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Ethnobotanical
Leaflets 13:611- 17, 2009. Antibacterial Activity of �Amchur (Dried Pulp of Unripe Mangifera indica) Extracts on Some Indigenous Oral Microbiota Causing Dental
�Caries Charu Gupta1, �Amar P. Garg1 �and �Ramesh
C. Uniyal 2 For correspondence: Dr. Charu Gupta, Lecturer, Amity Institute for Herbal Research & Studies, E- 3 block, 4th floor, Tel: +91-9958905744; �Email: cgupta@amity.edu Issued ��������������������������������������������������������������������
Abstract �The antibacterial
activity of amchur (dried pulp of unripe Mangifera
indica) extract (50% ethanol) was tested against ten bacterial strains causing
dental plaque by agar well diffusion method. The crude extract showed a broad
spectrum of antibacterial activity inhibiting both the groups of Gram-positive
& Gram-negative bacteria. The
extract was most effective against Bacillus sp., followed by Staphylococcus mutans and Pseudomonas
sp., whereas Halobacterium sp. was found to be the most resistant. Chlorhexidine (present in
mouthwashes to prevent infection of dental caries) was used as a positive
control. Natural
extract of amchur was found to be more effective as compared to chlorhexidine. This study shows
the potential of amchur in the treatment of dental caries. Keywords: Amchur, Mangifera indica, antibacterial
activity, dental
caries, agar well diffusion method Introduction ���� �Dental
caries is the localized destruction of the tissues of the tooth by acid
produced from the bacterial degradation of fermentable sugars. Gnotobiotic
animal studies showed that caries could be induced by specific bacteria,
especially members of the mutans Streptococci-
group (e.g Streptococcus mutans and
Streptococcus sobrinus), but only
when fed a cariogenic (high sucrose) diet. These studies also showed the
potential for transmission from animal to animal, and that protection could
be achieved by antimicrobial agents & vaccination. Advanced lesions often
have a high proportion of lactobacilli, while dentinal lesions have a diverse
microflora with many fastidious Gram positive (Actinomyces naeslundii, A. odontolyticus, Propionibacterium spp., Eubacterium spp.) and Gram negative (Fusobacterium
spp., Capnocytophage spp., Veillonella spp.) bacteria. ���� �Many natural substances of plant origin are
reported to be biologically active, endowed with antimicrobial, allelopathic
and antioxidant properties (Beuchat and Golden, 1989). Mango is considered
as a king of fruits in Indian delicacy. The roots and bark of
mango Mangifera indica
(Anacardiaceae) are astringent, acrid, anti-inflammatory, and constipating.
The leaves and flowers are refrigerant, styptic, vulnerary and constipating. Amchur (dried or dehydrated product of unripe
mango flesh in the form of peeled slices or powder) is used as an acidulate
or a souring agent to provide the desired acidity in the various food
recipes. �Amchur is rich in citric
acid. ���� �Very limited literature is available on the
antimicrobial activity of amchur extract. In the present study, we have
investigated the antibacterial activity of dried pulp of unripe
Mangifera indica against dental
caries for the first time. Materials and Methods ����� Materials: All chemicals used were of
analytical-reagent grade and obtained from E. Merck ( ����� Bacterial Strains: Ten bacterial strains (6 Gram
positive and 4 Gram negative), involved in
dental caries, were selected for the study. Gram positives were Streptococcus mutans, Streptococcus salivarius, Lactobacillus sp., Bacillus
sp., Micrococcus sp., Staphylococcus aureus, Halobacterium sp., Veilonella sp., Pseudomonas
aeruginosa, Pseudomonas sp. The bacterial stock cultures
were obtained from the culture collection unit of Department of Microbiology,
� ����Extraction: The pulp of unripe mango (Mangifera
indica) was dried and powdered in milling
machine (Inalsa Mixer Grinder) to obtain fine dry powder called amchur. The
powder was weighed using single pan electronic weighing balance (Ohaus model).
The herbal extract was prepared at the rate of 1g/5ml of solvent (50% ethanol)
in a 250mL Erlenmeyer flasks. The flasks were closed with cotton plug and
aluminium foil. The spice powder was soaked in 50% ethanol for 48h at room
temperature with intermittent shaking. The mixture was centrifuged at 3500xg
for 20min and finally filtered through Whatmann filter paper No.1 (Azoro,
2000). The pellet was discarded and the supernatant was collected and
concentrated under reduced pressure in a rotary vacuum evaporator (Buchi
Type) until semisolid substance was obtained. This was dried inside the
crucible under a controlled temperature (45�C) to obtain solid powder (Jonathan
and Fasidi, 2003). The process of extraction was repeated until the weight of
500mg was obtained. The powder was weighed and reconstituted in dimethyl
sulfoxide (DMSO). These were stored in the refrigerator at 4�C for testing
antimicrobial sensitivity. The extract was exposed to UV rays for 24h and
checked for sterility by streaking on ����� Antibacterial assay: The antimicrobial activity of amchur extract
was determined by agar well diffusion method against different bacteria as
described by Okeke �et al., (2001).
In this method, pure isolate of each bacterium was sub-cultured in nutrient
broth at 37�C for 24h. One hundred microlitres (about 106CFU/mL, standardized by 0.5 Mac-Farland) of
each test bacterium was spread with the help of sterile spreader on to a
sterile Muller-Hinton Agar plate (Hi Media, Mumbai, India) so as to achieve a
confluent growth. The plates were allowed to dry and a sterile cork borer of
diameter 6.0mm was used to bore wells in the agar plates. Subsequently, a
50L volume of the extract was introduced in triplicate wells into Muller-Hinton
Agar plate. Sterile DMSO served as negative control. Chlorhexidine (standard
chemotherapeutic agent in mouth washes) was also used as positive control. �The plates were allowed to stand for 1h or
more for diffusion to take place and then incubated at 37�C for 24h. The zone
of inhibition was recorded to the nearest size in mm (Norrel and Messely,
1997). Results ����� Following the extraction of the dried
unripe pulp of Mangifera indica
(Amchur) using 50% ethanol by maceration method, the antimicrobial activity
of the extract was determined by agar well diffusion method. Table 1 shows
the antimicrobial activity of the amchur extract on the indigenous oral
microbiota that cause dental caries. The extract was effective against both Gram
positive and Gram negative �bacteria.
However the ethanolic extract was most effective against Bacillus
sp. with diameter of zone of inhibition 19.0mm followed by Streptococcus mutans (main causative
organism of dental caries).
Chlorhexidine, on the other hand was less effective producing an inhibition
zone of diameter 14mm. Amongst the Gram negative bacteria, the extract showed
highest activity against Pseudomonas sp. with diameter of zone of
inhibition 18.0mm and was least effective against Halobacterium sp. with diameter of zone of
inhibition 10.0mm. Table 1. �Zone of inhibition (mm) of ethanolic extract of
amchur (Mangifera indica) on
selected bacteria that cause dental plaque.
Incubation temperature: 37�C; Incubation
period: 24h Negative control- Dimethyl sulfoxide Positive control- Chlorhexidine Volume of extract in each well = 50mL Discussion ����� From
this investigation, it was
observed that amchur extract was more effective than chlorhexidine against
both groups of bacteria. It may possibly be due to the change of pH of the
medium due to amchur which cause the pH to bring down in acidic range. pH is
known to control the growth, development and sporulation of all microbes
including bacteria.�� ���� Amchur
contains citric acid related compounds which is responsible for its sour
taste. Several terpenes (ocimene, myrcene, limonene), aldehydes and esters
have been found in dried unripe mango fruit. They also contain proteolytic
enzymes (Gernot Katzer�s Spice Pages- An encyclopedia of Spices). ����� Investigations
into the effects of terpenoids upon isolated bacterial membranes have suggested
that their activity is a function of the lipophilic properties of the
constituent terpenes, the potency of their functional groups and their
aqueous solubility (Knobloch et al., 1989, Elgayyar et al., 2001).
Their site of action is at the phospholipid bilayer, & the biochemical
mechanisms include the inhibition of electron transport, protein
translocation, phosphorylation steps and other enzyme � dependent reactions (Knobloch
et al., 1989). These activities suggest their potential use as food
preserving agents, chemotherapeutic agents and disinfectants. Conclusion ����� In
conclusion, amchur extract can be used as an inexpensive source for the
treatment of dental caries caused by the bacteria. Further research on
the use of other botanical extracts can be rewarding to pursue in hunt for
new herbal therapeutic agent. Acknowledgement We
would like to place special thanks to Dr C.M Govil, Professor, Department of
Botany, References Azoro, C.
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