Tropical Biomedicine 39(4): 552-558 (2022) htps://doi.org/10.47665/tb.39.4.011 RESEARCH ARTICLE Steroids from Diplazium esculentum: Antplasmodial actvity and molecular docking studies to investgate their binding modes Safar, H.F. 1 , Ali, A.H. 1 , Zakaria, N.H. 1 , Kamal, N. 2 , Hassan, N.I. 1 , Agustar, H.K. 3 , Talip, N. 4 , Latp, J. 1* 1 Department of Chemical Sciences, Faculty of Science and Technology, Universit Kebangsaan Malaysia, 43600 UKM, Bangi, Selangor, Malaysia 2 Insttute of Systems Biology (INBIOSIS), Universit Kebangsaan Malaysia, 43600 UKM, Bangi, Selangor, Malaysia 3 Department of Earth Sciences and Environment, Faculty of Science and Technology, Universit Kebangsaan Malaysia, 43600 UKM, Bangi, Selangor, Malaysia 4 Department of Biological Sciences and Biotechnology, Faculty of Science and Technology, Universit Kebangsaan Malaysia, 43600 UKM, Bangi, Selangor, Malaysia *Corresponding author: jalifah@ukm.edu.my Published by Malaysian Society of Parasitology and Tropical Medicine. All rights reserved. ARTICLE HISTORY ABSTRACT Received: 15 May 2022 Revised: 10 November 2022 Accepted: 10 November 2022 Published: 31 December 2022 Diplazium esculentum is an edible fern commonly consumed by the local community in Malaysia either as food or medicine. Isolaton work on the ethyl acetate extract of the stem of D. esculentum resulted in the purifcaton of two steroids, subsequently identfed as stgmasterol (compound 1) and ergosterol- 5,8-endoperoxide (compound 2). Upon further testng, compound 2 displayed strong inhibitory actvity against the Plasmodium falciparum 3D7 (chloroquine-sensitve) strain, with an IC 50 of 4.27±1.15 µM, while compound 1 was inactve. In silico data revealed that compound 2 showed good binding afnity to P. falciparum-Sarco endoplasmic retculum calcium-dependent ATPase (PfATP6); however, compound 1 did not show an antplasmodial efect due to the lack of a peroxide moiety in the chemical structure. Our data suggested that the antplasmodial actvity of compound 2 from D. esculentum might be due to the inhibiton of PfATP6, which resulted in both in vitro and in silico inhibitory propertes. Keywords: Antplasmodial actvity; Diplazium esculentum; ergosterol-5,8-endoperoxide; in silico; PfATP6. INTRODUCTION Malaria is an infectious disease that caused mortalities and morbidites worldwide. Increasing malaria-related deaths, especially in the tropics in recent years, are primarily associated with the most virulent P. falciparum infecton (WHO, 2021). The alarming morbidity is atributed mainly to the development of parasite resistance towards antmalarial agents, including artemisinin, the current front-line drug against the disease (Ouji et al., 2018). The emergence of artemisinin resistance cases in the Greater Mekong regions urged a serious strategy to be taken to curb the transmission of the disease to other neighbouring countries (Imwong et al., 2017). The most efectve means to prevent mortality and morbidity due to the resistant parasite infecton is through chemotherapeutcs since there is no fully licensed vaccine has been developed for malaria so far. Therapeutcs for malaria derived from plants have high efcacy, and availability, low cost, and are generally safe for human consumpton. Medicinal plants have been widely used to reduce malarial infectons and symptoms for a long tme, for example Artemisia annua (Septembre-Malaterre et al., 2020). Diplazium esculentum (Retz.) Sw. (synonym Diplazium malabaricum Spreng., common name Paku Tanjung) belongs to the family Athyriaceae. It is a relatvely large fern, commonly found in South East Asia farms and villages (Nur et al., 2018). Most people in South East Asia consume this fern either as food or medicine (Cicuzza, 2021). In Malaysia, several indigenous tribes in Borneo and Perak eat this fern as a traditonal vegetable salad or “ulam” (Awang-Kanak & Abu Bakar, 2020). Local people in Malaysia traditonally used it to treat fever, diabetes, acne, asthma, scars, hemoptysis, and cough, and as a post-partum health tonic (Tag et al., 2012; Jasim et al., 2014). Phytochemical screening of D. esculentum revealed numerous bioactve components, such as steroids, phenolic compounds, alkaloids, favonoids, tannins, glycosides, saponins, carbohydrates, and protein (Tongco et al., 2014; Junejo et al., 2015). Previous studies demonstrated that D. esculentum exhibited antinfammatory (Kaushik et al., 2011), antoxidant (Roy et al., 2013), antdiabetc (Tag et al., 2012), and antbacterial actvites (Amit et al., 2011). A recent antmalarial efect of D. esculentum aqueous extract in Plasmodium berghei-infected mice had been reported (Ramli et al., 2021). However, the antmalarial efcacy of this plant and its bioactve components against human malarial parasite has yet to be reported. P. falciparum-Sarco endoplasmic retculum calcium-dependent ATPase (PfATP6) is homologous to mammalian Sarco endoplasmic retculum calcium ATPase (SERCA) with all essental motfs and sequences for the structure and functon of a SERCA are conserved, for example, high-afnity Ca 2+ -binding sites, a phosphorylaton site and a nucleotde-binding site (Arnou et al., 2011). PfATP6 is the only SERCA-type Ca 2+ ATPase enzyme that exists in the Plasmodium sp. One suggested mechanism of acton of artemisinin that is commonly crucial for its actvity is through the interacton of peroxide bonds with haem (Klayman, 1985; Meshnick et al., 1996). An oxy free radical generated upon hydrolysis of the peroxide linkage by the alkylaton process in artemisinin is responsible for the lethal damage of the parasite (Jeford, 2001). Artemisinin also targets the specifc plasmodial protein, PfATP6, which is known as another mechanism