Evaluation of antimicrobial activity of orange peel extract against oral biofilm forming organisms: an in vitro microbial study and scanning electron microscopic assessment

Chetana Dilip Mankar, Mona Udayan Shah, Yogesh Sharad Doshi, Monika Bajaj, Vidhi Kevadia, Roshini Vinod


Background: To evaluate in vitro antimicrobial activity of orange peel extract, 0.2% chlorhexidine (CHX) and saline against dental biofilm-forming bacteria and their effect on plaque structure under SEM.

Methods: Present study included 3 groups; Group A: Orange peel extract; Group B: 0.2% CHX and Group C: Saline.  MIC of each group was determined by broth and well diffusion method. Reference strains of Porphyromonas gingivalis (P.g.), Prevotella intermedia (P.i.) and Aggregatibacter actinomycetemcomitans (A.a.) were selected as being colonizers in dental biofilm. For SEM analysis, plaque retained orthodontic e-chains were subjected to each group for 60s and studied under SEM.

Results: The MIC of Group A by broth diffusion method is 66.66 µg/ml for P. intermedia, 75 µg/ml for P. gingivalis and 33.33 µg/ml for A.a. MIC of Group B was 14.58 µg/ml for P.i., 7.29 µg/ml for P.g. and 20.83 µg/ml for A.a. MIC of Group C was 83.33 µg/ml for A.a. while P.i. and P.g. showed resistance to Group C. The determination of MIC by well diffusion showed increase in clear zone diameter with increasing concentrations.  Greater zone of inhibition was seen in Group B with statistical significance for all 3 microorganisms. Morphologic changes in the SEM study showed maximum change in the surface characteristics and loss of adhesion of the clusters of microorganisms subjected to Group B.

Conclusions: CHX and orange peel extract exhibited notable anti-microbial activity against biofilm organisms, but at varying concentrations. The plaque samples subjected to orange peel extract showed slightly shrunken and coarse form indicating its ability to disrupt the plaque biofilm.


Orange peel extract, Chlorhexidine, Saline, Well diffusion, Broth diffusion, Scanning electron microscope

Full Text:



Marsh PD. Dental Biofilms, In: Lang N, Lindhe J(eds). Clinical Periodontology and Implant Dentistry 6th Edn. Oxford U.K. John Wiley and Sons; 2015: 169.

Hioe K, Van der Weijden G. The effectiveness of self performed mechanical plaque control with triclosan containing dentifrices. Int J Dent Hyg. 2005;3:192-204.

Gunsolley J. A meta- analysis of six-month studies of antiplaque and antigingivitis agents. J Am Dental Assoc. 2010;137:1649-57.

Paraskevas, Van der Weijden G. A review of the effects of Stannous Flouride on gingivitis. J Clin Periodontol. 2006;33:1-13.

Addy M, Moran J, Newcombe R. Meta-analyses of studies of 0.2% delmopinol mouthrinse as an adjunct to gingival health and plaque control measures. J Clin Periodontol. 2007;34:58-65.

Stoeken J, Paraskevas S, Van der Weijden G. The long term effect of a mouthrinse containing essential oils on dental plaque and gingivitis: a systematic review. J Periodontol. 2007;78:1218-28.

Gunsolley J. Clinical efficacy of anti-microbial mouthrinses. J Dent. 2010;38(1):6-10.

Sahrmann P, Puhan M, Attin T, Schmidlin P. Systematic review on the effect of rinsing with povidone-iodine during non-surgical periodontal therapy. J Periodontol Res. 2010;45:153-64.

Afennich F, Slot D, Hossainian N, Van der Weijden G. The effect of hexetidine mouthwash on prevention of plaque and gingival inflammation: A Systematic Review. Int J Dent Hyg. 2011;9:182-90.

Hossainian N, Slot D. Affenich F, Van der Weijden G. The effect of hydrogen peroxide mouthwashes on the prevention of plaque and gingival inflammation: a systematic review. Int J Dent Hyg. 2011;9:171-81.

Jones C. Chlorhexidine: Is it still the gold standard? Perio 2000. 1997;15:55-62.

Flotra L. Different modes of chlorhexidine application and related local side effects. J Periodontal Res Suppl. 1973;8:41-4.

Malhotra R, Grover V, Kapoor A, Saxena D. Comparison of the effectiveness of a commercially available herbal mouth rinse with chlorhexidine gluconate at the clinical and patient level. J Indian Soc Periodontol. 2011;15(4):349-52.

Amruthesh S. Dentistry and ayurveda III (basics-ama, immunity, ojas, rasa, etiopathogenesis and prevention). Indian J Dent Res. 2007;18:112-9.

Jagdish L, Anand V, Kaviyarasan V. Effect of Triphala on dental biofilm. Indian J Sci Technol. 2009;1:93-9.

Biswas N, Biswas A. Evaluation of some leaf dusts as grain protectant against rice weevil sitophlis oryzae (Linn). Environ Ecol. 2005;23(3):485-8.

Moghbel A, Farajzzadeh A, Aghel N, Agheli H, Raisi N. Toxicology letters. 2009;189(1):257.

Wolinsky L, Mania S, Nachnani S, Ling S. The inhibiting effect of aqueous Azadirachta indica (Neem) extract upon bacterial properties influencing in vitro plaque formation. J Dent Res. 1996;75(2):816-22.

Nascimento G, Locatelli J, Freitas P, Silva G. Antibacterial activity of plant extracts and phytochemicals on antibiotic-resistant bacteria. Braz J Microbiol. 2000;31:247–56.

Wynn RL. Aloe vera gel: Update for dentistry. Gen Dent. 2005;53:6-9.

Menezes S, Corderio L, Viana G. Punica granatum (pomegranate) extract is active against dental plaque. J Herb Pharmacother. 2006;6:79-92.

Nihad A. Evaluation of Antibacterial Activity of Cinnamomum zeylanicum Extract on Cariogenic Microorganisms of the Dental plaque. In-vitro Study and Scanning Electron Microscopic Assessment. MDJ. 2009;6(4):334-48.

Hussain T, Kandy. Antimicrobial effects of Citrus Sinensis Peel Extracts Against Periodontopathic Bacteria: An in vitro Study. Rocz Panstw Zakl Hig. 2015;66(2):173-8.

Zhenbin W, Zhongli P, Haile M, Griffith G. Extract of Phenolics from Peels. J Food Sci. 2015;5:17-25.

Schwalbe R, Steele-Moore L, Goodwin AC. Antimicrobial susceptibility testing protocols. New York. CRC Press; 2007.

Hudzicki J. 8 December 2009, posting date. Kirby-Bauer disk diffusion susceptibility test protocol. American Society for Microbiology, Washington, DC.


Aboaba O, Efuwape B. Antibacterial Properties of Some Nigerian Species. Bio Res Comm. 2001;13:183-8.

Shimada T. Salivary proteins as a defense against dietary tannins. J Chem Ecol. 2006;32(6):1149-63.

Alams K, Skaug N, Ahmad I. An in vitro antimicrobial comparison of miswak extract with commercially available non-alcohol mouthrinses. Int J Dent Hyg. 2005;3(1):18-24.

Jardim E, Landucci L, Kidudsi Arafat O, Ranieri R, Ramos M., Ciesielski F, et al. Antimicrobial Activity of Six Plant Extracts from the Brazilian Savanna on Periodontal Pathogens. Int J Odontostomat. 2011;5(3):249-56.

Vahabi S, Najafi E, Alizadeh S. In-vitro antimicrobial effects of some herbal essences against oral pathogens. Planta Med. 2011;5(19):4870-8.

Madhuri S, Hegde A, Srilaxmi T, Prashith. Antimicrobial activity of Citrus sinensis and Citrus aurantium peel extracts. JPSI. 2014;3(4):366-9.

Lanciotti R, Gianotti A, Patrignani F, Belletti N, Guerzoni E, Gardini F. Use of natural aromacompounds to improve shelf-life and safety of minimally processed fruits. Trends Food Sci Tech. 2004;15:201-8.

Caccioni D, Guizzardi, M, Biondi, D, Renda A, Ruberto G. Relationship between volatile components of citrus fruit essential oils and antimicrobial action on Penicillium digitatum and Penicillium italicum. Int J Food Microbiol. 1998;43:73-9.

Dabbah R, Edwards M, Moats A. Antimicrobial action of some citrus fruit oils on selected food borne bacteria. J Appl Microbiol. 1970;19:27-31.

Lawal D, Bala J, Aliyu S, Huguma M. Phytochemical Screening and invitro Antibacterial Studies of the Ethanolic Extract of Citrus sinensis Peel against some Clinical Bacterial Isolates. IJIAS. 2013;2(2):138-45.

Vivek K, Nandin, Shashadhara S, Anitha S. Anti-Typhoid activity of Aqueous extract of Fruit Peel Citrus sinensis. IJPRD. 2010;2(9):31.

Nisha S, Swedha A, Rahaman J. Antibacterial activity of citrus sinensis peel against enteric pathogens. IJPRBS. 2013;2(5):1-13.

Jeyaseelan E, Jashothan P. In vitro control of Staphylococcus aureus (NCTC 6571) and Escherichia coli (ATCC 25922) by Ricinus communis L. Asian Pac J Trop Biomed. 2012;2(9):717-21.

Lakhdar L, Farah A, Bajjou T, Rida S. In vitro Antibacterial Activity of Essentials Oils from Mentha pulegium, Citrus aurantium and Cymbopogon citratus on Virulent Strains of Aggregatibacter actinomycetemcomitans. IJPPR. 2015;6(4):1035-42.