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Ethnobotanical Leaflets 14: 402-412, 2010. In Vitro Antimicrobial Activity of Sapindus mukorossi and Emblica officinalis Against Dental
Caries Pathogens Kamal Rai Aneja, Radhika Joshi*
and Chetan Sharma Department of Microbiology, Kurukshetra University, Kurukshetra
-136119. Corresponding author Email: joshi_radhika31282@yahoo.com
Issued: April 01, 2010 Abstract The in vitro
antimicrobial activity of Sapindus
mukorossi and Emblica officinalis
fruit extracts were studied against Streptococcus
mutans, Staphylococcus aureus, Lactobacillus acidophilus, Candida albicans and
Saccharomyces cerevisiae. The
acetone, ethanol, methanol, hot water and cold water extracts of S.mukorossi exhibited antimicrobial
activity against one of the tested microorganisms i.e. S.cerevisiae. All the five extracts of E.officinalis showed inhibitory activity against S.mutans while the acetonic, hot and
cold aqueous extracts showed inhibitory activity against S.aureus also. The largest zone of inhibition was obtained with
the acetonic extract of S.mukorossi against
S.cerevisiae (29.65mm) and hot
water extract of E.officinalis
against S.aureus (40.32mm). Minimum
inhibitory concentrations (MIC) of the extracts were also determined against
the selected microorganisms showing zones of inhibition ≥8mm. This
study depicts that the fruits of Sapindus
mukorossi and Emblica officinalis
possess very good antifungal and antibacterial activities respectively and
can be used as a potential source of novel antimicrobial agents used to cure
dental caries. Key
words : Dental caries, Sapindus
mukorossi, Emblica officinalis, antimicrobial activity, zone of
inhibition, minimum inhibitory concentration. Introduction Dental caries is a very common
problem that affects all age groups. It is a process in which the enamel and
the dentine are demineralised by acids produced by bacterial fermentation of
carbohydrates (de Soet and de Graff, 1998). In real life, it is the most
common infectious disease affecting human beings (Balakrishnan et al., 2000). Medicinal plants since
ancient times have been employed for prophylactic and curative purposes
(Joshi and Joshi, 2005; Amadi et al., 2007).
The present study reports the antimicrobial activity of five different
solvent extracts of Sapindus mukorossi
and Emblica officinalis on dental
caries causing microorganisms. Sapindus mukorossi
Gaertn., a member of the family Sapindaceae,
is commonly known by several names such as soapnut, soapberry, washnut,
ritha, reetha, aritha, dodan and doadni. It is a deciduous tree widely grown in upper reaches of Indo-Gangetic
plains, Shivaliks and sub Himalayan tracts at altitudes from 200m to 1500m.
It is one of the most important trees of tropical and sub-tropical regions of
Emblica officinalis Gaertn., a member of the family Euphorbiaceae, is commonly called by
several names such as amalaka, aavalaa, amla, amlaki and Indian gooseberry.
It is found throughout Materials
and Methods Sapindus mukorossi
and Emblica officinalis fruits were
obtained from the local market of Delhi, India. Dr. B.D.Vashistha, Botany
Department, Extraction
of plant material The samples were carefully washed
under running tap water followed by sterile distilled water. These were air
dried at room temperature (40OC) for five 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 fruits was separately soaked in 100ml of acetone,
ethanol, methanol (100% each) and cold sterile distilled water for 24h. Also
the same amount (i.e. 10g) of pulverized fruits was immersed in 100ml of hot
sterile distilled water 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 (Ogundiya et al., 2006) and the filtered extract
was concentrated under vacuum below 40oC using Heidolph, VE-11
rotaevaporator (Bag et al., 2009).
The dried extract thus obtained was exposed to UV rays for 24hrs and checked
for sterility on nutrient agar plates and stored in labelled 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., Antimicrobial
Assay The acetone, methanol, ethanol, cold
and hot water 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 24hrs. 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 (Aneja et al., 2010). One hundred microlitre (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 (Aneja et al., 2009; Khokra et al., 2008; Rios et al., 1980). Sterile DMSO (20%)
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 was determined by following the modified agar well
diffusion method (Rajasekaran et al., 2008). A twofold serial dilution of each extract
was prepared by first reconstituting the powder (100:1mg/ml) in 20%
dimethylsulphoxide (DMSO) followed by dilution in sterile distilled water
(1:1) 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 microbial inoculum adjusted
with sterile distilled water equal to that of 106 cfu/ml
(standardized by 0.5McFarland standard) 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 (>8mm), considered as the MIC, was
recorded for each test organism (Nkere and Iroegbu, 2005; Aneja et al., 2009). Results
and Discussion The results of antimicrobial
activity of the five extracts of S.mukorossi
and E.officinalis by
agar well diffusion method have been shown in Table 1 and Table 2
respectively. From the data presented in the table 1, it is evident that all
the five extracts of S.mukorossi i.e.
hot aqueous, cold aqueous, acetonic, methanolic and ethanolic showed
antimicrobial inhibitory activity against only one of the five tested dental
caries causing microorganism S.cerevisiae,
with the mean diameter of the highest zone of inhibition being 29.65mm.
S.cerevisiae survived upto 12.5mg/ml in the acetonic extract, thus having a
MIC of 25mg/ml (Table 3). Although no inhibitory activity of S.mukorossi was shown against S.mutans, S.aureus, L.acidophilus and C.albicans, the antifungal activity
was much higher than the control (amphotericin-B, with the mean diameter
being 11.94mm), thus S.mukorossi
possesses very good antifungal properties against S.cerevisiae. The major active ingredient responsible for
antifungal activity of S.mukorossi might
be saponin. Saponins are generally known as non–volatile, surface–active
compounds that are widely distributed in nature, occurring primarily in the
plant kingdom (Oleszek, 2002). Saponins have a diverse range of properties,
including foaming and emulsifying (Price et
al., 1987), pharmacological and medicinal properties (Attele et al., 1999), haemolytic properties
(Sparg et al., 2004), as well as
antimicrobial, insecticidal, spermicidal and molluscicidal activities (Saxena
et al., 2004; Shiau et al., 2009). All
the five tested extracts of E.officinalis
as presented in table 2 showed antimicrobial activity against S.mutans with the mean diameter of the
highest zone of inhibition being 18.96mm and an MIC of 50mg/ml (table 4), as
it survived upto 25mg/ml, while the acetonic, hot and cold aqueous extracts
of E.officinalis showed excellent
inhibitory activity against S.aureus
with the mean diameter of highest zone of inhibition being 40.32mm produced
by hot aqueous extract (much higher than the positive control ciprofloxacin
34.66mm) and an MIC of 12.5mg/ml, as it survived upto 6.25mg/ml. L.acidophilus, C.albicans and S.cerevisiae did not show any
inhibitory activity when assayed against E.officinalis
fruit extracts. Thus E.officinalis possesses
good antibacterial activity but no antifungal activity. The excellent
activity of E.officinalis against S.aureus shows a very good potential
of E.officinalis to treat the
diseases caused by S.aureus. The possible reason for the antibacterial
activity of E.officinalis might be
due to the tannins present in its fruits.
The fruits have 28% of the total tannins distributed in the whole plant. The
fruit contains two hydrolysable tannins Emblicanin A and B, which has
antioxidant properties, one on hydrolysis gives gallic acid, ellagic acid and
glucose wherein the other gives ellagic acid and glucose. The fruit also
contains Phyllemblin (Wealth of Asia, 1998; Ghosal, 1996; Dictionary of
Indian Medicinal Plants, 1988). Table 1: Antimicrobial
activity of fruit extracts of Sapindus
mukorossi on dental caries causing microorganisms determined by agar well
diffusion method on specific media for each test microorganism.
(-) = no
activity, nt = not tested, S.m. = Streptococcus mutans, S.a.= Staphylococcus aureus, L.a.= Lactobacillus
acidophilus, C.a.= Candida albicans, S.c.= Saccharomyces cerevisiae, * Values, including diameter of
the well (8 mm), are means of three replicates, † ± Standard
deviation. Table 2: Antimicrobial
activity of fruit extracts of Emblica
officinalis on dental caries causing microorganisms determined by agar
well diffusion method on specific media for each test microorganism.
(-) = no activity, nt
= not tested, S.m. = Streptococcus mutans, S.a.= Staphylococcus
aureus, L.a.= Lactobacillus acidophilus, C.a.= Candida albicans, S.c.=
Saccharomyces cerevisiae, * Values,
including diameter of the well (8 mm), are means of three replicates, †
± Standard deviation. Table 3:
MIC of fruit extracts of Sapindus
mukorossi against dental caries causing microorganisms on specific media
for each microorganism, determined by modified agar well diffusion method.
Table 4: MIC
of fruit extracts of Emblica
officinalis against dental caries causing microorganisms on specific
media for each microorganism, determined by modified agar well diffusion
method.
Conclusion Since
the acetonic and hot water extracts of fruits of Sapindus mukorossi and Emblica
officinalis were most effective against the tested dental caries causing
microorganisms, purification and toxicological studies of the plants and in vivo trials should be carried out
so that it can be used as a potential source for the development of a
phytomedicine to act against dental caries causing microbes. The
antimicrobial activities can be enhanced if the phytoactive components are
purified and adequate dosage determined for proper administration. Acknowledgement We would like to thank Dr. B.D.
Vashishta, Department of Botany, Kurukshetra University, Kurukshetra, for
rending help for the confirmation of identification of the plants, Dr. Tapan Chakrabarti,
Institute of Microbial Technology, Chandigarh, for providing the microbial
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