Abstract
Antimicrobial resistance is considered a threat to global health. Given the use and benefits of herbal medicine, the healthcare community has been investigating plant-derived compounds for their potential antimicrobial activity. Susong kalabaw (Uvaria rufa) was studied for its antibacterial activity using fractionated leaf extracts. U. rufa leaves were extracted using 70% ethanol and fractionated using five solvents, including water, methanol/water, sec-butanol, DCM, and hexane. Phytochemical screening was performed on crude extracts. Moreover, the inhibitory activity of crude and fractionated leaf extracts of U. rufa was evaluated using the Kirby-Bauer test and MIC assay. Phytochemical screening confirmed the presence of alkaloids, flavonoids, saponins, tannins, and terpenoids in crude extracts. The Kirby-Bauer test showed that crude extracts inhibited the growth of Escherichia coli (8.3 mm), Bacillus subtilis (10.0 mm), and Staphylococcus aureus (14.5 mm). The fractions, including water (10.3 mm), methanol/water (11.2 mm), and sec-butanol (17.8 mm), also revealed inhibitory activity against S. aureus. Moreover, Pseudomonas aeruginosa was susceptible to the sec-butanol fraction, showing an average zone of inhibition of 9.3 mm. For the MIC assay, the methanol/water fraction inhibited the growth of S. aureus at a substantially lower concentration of 67.35 μg/mL. On the other hand, B. subtilis and S. aureus were susceptible to water and sec-butanol fractions, showing lower concentrations of 68.1 μg/mL and 26.21 μg/mL, respectively. These results emphasized that crude and fractionated leaf extracts of U. rufa demonstrated promising antibacterial activity against bacterial samples in the Kirby-Bauer test and MIC assay.
References
Abubakar, A. R., & Haque, M. (2020). Preparation of medicinal plants: Basic extraction and fractionation procedures for experimental purposes. Journal of Pharmacy & BioAllied Sciences, 12(1), 1-10. https://doi.org/10.4103/jpbs.JPBS_175_19
Alastruey‐Izquierdo, A., De Souza Carvalho Melhem, M., Bonfietti, L. X., & Rodríguez-Tudela, J. L. (2015). Susceptibility test for fungi: Clinical and laboratorial correlations in medical mycology. Revista Do Instituto De Medicina Tropical De Sao Paulo, 57(suppl 19), 57–64. https://doi.org/10.1590/s0036-46652015000700011
Aly, M., & Balkhy, H. H. (2012). The prevalence of antimicrobial resistance in clinical isolates from Gulf Corporation Council countries. Antimicrobial Resistance and Infection Control, 1(1), 1-5. https://doi.org/10.1186/2047-2994-1-26
Auwal, M. S., Saka, S., Mairiga, I. A., Sanda, K. A., Shuaibu, A., & Ibrahim, A. (2014). Preliminary phytochemical and elemental analysis of aqueous and fractionated pod extracts of Acacia nilotica (Thorn mimosa). Veterinary Research Forum, 5(2), 95–100. https://tinyurl.com/27wkfre8
BaoDuy, N. L., Pham, N. K. T., & Trang, M. (2015). Preliminary phytochemical analysis of leaf extracts of Thuja orientalis (L.) Endl. International Journal of Research Science & Management, 2(1), 21–25. https://tinyurl.com/bd83xw2h
Barbieri, R., Coppo, E., Marchese, A., Daglia, M., Sobarzo-Sánchez, E., Nabavi, S. F., & Nabavi, S. M. (2017). Phytochemicals for human disease: An update on plant-derived compounds antibacterial activity. Microbiological Research, 196, 44–68. https://doi.org/10.1016/j.micres.2016.12.003
Berkow, E. L., Lockhart, S. R., & Ostrosky-Zeichner, L. (2020). Antifungal susceptibility testing: current approaches. Clinical Microbiology Reviews, 33(3). https://doi.org/10.1128/cmr.00069-19
Bhalodia, N. R., & Shukla, V. J. (2011). Antibacterial and antifungal activities from leaf extracts of Cassia fistula l.: An ethnomedicinal plant. Journal of Advanced Pharmaceutical Technology & Research, 2(2), 104-109. https://doi.org/10.4103/2231-4040.82956
Breijyeh, Z., Jubeh, B., & Karaman, R. (2020). Resistance of Gram-Negative bacteria to current antibacterial agents and approaches to resolve it. Molecules, 25(6), 1340. https://doi.org/10.3390/molecules25061340
Buncharoen, W., Saenphet, K., Saenphet, S., & Thitaram, C. (2016). Uvaria rufa Blume attenuates benign prostatic hyperplasia via inhibiting 5α-reductase and enhancing antioxidant status. Journal of Ethnopharmacology, 194, 483-494. https://doi.org/10.1016/j.jep.2016.10.036
Buncharoen, W., Saenphet, S., & Saenphet, K. (2019). Relaxant activities of extracts from Uvaria rufa Blume and Caesalpinia sappan L. on excised rat’s prostate strips. Journal of Pharmaceutical Research International, 29(1), 1–12. https://doi.org/10.9734/jpri/2019/v29i130228
Chua, L. S., Lau, C. H., Chew, C. Y., & Dawood, A. S. (2019). Solvent fractionation and acetone precipitation for crude saponins from Eurycoma longifolia extract. Molecules, 24(7), 1416. https://doi.org/10.3390/molecules24071416
Clinical and Laboratory Standards Institute. (2012). Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; Approved standard (9th edition). CLSI document M07-A9.
Cockerill, F. R., & Clinical. (2008). Performance standards for antimicrobial susceptibility testing; Twenty-second informational supplement. http://ci.nii.ac.jp/ncid/BB08787049
Cunha, L., de Morais, S., Aquinoa, F., Changa, R., de Oliveira, A., Martins, M., Martins, C., Sousa, L., Barros, T., da Silva, C., & do Nascimento, E. (2016). Bioassay-guided fractionation and antimicrobial and cytotoxic activities of Cassia bakeriana extracts. Brazilian Journal of Pharmacognosy, 27(1), 91-98. https://doi.org/10.1016/j.bjp.2016.08.002
Czerkas, K., Olchowik-Grabarek, E., Łomanowska, M., Abdulladjanova, N., & Sękowski, S. (2024). Antibacterial activity of plant polyphenols belonging to the tannins against Streptococcus mutans—Potential against dental caries. Molecules, 29(4), 879s. https://doi.org/10.3390/molecules29040879
Farha, A. K., Yang, Q. Q., Kim, G., Li, H. Bin, Zhu, F., Liu, H. Y., Gan, R. Y., & Corke, H. (2020). Tannins as an alternative to antibiotics. Food Bioscience, 38, 100751. https://doi.org/10.1016/j.fbio.2020.100751
Gauba, A., & Rahman, K. M. (2023). Evaluation of antibiotic resistance mechanisms in Gram-Negative bacteria. Antibiotics, 12, 1590. https://doi.org/10.3390/antibiotics12111590
Ginovyan, M., Petrosyan, M., & Trchounian, A. (2017). Antimicrobial activity of some plant materials used in Armenian traditional medicine. BMC Complementary and Alternative Medicine, 17(1), 50. https://doi.org/10.1186/s12906-017-1573-y
Hamed, A. A., Soldatou, S., Qader, M. M., Arjunan, S., Miranda, K. J., Casolari, F., Pavesi, C., Diyaolu, O. A., Thissera, B., Eshelli, M., Belbahri, L., Luptakova, L., Ibrahim, N. A., Abdel-Aziz, M. S., Eid, B. M., Ghareeb, M. A., Rateb, M. E., & Ebel, R. (2020). Screening fungal endophytes derived from under-explored Egyptian marine habitats for antimicrobial and antioxidant properties in factionalised textiles. Microorganisms, 8(10), 1617. https://doi.org/10.3390/microorganisms8101617
Hilaria, M., Elisma, Dapa, M. Y., & Teti, M. (2016). Antioxidant activities of ethyl acetate extract and hexane extract of Lelak (Uvaria rufa Blume) leaves. Asian Journal of Applied Sciences, 4(4), 930–934. https://tinyurl.com/44n479mv
Jamkhande, P. G., Wattamwar, A. S., Kankudta, A. D., Tidke, P. S., & Kalaskar, M. G. (2015). Assessment of Annona reticulata Linn. leaves fractions for in vitro antioxidative effect and antimicrobial potential against standard human pathogenic strains. Alexandria Journal of Medicine, 52(1), 19-25. http://dx.doi.org/10.1016/j.ajme.2014.12.007
Kaczmarek, B. (2020). Tannic acid with antiviral and antibacterial activity as a promising component of biomaterials-A minireview. Materials, 13(14), 3224. https://doi.org/10.3390/ma13143224
Khameneh, B., Eskin, N. A. M., Iranshahy, M., & Fazly Bazzaz, B. S. (2021). Phytochemicals: A promising weapon in the arsenal against antibiotic‐resistant bacteria. Antibiotics, 10(9), 1–33. https://doi.org/10.3390/antibiotics10091044
Kibungu, W. C., Fri, J., Clarke, A. M., Otigbu, A., & Njom, H. A. (2021). Seasonal variation in antimicrobial activity of crude extracts of Psammaplysilla sp. 1 from Phillips Reef, South Africa. International Journal of Microbiology. https://doi.org/10.1155/2021/7568493
Klionsky, D. J., Abdel-Aziz, A. K., Abdelfatah, S., Abdellatif, M., Abdoli, A., Abel, S., Abeliovich, H., Abildgaard, M. H., Abudu, Y. P., Acevedo-Arozena, A., Adamopoulos, I. E., Adeli, K., Adolph, T. E., Adornetto, A., Aflaki, E., Agam, G., Agarwal, A., Aggarwal, B. B., Agnello, M., . . . Tong, C. (2021). Guidelines for the use and interpretation of assays for monitoring autophagy (4th ed.). Autophagy, 17(1), 1–382. https://doi.org/10.1080/15548627.2020.1797280
Macabeo, A. P. G., Tudla, F. A., Krohn, K., & Franzblau, S. G. (2012). Antitubercular activity of the semi–polar extractives of Uvaria rufa. Asian Pacific Journal of Tropical Medicine, 5(10), 777-780. https://doi.org/10.1016/s1995-7645(12)60142-4
Marasini, B. P., Baral, P., Aryal, P., Ghimire, K. R., Neupane, S., Dahal, N., Singh, A., Ghimire, L., & Shrestha, K. (2015). Evaluation of antibacterial activity of some traditionally used medicinal plants against human pathogenic bacteria. BioMed Research International, 2015, 1–6. https://doi.org/10.1155/2015/265425
Masyita, A., Mustika Sari, R., Dwi Astuti, A., Yasir, B., Rahma Rumata, N., Emran, T. Bin, Nainu, F., & Simal-Gandara, J. (2022). Terpenes and terpenoids as main bioactive compounds of essential oils, their roles in human health and potential application as natural food preservatives. Food Chemistry: X, 13, 100217. https://doi.org/10.1016/j.fochx.2022.100217
O’Neill, J. (2014) Antimicrobial resistance: Tackling a crisis for the health and wealth of nations. Review on Antimicrobial Resistance.
Padma, K. R., Don, K. R., & Josthna, P. (2014). Herbal plant Uvaria species and its therapeutic potentiality. World Journal of Environmental Biosciences, 9(1), 33–38. https://tinyurl.com/7adr33j5
Pamok, S., Saenphet, K., & Buncharoen, W. (2018). Effects of Uvaria rufa Blume on the histology of male reproductive organs of testosterone induced alopecia rats. Microscopy and Microanalysis Research, 31(1), 9-17. https://doi.org/10.14456/microsc-microanal-res.2018.2
Paul, S., Dash, B., Bora, A.J., & Gupta, B. (2018). Preliminary phytochemical screening and in vitro antimicrobial activity of ethanolic extracts of fruits of Annona reticulata against standard pathogenic strains. International Journal of Current Pharmaceutical Research, 10(4), 59-63. http://dx.doi.org/10.22159/ijcpr.2018v10i4.28466
Pieroni, A. (2002). Trease and Evans pharmacognosy. Fitoterapia, 73(7-8). https://doi.org/10.1016/s0367-326x(02)00228-9
Reygaert, W. (2018). An overview of the antimicrobial resistance mechanisms of bacteria. AIMS Microbiology, 4(3), 482–501. https://doi.org/10.3934/microbiol.2018.3.482
Rosandy, A. R., Din, L. B., Yaacob, W. Z. W., Yusoff, N. M. I. N., Sahidin, I., Latip, J., Nataqain, S., & Noor, N. M. (2013). Isolation and characterization of compounds from the stem bark of Uvaria rufa (Annonaceae). The Malaysian Journal of Analytical Sciences, 17(1), 50–58. https://tinyurl.com/2n3jjdf8
Saptarini, N. M., & Wardati, Y. (2020). Effect of extraction methods on antioxidant activity of papery skin extracts and fractions of Maja Cipanas onion (Allium cepa L. var. ascalonicum). The Scientific World Journal, 2020. https://doi.org/10.1155/2020/3280534
Saptarini, N. M., Herawati, I. E., & Permatasari, U. Y. (2016). Total flavonoids content in acidified extract of flowers and leaves of Gardenia (Gardenia jasminoides Ellis). Asian Journal of Pharmaceutical and Clinical Research, 9(7), 213–215. https://tinyurl.com/2mnvekvw
Savoia, D. (2012). Plant-derived antimicrobial compounds: Alternatives to antibiotics. Future Microbiology, 7(8), 979-990. https://doi.org/10.2217/fmb.12.68
Shami, A. M. (2017). The effect of alkaloidal fraction from Annona squamosa L. against pathogenic bacteria with antioxidant activities. Pharmaceutical Sciences, 23(4), 301–307. https://doi.org/10.15171/ps.2017.44
Sulaiman, M., Jannat, K., Nissapatorn, V., Rahmatullah, M., Paul, A. K., de Lourdes Pereira, M., Rajagopal, M., Suleiman, M., Butler, M. S., Break, M. K. Bin, Weber, J. F., Wilairatana, P., & Wiart, C. (2022). Antibacterial and antifungal alkaloids from Asian angiosperms: Distribution, mechanisms of action, structure-activity, and clinical potentials. Antibiotics, 11(9), 1146. https://doi.org/10.3390/antibiotics11091146
Thawabteh, A. M., Ghanem, A. W., AbuMadi, S., Thaher, D., Jaghama, W., Karaman, R., Scrano, L., & Bufo, S. A. (2024). Antibacterial activity and antifungal activity of monomeric alkaloids. Toxins, 16(11). https://doi.org/10.3390/toxins16110489
Tsui, C., Kong, E. F., & Jabra‐Rizk, M. A. (2016). Pathogenesis of Candida albicans biofilm. Pathogens and Disease, 74(4), ftw018. https://doi.org/10.1093/femspd/ftw018
Uddin, T. M., Chakraborty, A. J., Khusro, A., Zidan, B. R. M., Mitra, S., Emran, T. B., Dhama, K., Ripon, K. H., Gajdacs, M., Sahibzada, M. U. K., Hossain, M. J., & Koirala, N. (2021). Antibiotic resistance in microbes: History, mechanisms, therapeutic strategies and future prospects. Journal of Infection and Public Health, 14(12), 1750-1766. https://doi.org/10.1016/j.jiph.2021.10.020
Ugwu Okechukwu, P. C., Nwodo Okwesili, F. C., Parker, E. J., Bawa Abubakar, Ossai, E. C., & Odo, C. E. (2013). Phytochemical and acute toxicity studies of Moringa oleifera ethanol leaf extract. International Journal of Life Sciences, Biotechnology, and Pharma Research, 2(2), 66–71.
Upadhyay, A., Upadhyaya, I., Kollanoor-Johny, A., & Venkitanarayanan, K. (2014). Combating pathogenic microorganisms using plant-derived antimicrobials: A minireview of the mechanistic basis. BioMed Research International, 2014, 1–18. https://doi.org/10.1155/2014/761741
Valle, D. L., Andrade, J. I., Puzon, J. J. M., Cabrera, E. C., & Rivera, W. L. (2015). Antibacterial activities of ethanol extracts of Philippine medicinal plants against multidrug-resistant bacteria. Asian Pacific Journal of Tropical Biomedicine, 5(7), 532-540. https://doi.org/10.1016/j.apjtb.2015.04.005
World Health Organization: WHO. (2021) Antimicrobial resistance. www.who.int. https://tinyurl.com/27cnzt2w
Wright, P. M., Seiple, I. B., & Myers, A. G. (2014). The evolving role of chemical synthesis in antibacterial drug discovery. Angewandte Chemie International Edition, 53(34), 8840–8869. https://doi.org/10.1002/anie.201310843
Yan, Y., Li, X., Zhang, C., Lv, L., Gao, B., & Li, M. (2021). Research progress on antibacterial activities and mechanisms of natural alkaloids: A review. Antibiotics, 10(3), 318. https://doi.org/10.3390/antibiotics10030318

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