Formulation and antimicrobial evaluation of mouthwash containing propolis and roselle extract

Putu Diah Apri Anjalikha; Amirah Adlia; Dhyan Ayu Kusumaningtyas; Deasy Dyah Retno Wulan; I Ketut Adnyana

doi:10.35814/jifi.v24i2.2024

Authors

Putu Diah Apri Anjalikha Graduate Program, School of Pharmacy, Bandung Institute of Technology, Bandung, 40132, Indonesia
Amirah Adlia Department of Pharmaceutics, School of Pharmacy, Bandung Insitute of Technology, Bandung, 40132, Indonesia
Dhyan Ayu Kusumaningtyas Department of Pharmacology-Clinical Pharmacy, School of Pharmacy, Bandung Insitute of Technology, Bandung, 40132, Indonesia
Deasy Dyah Retno Wulan Center of Excellence for Innovative Cosmeceuticals and Natural Medicines for Degenerative Disease, Center for Pharma Valorisation, School of Pharmacy, Bandung Insitute of Technology, Bandung, 40132, Indonesia
I Ketut Adnyana Department of Pharmacology-Clinical Pharmacy, School of Pharmacy, Bandung Insitute of Technology, Bandung, 40132, Indonesia

DOI:

https://doi.org/10.35814/jifi.v24i2.2024

Keywords:

Antimicrobial, mouthwash, oral disease, propolis, roselle

Abstract

Poor oral hygiene can promote the transition of commensal microorganisms into opportunistic pathogens and contribute to the development of oral diseases. Antimicrobial mouthwashes may serve as preventive or adjunctive therapies in the early stages of oral infections. On the other hand, propolis and roselle (Hibiscus sabdariffa), both rich in phenolic compounds, possess well-documented antimicrobial activities and are promising candidates for natural mouthwash formulations. This study evaluated the antimicrobial activity of the combination of ethanolic extract of propolis (EEP) and aqueous extract of roselle (AER) against Staphylococcus aureus, Streptococcus mutans, and Candida albicans, and developed a combined-extract mouthwash formulation. The MIC values of EEP against S. aureus, S. mutans, and C. albicans were 1280, 640, and >20,480 μg/mL, respectively, whereas AER exhibited MIC values of 320, 1280, and 10,240 μg/mL. The MBC values of EEP were 1280 μg/mL for S. aureus and 2560 μg/mL for S. mutans, while AER showed MBC/MFC values of 1280, 10,240, and 10,240 μg/mL against S. aureus, S. mutans, and C. albicans, respectively. Checkerboard assays indicated additive interactions against S. aureus (FICI = 2) and S. mutans (FICI = 3). A mouthwash containing 2% EEP and 1% AER was successfully formulated, exhibiting a transparent light-brown appearance, menthol aroma, homogeneous characteristics, pH of 5.44 ± 0.03, and viscosity of 1.36 mPa·s. The undiluted formulation achieved complete microbial reduction within 2–10 min of contact. These findings suggest that the EEP–AER combination has potential as a natural antimicrobial mouthwash for oral health management.

References

[1] X. Li, Y. Liu, X. Yang, C. Li, and Z. Song, “The oral microbiota: community composition, ınfluencing factors, pathogenesis, and ınterventions,” Front. Microbiol., vol. 13, p. 895537, Apr. 2022, doi: 10.3389/FMICB.2022.895537.

[2] M. Lu, S. Xuan, and Z. Wang, “Oral microbiota: a new view of body health,” Food Science and Human Wellness, vol. 8, no. 1, pp. 8–15, Mar. 2019, doi: 10.1016/J.FSHW.2018.12.001.

[3] F. M. Georges, N. T. Do, and D. Seleem, “Oral dysbiosis and systemic diseases,” Frontiers in Dental Medicine, vol. 3, p. 995423, Sep. 2022, doi: 10.3389/FDMED.2022.995423/BIBTEX.

[4] N. Jain, U. Dutt, I. Radenkov, and S. Jain, “WHO’s global oral health status report 2022: actions, discussion and implementation,” Oral Dis., vol. 30, no. 2, pp. 73–79, Mar. 2024, doi: 10.1111/ODI.14516.

[5] “Oral health.” Accessed: Sep. 10, 2025. [Online]. Available: https://www.who.int/news-room/fact-sheets/detail/oral-health

[6] Z. L. S. Brookes, M. McCullough, P. Kumar, and C. McGrath, “Mouthwashes: ımplications for practice,” Int. Dent. J., vol. 73, no. Suppl 2, p. S98, Nov. 2023, doi: 10.1016/J.IDENTJ.2023.08.013.

[7] C. McGrath, J. Clarkson, A. M. Glenny, L. J. Walsh, and F. Hua, “Effectiveness of mouthwashes in managing oral diseases and conditions: do they have a role?,” Int. Dent. J., vol. 73, no. Suppl 2, p. S69, Nov. 2023, doi: 10.1016/J.IDENTJ.2023.08.014.

[8] S. Tidke, G. K. Chhabra, P. P. Madhu, A. Reche, S. Wazurkar, and S. R. Singi, “The effectiveness of herbal versus non-herbal mouthwash for periodontal health: a literature review,” Cureus, vol. 14, no. 8, Aug. 2022, doi: 10.7759/CUREUS.27956.

[9] H. M. Al Marzooqi et al., “Antioxidant and antimicrobial properties of propolis from different geographic regions in UAE and its applications in shelf-life extension of beef burger,” Front. Sustain. Food Syst., vol. 9, p. 1574880, Apr. 2025, doi: 10.3389/FSUFS.2025.1574880/BIBTEX.

[10] G. Albanese et al., “Functional and antimicrobial properties of propolis from different areas of Romania,” Applied Sciences (Switzerland), vol. 15, no. 2, p. 898, Jan. 2025, doi: 10.3390/APP15020898/S1.

[11] A. A. Abass et al., “Antimicrobial effect of Red Roselle (Hibiscus Sabdariffa) against different types of oral bacteria,” J. Med. Life, vol. 15, no. 1, p. 89, 2022, doi: 10.25122/JML-2021-0184.

[12] M. A. Saeed, A. Khabeer, M. A. Faridi, and G. Makhdoom, “Effectiveness of propolis in maintaining oral health: a scoping review,” Canadian Journal of Dental Hygiene, vol. 55, no. 3, p. 167, Oct. 2021, Accessed: Sep. 10, 2025. [Online]. Available: https://pmc.ncbi.nlm.nih.gov/articles/PMC8641552/

[13] Badan Standardisasi Nasional (BSN), SNI 8490:2018. Jakarta, 2018.

[14] M. P. . Weinstein, Performance standards for antimicrobial susceptibility testing. Clinical and Laboratory Standards Institute, 2019.

[15] P. Bellio, L. Fagnani, L. Nazzicone, and G. Celenza, “New and simplified method for drug combination studies by checkerboard assay,” MethodsX, vol. 8, p. 101543, Jan. 2021, doi: 10.1016/J.MEX.2021.101543.

[16] C. Ononugbo, E. Reward, and A. Ike, “The Effect of pH and temperature on phenol coefficients of two common disinfectants using clinical ısolates of Escherichia coli and Staphylococcus aureus,” J. Adv. Microbiol., vol. 10, no. 2, pp. 1–7, May 2018, doi: 10.9734/JAMB/2018/41376.

[17] Aminu, A.I. and Abdullahi, M.S., “Evaluation of the antibacterial effectiveness of some antiseptics and disinfectants,” UMYU Journal of Microbiology Research (UJMR), vol. 6, no. 1, pp. 175–181, Jun. 2021, doi: 10.47430/UJMR.2161.023.

[18] A. Lobiuc et al., “Future antimicrobials: natural and functionalized phenolics,” Molecules, vol. 28, no. 3, p. 1114, Feb. 2023, doi: 10.3390/MOLECULES28031114.

[19] S. M. Al-haliem, M. J. Mohammed, M. A. Hesarinejad, and T. G. Abedelmaksoud, “antimicrobial, anti‐biofilm activity and antioxidants of phenolic compounds ısolated from hypericum perforatum on periodontal pathogenic oral bacteria,” Food Sci. Nutr., vol. 13, no. 6, p. e70336, Jun. 2025, doi: 10.1002/FSN3.70336.

[20] S. Nubla, R. Adawiyah, M. Sahlan, M. Tugiran, N. M. C. Widyantari, and C. Harlim, “phytochemical analysis and antifungal activity propolis lombok against Candida sp and Cryptococcus sp,” Journal of Research in Pharmacy, vol. 28, no. 5, pp. 1391–1399, 2024, doi: 10.29228/JRP.817.

[21] I. Sitoresmi, M. Purbowati, K. Syamsu, and E. Warsiki, “Optimization of phenols extraction from roselle (Hibiscus sabdariffa) By microwave assisted extraction as antibacterial and antioxidant agents,” Herastuti Sri R Jurnal Teknologi Industri Pertanian, vol. 26, no. 1, pp. 23–30.

[22] M. Balouiri, M. Sadiki, and S. K. Ibnsouda, “Methods for in vitro evaluating antimicrobial activity: A review,” J. Pharm. Anal., vol. 6, no. 2, pp. 71–79, Apr. 2016, doi: 10.1016/J.JPHA.2015.11.005.

[23] A. C. O. Silva et al., “Which approach ıs more effective in the selection of plants with antimicrobial activity?,” Evidence-Based Complementary and Alternative Medicine, vol. 2013, no. 1, p. 308980, Jan. 2013, doi: 10.1155/2013/308980.

[24] R. Bridi et al., “International regulations of propolis quality: required assays do not necessarily reflect their polyphenolic-related ın vitro activities,” J. Food Sci., vol. 80, no. 6, pp. C1188–C1195, Jun. 2015, doi: 10.1111/1750-3841.12881;ISSUE:ISSUE:DOI.

[25] L. C. Lu, Y. W. Chen, and C. C. Chou, “Antibacterial activity of propolis against Staphylococcus aureus,” Int. J. Food Microbiol., vol. 102, no. 2, pp. 213–220, Jul. 2005, doi: 10.1016/J.IJFOODMICRO.2004.12.017.

[26] K. Grecka, Z. R. Xiong, H. Chen, K. Pełka, R. W. Worobo, and P. Szweda, “Effect of ethanol extracts of propolis (EEPs) against Staphylococcal Biofilm—Microscopic Studies,” Pathogens, vol. 9, no. 8, p. 646, Aug. 2020, doi: 10.3390/PATHOGENS9080646.

[27] A. Dziedzic, R. Kubina, R. D. Wojtyczka, A. Kabała-Dzik, M. Tanasiewicz, and T. Morawiec, “The antibacterial effect of ethanol extract of polish propolis on mutans Streptococci and Lactobacilli Isolated from Saliva,” Evid. Based. Complement. Alternat. Med., vol. 2013, p. 681891, 2013, doi: 10.1155/2013/681891.

[28] M. Dwivedi, S. Muralidhar, and D. Saluja, “Hibiscus sabdariffa Extract Inhibits Adhesion, Biofilm Initiation and Formation in Candida albicans,” Indian J. Microbiol., vol. 60, no. 1, p. 96, Mar. 2019, doi: 10.1007/S12088-019-00835-9.

[29] A. Ishak, N. Mazonakis, N. Spernovasilis, K. Akinosoglou, and C. Tsioutis, “Bactericidal versus bacteriostatic antibacterials: clinical significance, differences and synergistic potential in clinical practice,” Journal of Antimicrobial Chemotherapy, vol. 80, no. 1, p. 1, Jan. 2024, doi: 10.1093/JAC/DKAE380.

[30] C. Bozkurt-Guzel, G. Inci, O. Oyardi, and P. B. Savage, “synergistic activity of ceragenins against carbapenem-resistant acinetobacter baumannii strains in both checkerboard and dynamic time-kill assays,” Curr. Microbiol., vol. 77, no. 8, pp. 1419–1428, Aug. 2020, doi: 10.1007/S00284-020-01949-W.

[31] S. Bouchelaghem, “Propolis characterization and antimicrobial activities against Staphylococcus aureus and Candida albicans: a review,” Saudi J. Biol. Sci., vol. 29, no. 4, p. 1936, Apr. 2021, doi: 10.1016/J.SJBS.2021.11.063.

[32] S. Deniz and N. Chaachouay, “Synergy, additive effects, and antagonism of drugs with plant bioactive compounds,” Drugs and Drug Candidates 2025, Vol. 4, Page 4, vol. 4, no. 1, p. 4, Feb. 2025, doi: 10.3390/DDC4010004.

[33] J. C. Jenny, P. M. Kuś, and P. Szweda, “Investigation of antifungal and antibacterial potential of green extracts of propolis,” Sci. Rep., vol. 14, no. 1, pp. 1–13, Dec. 2024, doi: 10.1038/S41598-024-64111-7;SUBJMETA.

[34] G. Schwerdt, M. C. Schulz, M. Kopf, S. Mildenberger, S. Reime, and M. Gekle, “Physiological regulation of oral saliva ion composition and flow rate are not coupled in healthy humans—Partial revision of our current knowledge required,” Pflugers Archiv, vol. 477, no. 1, p. 55, Jan. 2024, doi: 10.1007/S00424-024-03025-9.

[35] B. W. M. van Swaaij, D. E. Slot, G. A. Van der Weijden, M. F. Timmerman, and J. Ruben, “Fluoride, pH Value, and titratable acidity of commercially available mouthwashes,” Int. Dent. J., vol. 74, no. 2, pp. 260–267, Apr. 2024, doi: 10.1016/J.IDENTJ.2023.09.002.

[36] S. Rahman and R. Ariastuti, “Formulation of mouthwash preparations ethanol extract of coffee beans roasted robusta (Coffea canephora) and effectiveness test on bacteria Streptococcus mutans,” Journal of Nutraceuticals and Herbal Medicine |, vol. 4, no. 1, pp. 53–65, 2021, [Online]. Available: http://journals.ums.ac.id/index.php/jnhm