Invitro Antibacterial Activity of  Acacia catechu ethanolic leaf extract against selected acidogenic oral bacteria

 

Aravind Kumar. S¹ , Lakshmi. T²*  and Arun. A.V³

¹Reader, Department of Orthodontics, Saveetha Dental College and Hospitals , Chennai.

²Assistant Professor, Department of Pharmacology, Saveetha Dental College and Hospitals , Chennai.

³Professor, Department of Orthodontics , Saveetha Dental College and Hospitals , Chennai.

*Corresponding author Email.: lakshmi085@gmail.com

 

Background: The background of our study is to evaluate the in vitro antibacterial activity of ethanolic  leaf extract of Acacia catechu against streptococcus mutans  ,streptococcus mitis ,streptococcus salivarius  and streptococcus sobrinus.

Materials and Methods: The inhibitory effect of the extract were tested against selected acidogenic oral bacteria like streptococcus mutans  ,streptococcus salivarius ,streptococcus mitis  ,streptococcus  sobrinus  that causes dental plaque in fixed appliances patients undergoing orthodontic treatment by using the broth culture method .

Results: The ethanolic leaf extract of Acacia catechu exhibited antibacterial activity against streptococcus mutans and streptococcus mitis with minimum bactericidal concentration of 62µg/ml and 5mg/ml against streptococcus salivarius and ,streptococcus sobrinus respectively.

Conclusion: The ethanolic leaf extract of Acacia catechu is found to be bactericidal in action against tested bacterial strains.

 

 

INTRODUCTION:

Control  of  dental plaque is an major factor in prevention of dental caries and Periodontal disease. ^[1] The viridans streptococci include S mitis, S mutans, S salivarius, S sanguis, and others surround the orthodontic appliances in patients undergoing orthodontic treatment.^[2,3]. Typically they are alpha-hemolytic, but they may be nonhemolytic. Their growth is not inhibited by Optochin, and colonies are not soluble in bile (deoxycholate). The viridans streptococci are the most prevalent members of the normal flora of the upper respiratory tract and are important for the healthy state of the mucous membranes there. They may reach the bloodstream as a result of trauma and are a principal cause of endocarditis on abnormal heart valves. Some viridans streptococci (eg, S mutans) synthesize large polysaccharides such as dextrans or levans from sucrose and contribute importantly to the genesis of dental caries/plaque.^[4]

 

Acacia catechu is highly valuable for its powerful astringent and antioxidant activities. It is useful in dental, oral, throat infections and as an astringent for reducing oozing from chronic ulcers and wounds. The extracts of Acacia catechu  exhibits  various pharmacological effects like antipyretic, anti-inflammatory, antidiarrhoeal, hypoglycemic, hepatoprotective, antioxidant and antimicrobial activities .^[5-9]

 

Main chemical constituents of Acacia catechu are catechin, (-) epicatechin, epigallocatechin, epicatechin gallate, epigallocatechin, phloroglucin, protocatechuic acid, quercetin, poriferasterol glucosides, poriferasterol acyglucosides, lupenone, kaempferol, dihydrokaempferol, taxifolin, (+)-afzelchin gum and minerals.^[10-15]The chief phytoconstituent  of the heartwood are catechin and epicatechin. Catechins have siginificant antioxidant and antimicrobial effects. ^[16,17]

The presence of  acidogenic  bacteria leads to oral health problems such as  primary lesions and progressive enamel loss after the removal of fixed orthodontic appliances.^[3] antiplaque approaches are based on the use of broad-spectrum microbicidal agents, e.g. chlorhexidine ,fluoride. Natural products offer a rich source of diverse substances with a broad range of biological activities, which could be useful for the development of alternative or adjunctive anticaries therapies. However, it is a challenging approach owing to complex chemistry and isolation procedures to derive active compounds from natural products.

 

Hence an attempt was made to evaluate the antibacterial activity of Acacia catechu Ethanolic leaf extract against selected acidogenic oral bacteria to prevent the dental plaque that occurs in fixed orthodontic appliances patients.

 

MATERIALS AND METHODS:    

Plant material

Acacia catechu ethanolic leaf extract were obtained from Green Chem. Herbal Extracts and Formulations. Bangalore, India

 

Test microorganisms

 Bacterial strains used were  streptococcus mutans (ATCC 25175) ,streptococcus salivarius (ATCC 25975), streptococcus mitis  (ATCC 9811),Streptococcus sobrinus (ATCC 27607).The organisms were obtained from Department of Microbiology , Saveetha Dental College and Hospitals, Chennai .

 

Methodology

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.100µl of these concentration were taken  and the plates were incubated at 37°C for 24hrs.

 

Screening of Antibactericial activity 

The tested organisms was grown in (TSB) Tryptic soy broth medium [Hi media ,Mumbai] for 24hrs at 37°C and concentration was adjusted to 0.5 McFarland    standard.^[18-20] The above concentration of  extracts were taken in 100µl quantities in a U bottom micro culture plates. 100µl of the bacterial suspension was added to each well. 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 duplicate to minimize the test error.

 

Minimum Inhibitory Concentration (MIC)

Minimum inhibitory concentration of herbal extracts against tested microorganism was determined by broth culture method .^[21] A series of two- fold dilution of each extract (62 µg/100µl to 10mg/100µl) was made in to which 100μl 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)^[22] 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 growth/no growth is detected, then the MBC were obtained.

 

Minimum Bactericidal Concentration (MBC)

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:

Dental caries is the most prevalent  oral infectious disease worldwide. Virulent biofilms firmly attached to tooth surfaces are primary biological factors associated with this disease.

 

One of the major and most common challenges in prevention within the field of oral health is the control of plaque and, consequently, the control of dental caries and gingival inflammation.^[23,24] Mechanical methods such as the use of toothbrush and dental floss, when applied effectively, can promote proper plaque control.^[25,26] Dentists  play a major role in guiding and encouraging patients to perform proper oral hygiene  effectively.

 

Various literatures reveals that the patients undergoing orthodontic treatment particularly find difficult to maintain proper oral hygiene owing to the presence of bands and wires. It is therefore has become vital to establish preventive motivation and guidance methods to ensure control   of dental plaque using safer herbal medicines other than mechanical devices.

 

Patients undergoing fixed orthodontic treatment are more prone to retaining dental plaque. Orthodontic accessories can lead to enamel demineralization, causing white spots, tooth decay and gingivitis.^[27,28,29] In fixed orthodontic treatment, plaque retention surfaces are increased and, as a result, most patients are confronted with hygiene difficulties, which eventually cause elevated plaque indices.^[29] Mechanical methods of plaque removal require time, motivation and manual skill.^[30] several anti-microbial studies have established that after a fixed orthodontic appliance has been placed the number of bacteria rises significantly, particularly lactobacilli and streptococci, subjecting the oral environment to an imbalance that predisposes to the emergence of diseases.^[31]

 

Several literature studies proves that plants as intact crude organs and their products (e.g., powdered plants, extracts, etc) have been widely used by different cultures to promote oral hygiene since antiquity.^[32]

 

Anitha and coworkers conducted a study to evaluate the Antibacterial efficacy of Aqueous and Ethanolic extract of Aesculus Hippocastanum against oral microbes Causing dental caries/plaque.the bacterial strains used in our study are Streptococcus mutans,  streptococcus salivarius,streptococcus mitis ,streptococcus sanguis,

 

 

Table 1 Antibacterial activity of Acacia catechu ethanolic leaf extract against selected oral microbes.

Acacia catechu ethanolic leaf extract	62µg/ml	125 µg/ml	250 µg/ml	500 µg/ml	1mg/ml	5mg/ml	10mg/ml	Control	MBC
Streptococcus mutans	--	--	--	--	--	--	--	++	2 µg/ml
Streptococcus mitis	--	--	--	--	--	--	--	++	62 µg/ml
Streptococcus salivarius	++	++	++	++	++	--	--	++	5mg/ml
Streptococcus sobrinus	++	++	++	++	++	--	--	++	5mg/ml

++ = Growth; --   = No Growth

 

 

Lactobacillus acidophilus .the antibacterial efficacy was significant against streptococcus mutans and streptococcus sanguis when compared to the other bacterial organism tested. Hence  it can be  concluded that Aesculus hippocastanum is highly efficient against dental plaques caused primarily by streptococcus mutans.^[33]

 

Jian HE et al in his study found that liquorice extract exhibits potent antimicrobial activity against streptococcus mutans and are now being used in lollipops to reduce caries/plaques.^[34]

 

Streptococcus mutans and Lactobacillus acidophilus are potent initiator for dental plaques that results in destruction of mineralised tissues in the teeth .Geetha and co workers in their study evaluated  the potency of Acacia catechu heartwood extract against dental caries/plaque causing microbes like streptococcus mutans, streptococcus salivarius, and Lactobacillus acidophilus  .^[35] 

 

In table-1  Acacia catechu leaf extract shows No growth (MBC) at a concentration of 62 µg/ml against streptococcus mutans and streptococcus mitis. It also shows No growth (MBC) at a concentration of 5mg/ml against streptococcus salivarius and Streptococcus sobrinus respectively.  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 1.

 

CONCLUSION:

The available data in our study suggest that ethanolic leaf extract of Acacia catechu extract is highly effective against oral microbes and it can also be applied in dental practice to treat dental caries/plaques in periodontal  and orthodontic fixed appliance patients where the dental plaques are difficult to be removed.

 

ACKNOWLEDGEMENTS:

We wish to thank Mr.Rajendran CEO  of Green Chem Herbal Extracts and Formulations, Bangalore, India for Providing me the ethanolic leaf extract of Acacia catechu  as a gift sample to conduct this In vitro Study  and Dr. Auxilia Hemamalini ,HOD of Microbiology, Saveetha Dental College and Hospitals, Chennai for providing the test organisms for the study.

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