• Nurul Izzati Azahar Faculty of Chemical & Process Engineering Technology, Universiti Malaysia Pahang, 26300 Gambang, Pahang, Malaysia https://orcid.org/0000-0002-2222-7703
  • Nadzirah Mohd Mokhtar Faculty of Civil Engineering Technology, Universiti Malaysia Pahang, 26300 Gambang, Pahang, Malaysia
  • Syed Mahmood Department of Pharmaceutical Technology, Faculty of Pharmacy, Universiti Malaya, 50603 Kuala Lumpur, Malaysia
  • ‪Mohd Aizudin Abd Aziz Faculty of Chemical & Process Engineering Technology, Universiti Malaysia Pahang, 26300 Gambang, Pahang, Malaysia
  • Mohd Azmir Arifin Faculty of Chemical & Process Engineering Technology, Universiti Malaysia Pahang, 26300 Gambang, Pahang, Malaysia https://orcid.org/0000-0002-7052-5078




Piper betle, P. aeruginosa, antivirulence, antibacterial, Pyoverdine assay


The virulence factor of bacteria such as P. aeruginosa causes severe problems affecting human health and environmental quality. In this study, Piper betle undergoes an extraction process yielding extract to diminish the virulence factor of P. aeruginosa. The efficiency of Piper betle treatment on P. aeruginosa was measured using Pyoverdine assay. The different factors affected the Piper betle extract yield such as leaves to a solvent ratio (1:6 and 1:10), extraction method (maceration and sonication) and different solvents (methanol, ethanol, ethyl acetate and hexane) were tested. Pyoverdine assay illustrates ethyl acetate exhibits the lowest peak (OD630 = 0.2320) compared to methanol, ethanol and hexane due to the presence of a bioactive compound reducing the virulence factor. The ratio of 1:10 has a higher yield of 4.53±0.05 g and the ratio of 1:6 yields 2.86±0.05 g of extracts because of a better contact area. Maceration with agitation indicated the highest yield of 0.5210±0.05 g followed by maceration without agitation at 0.2660±0.05 g and 0.2792±0.05 g for sonication. The yield of Piper betle with different solvents showed the lowest yield is hexane 0.4741±0.05 g followed by ethyl acetate 2.4975±0.05 g, ethanol 3.7658±0.05 g and methanol 6.3331±0.05 g due to solvent polarity. This study aims to provide insightful knowledge of applied factor affecting Piper betle extracts and the ability of Piper betle as antivirulence and antibacterial agent against P. aeruginosa.


Wendy Voon, W. Y., Ghali, N. A., Rukayadi, Y., Meor Hussin, A. S. 2014. Application of Betel Leaves (Piper betle L.) Extract for Preservation of Homemade Chili Bo. International Food Research Journal. 21(6): 2399-2403.

Ravindran, P. N., Pillai, G. S. and Nirmal Babu, K. 2004. Under-utilized Herbs and Spices. Handbook of Herbs and Spices. 53-103.

Doi: https://doi.org/10.1533/9781855738355.1.53.

Shah, S. S., Garg, G., Jhade, D. & Patel, N. 2016. Piper betle: Phytochemical, Pharmacological and Nutritional Value in Health Management. Int. J. Pharm. Sci. Rev. Res. 38(2): 181-9.

Azahar, N. I., Mokhtar, N. M., & Arifin, M. A. 2020. Piper betle: A Review on Its Bioactive Compounds, Pharmacological Properties, and Extraction Process. IOP Conference Series: Materials Science and Engineering. 991: 012044.

Doi: https://doi.org/10.1088/1757-899X/991/1/012044.

Chan, E. and Wong, S. 2014. Phytochemistry and Pharmacology of Three Piper Species: An Update. International Journal of Pharmacognosy. 1: 534-54.

Doi: https://doi.org/10.13040/IJP.

Pin, K. Y., Chuah, T. G., Abdull Rashih, A., Rasadah, M. A., Law, C. L. and Choong, T. S. Y. 2006. Aqueous Extraction of Hydroxychavicol from Piper betle L. Leaves. Proceedings of the 1st International Conference on Natural Resources Engineering & Technology 2006. 146-152.

Sarma. C., Rasane, P., Kaur, S., Singh, J., Singh, J., Gat, Y., … Dhawan, K. 2018. Antioxidant and Antimicrobial Potential of Selected Varieties of Piper betle L. (betel leaf). Anais Da Academia Brasileira de Ciências.

Doi: https://doi.org/10.1590/0001-3765201820180285.

Hoque, M. M., Rattila, S., Shishir, M. A., Bari, M. L., Inatsu, Y. and Kawamoto, S. 2012. Antibacterial Activity of Ethanol Extract of Betel Leaf (Piper betle L.) against some Food Borne Pathogens. Bangladesh Journal of Microbiology. 28(2). Doi: https://doi.org/10.3329/bjm.v28i2.11817.

Mohanto, S., Datta, S., and Mandal, S. 2017. Piper betel Linn: A Brief Study. International Journal of Current Medical and Pharmaceutical. 3(2): 1290-96.

Thamaraikani, I. and Kulandhaivel, M. 2017. Purification of Hydroxychavicol from Piper betle linn and Evaluation of Antimicrobial Activity Against Some Food Poison Causing Bacteria. Journal of Pure and Applied Microbiology.

Doi: https://doi.org/10.22207/JPAM.11.4.28.

M. Guha, P. and Nag, A. 2019. Extraction of Betel Leaves (Piper betle l.) Essential Oil and Its Bio-actives Identification: Process Optimization, GC-MS Analysis and Anti-microbial Activity. Industrial Crops and Products. 138: 111578.

Doi: https://doi.org/10.1016/j.indcrop.2019.111578.

Nayaka, N., Sasadara, M., Sanjaya, D. A., Yuda, P., Dewi, N., Cahyaningsih, E., & Hartati, R. 2021. Piper betle (L): Recent Review of Antibacterial and Antifungal Properties, Safety Profiles, and Commercial Applications. Molecules (Basel, Switzerland). 26(8): 2321.

Doi: https://doi.org/10.3390/molecules26082321.

Widyaningtias, N. M. S. R., Yustiantara, P. S., Paramita, N. L. P. V. 2014. Uji Aktivitas Antibakteri Ekstrak Terpurifikasi Daun Sirih Hijau (Piper betle L.) Terhadap Bakteri Propionibacterium acnes, J. Farm. 50-53.

Datta, A., Ghoshdastidar, S., & Singh, M. 2011. Antimicrobial Property of Piper betel Leaf against Clinical Isolates of Bacteria. International Journal of Pharma Sciences and Research. 2(3): 104-109.

Kaveti, B., Sarnnia, L. T., Tan, S. K., Baig, M. 2011. Antibacterial Activity of Piper Betel Leaves. IJPTP. 2(3):129-132.

Zhang, Q. W., Lin, L. G. and Ye, W. C. 2018. Techniques for Extraction and Isolation of Natural Products: A Comprehensive Review. Chinese Medicine. 13(1).

Doi: https://doi.org/10.1186/s13020-018-0177-x.

Azwanida, N. N. 2015. A Review on the Extraction Methods Use in Medicinal Plants, Principle, Strength and Limitation. Medicinal & Aromatic Plants. 04(03).

Doi: https://doi.org/10.4172/2167-0412.1000196.

Ingle, K. P., Deshmukh, A. G., Padole, D. A., Dudhare, M. S., Moharil, M. P. and Khelurkar, V. C. 2017. Phytochemicals: Extraction Methods, Identification and Detection of Bioactive Compounds from Plant Extracts. Journal of Pharmacognosy and Phytochemistry. 6(1): 32-6.

Ali, A., Lim, X. Y., Chong, C. H., Mah, S. H. and Chua, B. L. 2018b. Ultrasound-assisted Extraction of Natural Antioxidants from Betel Leaves (Piper betle): Extraction Kinetics and Modeling. Separation Science and Technology. 53(14): 2192-2205.

Doi: https://doi.org/10.1080/01496395.2018.1443137.

Das, S., Ray, A., Nasim, N., Nayak, S., and Mohanty, S. 2019. Effect of Different Extraction Techniques on Total Phenolic and Flavonoid Contents, and Antioxidant Activity of Betel Vine and Quantification of its Phenolic Constituents by validated HPTLC Method. Biotech. 9(1).

Doi: https://doi.org/10.1007/s13205-018-1565-8.

Foo, L. W., Salleh, E. and Hana, S. N. 2017. Green Extraction of Antimicrobial Bioactive Compound from Piper betle Leaves: Probe Type Ultrasound-assisted Extraction vs Supercritical Carbon Dioxide Extraction. Chemical Engineering Transactions. 56: 109-14.

Doi: https://doi.org/10.3303/CET1756019.

Ali, A., Lim, X. Y., Chong, C. H., Mah, S. H. and Chua, B. L. 2018a. Optimization of Ultrasound-assisted Extraction of Natural Antioxidants from Piper betle using Response Surface Methodology. LWT. 89: 681-8.

DOI: https://doi.org/10.1016/j.lwt.2017.11.033.

Dhanani, T., Shah, S., Gajbhiye, N. A., & Kumar, S. 2017. Effect of Extraction Methods on Yield, Phytochemical Constituents and Antioxidant Activity of Withania somnifera. Arabian Journal of Chemistry. 10: S1193-S1199. Doi: https://doi.org/10.1016/j.arabjc.2013.02.015.

Ghasemzadeh, A., Jaafar, H., Juraimi, A., & Tayebi-Meigooni, A. 2015. Comparative Evaluation of Different Extraction Techniques and Solvents for the Assay of Phytochemicals and Antioxidant Activity of Hashemi Rice Bran. Molecules. 20(6): 10822-10838.

Doi: https://doi.org/10.3390/molecules200610822.

Ghasemzadeh, A., Jaafar, H. Z., Rahmat, A. 2011. Effects of Solvent Type on Phenolics and Flavonoids Content and Antioxidant Activities in Two Varieties of Young Ginger (Zingiber officinale Roscoe) Extracts. J Med Plants Res. 5(7): 1147-1154.

Doi: https://doi.org/10.5897/JMPR.9000540.

Barchan, A., Bakkali, M., Arakrak, A., Pagán, R., Laglaoui, A. 2014. The Effects of Solvents Polaritiy on the Phenolic Contents and Antioxidant Activity of Three Mentha Species Extracts. Int J Curr Microbiol App Sci. 3(11): 399-412.

Nawaz, H., Shad, M. A., Rehman, N., Andaleeb, H., & Ullah, N. 2020. Effect of Solvent Polarity on Extraction Yield and Antioxidant Properties of Phytochemicals from Bean (Phaseolus vulgaris) Seeds. Brazilian Journal of Pharmaceutical Sciences. 56.

Doi: https://doi.org/10.1590/s2175-97902019000417129.

Taukoorah, U., Lall, N. and Mahomoodally, F. 2016. Piper betle L. (betel quid) Shows Bacteriostatic, Additive, and Synergistic Antimicrobial Action when Combined with Conventional Antibiotics. South African Journal of Botany. 105: 133-140.

Dwivedi, V. and Tripathi, S. M. 2014. Review Study on Potential Activity of Piper betle. Journal of Pharmacognosy and Phytochemistry. 3(4): 93-8.

Patil, R. S., Harale, P. M., Shivangekar, K. V., Kumbhar, P. P. and Desai, R. R. 2015. Phytochemical Potential and In Vitro Antimicrobial Activity of Piper betle Linn. Leaf Extracts. Journal of Chemical and Pharmaceutical Research. 7(5): 1095-1101.

Pradhan, D., Suri, K. A., Pradhan, D. K. and Biswasroy, P. 2013. Golden Heart of the Nature: Piper betle L. J Pharmacog Phytochem. 1(6): 147-67.

Budiman, A., Rusnawan, D. W., Yuliana, A. 2018. Antibacterial Activity of Piper betle L. Extract in Cream Dosage forms against Staphylococcus aureus and Propionibacterium acne. J. Pharm. Sci. & Res. 10(3): 493-496.

LaBauve, A. E., and Wargo, M. J. 2012. Growth and Laboratory Maintenance of Pseudomonas aeruginosa. Current Protocols in Microbiology. 25(1): 1-8. Doi: https://doi.org/10.1002/9780471729259.mc06e01s25.

Sykes, J. E. 2014. Gram-negative Bacterial Infections. Canine and Feline Infectious Diseases. 355-363.

Doi: https://doi.org/10.1016/B978-1-4377-0795-3.00036-3.

Vukić Lušić, D., Maestro, N., Cenov, A., Lušić, D., Smolčić, K., Tolić, S., Maestro, D., Kapetanović, D., Marinac-Pupavac, S., Tomić Linšak, D., Linšak, Ž., & Glad, M. 2021. Occurrence of P. aeruginosa in Water Intended for Human Consumption and in Swimming Pool Water. Environments. 8(12): 132.

DOI: https://doi.org/10.3390/environments8120132.

Samanta, I. and Bandyopadhyay, S. 2020. Alternative Antiinfective Therapy. Antimicrobial Resistance in Agriculture. 343-355.

Doi: https://doi.org/10.1016/B978-0-12-815770-1.00030-4.

Frederix, M., & Downie, J. A. 2011. Quorum Sensing. Advances in Microbial Physiology. 23-80.

Doi: https://doi.org/10.1016/B978-0-12-381043-4.00002-7.

Kang, D., Kirienko, D. R., Webster, P., Fisher, A. L., & Kirienko, N. V. 2018. Pyoverdine, a Siderophore from Pseudomonas aeruginosa, Translocates into C. elegans, removes iron, and Activates a Distinct Host Response. Virulence. 9(1): 804-817.

Doi: https://doi.org/10.1080/21505594.2018.1449508.

Lyczak, J. B., Cannon, C. L., & Pier, G. B. 2000. Establishment of Pseudomonas aeruginosa Infection: Lessons from a Versatile Opportunist. Microbes and Infection. 2(9): 1051-1060.

Doi: https://doi.org/10.1016/S1286-4579(00)01259-4.

Lamont, I. L., Beare, P. A., Ochsner, U., Vasil, A. I., & Vasil, M. L. 2002. Siderophore-mediated Signaling Regulates Virulence Factor Production in Pseudomonas aeruginosa. Proceedings of the National Academy of Sciences. 99(10): 7072-7077.

Doi: https://doi.org/10.1073/pnas.092016999.

Meyer, J. M., Neely, A., Stintzi, A., Georges, C., & Holder, I. A. 1996. Pyoverdin is Essential for Virulence of Pseudomonas aeruginosa. Infection and Immunity. 64(2): 518-523.

Doi: https://doi.org/10.1128/iai.64.2.518-523.1996z.

Hider, R. C. 1984. Siderophore Mediated Absorption of Iron. Siderophores from Microorganisms and Plants. 58: 25-87. Springer Berlin Heidelberg.

Doi: https://doi.org/10.1007/BFb0111310.

Bonneau, A., Roche, B., & Schalk, I. J. 2020. Iron Acquisition in Pseudomonas aeruginosa by the Siderophore Pyoverdine: An Intricate Interacting Network Including Periplasmic and Membrane Proteins. Scientific Reports. 10(1).

Doi: https://doi.org/10.1038/s41598-019-56913-x.

Saha, J., and Deka, S. C. 2016. Functional Properties of Sonicated and Non-sonicated Extracted Leaf Protein Concentrate from Diplazium esculentum. International Journal of Food Properties. 20(5): 1051-1061.

Doi: https://doi.org/10.1080/10942912.2016.1199034.

Lamichhane, J. R. and Varvaro, L. 2012. A New Medium for the Detection of Fluorescent Pigment Production by Pseudomonads. Plant Pathology. 62(3): 624-632.

Doi: https://doi.org/10.1111/j.1365-3059.2012.02670.x.

Louden, B. C., Lynne, A. M., & Haarmann, D. 2011. Use of Blue Agar CAS Assay for Siderophore Detection. Journal of Microbiology & Biology Education. 12(1): 51-53.

Doi: https://doi.org/10.1128/jmbe.v12i1.249.

Srimathi, K. & Ann Suji, H. 2018. Siderophores Detection by using Blue Agar CAS Assay Methods. International Journal of Scientific Research in Biological Sciences. 5(6): 180-5.

Virpiranta, H., Banasik, M., Taskila, S., Leiviskä, T., Halttu, M., Sotaniemi, V.-H., & Tanskanen, J. 2020. Isolation of Efficient Metal-binding Bacteria from Boreal Peat Soils and Development of Microbial Biosorbents for Improved Nickel Scavenging. Water. 12(7): 2000.

Doi: https://doi.org/10.3390/w12072000.

Nawas, T. 2018. Extraction and Purification of Pyocyanin: A Simpler and More Reliable Method. MOJ Toxicology. 4(6).

Doi: https://doi.org/10.15406/mojt.2018.04.00139.

Krieg, N. R. and Padgett, P. J. 2011. Phenotypic and Physiological Characterization Methods. Taxonomy of Prokaryotes. 15-60.

Doi: https://doi.org/10.1016/B978-0-12-387730-7.00003-6.

Sayyar, S., Abidin, Z. Z., Yunus, R., & Muhammad, A. 2009. Extraction of Oil from Jatropha Seeds-Optimization and Kinetics. American Journal of Applied Sciences. 6(7): 1390-1395.

Doi: https://doi.org/10.3844/ajassp.2009.1390.1395.






Science and Engineering

How to Cite

EVALUATION OF PIPER BETLE L. EXTRACTS AND ITS ANTIVIRULENCE ACTIVITY TOWARDS P. AERUGINOSA . (2022). Jurnal Teknologi, 85(1), 133-140. https://doi.org/10.11113/jurnalteknologi.v85.18892