Comparative in-vitro evaluation of antimicrobial efficacy of herbal extracts versus chlorhexidine against oral Staphylococcus aureus isolates in smokers

Saad Shahid; Muhammad Munir; Mirza Ameer Faizan Ali

doi:10.53685/jshmdc.v7i1.454

Authors

Saad Shahid Azra Naheed Medical College, Superior University Lahore
Muhammad Munir Azra Naheed Medical College, Superior University, Lahore
Mirza Ameer Faizan Ali Azra Naheed Medical College, Superior University, Lahore

DOI:

https://doi.org/10.53685/jshmdc.v7i1.454

Keywords:

Antimicrobial efficacy, Chlorhexidine, Herbal Products, Smoking, Staphylococcus aureus

Abstract

Background: Staphylococcus aureus is an opportunistic oral pathogen that can colonize in smokers due to alterations in the oral microbiome.

Objective: To assess and compare the antimicrobial effectiveness of herbal extracts (Azadirachta indica (neem), Syzygium aromaticum (clove oil), Ocimum tenuiflorum (Tulsi), and Camellia sinensis (green tea)) versus chlorhexidine against Staphylococcus aureus in smokers.

Methods: This in vitro experimental study was conducted at Azra Naheed Medical and Dental College from December 2025 to March 2026 on 100 adult smokers. Dental plaque samples were collected from 100 adult smokers, from which Staphylococcus aureus isolates were obtained and tested. Herbal extracts of Azadirachta indica (neem), Syzygium aromaticum (clove oil), Ocimum tenuiflorum (Tulsi), and Camellia sinensis (green tea) were prepared using ethanol extraction and compared for antimicrobial activity using the agar well diffusion method. Chlorhexidine (0.2%) was used as a control. Mean inhibition zones were compared with one-way-ANOVA.

Results: Chlorhexidine showed the largest inhibition zone (24.70±3.57), followed by clove oil (19.40±4.11), green tea (14.10±3.92), Tulsi (15.30±3.25), and Neem showed the smallest zone (12.01±2.58mm) (p<0.001). There was a significant difference in mean inhibition zones between chlorhexidine and clove (p<0.001), whereas no significant difference was observed between green tea and Tulsi (p=0.116). An inverse correlation was observed between the number of cigarettes smoked per day and the zone of inhibition for green tea (r=−0.213, p=0.033), but no significant correlation was observed with chlorhexidine (r=0.029, p=0.778).

Conclusion: Chlorhexidine demonstrated the greatest antimicrobial efficacy against oral S. aureus isolates from smokers, followed by clove oil, green tea, Tulsi, and neem. Further optimization of herbal agent concentrations may enhance their clinical applicability.

Author Biographies

Muhammad Munir, Azra Naheed Medical College, Superior University, Lahore

Professor Microbiology

Mirza Ameer Faizan Ali, Azra Naheed Medical College, Superior University, Lahore

Associate professor Microbiology

References

Antimicrobial Resistance Collaborators. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet. 2022; 399(10325): 629-655.

Razzaq S, Nagi MLF, Athar U, Kazmi T, Alslamah T, Naz S, et al. Prevalence of tobacco consumption and the associated factors among the adults in an urban slum: findings from the WHO STEPwise survey. Tob Induc Dis. 2022; 20: 91.

Cátala-Valentín AR, Almeda J, Bernard JN, Cole AM, Cole AL, Moore SD, et al. E-cigarette aerosols promote oral S. aureus colonization by delaying an immune response and bacterial clearing. Cells. 2022; 11(5): 773.

Buonavoglia A, Leone P, Solimando AG, Fasano R, Malerba E, Prete M, et al. Antibiotics or no antibiotics, that is the question: an update on efficient and effective use of antibiotics in dental practice. Antibiotics. 2021; 10(5): 550.

Tan S, Guo H, Chow A. Complementary and alternative medicine (CAM) use and its association with antibiotic usage practices: A nationally representative population study. Int J Antimicrob Agents. 2023; 61(6): 106796.

Subramanian S, Shenoy PA, Pai V. Antimicrobial activity of some Essential oils and Extracts from Natural sources on Skin and Soft tissue infection causing microbes: An In-vitro Study. Res J Pharm Technol. 2021; 14(7): 3603-3609.

Touati A, Mairi A, Ibrahim NA, Idres T. Essential Oils for Biofilm Control: Mechanisms, Synergies, and Translational Challenges in the Era of Antimicrobial Resistance. Antibiotics. 2025; 14(5): 503.

Arzani V, Soleimani M, Fritsch T, Jacob UM, Calabrese V, Arzani A. Plant polyphenols, terpenes, and terpenoids in oral health. Open Med (Wars). 2025; 20(1): 20251183.

Deepika B, Ramamurthy J, Jayakumar ND, Kumar SR. Comparative clinical data for gingivitis treatment using gels from Ocimum sanctum (Tulsi) and chlorhexidine (CHX). Bioinformation. 2021; 17(12): 1091-1098.

Nagappan N, Palaneeswaran K, Nalankilli AV, Nyklesh V, Mani M, Aziz MBA. Antibacterial efficacy of herbal and 0.2% chlorhexidine mouth rinse against oral pathogens–An in vitro study. J Pharm Bioallied Sci. 2024; 16(Suppl 2): S1453-S1455.

Wylie MR, Merrell DS. The antimicrobial potential of the neem tree Azadirachta indica. Front Pharmacol. 2022; 13: 891535.

Shakhatreh MAK, Khabour OF, Alzoubi KH, Masadeh MM, Hussein EI, Bshara GN. Alterations in oral microbial flora induced by waterpipe tobacco smoking. Int J Gen Med. 2018: 11: 47-54.

Bartsch S, Kohnert E, Kreutz C, Woelber JP, Anderson A, Burkhardt AS, et al. Chlorhexidine digluconate mouthwash alters the oral microbial composition and affects the prevalence of antimicrobial resistance genes. Front Microbiol. 2024; 15: 1429692.

Chavarría-Bolaños D, Esparza-Villalpando V, Ramírez K. Antibacterial and antifungal capacity of three commercially available mouthwashes with different concentrations of chlorhexidine. Odovtos Int J Dent Sci. 2022; 24(2): 69-80.

Kamdem GSJN, Toukam M, Ntep DBN, Kwedi KGG, Brian NZ, Fokam ST, et al. Comparison of the effect of saline mouthwash versus chlorhexidine on oral flora. Adv Oral Maxillofac Surg. 2022; 6: 100273.

Lee J-S, Choi YS, Lee HG. Synergistic antimicrobial properties of nanoencapsulated clove oil and thymol against oral bacteria. Food Sci Biotechnol. 2020; 29(11): 1597-1604.

Li J, Li C, Shi C, Aliakbarlu J, Cui H, Lin L. Antibacterial mechanisms of clove essential oil against Staphylococcus aureus and its application in pork. Int J Food Microbiol. 2022; 380: 109864.

Fatima S, Saba S, Hussain A, Aslam L, Naveed A. Antibacterial and anti-biofilm profiling of Syzygium aromaticum plant extracts against multi-drug resistant human pathogens. Pak J Biochem Biotechnol. 2023; 4(2): 71-82.

Hiwandika N, Sudrajat SE, Rahayu I. Antibacterial and antifungal activity of clove extract (Syzygium aromaticum). Eureka Herba Indones. 2021; 2(2): 86-94.

Hussain M, Riasat M, Nisa WU, Khattak I, Ullah F, Shaid F. Assessment of effect of cigarette smoking on periodontal health of patients reporting to dental section of Bacha Khan Medical College Mardan. Pak J Public Health. 2020; 10(3): 185-189.

Kong C, Zhang H, Li L, Liu Z. Effects of green tea extract epigallocatechin-3-gallate (EGCG) on oral disease-associated microbes: A review. J Oral Microbiol. 2022; 14(1): 2131117.

Sartini S, Djide MN, Amir MN, Permana AD. Phenolic-rich green tea extract increases the antibacterial activity of amoxicillin against Staphylococcus aureus by in vitro and ex vivo studies. J Pharm Pharmacogn Res. 2020; 8(6): 491-500.

Stapleton EM, Manges R, Parker G, Stone EA, Peters TM, Blount RJ, et al. Indoor particulate matter from smoker homes induces bacterial growth, biofilm formation, and impairs airway antimicrobial activity. A pilot study. Front Public Health. 2020; 7: 418.

Kowalewska A, Majewska-Smolarek K. Eugenol-based polymeric materials antibacterial activity and applications. Antibiotics. 2023; 12(11): 1570.

Rahman RTA, Kamal N, Mediani A, Farag MA. How do herbal cigarettes compare to tobacco? A comprehensive review of their sensory characters, phytochemicals, and functional properties. ACS Omega. 2022; 7(50): 45797-45809.