The objective of our study is to investigate the in vitro antibacterial activity of ethanolic  bark extract of Acacia catechu willd against selected oral microbes and to investigate the phytochemicals present in it. The inhibitory effect of the extract were tested against selected oral microbes by using the Macro broth dilution method .The ethanolic bark extract of Acacia catechu exhibited antibacterial activity against  streptococcus mitis with minimum bactericidal concentration of  500µg/ml whereas the MBC for streptococcus sanguis and Lactobacillus acidophilus were found to be 1mg/ml, 5mg/ml,10mg/ml and the phytochemical analysis revealed the presence of tannins, flavonoids, amino acids , saponins, triterpenoids. The ethanolic  bark extract of Acacia catechu willd was found to be bactericidal  in action against tested bacterial strains and these action may be due to the presence of phytochemical constituents in it. 

S, Aravind kumar

T, Lakshmi

English

International Journal of Phytomedicine

International Journal of Phytomedicine 3 (2011) 579-584 
http://www.arjournals.org/index.php/ijpm/index 
 
Original Research Article 
           
Preliminary phytochemical analysis & Invitro Antibacterial activity of 
Acacia catechu willd Bark against Streptococcus mitis, Streptococcus 
sanguis & Lactobacillus acidophilus. 
Lakshmi T1*, Aravind kumar S2 
 
*Corresponding author: 
 
Lakshmi T  
 
1Department of 
Pharmacology, Saveetha 
Dental College & Hospitals, 
Chennai. 
2Department of Orthodontics,   
Saveetha Dental College & 
Hospitals, Chennai. 
 
 
 
 
 
 
 
A B S T R A C T  
The objective of our study is to investigate the in vitro antibacterial 
activity of ethanolic bark extract of Acacia catechu willd against 
selected oral microbes and to investigate the phytochemicals present in 
it. The inhibitory effect of the extract were tested against selected oral 
microbes by using the Macro broth dilution method .The ethanolic 
bark extract of Acacia catechu exhibited antibacterial activity against  
streptococcus mitis with minimum bactericidal concentration of  
500µg/ml whereas the MBC for streptococcus sanguis and 
Lactobacillus acidophilus were found to be 1mg/ml, 5mg/ml,10mg/ml 
and the phytochemical analysis revealed the presence of tannins, 
flavonoids, amino acids , saponins, triterpenoids. The ethanolic  bark 
extract of Acacia catechu willd was found to be bactericidal  in action 
against tested bacterial strains and these action may be due to the 
presence of phytochemical constituents in it. 
Keywords: Acacia catechu, Anti bacterial activity, oral microbes, 
MBC, Phytochemicals. 
Introduction 
Acacia catechu Willd (Family: Fabaceae and 
subfamily: Mimosoideae.) is highly valuable for 
its powerful astringent and antioxidant activities. 
The bark, wood,  fruits , Gum and flowering tops 
of  Acacia catechu are used for medicinal 
purpose. The plant is useful, internally as well as 
externally. Used externally as a powder by itself, 
it arrests the bleeding in gums. The decoction is 
an effective gargle in sore throat, cough and 
hoarseness of voice. The paste is beneficial, 
externally, in skin diseases and wounds.  
 
 
 
The bath of its decoction is an effective panacea 
for various skin affection. In stomatitis, halitosis, 
dental caries and cavities, halitosis, dental caries 
and cavities, khadira(Acacia catechu in Sanskrit 
is known)  is used with great benefit. The popular 
preparation Khadiradi guti in ayurveda is 
extremely helpful for chewing in sore throat, 
hoarseness of voice, and tonsillitis etc. due to 
vitiation of kapha doshas. It dries up the mucous 
secretions and regains the taste sensation.  
The extracts of Acacia catechu  exhibits  various 
pharmacological effects like antipyretic, anti-
inflammatory, antidiarrhoeal,  hypoglycaemic, 
ISSN: 0975-0185 
 
  
This work is licensed under a Creative Commons Attribution 3.0 License.  
  Lakshmi et al. International Journal of Phytomedicine 3 (2011) 579-584 
 
580 
 
hepatoprotective, antioxidant and antimicrobial 
activities. [1-10] 
Oral microbial flora is dominated by gram 
positive micro organisms and hence dental plaque 
which is formed on the tooth surface contains 
gram positive cocci and bacilli. Oral health also 
influences the general quality of life and poor 
oral health is linked to chronic conditions and 
systemic diseases. oral bacteria like 
Streptococcus mutans ,Streptococcus sobrinus, 
Streptococcus oralis, Streptococcus intermedius, 
Streptococcus anginosus, Lactobacillus 
acidophilus, streptococcus salivarius, 
Streptococcus mitis, Streptococcus sanguis  is an 
potent initiator that causes dental caries/plaques  .  
These dental plaques are more difficult to be 
removed in the fixed appliances patients 
undergoing orthodontic treatment. Hence an 
attempt was taken by us to evaluate the invitro 
Antibacterial activity of Ethanolic Bark extract 
Of Acacia catechu   against selected oral 
microbes causing dental caries/plaque. moreover, 
The review of literature revealed that no method 
is yet reported for the estimation of 
phytochemicals in Acacia catechu Ethanolic Bark 
extract. This prompted us to conduct the 
preliminary study to identify the presence of 
phytochemical constituents in the plant extract 
responsible for the antibacterial action. 
 
 
MATERIALS AND METHODS 
Extract and Chemicals 
The Ethanolic Bark extract of Acacia catechu willd 
was obtained from Green Chem, Herbal Extracts & 
Formulations, Bangalore.  
Phytochemical analysis 
Phytochemical analysis was carried out according 
to the methods specified by Siddiqui , [11] 
Edeoga [12],Iyenger(1995)[13] and Trease and 
evans (2002).[14] The phytochemicals analyzed 
were alkaloids, tannins, saponins, flavonoids, 
steroids, phenols,  glycosides, carbohydrates, 
amino acids, proteins, terpenoids and 
phlobatannins. 
 
Test microorganisms 
 
       Bacterial strains used were streptococcus 
mitis(ATCC 9811), streptococcus sanguis(ATCC 
10557) and Lactobacillus acidophilus(ATCC 
4356)The organisms were obtained from 
Department of Microbiology , Saveetha Dental 
College & Hospitals, Chennai 
Anti Bacterial Assay 
The plant extract 200mg were weighed 
aseptically into a sterile tube and dissolved in 2ml 
of sterile Tryptic soy Broth (TSB).From the stock 
solution various concentrations were 
prepared,viz.,62µg,125 µg,250 µg,500 µg/100µl 
,1mg,5mg,10mg/100µl respectively in to wells of 
micro plates.The tested organism was grown in 
(TSB)Tryptic soy broth medium [MHA-Hi media 
,Mumbai] for 24hrs at 37°C and concentration 
was adjusted to 0.5 Macfarland 
 Standard.[15-17]  The above concentration of 
extracts were taken in 100µl quantities in a U 
bottom micro culture plates. control well received 
plain broth without plant extract. the plates were 
kept in sealed covers and incubated at 37°C 
overnight and growth/no growth was detected. 
All the tests were done in triplicate to minimize 
the test error. 
 
Minimum Inhibitory Concentration (MIC) 
   Minimum inhibitory concentration of herbal 
extracts against tested microorganism was 
determined by macro broth dilution method [18]. 
A series of two- fold dilution of each extract (62 
µg/100µl to 10mg/100µl) was made in to 
which100μl of the standardized bacterial 
suspension containing 106   organisms was made 
in Tryptic soy  broth as specified by National 
Committee for Clinical Laboratory Standards 
(NCCLS, 1990) [19].The control well received 
plain broth without herbal extract .The plates 
were incubated at 37°C for 24 hours and 
observed for visible growth. As the extracts were 
colored, MIC could not be read directly by visual 
methods.Hence subcultures from all the wells 
were made and turbidity/nogrowth is detected, 
then the MBC was obtained. 
 
Minimum Bactericidal Concentration (MBC) 
  Lakshmi et al. International Journal of Phytomedicine 3 (2011) 579-584 
 
581 
 
    The MBCs were determined by selecting wells 
that showed no growth . The least concentration, 
at which no growth was observed, were noted as 
the MBC. 
RESULT AND DISCUSSION 
          Oral cavity is a complex ecosystem with 
higly divergent acid tolerant and acid-producing 
microbiota. The mouth harbors a diverse, 
abundant and complex microbial community. 
This highly diverse micro flora inhabits the 
various surfaces of the normal mouth. Bacteria 
accumulate on both the hard and soft oral tissues 
in biofilms.  
Bacterial adhesion is particularly important for 
oral bacteria. Dental plaque is the material that 
adheres to the teeth and consists of bacterial cells 
(mainly S. mutans and S. sanguis), salivary 
polymers and bacterial extracellular products. 
Plaque is a biofilm on the surfaces of the teeth. 
This accumulation of microorganisms subject the 
teeth and gingival tissues to high concentrations 
of bacterial metabolites which results in dental 
disease. If not taken care of, via brushing or 
flossing, the plaque can turn into tartar (its 
hardened form) and lead to gingivitis or 
periodontal disease.21 
Acidogenic oral microbes is the key factor of 
Dental plaques.the primary acid tolerant bacteria 
associated with Dental plaque  includes 
streptococcus mutans, Streptococcus oralis 
streptococcus sobrinus,Lactobacillus 
acidophilus, streptococcus salivarius, 
Streptococcus mitis, Streptococcus sanguis 
,Streptococcus intermedius, Streptococcus 
anginosus that surround orthodontic appliances 
are a common orthodontic problem in many 
patients undergoing Orthodontic treatment. 
[20,22,23] 
It also has been reported thet presence of fixed 
orthodontic appliance  greatly inhibitis oral 
hygiene and creates new retentive areas for 
plaque and debris  ,which in turn predisposes to 
increased carriage of microbes and subsequent 
infection .Therefore, prevention of bacterial 
attachment to orthodontic wires is a critical 
concern for  orthodontists.24,25 
The occurrence of mutans streptocooci and 
streptococcus sobrinus together makes the oral 
environment more conductive to caries/plaque. 
These cariogenic pathogens utilise dietary 
sucrose and produce adhesive exopolysaccharides 
and acids which lead to plaque formation and 
carious lesions on susceptible tooth surfaces .  
 
 
 Dental plaque plays the primary role in the 
pathogenesis of the dental caries. Dental plaque is 
a general term for the diverse microbial 
community  found on the tooth surface, 
embedded in a matrix of polymers of bacterial 
and salivary origin. Plaque is an example of a 
biofilm; current researches are showing that the 
properties of bacteria associated with a surface in 
a biofilm can be markedly different than those of 
the same cells growing in liquid broth . Plaque is 
found preferentially at protected and stagnant 
surfaces, and these are at the greatest threat of 
disease . 
 
The preliminary phytochemical analysis was 
carried out for the plant extract by standard 
procedure.the extract showed the presence of 
Tannins ,phenol ,saponins,carbohydrates 
,flavonoids,alkaloids and glycosides.the extract 
lacks the presence of phlobatanins ,proteins and 
aminoacids.the data is represented in Table 1. 
The findings of the Antibacterial study reveals 
that the extract at different concentrations has 
exhibited antibacterial activity against the 
bacterial strain tested. The ethanolic bark extract 
of Acacia catechu exhibited antibacterial activity 
against streptococcus mitis with minimum 
bactericidal concentration of 500µg/ml whereas 
the MBC for streptococcus sanguis and 
Lactobacillus acidophilus were found to be 
1mg/ml, 5mg/ml and 10mg/ml. 
  Lakshmi et al. International Journal of Phytomedicine 3 (2011) 579-584 
 
582 
 
The presence of No growth is an indication of 
high effectiveness of the extract whereas 
presence of Growth indicates the less 
effectiveness of the extract, which was 
represented in Table 2. 
    Current  research indicate that the polyphenols, 
being secondary metabolites, are present in rich 
amount in various plants. Many of them possess 
antioxidant, anti-inflammatory and  several others 
therapeutic properties Acacia catechu willd 
contains many active constituents in it like 
Catechin, epigallocatechin, epicatechin gallate, 
epigallocatechin gallate ,phloroglucin, 
protocatechuic acid,  poriferasterol glucosides, 
poriferasterol acyglucosides,  lupenone, 
kaempferol, dihydrokaemferol ,Quercetin, 
Taxifolin etc., 
 
 
 
 
Table 1 Phytochemical analysis of Acacia catechu willd bark extract 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
++ =high concentration  + =moderate concentration   - =Absence 
 
 
 
 
 
 
Phytochemical constituents   
 
Acacia catechu 
Ethanolic bark extract 
Saponins 
 
          ++ 
Tannins 
 
          ++ 
Phenol 
 
          + 
Carbohydrates 
 
          + 
Proteins 
 
           - 
Amino Acids 
 
           - 
Flavonoids 
 
         ++ 
Phlobatanins 
 
           - 
Alkaloids 
 
           + 
 Glycosides 
 
           + 
  Lakshmi et al. International Journal of Phytomedicine 3 (2011) 579-584 
 
583 
 
 
 
TABLE – II   DETERMINATION OF THE MBC  
 
 
Strain 
 
                                 Concentrations of Acacia catechu willd  bark extract 
 
62µg/ml 
 
125µg/ml 
 
250µg/ml 
 
500µg/ml 
 
1mg/ml 
 
5mg/ml 
 
10mg/ml 
Streptococcus 
mitis 
Turbidity Turbidity Turbidity NG NG NG NG 
Streptococcus 
sanguis 
Turbidity Turbidity Turbidity Turbidity NG NG NG 
Lactobacillus 
acidophilus 
Turbidity Turbidity Turbidity Turbidity Turbidity NG NG 
 
NG –No Growth(Indicates MBC) 
Turbidity –Least effectiveness of extract 
 
 
Conclusion 
 
In conclusion, our study provides new scientific 
information about Acacia catechu ethanolic bark 
extract, based on its antibacterial potential and 
chemical profiling that has never been reported. 
The antibacterial activity of Acacia catechu bark 
extract against oral microbes may be attributed to 
the various phytochemical constituents present in 
the refined extract. The purified components have  
more potency with respect to inhibition of 
Selected oral microbes. Further work on the types 
of phytoconstituents and purification of 
individual groups of bioactive components can 
reveal the exact potential of the plant to inhibit 
several pathogenic microbes and encourage in 
developing a noval broad spectrum antibacterial 
herbal formulation in future to prevent the dental 
caries/plaque in fixed appliances patients 
undergoing orthodontic Treatment 
. 
ACKNOWLEDGEMENTS 
 
Our heartfelt thanks to Mr. Rajendran, Green 
Chem Herbal Extracts & Formulations, 
Bangalore for Providing me the Ethanolic Bark 
Extract of Acacia Catchu Willd  as a Gift sample 
to conduct this In vitro Study and we thank 
Dr.Auxilia Hemamalini ,HOD of Microbiology , 
Saveetha Dental College & Hospitals,Chennai for 
providing the test organism for the study. 
 
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Preliminary phytochemical analysis & Invitro Antibacterial activity of Acacia catechu willd Bark against Streptococcus mitis, Streptococcus sanguis & Lactobacillus acidophilus

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Preliminary phytochemical analysis & Invitro Antibacterial activity of Acacia catechu willd Bark against Streptococcus mitis, Streptococcus sanguis & Lactobacillus acidophilus

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