• R Ejaz Department of Biotechnology, Kinnaird College for Women, 93 Jail Road, Lahore, Punjab, Pakistan
  • S Malik 2Center of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan
  • M Ahmad Department of Biotechnology, Kinnaird College for Women, 93 Jail Road, Lahore, Punjab, Pakistan
  • H ALi College of Plant Protection, Nanjing Agricultural University, Nanjing, 210095, China
  • S Choudhry Department of Biotechnology, Kinnaird College for Women, 93 Jail Road, Lahore, Punjab, Pakistan



menthol, Mentha piperita, anti-bioflim, Pseudomonas aeruginosa, inhibition


Menthol, a bioactive compound of Mentha piperita (mint) with antibacterial properties was purified by column chromatography to determine its anti-biofilm potential. After phytochemical analysis, TLC was carried out using n-hexane: ethyl acetate: methanol: water (2:2:2:1) as the solvent system for ethanolic extract of mint. TLC achieved the maximum separation of mint constituents with Rf value of 0.68. A purified menthol fraction was obtained after silica gel column chromatography using four different eluting solvents. The menthol obtained was then used to perform biofilm inhibition assay to establish its antibacterial potential. Percentage inhibition was highest for bacillus subtilis (79.4%), as opposed to Pseudomonas aeruginosa (33.6%) and the combination of both bacteria (20%). ELISA reader was used to measure absorbance at 450-620nm and 630 nm. Using 450-620nm filter the values for percentage inhibition lies between 48.6-95% for standard and crude menthol samples. Similarly, at 630nm the values of inhibition lie between 23.4-70.6%. This anti-biofilm property of menthol can be utilized in antibacterial drug formulations.


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Al-Bayati, F. A. (2009). Isolation and identification of antimicrobial compound from Mentha longifolia L. leaves grown wild in Iraq. Annals of clinical microbiology and antimicrobials 8, 20.

Bupesh, G., Amutha, C., Nandagopal, S., Ganeshkumar, A., Sureshkumar, P., and Murali, K. (2007). Antibacterial activity of Mentha piperita L.(peppermint) from leaf extracts-a medicinal plant. Acta Agriculturae Slovenica 89, 73.

Chandki, R., Banthia, P., and Banthia, R. (2011). Biofilms: A microbial home. Journal of Indian Society of Periodontology 15, 111.

Chusri, S., Sompetch, K., Mukdee, S., Jansrisewangwong, S., Srichai, T., Maneenoon, K., Limsuwan, S., and Voravuthikunchai, S. (2012). Inhibition of Staphylococcus epidermidis biofilm formation by traditional Thai herbal recipes used for wound treatment. Evidence-Based Complementary and Alternative Medicine 2012.

Çitoğlu, G. S., and Acıkara, Ö. B. (2012). Column chromatography for terpenoids and flavonoids. Chromatography and its applications: INTECH Rijeka, 13-49.

Delwiche, S., and Gaines, C. (2005). Wavelength selection for monochromatic and bichromatic sorting of Fusarium-damaged wheat. Applied engineering in agriculture 21, 681-688.

Djordjevic, D., Wiedmann, M., and McLandsborough, L. (2002). Microtiter plate assay for assessment of Listeria monocytogenes biofilm formation. Applied and environmental microbiology 68, 2950-2958.

Donlan, R. M. (2001). Biofilm formation: a clinically relevant microbiological process. Clinical Infectious Diseases 33, 1387-1392.

Doughari, J. H. (2012). Phytochemicals: extraction methods, basic structures and mode of action as potential chemotherapeutic agents. In "Phytochemicals-A global perspective of their role in nutrition and health". InTechOpen.

Elvers, K., Leeming, K., Moore, C., and Lappin‐Scott, H. (1998). Bacterial‐fungal biofilms in flowing water photo‐processing tanks. Journal of Applied Microbiology 84, 607-618.

Husain, F. M., Ahmad, I., Khan, M. S., Ahmad, E., Tahseen, Q., Khan, M. S., and Alshabib, N. A. (2015). Sub-MICs of Mentha piperita essential oil and menthol inhibits AHL mediated quorum sensing and biofilm of Gram-negative bacteria. Frontiers in microbiology 6, 420.

Kelly, A., McKenna, J. P., McLelland, A., Percy, R. A., and Spooner, R. J. (1979). A bichromatic method for total bilirubin with a CentrifiChem 400. Clinical chemistry 25, 1482-1484.

Kuehler, T. C., and Lindsten, G. R. (1983). Preparative reversed-phase flash chromatography, a convenient method for the workup of reaction mixtures. The Journal of Organic Chemistry 48, 3589-3591.

Liu, X., Sun, Z.-L., Jia, A.-R., Shi, Y.-P., Li, R.-H., and Yang, P.-M. (2014). Extraction, preliminary characterization and evaluation of in vitro antitumor and antioxidant activities of polysaccharides from Mentha piperita. International journal of molecular sciences 15, 16302-16319.

Lugemwa, F. N. (2012). Extraction of betulin, trimyristin, eugenol and carnosic acid using water-organic solvent mixtures. Molecules 17, 9274-9282.

Mikaili, P., Mojaverrostami, S., Moloudizargari, M., and Aghajanshakeri, S. (2013). Pharmacological and therapeutic effects of Mentha Longifolia L. and its main constituent, menthol. Ancient science of life 33, 131.

Odeyemi, O. A., and Oluwajoba, S. O. (2011). Synergistic effect of menthol on in-vitro antibacterial activity of Garcinia kola against Gram positive Bacteria: A preliminary study. Journal of Applied Pharmaceutical Science 1, 98.

Parkin, J. (1984). High-performance liquid chromatographic assay of menthol using indirect photometric detection. Journal of Chromatography A 303, 436-439.

Qiu, J., Luo, M., Dong, J., Wang, J., Li, H., Wang, X., Deng, Y., Feng, H., and Deng, X. (2011). Menthol diminishes Staphylococcus aureus virulence-associated extracellular proteins expression. Applied microbiology and biotechnology 90, 705-712.

Rao, M. V., Al‐Marzouqi, A. H., Kaneez, F. S., Ashraf, S. S., and Adem, A. (2007). Comparative evaluation of SFE and solvent extraction methods on the yield and composition of black seeds (Nigella Sativa). Journal of liquid chromatography & related technologies 30, 2545-2555.

Saharkhiz, M. J., Motamedi, M., Zomorodian, K., Pakshir, K., Miri, R., and Hemyari, K. (2012). Chemical composition, antifungal and antibiofilm activities of the essential oil of Mentha piperita L. International Scholarly Research Notices 2012.

Sasidharan, S., Chen, Y., Saravanan, D., Sundram, K., and Latha, L. Y. (2011). Extraction, isolation and characterization of bioactive compounds from plants’ extracts. African Journal of Traditional, Complementary and Alternative Medicines 8.

Shaikh, K., and Patil, S. D. (2010). Sensitive and selective method for the analysis of menthol from pharmaceutical products by RP-HPLC with refractive index detector. Journal of Pharmacy and Bioallied Sciences 2, 360.

Skalicka-Woźniak, K., and Walasek, M. (2014). Preparative separation of menthol and pulegone from peppermint oil (Mentha piperita L.) by high-performance counter-current chromatography. Phytochemistry Letters 10, xciv-xcviii.

Snoussi, M., Noumi, E., Trabelsi, N., Flamini, G., Papetti, A., and De Feo, V. (2015). Mentha spicata essential oil: chemical composition, antioxidant and antibacterial activities against planktonic and biofilm cultures of Vibrio spp. strains. Molecules 20, 14402-14424.

Still, W. C., Kahn, M., and Mitra, A. (1978). Rapid chromatographic technique for preparative separations with moderate resolution. The Journal of Organic Chemistry 43, 2923-2925.

Sun, F., Qu, F., Ling, Y., Mao, P., Xia, P., Chen, H., and Zhou, D. (2013). Biofilm-associated infections: antibiotic resistance and novel therapeutic strategies. Future Microbiology 8, 877-886.

Tang, Z., Qin, J., Xu, X., Shi, G., Yang, H., and Liang, Y. (2011). Applying silica gel column chromatography purify resveratrol from extracts of Morus alba L. Leaf. Journal of Medicinal Plants Research 5, 3020-3027.

Uzair, B., Ahmed, N., Ahmad, V. U., Mohammad, F. V., and Edwards, D. H. (2008). The isolation, purification and biological activity of a novel antibacterial compound produced by Pseudomonas stutzeri. FEMS microbiology letters 279, 243-250.

Wakimoto, N., Nishi, J., Sheikh, J., Nataro, J. P., Sarantuya, J., Iwashita, M., Manago, K., Tokuda, K., Yoshinaga, M., and Kawano, Y. (2004). Quantitative biofilm assay using a microtiter plate to screen for enteroaggregative Escherichia coli. The American journal of tropical medicine and hygiene 71, 687-690.

Zahra, N., Jahan, N., Nosheen, S., and Khalil-ur-Rehman (2011). Antimicrobial activity of aqueous, ethanolic extracts and crude extracted phytoconstituents of Nigella sativa seeds. Bioscience Research 8, 19-25.




How to Cite

Ejaz, R., Malik, S., Ahmad, M., ALi, H., & Choudhry, S. (2020). ANTI-BIOFILM POTENTIAL OF MENTHOL PURIFIED FROM MENTHA PIPERITA L. (MINT). Biological and Clinical Sciences Research Journal, 2020(1).



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