In vitro antifungal activity of extracts and alkaloid compounds from Piper arboreum against dermatophytes

Abstract

Piper is widely distributed in subtropical regions and species of this genus are known for their potent pharmacological activities. Piper arboreum Aubl. is a traditional medicinal plant popularly known as "pau-de-angola", "jaborandi", and chili pepper, demonstrating antifungal, trypanocidal, antibacterial, and antioxidant activities. The leaves of P. arboreum were extracted using Soxhlet and dichloromethane to obtain the extract, which was then fractionated using solvents of different polarities. Samples were analyzed using ultra-high-performance liquid chromatography coupled with mass spectrometry equipped with an electrospray ionization source. Antifungal microdilution assays were performed, and scanning and transmission electron microscopy were used to assess the invasion of the pretreated nail. The minimum inhibitory concentration (MIC) values of the extract and a dichloromethane fraction were, respectively, 62.5 μg/ml and 16.0 μg/ml against Trichophyton rubrum , and 125 μg/ml and 62.5 μg/ml, and 500 μg/ml and 500 μg/ml against T. mentagrophytes , and Microsporum gypseum , respectively. No growth was observed on nail fragments exposed to the extract (at concentrations > 64 µg/ml and then inoculated with spore suspension. Transmission electron microscopy revealed strong inhibition of hyphal growth and an irregular growth pattern following treatment with the extract and the dichloromethane fraction. Results demonstrated the antifungal properties of the P. arboreum extract and its dichloromethane fraction against dermatophytes, with the identification of three different alkaloid compounds. The cytotoxicity was specific towards the fungal cells, and morphological and ultrastructural analyses indicated damage to the cell wall and cytoplasmic membrane as the potential mechanism of action. The leaf material used to generate the extract can be taken from the plant without any detrimental effect thus enabling strategies to be implemented for the exploitation of this species.

Keywords

References

1. Achenbach, H., Grob, J., Dominguez, X. A., Cano, G., Star, J. V., Brussolo, L. D. C., Muñoz, G., Salgado, F., & López, L. (1987). Lignans neolignans and norneolignans from Krameria cystisoides. Phytochemistry, 26(4), 1159-1166. https://doi.org/10.1016/S0031-9422(00)82370-5 DOI: https://doi.org/10.1016/S0031-9422(00)82370-5
2. Andrade, A., Kuah, C. Y., Martin‐Lopez, J. E., Chua, S., Shpadaruk, V., Sanclemente, G., Franco, J. V., & Group, C. S. (1996). Interventions for chronic pruritus of unknown origin. Cochrane Database of Systematic Reviews, 2020(1), CD013128. https://doi.org//10.1002/14651858.CD013128.pub2 DOI: https://doi.org/10.1002/14651858.CD013128.pub2
3. Baghi, N., Shokohi, T., Badali, H., Makimura, K., Rezaei-Matehkolaei, A., Abdollahi, M., Didehdar, M., Haghani, I., & Abastabar, M. (2016). In vitro activity of new azoles luliconazole and lanoconazole compared with ten other antifungal drugs against clinical dermatophyte isolates. Medical Mycology, 54(7), 757-763. https://doi.org/10.1093/mmy/myw016 DOI: https://doi.org/10.1093/mmy/myw016
4. Benassi-Zanqueta, É., Marques, C. F., Nocchi, S. R., Dias Filho, B. P., Nakamura, C. V., & Ueda-Nakamura, T. (2018). Parthenolide Influences Herpes simplex virus 1 Replication in vitro. Intervirology, 61(1), 14-22. https://doi.org/10.1159/000490055 DOI: https://doi.org/10.1159/000490055
5. Bennett, M. L., Fleischer Jr, A. B., Loveless, J. W., & Feldman, S. R. (2000). Oral griseofulvin remains the treatment of choice for tinea capitis in children. Pediatric Dermatology, 17(4), 304-309. https://doi.org/10.1046/j.1525-1470.2000.01784.x DOI: https://doi.org/10.1046/j.1525-1470.2000.01784.x
6. Bernuci, K. Z., Iwanaga, C. C., Fernandez-Andrade, C. M. M., Lorenzetti, F. B., Torres-Santos, E. C., Faioes, V. D. S., Gonçalves, J. E., Do Amaral, W., Deschamps, C., & Scodro, R. B. D. L. (2016). Evaluation of chemical composition and antileishmanial and antituberculosis activities of essential oils of Piper species. Molecules, 21(12), 1698. https://doi.org/10.3390/molecules21121698 DOI: https://doi.org/10.3390/molecules21121698
7. Biswas, P., Ghorai, M., Mishra, T., Gopalakrishnan, A. V., Roy, D., Mane, A. B., Mundhra, A., Das, N., Mohture, V. M., & Patil, M. T. (2022). Piper longum L.: A comprehensive review on traditional uses, phytochemistry, pharmacology, and health‐promoting activities. Phytotherapy Research, 36(12), 4425-4476. https://doi.org/10.1002/ptr.7649 DOI: https://doi.org/10.1002/ptr.7649
8. Brenton, A. G., & Godfrey, A. R. (2010). Accurate mass measurement: terminology and treatment of data. Journal of the American Society for Mass Spectrometry, 21(11), 1821-1835. https://doi.org/10.1016/j.jasms.2010.06.006 DOI: https://doi.org/10.1016/j.jasms.2010.06.006
9. Butler, D. C., Berger, T., Elmariah, S., Kim, B., Chisolm, S., Kwatra, S. G., Mollanazar, N., & Yosipovitch, G. (2024). Chronic Pruritus: A Review. JAMA, 331(24), 2114-2124. https://doi.org/10.1001/jama.2024.4899 DOI: https://doi.org/10.1001/jama.2024.4899
10. Chauret, D. C., Bernard, C. B., Arnason, J. T., Durst, T., Krishnamurty, H., Sanchez-Vindas, P., Moreno, N., San Roman, L., & Poveda, L. (1996). Insecticidal neolignans from Piper decurrens. Journal of Natural Products, 59(2), 152-155. https://doi.org/10.1021/np960036y DOI: https://doi.org/10.1021/np960036y
11. Clinical and Laboratory Standards Institute-CLSI. (2008a). Reference Method for Broth dilution antifungal susceptibility testing of yeasts. Approved standard M27-A3. Clinical and Laboratory Standards Institute, Available from: https://www.astm.org/products-services/standards-and-publications/technical-reports.html: Villanova, PA.
12. Clinical and Laboratory Standards Institute-CLSI. (2008b). Reference method for broth dilution antifungals susceptibility testing of bconidia-forming filamentous fungi. Approved standard, 2nd ed. M38-A2. Available from: https://www.astm.org/products-services/standards-and-publications/technical-reports.html: Wayne, PA.
13. Cysne, J. B., Canuto, K. M., Pessoa, O. D. L., Nunes, E. P., & Silveira, E. R. (2005). Leaf essential oils of four Piper species from the State of Ceará-Northeast of Brazil. Journal of the Brazilian Chemical Society, 16(6b), 1378-1381. https://doi.org/10.1590/S0103-50532005000800012 DOI: https://doi.org/10.1590/S0103-50532005000800012
14. da Silva, R. V., Navickiene, H. M. D., Kato, M. J., Bolzani, V. D. S., Méda, C. I., Young, M. C. M., & Furlan, M. (2002). Antifungal amides from Piper arboreum and Piper tuberculatum. Phytochemistry, 59(5), 521-527. https://doi.org/10.1016/S0031-9422(01)00431-9 DOI: https://doi.org/10.1016/S0031-9422(01)00431-9
15. Dalla Lana, D., Batista, B., Alves, S., & Fuentefria, A. (2016). Dermatophytoses: etiologic agents, clinical forms, therapy and new perspectives of treatment. Clinical and Biomedical Research, 36, 230-241. https://doi.org/10.4322/2357-9730.68880 DOI: https://doi.org/10.4322/2357-9730.68880
16. de Pauw, B. (2000). Is there a need for new antifungal agents? Clinical Microbiology and Infection, 6, 23-28. https://doi.org/10.1046/j.1469-0691.2000.00006.x DOI: https://doi.org/10.1046/j.1469-0691.2000.00006.x
17. Durant-Archibold, A. A., Santana, A. I., & Gupta, M. P. (2018). Ethnomedical uses and pharmacological activities of most prevalent species of genus Piper in Panama: A review. Journal of Ethnopharmacology, 217, 63-82. https://doi.org/10.1016/j.jep.2018.02.008 DOI: https://doi.org/10.1016/j.jep.2018.02.008
18. Dyer, L. A., & Palmer, A. D. (2004). Piper: a model genus for studies of phytochemistry, ecology, and evolution (Vol. 25): Springer New York, NY. https://doi.org/10.1007/978-0-387-30599-8 DOI: https://doi.org/10.1007/978-0-387-30599-8
19. Freixa, B., Vila, R., Ferro, E. A., Adzet, T., & Cañigueral, S. (2001). Antifungal principles from Piper fulvescens. Planta Medica, 67(9), 873-875. https://doi.org/10.1055/s-2001-18838 DOI: https://doi.org/10.1055/s-2001-18838
20. Guerrini, A., Sacchetti, G., Rossi, D., Paganetto, G., Muzzoli, M., Andreotti, E., Tognolini, M., Maldonado, M. E., & Bruni, R. (2009). Bioactivities of Piper aduncum L. and Piper obliquum Ruiz & Pavon (Piperaceae) essential oils from eastern Ecuador. Environmental Toxicology and Pharmacology, 27(1), 39-48. https://doi.org/10.1016/j.etap.2008.08.002 DOI: https://doi.org/10.1016/j.etap.2008.08.002
21. Gupta, A. K., Shemer, A., Economopoulos, V., & Talukder, M. (2024). Diabetic Foot and Fungal Infections: Etiology and Management from a Dermatologic Perspective. Journal of Fungi, 10(8), 577. https://doi.org/10.3390/jof10080577 DOI: https://doi.org/10.3390/jof10080577
22. Hay, R. J., Johns, N. E., Williams, H. C., Bolliger, I. W., Dellavalle, R. P., Margolis, D. J., Marks, R., Naldi, L., Weinstock, M. A., & Wulf, S. K. (2014). The global burden of skin disease in 2010: an analysis of the prevalence and impact of skin conditions. Journal of Investigative Dermatology, 134(6), 1527-1534. https://doi.org/10.1038/jid.2013.446 DOI: https://doi.org/10.1038/jid.2013.446
23. Holetz, F. B., Pessini, G. L., Sanches, N. R., Cortez, D. A. G., Nakamura, C. V., & Dias Filho, B. P. (2002). Screening of some plants used in the Brazilian folk medicine for the treatment of infectious diseases. Memórias do Instituto Oswaldo Cruz, 97, 1027-1031. https://doi.org/10.1590/S0074-02762002000700017 DOI: https://doi.org/10.1590/S0074-02762002000700017
24. Jones, H. E., Reinhardt, J. H., & Rinaldi, M. G. (1973). A clinical, mycological, and immunological survey for dermatophytosis. Archives of Dermatology, 108(1), 61-65. https://doi.org/10.1001/archderm.1973.01620220033008 DOI: https://doi.org/10.1001/archderm.108.1.61
25. Lahmer, M., Grari, O., Beyyoudh, S., Amrani, A., Faiz, I., & Hami, A. (2024). Epidemiological profile of dermatophytes at the parasitology-mycology laboratory at Mohammed VI University Hospital in Oujda. La Tunisie Medicale, 102(8), 447-451. https://doi.org/10.62438/tunismed.v102i8.4862 DOI: https://doi.org/10.62438/tunismed.v102i8.4862
26. Lesher Jr, J. L. (1999). Oral therapy of common superficial fungal infections of the skin. Journal of the American Academy of Dermatology, 40(6), S31-S34. https://doi.org/10.1016/S0190-9622(99)70395-6 DOI: https://doi.org/10.1016/S0190-9622(99)70395-6
27. Macura, A. B., Macura-Biegun, A., & Pawlik, B. (2003). Susceptibility to fungal infections of nails in patients with primary antibody deficiency. Comparative Immunology, Microbiology and Infectious Diseases, 26(4), 223-232. https://doi.org/10.1016/S0147-9571(02)00051-6 DOI: https://doi.org/10.1016/S0147-9571(02)00051-6
28. Mayorga, J., Esquivel-González, P. L., Prado-Trillo, A., & Barba-Gómez, J. F. (2016). Características clínicas y epidemiológicas de pacientes con infección por Microsporum canis. Dermatología Revista Mexicana, 60(1), 18-23.
29. Nakamura, M. T., Endo, E. H., de Sousa, J. P. B., Callejon, D. R., Ueda-Nakamura, T., Dias, B. P., de Freitas, O., Nakamura, C. V., & Lopes, N. P. (2017). Copaiba oil and its constituent copalic acid as chemotherapeutic agents against dermatophytes. Journal of the Brazilian Chemical Society, 28, 1377-1383. https://doi.org/10.21577/0103-5053.20160309 DOI: https://doi.org/10.21577/0103-5053.20160309
30. Nascimento, S. A., Araujo, E. A., Da Silva, J. M., & Ramos, C. S. (2015). Chemical study and antimicrobial activities of Piper arboreum (Piperaceae). Journal of the Chilean Chemical Society, 60(1), 2837-2839. http://dx.doi.org/10.4067/S0717-97072015000100013 DOI: https://doi.org/10.4067/S0717-97072015000100013
31. Navickiene, H. M. D., Alécio, A. C., Kato, M. J., Bolzani, V. D. S., Young, M. C. M., Cavalheiro, A. J., & Furlan, M. (2000). Antifungal amides from Piper hispidum and Piper tuberculatum. Phytochemistry, 55(6), 621-626. https://doi.org/10.1016/S0031-9422(00)00226-0 DOI: https://doi.org/10.1016/S0031-9422(00)00226-0
32. Navickiene, H. M. D., Morandim, A. D. A., Alécio, A. C., Regasini, L. O., Bergamo, D. C. B., Telascrea, M., Cavalheiro, A. J., Lopes, M. N., Bolzani, V. D. S., & Furlan, M. (2006). Composition and antifungal activity of essential oils from Piper aduncum, Piper arboreum and Piper tuberculatum. Química Nova, 29(3), 467-470. https://doi.org/10.1590/S0100-40422006000300012 DOI: https://doi.org/10.1590/S0100-40422006000300012
33. Obici, S., Otobone, F. J., da Silva Sela, V. R., Ishida, K., da Silva, J. C., Nakamura, C. V., Cortez, D. A. G., & Audi, E. A. (2008). Preliminary toxicity study of dichloromethane extract of Kielmeyera coriacea stems in mice and rats. Journal of Ethnopharmacology, 115(1), 131-139. https://doi.org/10.1016/j.jep.2007.09.013 DOI: https://doi.org/10.1016/j.jep.2007.09.013
34. Odds, F. C. (2003). Synergy, antagonism, and what the chequerboard puts between them. Journal of Antimicrobial Chemotherapy, 52(1), 1-1. https://doi.org/10.1093/jac/dkg301 DOI: https://doi.org/10.1093/jac/dkg301
35. Olson, J., & Troxell, T. (2023). Griseofulvin: Treasure Island (FL): StatPearls [Internet]. PMID: 30726008, Bookshelf ID: NBK537323
36. Polak, A. (1990). Combination Therapy in Systemic Mycosis. Journal of Chemotherapy, 2(4), 211-217. https://doi.org/10.1080/1120009X.1990.11739020 DOI: https://doi.org/10.1080/1120009X.1990.11739020
37. Quijano‐Abril, M. A., Callejas‐Posada, R., & Miranda‐Esquivel, D. R. (2006). Areas of endemism and distribution patterns for Neotropical Piper species (Piperaceae). Journal of Biogeography, 33(7), 1266-1278. https://doi.org/10.1111/j.1365-2699.2006.01501.x DOI: https://doi.org/10.1111/j.1365-2699.2006.01501.x
38. Rani, N., Sharma, A., & Singh, R. (2013). Imidazoles as promising scaffolds for antibacterial activity: a review. Mini Reviews in Medicinal Chemistry, 13(12), 1812-1835. DOI: https://doi.org/10.2174/13895575113136660091
39. Regasini, L. O., Cotinguiba, F., Morandim, A. D. A., Kato, M. J., Scorzoni, L., Mendes-Giannini, M. J., Bolzani, V. D. S., & Furlan, M. (2009). Antimicrobial activity of Piper arboreum and Piper tuberculatum (Piperaceae) against opportunistic yeasts. African Journal of Biotechnology, 8(12), 2866-2870.
40. Regasini, L. O., Cotinguiba, F., Siqueira, J. R., Bolzani, V. S., Silva, D., Furlan, M., & Massuo, J. (2008). Radical scavenging capacity of Piper arboreum and Piper tuberculatum (Piperaceae). Latin American Journal of Pharmacy, 27(6), 900-903.
41. Ridzuan, P., Mohamad, N., Ismail, S., Rahman, N. I. A., Zunariah, B., Norazian, M., & Roesnita, B. (2018). Cytotoxicity and antifungal properties of hydroxychavicol against Trichophyton rubrum. IIUM Medical Journal Malaysia, 17(1), 31-36. https://doi.org/10.31436/imjm.v17i1.288 DOI: https://doi.org/10.31436/imjm.v17i1.288
42. Rinaldi, M. G. (2000). Dermatophytosis: epidemiological and microbiological update. Journal of the American Academy of Dermatology, 43(5), S120-S124. https://doi.org/10.1067/mjd.2000.110378 DOI: https://doi.org/10.1067/mjd.2000.110378
43. Rouzaud, C., Chosidow, O., Brocard, A., Fraitag, S., Scemla, A., Anglicheau, D., Bouaziz, J. D., Dupin, N., Bougnoux, M. E., & Hay, R. (2018). Severe dermatophytosis in solid organ transplant recipients: a French retrospective series and literature review. Transplant Infectious Disease, 20(1), e12799. https://doi.org/10.1111/tid.12799 DOI: https://doi.org/10.1111/tid.12799
44. Rupérez, M. B. H., Domínguez, M. C., & Saavedra-Lozano, J. (2013). Infecciones fúngicas superficiales. Anales de Pediatría Continuada, 11(5), 254-266. https://doi.org/10.1016/S1696-2818(13)70146-X DOI: https://doi.org/10.1016/S1696-2818(13)70146-X
45. Seebacher, C., Bouchara, J. P., & Mignon, B. (2008). Updates on the epidemiology of dermatophyte infections. Mycopathologia, 166, 335-352. https://doi.org/10.1007/s11046-008-9100-9 DOI: https://doi.org/10.1007/s11046-008-9100-9
46. Silva, R. (2004). Estudo químico e biossintético em Piper arboreum L (Piperaceae). (Ph. D. Doutorado em Química). Universidade Estadual Paulista, Araraquara, Retrieved from https://oasisbr.ibict.br/vufind/DataSources/Datasource?name=PortaldeDadosAbertosdaCAPES. Retrieved from https://oasisbr.ibict.br/vufind/DataSources/Datasource?name=PortaldeDadosAbertosdaCAPES
47. Tran, T. T. P., Dang, N. H., Nguyen, X. N., Pham, H. T., Phan, U. T. T., & Le, T. H. (2024). Alkaloids from Piper longum L and Their Anti‐inflammatory Properties. Chemistry & Biodiversity, 2024, e202401224. https://doi.org/10.1002/cbdv.202401224 DOI: https://doi.org/10.1002/cbdv.202401224
48. Walters, K. A., Flynn, G. L., & Marvel, J. R. (1981). Physiocochemical characterization of the human nail: I. Pressure sealed apparatus for measuring nail plate permeabilities. Journal of Investigative Dermatology, 76(2), 76-79. https://doi.org/10.1111/1523-1747.ep12525318 DOI: https://doi.org/10.1111/1523-1747.ep12525318
49. Watanabe, S. (1999). Present state and future direction of topical antifungals. Nippon Ishinkin Gakkai Zasshi, 40(3), 151-155. https://doi.org/10.3314/jjmm.40.151 DOI: https://doi.org/10.3314/jjmm.40.151
50. White, T. C., Findley, K., Dawson, T. L., Scheynius, A., Boekhout, T., Cuomo, C. A., Xu, J., & Saunders, C. W. (2014). Fungi on the skin: dermatophytes and Malassezia. Cold Spring Harbor Perspectives in Medicine, 4(8), a019802. https://doi.org/10.1101/cshperspect.a019802 DOI: https://doi.org/10.1101/cshperspect.a019802
51. Worek, M., Kwiatkowska, A., Ciesielska, A., Jaworski, A., Kaplan, J., Miedziak, B., Deregowska, A., Lewinska, A., & Wnuk, M. (2014). Identification of dermatophyte species using genomic in situ hybridization (GISH). Journal of Microbiological Methods, 100, 32-41. https://doi.org/10.1016/j.mimet.2014.02.012 DOI: https://doi.org/10.1016/j.mimet.2014.02.012
52. Yadav, V., Krishnan, A., & Vohora, D. (2020). A systematic review on Piper longum L.: Bridging traditional knowledge and pharmacological evidence for future translational research. Journal of Ethnopharmacology, 247, 112255. https://doi.org/10.1016/j.jep.2019.112255 DOI: https://doi.org/10.1016/j.jep.2019.112255
53. Yousefian, F., Smythe, C., Han, H., Elewski, B. E., & Nestor, M. (2024). Treatment Options for Onychomycosis: Efficacy, Side Effects, Adherence, Financial Considerations, and Ethics. The Journal of Clinical and Aesthetic Dermatology, 17(3), 24-33.

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Copyright (c) 2024 Fabiana Brusco Lorenzetti, Carla Maria Mariano Fernandez, Eliana Harue Endo, Regina Yasuko Makimori, Mariza Barion Romagnolo, César Armando Contreras Lancheros, Marcia Regina Pereira Cabral, Maria Helena Sarragiotto, Celso Vataru Nakamura, Tânia Ueda Nakamura, Ludmila Pini Simões, Benedito Prado Dias Filho