In vitro Evaluation of Antibiofilm Activity of Methanolic Leaf Extract of Azadirachta indica on Cariogenic Streptococcus mutans

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  • Department of Life Sciences, Nucleobase Life Sciences Research Laboratory, Bengaluru - 560043, Karnataka
  • Department of Microbiology, Krupanidhi Degree College, Bengaluru - 560035, Karnataka
  • Department of Life Sciences (Zoology), Manipur University, Imphal - 795003, Manipur
  • Department of Pharmacognosy and Phytochemistry, Krupanidhi College of Pharmacy, Bengaluru - 560035, Karnataka
  • Krupanidhi Degree College, Bengaluru - 560035, Karnataka



Dental Caries, Microtiter Plate Assay, Neem, Phytochemicals, Antibacterial Compounds.


Dental caries is a biofilm-dependent disease, and Streptococcus mutans is the primary etiological agent involved in the initiation of the disease. The extensive use of a limited range of antimicrobial drugs in dentistry has led to the development of drug-resistant bacteria. There is an increasing need to find new alternatives against drug-resistant bacteria. Globally, there is a continuous effort towards identifying natural anti-caries agents for the prevention and better management of caries. The objective of the present study was to evaluate the antibiofilm potential of Azadirachta indica leaf methanolic (ALM) extract against S. mutans biofilm. The study employed a standard reference strain of S. mutans MTCC 497, for in vitro standardisation of biofilm by microtiter plate assay. The antibiofilm activity of the ALM extract was evaluated against the S. mutans strain, and the same was confirmed by light and scanning electron microscopy (SEM). The in vitro biofilm standardisation results demonstrated that 50 µl/ml of S. mutans inoculum concentration exhibited a much superior biofilm formation than the other concentrations employed. Light microscopy and SEM images revealed that ALM extract at 100 mg/ml concentration significantly inhibited the S. mutans biofilm. To conclude, the study reports that the A. indica leaf extract is a potential source to inhibit the S. mutans biofilm. Further studies are warranted to identify the phytochemicals responsible for the antibiofilm activity of ALM extract against S. mutans biofilm that aid in the design of natural anti-caries products.


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Yadav K, Prakash S. Dental Caries: A Microbiological Approach. J Clin Infect Dis Pract. 2017; 2(1):1-5. https://

Oral Health Database. Available online: https://www.mah. se/CAPP/ (accessed on 14 April 2020).

Loesche WJ. Role of Streptococcus mutans in human dental decay. Microbiological Reviews. 1986; 50(4):353-80. https://

Koo H, Duarte S, Murata RM, Scott-Anne K, Gregoire S, Watson GE, Singh AP, Vorsa N. Influence of cranberry proanthocyanidins on formation of biofilms by Streptococcus mutans on saliva-coated apatitic surface and on dental caries development in vivo. Caries Research. 2010; 44(2):116-26.

Forssten SD, Bjorklund M, Ouwehand AC. Streptococcus mutans, Caries and Simulation Models. Nutrients. 2010; 2:290–298.

Pourhajibagher M, Alaeddini M, Etemad-Moghadam S, Rahimi Esboei B, Bahrami R, Mousavi M, Bahador A. Quorum quenching of Streptococcus mutans via the nanoquercetin-based antimicrobial photodynamic therapy as a potential target for cariogenic biofilm. BMC Microbiology. 2022; 22(1):1-8. 02544-8

Haque M, Sartelli M, Haque SZ. Dental infection and resistance - global health consequences. Dentistry Journal. 2019; 7(1):22.

Oyebode O, Kandala NB, Chilton PJ, Lilford RJ. Use of traditional medicine in middle-income countries: A WHOSAGE study. Health Policy Plan. 2016; 31:984–991. https://

Sarkar S, Singh RP, Bhattacharya G. Exploring the role of Azadirachta indica (neem) and its active compounds in the regulation of biological pathways: An update on molecular approach. 3 Biotech. 2021; 11(4):1-2. https://doi. org/10.1007/s13205-021-02745-4

Saleem S, Muhammad G, Hussain MA, Bukhari SN. A comprehensive review of phytochemical profile, bioactives for pharmaceuticals, and pharmacological attributes of Azadirachta indica. Phytotherapy research. 2018; 32(7):1241-72.

Wylie MR, Merrell DS. The Antimicrobial Potential of the Neem Tree Azadirachta indica. Frontiers in Pharmacology. 2022; 13.

Braga TM, Rocha L, Chung TY, Oliveira RF, Pinho C, Oliveira AI, et al. Biological Activities of Gedunin-A Limonoid from the Meliaceae Family. Molecules. 2020; 25(3).

Nagini S, Nivetha R, Palrasu M, Mishra R. Nimbolide, a Neem Limonoid, is a Promising Candidate for the Anticancer Drug Arsenal. J. Med. Chem. 2021; 64(7):3560– 3577.

Brahmachari G. Neem - An Omnipotent Plant: A Retrospection. Chembiochem. 2004; 5(4):408–421. https://

Gupta SC, Prasad S, Tyagi AK, Kunnumakkara AB, Aggarwal BB. Neem (Azadirachta indica): An Indian Traditional Panacea with Modern Molecular Basis. Phytomedicine. 2017; 34:14–20. phymed.2017.07.001

Reichling J. Anti-biofilm and virulence factor-reducing activities of essential oils and oil components as a possible option for bacterial infection control. Planta Medica. 2020; 86(8):520–537.

Guzzo F, Scognamiglio M, Fiorentino A, Buommino E, D’Abrosca B. Plant derived natural products against Pseudomonas aeruginosa and Staphylococcus aureus: Antibiofilm activity and molecular mechanisms. Molecules. 2020; 25(21):5024.

Freires IA, Denny C, Benso B, de Alencar SM, Rosalen PL. Antibacterial activity of essential oils and their isolated constituents against cariogenic bacteria: A systematic review. Molecules. 2015; 20(4):7329–7358. molecules20047329

Vestby LK, Gronseth T, Simm R, Nesse LL. Bacterial biofilm and its role in the pathogenesis of disease. Antibiotics. 2020; 9(2):59.

Guchhait KC, Manna T, Barai M, Karmakar M, Nandi SK, Jana D, Dey A, Panda S, Raul P, Patra A, Bhattacharya R. Antibiofilm and anticancer activities of unripe and ripe Azadirachta indica (neem) seed extracts. BMC complementary medicine and therapies. 2022; 22(1):1-8. https://doi. org/10.1186/s12906-022-03513-4

Wylie MR, Windham IH, Blum FC, Wu H, Merrell DS. In vitro antibacterial activity of nimbolide against Helicobacter pylori. Journal of Ethnopharmacology. 2022; 285:114828.

Christensen GD, Simpson WA, Bisno AL, Beachey EH. Adherence of slime-producing strains of Staphylococcus epidermidis to smooth surfaces. Infection and immunity. 1982; 37(1):318-26.

Hasan S, Danishuddin M, Adil M, Singh K, Verma PK, Khan AU. Efficacy of E. officinalis on the cariogenic properties of Streptococcus mutans: A novel and alternative approach to suppress quorum-sensing mechanism. Plos one. 2012; 7(7):e40319.

Gowrishankar S, Duncun Mosioma N, Karutha Pandian S. Coral-associated bacteria as a promising antibiofilm agent against methicillin-resistant and - susceptible Staphylococcus aureus biofilms. Evidence-Based Complementary and Alternative Medicine. 2012; 2012.

Yoshida A, Kuramitsu HK. Multiple Streptococcus mutans genes are involved in biofilm formation. Applied and environmental microbiology. 2002; 68(12):6283-91. https://doi. org/10.1128/AEM.68.12.6283-6291.2002

Lee HJ, Kang SM, Jeong SH, Chung KH, Kim BI. Antibacterial photodynamic therapy with curcumin and Curcuma xanthorrhiza extract against Streptococcus mutans. Photodiagn Photodyn Ther. 2017; 20:116–119. https://doi. org/10.1016/j.pdpdt.2017.09.003

Xu X, Zhou XD, Wu CD. The tea catechin epigallocatechin gallate suppresses cariogenic virulence factors of Streptococcus mutans. Antimicrob Agents Chemother. 2011; 55:1229–1236.

Santiago KB, Piana GM, Conti BJ, Cardoso EO, Murbach Teles Andrade BF, Zanutto MR, Mores Rall VL, Fernandes A Jr, Sforcin JM. Microbiological control and antibacterial action of a propoliscontaining mouthwash and control of dental plaque in humans. Nat Prod Res. 2017; 32:1441– 1445.

Lemos JA, Palmer SR, Zeng L, Wen ZT, Kajfasz JK, Freires IA, Abranches J, Brady LJ. The biology of Streptococcus mutans. Microbiology Spectrum. 2019; 7(1):7-1. https://

Pai MR, Acharya LD, Udupa N. Evaluation of antiplaque activity of Azadirachta indica leaf extract gel - a 6-week clinical study. Journal of ethnopharmacology. 2004; 90(1):99-103.




How to Cite

Dinesh, D., Imran, K., Vijayaraghavalu, S., Saifulla Khan, M., & Nikesh, V. V. (2023). <i>In vitro</i> Evaluation of Antibiofilm Activity of Methanolic Leaf Extract of <i>Azadirachta indica</i> on Cariogenic <i>Streptococcus mutans</i>. Journal of Natural Remedies, 23(1), 275–281.



Short Communication
Received 2022-12-27
Accepted 2023-01-13
Published 2023-03-23