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: firstname.lastname@example.org
�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
���� �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).
����� 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
����� 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.
����� 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.
would like to place special thanks to Dr C.M Govil, Professor, Department of
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Katzer, G. Gernot Ketzer�s Spice pages. Internet: http:// www-ang.kfunigraz.ac.at/katzer/engl/index.html (accessed 18 march 2002).
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Norrel, S. A.
and Messley, K. E., 1997. Microbiology
Laboratory Manual Principles and Applications �Prentice Hall,
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