staphylococcus aureus
(MTCC 740), Lactobacillus acidophilus
(MTCC *447) and two yeasts Candida
albicans (MTCC 227) and Saccharomyces
cerevisiae (MTCC 170) were procured from Microbial Type Culture
Collection, IMTECH, Chandigarh. The microorganisms were subcultured on the
specific media recommended for different microorganisms such as Brain heart
infusion agar (S.mutans), Nutrient
agar (S.aureus), Lactobacillus MRS
agar (L.acidophilus), Malt yeast agar (C.albicans and S.cerevisiae)
and incubated aerobically at 37OC. The media were procured from
Himedia Laboratory Pvt. Ltd., Screening
for Antimicrobial Activity The acetone, methanol, ethanol,
cold and hot water dried clove bud extracts were used for the screening.
Antimicrobial activity of various extracts was determined by the agar well
diffusion method (Okeke et al., 2001). In this method, pure isolate of each
microbe was subcultured on the recommended specific media for each
microorganism at 37OC for 24h. For testing clove oil, a final
concentration of 0.5% (v/v) Tween-20 (Sigma) was incorporated into the agar
after autoclaving to enhance oil solubility (Bansad, 2008). A plate of each
microorganism was taken and a minimum of four colonies were touched with a
sterile loop and transferred into normal saline (0.85%) under aseptic
conditions. Density of each microbial suspension was adjusted equal to that
of 106 cfu/ml (standardized by 0.5McFarland standard) and used as
the inoculum for performing agar well diffusion assay. One hundred microlitres (100µl) of inoculum
of each test organism was spread onto the specific media plates so as to
achieve a confluent growth. The agar plates were allowed to dry and wells or
cups of 8mm were made with a sterile borer in the inoculated agar plates and
the lower portion of each well was sealed with a little specific molten agar
medium. The extracts were reconstituted in 20% DMSO for the bioassay analysis
(Rajasekaran et al., 2008). A 100µl volume of each extract was propelled
directly into the wells (in triplicates) of the inoculated specific media
agar plates for each test organism. The plates were allowed to stand for 10
minutes for diffusion of the extract to take place and incubated at 37OC
for 24h (Khokra et al., 2008; Rios et al., 1980). Sterile DMSO served as the
negative control and ciprofloxacin (for bacteria) and amphotericin-B (for
fungi) served as the positive control. The antimicrobial activity, indicated
by an inhibition zone surrounding the well containing the extract, was
recorded if the zone of inhibition was greater than 8mm (Hammer et al.,
1999). The experiments were performed in triplicates and the mean values of the
diameter of inhibition zones with ± standard deviation were calculated (Aneja
and Joshi, 2009a, b). Determination
of Minimum Inhibitory Concentration (MIC) MIC is defined as the lowest
concentration of a compound/extract/drug that completely inhibits the growth
of the microorganism in 24h (Thongson et al., 2004). The MIC for the
acetonic, methanolic and ethanolic extract was determined by following the
modified agar well diffusion method (Okeke et al., 2001). A twofold serial dilution of each extract
was prepared by first reconstituting the powder in 20% dimethylsulphoxide
(DMSO) followed by dilution in sterile distilled water to achieve a
decreasing concentration range of 50mg/ml to 0.39mg/ml. A 100 µl volume of
each dilution was introduced into wells (triplicate) in the specific media
agar plates already seeded with 100µl of standardized inoculum (106
cfu/ml) of the test microbial strain. All test plates were incubated
aerobically at 37oC for 24 hrs and observed for the inhibition
zones. The lowest concentration of each extract showing a clear zone of
inhibition, considered as the MIC, was recorded for each test organism (Nkere and Iroegbu, 2005; Aneja et al., 2009). Results and Discussion Antibacterial properties of
clove have already been reported (Chopra et al, 1982, Ueda et al, 1982,
Watanabe et al., 1985, Briozzo et al, 1989 and Islam et al.,
1990; Hoque et al., 2008), but in the present study we tested the
antibacterial as well as the antifungal activity of clove and its oil against
dental caries causing microorganisms. The
results of antimicrobial activities of ethanol, methanol, acetone and aqueous
(hot and cold) extracts of clove buds and clove oil as well as the positive
control ciprofloxacin (for bacteria) and amphotericin-B (for fungi) are
presented in Table 1 and the MIC of the five
extracts as well as clove oil against the test pathogens are presented
in Table 2 . The antimicrobial activity of clove bud extracts on the agar
plates varied greatly in different solvents. Both the positive controls
produced significantly sized inhibition zones against the test bacteria
(ciprofloxacin) and yeasts (amphotericin-B). However, the negative control
produced no observable inhibitory effect. Of the five extracts screened for
antibacterial activity, all the five showed antibacterial activity against S.mutans. The hot aqueous extract was
most effective against S.mutans
showing the highest zone of inhibition (22.65mm) followed by the cold aqueous
(20.32mm), acetonic extract (14.65mm), ethanolic extract (13.95mm) and
methanolic extract (11.64mm). Clove oil showed the highest zone of inhibition
34.32mm against S.mutans (Figure
1a), which was even much higher than the positive control i.e. ciprofloxacin
(27.32mm). S.mutans was found to be
most sensitive pathogen which survived upto 1.56mg/ml in clove oil, thus
having a MIC of 3.125mg/ml followed by the hot and cold aqueous extract
(12.5mg/ml) and the acetonic, methanolic and ethanolic extracts (25mg/ml). S.aureus was found to be comparatively
more resistant than S.mutans as
only the methanolic (21.32mm) and ethanolic (19.32mm) clove bud extracts
showed antibacterial activity against it and it survived upto 12.5mg/ml thus
having a MIC of 25mg/ml. These results are in agreement with those of another study reporting
that clove essential oil exhibited antibacterial activity against a large
number of methicillin-resistant S. epidermidis and S. aureus (Enzo
and Susan, 2002). L.acidophilus was found to be completely
resistant to the five solvent extracts of clove buds while the clove oil
produced quite big inhibition zone with a diameter of 29.97mm which was
greater than the diameter of positive control (25.65mm). L.acidophilus survived upto 3.125mg/ml concentration of clove oil
thus having a MIC of 6.25mg/ml. Out of the five extracts screened for
antifungal activity, acetone, methanol and ethanol showed antifungal activity
against Candida albicans while
methanol and ethanol showed antifungal activity against Saccharomyces cerevisiae.
However, water extracts, both hot and cold, showed no activity against
the test strains. The inhibition zones produced by the three solvents against
C.albicans ranged between 20mm and
24mm. The methanolic extract produced the largest zone of inhibition, among
the various clove extracts tested, i.e. 25.32mm zone against S.cerevisiae (Figure 1b), while the
ethanolic extract produced a 17.96mm zone. Clove oil showed excellent
antifungal activity against S.cerevisiae
with a mean diameter of inhibition zone being 28.97mm (much greater than
the positive control i.e. amphotericin-B, which produced a zone of 11.94mm)
but no activity against C.albicans.
S.cerevisiae was found to be comparatively more resistant than C.albicans as it survived upto
25mg/ml, thus having a MIC of 50mg/ml in all the three extracts tested. Knowledge of the chemical
constituents of plants is desirable, not only for the discovery of
therapeutic agents, but also because such information may be of value in
disclosing new sources of such economic materials as tannins, oils, gums,
precursors for the synthesis of complex chemical substances. In addition, the
knowledge of the chemical constituents of plants would further be valuable in
discovering the actual value of folkloric remedies (Mojab et al., 2003).
Chemically constituents may be therapeutically active or inactive. The ones
which are active are called active constituents and the inactive ones are
called inert chemical constituents (Iyengar, 1995). Several constituents of clove have been
identified, mainly eugenol, eugenyl acetate, beta-caryophyllene, 2-heptanone
(Chaieb et al, 2007a; Cowan, 1999), acetyleugenol, alpha-humulene, methyl
salicylate, isoeugenol, methyleugenol (Yang et al., 2003), phenyl
propanoides, dehydrodieugenol, trans-confireryl aldehyde, biflorin,
kaempferol, rhamnocitrin, myricetin, gallic acid, ellagic acid and oleanolic
acid (Cai and Wu, 1996). The main constituents of essential oil are
phenylpropanoides such as carvacrol, thymol, eugenol and cinnamaldehyde
(Chaieb et al., 2007b). GC-MS
analysis of the oil extract shows eugenol (88.58%), eugenyl acetate (5.62%),
β-caryophyllene (1.39%), 2-heptanone (0.93%), ethyl hexanoate (0.66%),
humulenol (0.27%), α-humulene (0.19%), calacorene (0.11%) and calamenene
(0.10%) as the major constituent. Eugenol and caryo-phyllene are known to
possess antibacterial and antifungal properties. Hence the antibacterial and
antifungal properties demonstrated by clove and clove oil can be attributed
to the compounds reported (Ayoola et al., 2008; Prashar et al., 2006; Pawar and Thaker,
2006; Lee and Shibamoto, 2002). The
potential for developing antimicrobial drugs from plants appears rewarding,
as it will lead to the development of a phytomedicine that will act more
effectively against microorganisms. Therefore such screening experiments form
a primary platform for further phytochemical and pharmacological studies that
may open the possibilities of finding new clinically effective antimicrobial
compounds. Conclusion On comparison of the antimicrobial
activities of all the five S.aromaticum
bud extracts and clove oil tested against the bacterial and fungal strains,
it was finally concluded that clove oil emerged as the potent agent
exhibiting even much higher antibacterial and antifungal activity than the
standard antibacterial and antifungal drugs ciprofloxacin and amphotericin-B
respectively. The need of the hour is to perform more and more screening of
the natural products or plant parts as such screening experiments form a
primary platform for further phytochemical and pharmacological studies that
may open the possibilities of finding new clinically effective antifungal and
antibacterial compounds against the dental caries pathogens and the resistant
bacterial and fungal pathogens. Table
1: Antimicrobial activity of Clove and clove oil against the test microorganisms.
(-) = no activity, Nt = not tested a
Values, including diameter of the well (8 mm), are means of
three replicates b ± Standard deviation Table
2: Minimum Inhibitory Concentration (MIC) of Clove and Clove oil.
(-) = no activity, Nt = not tested
a. b. Figure 1: Zone of
inhibition shown by (a) clove oil against Streptococcus
mutans , (b) methanolic extract of clove against Saccharomyces cerevisiae and negative control (DMSO). Acknowledgement We acknowledge the Chairperson
of Microbiology Department for providing us research facilities in the
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