Chemical Constituents of Excoecaria cochinchinensis Exhibiting Cytotoxic, Antioxidant, Antityrosinase, and α-Glucosidase Inhibitory Activities

Chemical Constituents of Excoecaria cochinchinensis Exhibiting Cytotoxic, Antioxidant, Antityrosinase, and α-Glucosidase Inhibitory Activities | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Chemical Constituents of Excoecaria cochinchinensis Exhibiting Cytotoxic, Antioxidant, Antityrosinase, and α-Glucosidase Inhibitory Activities Van-Kieu Nguyen, Phan-Si-Nguyen Dong, Hoang-Vinh-Truong Phan, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7268944/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Dec, 2025 Read the published version in Revista Brasileira de Farmacognosia → Version 1 posted 5 You are reading this latest preprint version Abstract Eleven known compounds, including four triterpenoids ( 1 – 4 ), four sterols ( 5 – 8 ), two phenolic compounds ( 9 – 10 ), and a dipeptide ( 11 ), were isolated from Excoecaria cochinchinensis . Their structures were confirmed through spectroscopic analysis and compared with the existing literature. Eight compounds ( 1 , 2 , 5 – 8 , 10 – 11 ) have been identified in this species for the first time. The cytotoxicity of isolated compounds against MCF-7, A549, and HeLa cancer cell lines, antioxidant, α- glucosidase, and tyrosinase inhibitory activities were evaluated. The results revealed moderate cytotoxic effects against MCF-7 cells ( 2 – 4 ), A549 cells ( 1 – 4 ), and HeLa cells ( 1 – 5 ), while compounds 6 – 8 , 10 , and 11 showed no activity in any of the cell lines tested. Compound 1 exhibited more potent antioxidant activity and tyrosinase inhibitory properties than the positive control. Meanwhile, compound 4 showed a significant inhibitory effect on the α- glucosidase. In silico study, the molecular docking simulation of compound 1 to antioxidant (3RP8) and tyrosinase proteins (2Y9X), and of compound 4 to MCF-7 (1H7K), A549 cells (4ZXT), and α -glucosidase proteins (MAL32) were performed to calculate their binding affinities and to identify the ligand-binding sites. Excoecaria cochinchinensis. Cytotoxicity. α-Glucosidase inhibitory. Antioxidant. Antityrosinase Figures Figure 1 Figure 2 Figure 3 Introduction The Euphorbiaceae family is a large group of higher plants that contains around 300 genera and 7,500 species. This family is primarily found in tropical regions (Vasas and Hohmann 2014 ). One notable genus within this family is Excoecaria , which comprises about 40 species in tropical Africa, Asia, and the Western Pacific (Karamsetty and Aluri 2018 ). In recent years, natural products derived from medicinal plants have attracted increasing attention as promising sources of therapeutic agents, particularly for cancer treatment and metabolic disorders (Rojas-Castano et al. 2024 ; Manjia et al. 2024). Excoecaria cochinchinensis Lour., belonging to the Euphorbiaceae family, is prevalent across Vietnam and was recognized as a traditional medicine plant to treat furuncles, pruritus, prolonged diarrhoea, urethrorrhagia, and dysentery (Giang et al. 2005 ; Hieu et al. 2020). The phytochemical studies on E. cochinchinensis have confirmed the presence of diverse secondary metabolites such as megastigmanes (Giang et al. 2005 ), diterpenoids (Yang et al. 2005 ), triterpenoids (Chen and Wang 2015 ), lignans (Hieu et al. 2020; Le et al. 2025 ), flavonoids (Li 2006), and steroids (Wang et al. 2009 ). Researchers have focused on the biological activities of extracts from this plant. In particular, ethanol extract from the leaves of E. cochinchinensis has been shown to inhibit the growth of MKN45 gastric cancer cells (Hung 2021 ), while the methanol extract has demonstrated potential antibacterial activity (Nurhasana et al. 2023 ). Additionally, water and ethanol extracts exhibited notable anti-inflammatory properties (Jiangcun et al. 2020 ). Our previous studies reported a series of enzyme inhibitions of lignans and flavonoids from the ethyl acetate extract of this plant (Le et al. 2025 ). The current work reports the isolation and biological activities of eleven known compounds ( 1 – 11 ) from E. cochinchinensis . Their structures were determined through comprehensive spectroscopic analysis and comparison with structures reported in the previous data. All the compounds were identified as triterpenoids, sterols, phenolic compounds, and dipeptides. Materials and Methods The aerial parts of E. cochinchinensis were collected and identified as detailed in our previous report (Le et al. 2025 ). The aerial parts of E. cochinchinensis (20.0 kg) were air-dried and ground into powder, which was then extracted with methanol (50 L, three times) at room temperature. After evaporating the solvent under vacuum, the residues were combined and suspended in water. The mixture was then sequentially partitioned with n -hexane, a mixture of n -hexane and ethyl acetate (1:1), and ethyl acetate. This extraction process resulted in three fractions: H (70.0 g), HE (255.0 g), and EA (217.0 g). The HE extract (255.0 g) underwent silica gel column chromatography, eluted with the gradient solvent system of n -hexane:EtOAc (from 10:1 to 1:1), to afford six fractions (HE1–HE6). Fraction HE2 (14.2 g) was further fractionated through silica gel column chromatography, using a solvent mixture of n -hexane:CH 2 Cl 2 :acetone (5:1:0.5), resulting in five subfractions (HE2.1–HE2.5). Subfraction HE2.2 (8.9 g) was subsequently fractionated by a Sephadex LH-20 gel column with a solvent mixture of CH 2 Cl 2 and MeOH (1:1), yielding three subfractions (HE2.2.1–HE2.2.3). Compounds 1 (7.0 mg), 2 (6.0 mg), 3 (14.0 mg), and 7 (7.5 mg) were isolated from HE2.2.2 (1.2 g) by employing silica gel chromatography with n -hexane:CH 2 Cl 2 :acetone (2:1:0.2). Subfraction HE3 (3.7 g) yielded compounds 4 (4.5 mg), 5 (10.5 mg), 6 (2.0 mg), and 8 (9.0 mg) via successive silica gel separations using n -hexane:EtOAc (4:1) and n -hexane:CH 2 Cl 2 :acetone (2:1:0.5). From HE5 (4.1 g), compounds 9 (2.0 mg), 10 (10.0 mg), and 11 (6.5 mg) were obtained using CH 2 Cl 2 :MeOH (4:0.1), followed by RP-C18 CC (MeOH:H 2 O 1:1, 2:1 and 4:1). The cytotoxic properties of the isolated compounds were assessed against MCF-7 and HeLa cancer cell lines utilizing the sulforhodamine B (SRB) assay (Monks et al. 1991) by established protocols (Tran et al. 2021 ). The DPPH radical scavenging activity was evaluated to identify antioxidants among the isolated compounds, in alignment with the methodology presented by Kim et al. (Kim, Son, and Lee 2017 ). The inhibitory effect of the isolated compounds on α -glucosidase activity was assessed following the procedure outlined by Duong et al. (Duong et al. 2024). The inhibition of tyrosinase activity was conducted according to the method of Chatatikun et al. (Chatatikun et al. 2023), using kojic acid as the positive control. Molecular Docking Molecular docking was utilized to assess protein-ligand binding affinities and determine ligand-binding sites (Nguyen et al. 2019 ; Trott and Olson 2010 ), which provided an enhanced understanding of biological characteristics. The docking simulations were conducted using AutoDock Vina (Trott and Olson 2010 ). Since the crystallographic structure of α -glucosidase of Saccharomyces cerevisiae was unavailable, the 3D model designated AF-P38158-F1 (MAL32), was retrieved from AlphaFold Protein Structure Database ( https://alphafold.ebi.ac.uk/entry/P38158 ). This model was validated as the best representation of S. cerevisiae -derived α -glucosidase prior to the availability of its crystal structure (Tian et al. 2023 ). Crystal structures of other target proteins were retrieved from the RCSB Protein Data Bank ( https://www.rcsb.org/pdb/ ). For cytotoxicity assessments, isolated compounds were evaluated against multiple cancer cell lines with associated protein structures: A549 (PDB ID: 4ZXT) (Shin et al. 2016 ) and MCF-7 (PDB ID: 1H7K) (Andreoletti et al. 2001). The crystal structure of Klebsiella pneumoniae R204Q HpxO complexed with FAD (PDB ID: 3RP8) (Hicks et al. 2013 ) and mushroom tyrosinase structure (PDB ID: 2Y9X) (Ismaya et al. 2011 ) were also selected. Prior to molecular docking, each protein crystal structure was refined by removing water molecules, heteroatoms, and cofactors. The docking grid box was set to 23 Å × 23 Å × 23 Å for PDB IDs 2Y9X and 3RP8, and 26 Å × 26 Å × 26 Å for PDB IDs 1H7K, 4ZXT, and MAL32 (AlphaFold ID: AF-P38158-F1). The molecular docking outcomes were visualized using Biovia Discovery Studio Visualizer 2021 (Systèmes 2021 ). Results and Discussion The chemical examination of the methanol extract from E. cochinchinensis led to the isolation of eleven compounds. Their structures were established through NMR analysis and comparing with previous references, identified as cycloart-25-ene-3 \(\:\beta\:\) ,24-diol ( 1 ) (Paula et al. 2002 ), euphorfistrine A ( 2 ) (Wei et al. 2021 ), lupeol ( 3 ) (Shwe et al. 2019 ), baccatin ( 4 ) (Wang, Zhang, and Pan 2003 ), ergosterol peroxide ( 5 ) (Nowak et al. 2016 ), 7 \(\:\beta\:\) -hydroxysitosterol ( 6 ) (Li, Wang, and Li 2005 ), 7 \(\:\alpha\:\) -hydroxysitosterol ( 7 ) (Li, Wang, and Li 2005 ), 7-oxo- \(\:\beta\:\) -sitosterol ( 8 ) (Pettit et al. 2000), gallic acid ( 9 ) (Hernández-García et al. 2019), emodin ( 10 ) (Chu, Sun, and Liu 2005 ), and aurantiamide acetate ( 11 ) (Zhou et al. 2017 ), as shown in Fig. 1 . Notably, all compounds, except for 3 , 4 , and 9 , were isolated from E. cochinchinensis for the first time. Among these isolated compounds, 1 – 8 , and 10–11 , including triterpenoids ( 1–4 ), steroids ( 5–8 ), anthraquinone ( 10 ), and dipeptide ( 11 ) were selected for biological activity investigation on cytotoxicity against human cancer cell lines MCF-7, A549, and HeLa, antioxidant, and enzyme inhibition against α -glucosidase and tyrosinase which were presented in Table 1 . Table 1 The cytotoxicity against MCF-7, A549, and HeLa cancer cell lines, antioxidant, and enzyme inhibition against α -glucosidase and tyrosinase of the tested compounds. Compounds IC 50 (µM) Cancer cell lines Antioxidant Enzyme inhibition MCF-7 A549 Hela DPPH α -glucosidase Tyrosinase 1 > 100 95.39 ± 3.06 61.08 ± 3.31 69.34 > 200 182.26 2 67.72 ± 3.28 59.80 ± 3.34 41.29 ± 1.26 > 200 > 200 > 300 3 92.01 ± 4.35 89.31 ± 3.68 67.32 ± 1.76 > 200 > 200 > 300 4 31.88 ± 2.68 45.17 ± 3.05 60.68 ± 3.73 163.66 155.08 > 300 5 > 100 > 100 54.50 ± 2.89 > 200 > 200 > 300 6 > 100 > 100 > 100 > 200 > 200 293.18 7 > 100 > 100 > 100 > 200 194.65 > 300 8 > 100 > 100 > 100 178.63 > 200 > 300 10 > 100 > 100 > 100 > 200 > 200 > 300 11 > 100 > 100 > 100 > 200 > 200 195.75 Ellipticine 0.33 ± 0.02 0.37 ± 0.03 0.30 ± 0.03 Ascorbic acid 82.33 Acarbose 168.0 Kojic acid 251.28 The cytotoxicity was evaluated using the sulforhodamine B assay, with ellipticine as the positive control. Compounds 1 – 5 displayed moderate cytotoxic activity against these cell lines. Notably, compound 4 exhibited the strongest cytotoxicity against the MCF-7 and A549 cell lines, with IC 50 values of 31.88 ± 2.68 µM and 45.17 ± 3.05 µM, respectively, followed by compounds 2 and 3 . Compound 2 showed the most significant effect for the HeLa cell line, with an IC 50 value of 41.29 ± 1.26 µM. In contrast, compounds 6 – 8 , 10 , and 11 showed no inhibitory effects on all cancer cell lines. Regarding the antioxidants, compounds 1, 4 , and 8 displayed effectiveness in the DPPH assay with IC 50 values in the range of 69–179 µM. Among them, 1 revealed the most potent activity with an IC 50 value of 69.34 µM in comparison with the positive control (ascorbic acid, IC 50 82.33 µM). Moreover, in the enzyme inhibition, compounds 4 and 7 (IC 50 value of 155.08 and 194.65 µM, respectively) showed good α -glucosidase inhibition, while 1 and 11 (IC 50 value of 182.26 and 195.75 µM, respectively) revealed good tyrosinase inhibitory activity, in comparison with those of acarbose (IC 50 value of 168.0 µM) and kojic acid (IC 50 value of 251.28 µM), respectively. Furthermore, the above results also indicated that the triterpenoids ( 1 – 4 ) displayed the most potent activities compared to those of steroids ( 5–8 ) and anthraquinone ( 10 ). Among the isolated triterpenoids, the cycloartane-type triterpenoid ( 1 ) showed more potential activity in the DPPH and tyrosinase assay, while nor-triterpene peroxide ( 4 ) revealed more potential activity in the cytotoxicity against MCF-7 and A549 cancer cell lines and α -glucosidase inhibition. To further understand the conformation of the most active compound for each bioassay in the target binding site of their proteins, the binding interactions of 1 with the tyrosinase (2Y9X) and DPPH (3RP8) proteins and of 4 with MCF-7 (1H7K), A549 (4ZXT), and α -glucosidase (MAL32) proteins were studied using molecular docking simulation via vina-docking. In the tyrosinase inhibition and antioxidant activities, compound 1 exhibited one H-bonding to each protein as an H-donor from 3-OH to 2Y9X (with Asn B:81, 2.9 Å) and 3RP8 proteins (with Ser A:156, 2.2 Å) (Fig. 2 , Table S1 ), indicating the importance of the 3-OH moiety in the cycloartane triterpenoids to the respective activities. Moreover, the presence of carbon-carbon double bond moiety at C-25-C-26 led to the formation of H- α (H-24 and H 3 -27), due to the hyper-conjugation effect, which can form the carbon-hydrogen bond (H-24 with Asn 260 of 2Y9X and with Tyr 216 of 3RP8) or pi-sigma bond (H 3 -27-His 263) and pi-alkyl bonds (H 3 -27-His 259 and H 3 -27-Val 283) of tyrosinase protein (2Y9X). Therefore, the presence of the C-25–C-26 double bond in the cycloartane structure may significantly contribute to its tyrosinase inhibitory and antioxidant activities compared to those of a related 3-hydroxycycloartane ( 2 ), lacking this feature. As to the cytotoxicity, compound 4 interacted with the active site of the MCF-7 (1H7K) and A549 (4ZXT) proteins with affinity values of − 9.0 kcal/mol for each protein (Table S1 ), explaining why this compound was the most active on the cytotoxicity against these cancer cell lines. The data was further supported by the formation of two hydrogen bonds of 4 with Asp 339 (2.4 Å) and Ser 336 (3.1 Å), for 1H7K, and two with Ser 153 (2.5 Å) and Lys 114 (2.7 Å), for 4ZXT (Fig. 3 ). In two hydrogen bonds of 4 with 1H7K or 4ZXT, one of these is H-donor ( 4 -Asp 339 or 4 -Ser 153, respectively), and another is H-acceptor ( 4 -Ser 336 or 4 -Lys 114, respectively), indicating that the highly oxygenated function could display more potential activity on the cytotoxicity. In addition, compound 4 formed one hydrogen bond (with Phe 157, 2.7 Å) with MAL32 as an H-donor, indicating the significant role of the hydroxyl moiety on the A-ring of nor-triterpene peroxide in inhibiting the enzyme α -glucosidase activity. Conclusion The phytochemical analysis of the aerial parts of E. cochinchinensis resulted in the isolation and identification of eleven compounds comprising triterpenoids, sterols, phenolic compounds, and a dipeptide. Notably, eight of these compounds were reported for the first time in this species. Their structures were determined through extensive spectroscopic analysis. Biological evaluations showed that the triterpenoids (compounds 1 – 4 ) exhibited moderate cytotoxicity against MCF-7 and HeLa cancer cell lines, with compound 4 presenting the most potent cytotoxic activity. Additionally, compound 1 displayed significant antioxidant and tyrosinase inhibitory activities, surpassing standard controls. Compounds 4 and 7 also illustrated considerable α -glucosidase inhibition. Molecular docking studies confirmed these findings by highlighting the important interactions that contribute to the observed bioactivities. Declarations Supplementary Information The online version contains supplementary material available at Acknowledgements We acknowledge Nguyen Tat Thanh University, Ho Chi Minh City, Vietnam for supporting this study. Author Contributions Isolation and purification: NVK, PDSN, VHGL; Biological tests: PTTM, BXH; Structure identification: NVK, LTTH, THNK; Molecular docking: HLTTT, TTN; Writing, review, and editing: NVK, PHVT, LLTH. All authors read and approved the manuscript. Funding This research is funded by the Viet Nam Ministry of Education and Training under grant number B2024-SPS-06. Project title: “Khao sat thanh phan hoa hoc va nghien cuu hoat tinh chong oxy hoa, hoat tinh khang mot so dong te bao ung thu cua cay don la do ( Excoecaria cochinchinensis Lour.)”. Data Availability All data generated or analyzed during this study are included in this published article. Ethics approval Not applicable. Competing interests The authors declare no competing interests. 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J Ethnopharmacol 199:60–67. https://doi.org/10.1016/j.jep.2017.01.038 Supplementary Files Graphicalabstract.png SupportingInformation.pdf Cite Share Download PDF Status: Published Journal Publication published 19 Dec, 2025 Read the published version in Revista Brasileira de Farmacognosia → Version 1 posted Reviewers agreed at journal 04 Sep, 2025 Reviewers invited by journal 04 Sep, 2025 Editor invited by journal 08 Aug, 2025 Editor assigned by journal 01 Aug, 2025 First submitted to journal 01 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7268944","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":510317098,"identity":"fbdea594-15ed-44ac-94f7-f52920f726cd","order_by":0,"name":"Van-Kieu Nguyen","email":"","orcid":"","institution":"Nguyen Tat Thanh University","correspondingAuthor":false,"prefix":"","firstName":"Van-Kieu","middleName":"","lastName":"Nguyen","suffix":""},{"id":510317099,"identity":"06469938-bb06-422c-99e4-9aa46ec26f3a","order_by":1,"name":"Phan-Si-Nguyen Dong","email":"","orcid":"","institution":"Duy Tan University: Dai Hoc Duy Tan","correspondingAuthor":false,"prefix":"","firstName":"Phan-Si-Nguyen","middleName":"","lastName":"Dong","suffix":""},{"id":510317100,"identity":"e93b13f1-e137-42e7-82ac-4e28aa0f1208","order_by":2,"name":"Hoang-Vinh-Truong Phan","email":"","orcid":"","institution":"Duy Tan University: Dai Hoc Duy Tan","correspondingAuthor":false,"prefix":"","firstName":"Hoang-Vinh-Truong","middleName":"","lastName":"Phan","suffix":""},{"id":510317101,"identity":"daa04d17-a98a-4030-8a89-f91c3cf53ac4","order_by":3,"name":"Thanh-Nha Tran","email":"","orcid":"","institution":"Thu Dau Mot University","correspondingAuthor":false,"prefix":"","firstName":"Thanh-Nha","middleName":"","lastName":"Tran","suffix":""},{"id":510317102,"identity":"3bbc6c05-5d05-40ba-9e2a-e436147b3ba5","order_by":4,"name":"Le-Thuy-Thuy-Trang Hoang","email":"","orcid":"","institution":"Ton Duc Thang University","correspondingAuthor":false,"prefix":"","firstName":"Le-Thuy-Thuy-Trang","middleName":"","lastName":"Hoang","suffix":""},{"id":510317103,"identity":"cf86db08-1176-4462-93e8-2e4885e6e0e7","order_by":5,"name":"Thi-Tuyet-Minh Phan","email":"","orcid":"","institution":"Ho Chi Minh City University of Education","correspondingAuthor":false,"prefix":"","firstName":"Thi-Tuyet-Minh","middleName":"","lastName":"Phan","suffix":""},{"id":510317104,"identity":"e006f2fd-e85b-4e4c-8b29-e9d7462618f4","order_by":6,"name":"Hoang-Gia-Lac Vo","email":"","orcid":"","institution":"Ho Chi Minh City University of Education","correspondingAuthor":false,"prefix":"","firstName":"Hoang-Gia-Lac","middleName":"","lastName":"Vo","suffix":""},{"id":510317105,"identity":"ae93bee5-736d-4de3-ae74-549f9dac315b","order_by":7,"name":"Xuan-Hao Bui","email":"","orcid":"","institution":"Ho Chi Minh City University of Education","correspondingAuthor":false,"prefix":"","firstName":"Xuan-Hao","middleName":"","lastName":"Bui","suffix":""},{"id":510317106,"identity":"5bcc68b3-12c2-4f0a-8c4c-07a04c6f6f24","order_by":8,"name":"Khanh Huynh Nguyen Tran","email":"","orcid":"","institution":"Vietnam National University Ho Chi Minh City University of Science: University of Science","correspondingAuthor":false,"prefix":"","firstName":"Khanh","middleName":"Huynh Nguyen","lastName":"Tran","suffix":""},{"id":510317107,"identity":"4ba614d8-9e67-4e39-ba4c-dfe4b1ab174b","order_by":9,"name":"Huong Thi Thu Le","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIiWNgGAWjYBACAxDB2AAk2BsYmEnSIsHAc4BkLRIJRGoxl0g+9vDrDrs6c8k3hp8LKmwY+Nu7E/BqsZyRlm4seyZZwnJ2jrH0jDNpDBJnzm7A77AbOWbSkm0HJAxu5xhI87YdZjCQyCVWy80zxr+J1iL5EaTlBo8ZkbaceZYmzdiWLLnhTFqZNc+ZNB7CfjmefEzyZ5sdv8Hxw5tv81TYyPG39+LXAgLMPGCKAxxHPASVgwDjDzDF/oAo1aNgFIyCUTDyAACdz0WHunw8hwAAAABJRU5ErkJggg==","orcid":"","institution":"Ho Chi Minh City University of Education","correspondingAuthor":true,"prefix":"","firstName":"Huong","middleName":"Thi Thu","lastName":"Le","suffix":""}],"badges":[],"createdAt":"2025-08-01 08:20:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7268944/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7268944/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s43450-025-00731-y","type":"published","date":"2025-12-19T15:57:15+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":91121260,"identity":"8f790ec8-cf97-4760-969a-8b769f88b25b","added_by":"auto","created_at":"2025-09-11 19:08:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":38518,"visible":true,"origin":"","legend":"\u003cp\u003eStructures of isolated compounds \u003cstrong\u003e1\u003c/strong\u003e–\u003cstrong\u003e11\u003c/strong\u003e from\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eE. cochinchinensis\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7268944/v1/620be74a8a95008f6d4e05af.png"},{"id":91122284,"identity":"467f34e8-c748-4187-afc2-9fd67d28b91d","added_by":"auto","created_at":"2025-09-11 19:16:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":66925,"visible":true,"origin":"","legend":"\u003cp\u003eCorresponding amino acid residues of tyrosinase (2Y9X) and DPPH (3RP8) proteins interacted with \u003cstrong\u003e1\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7268944/v1/aabd7b8cb51419badcf6e648.png"},{"id":91121264,"identity":"94635040-8ecc-40f9-82e1-701cc5f967f2","added_by":"auto","created_at":"2025-09-11 19:08:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":83127,"visible":true,"origin":"","legend":"\u003cp\u003eCorresponding amino acid residues of MCF-7 (1H7K), A549 (4ZXT), and \u003cem\u003eα\u003c/em\u003e-glucosidase (MAL 32) proteins interacted with \u003cstrong\u003e4\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7268944/v1/116972b6f5888ec1218ad4a5.png"},{"id":98813937,"identity":"beec46ad-12c5-423c-8be2-822ec880f220","added_by":"auto","created_at":"2025-12-22 16:08:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":905115,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7268944/v1/8b8ba476-17e1-4a9d-8034-7b2a5cdad09f.pdf"},{"id":91121266,"identity":"ac9ae2b6-704b-4641-91ea-099141241bad","added_by":"auto","created_at":"2025-09-11 19:08:56","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":275166,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.png","url":"https://assets-eu.researchsquare.com/files/rs-7268944/v1/dda635aefb27f2d39940ed75.png"},{"id":91122287,"identity":"44133ff0-4bfd-49a4-9263-339b2d9fb9d2","added_by":"auto","created_at":"2025-09-11 19:16:56","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2440562,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7268944/v1/9eaf72b7d3aed73cbf526179.pdf"}],"financialInterests":"","formattedTitle":"Chemical Constituents of Excoecaria cochinchinensis Exhibiting Cytotoxic, Antioxidant, Antityrosinase, and α-Glucosidase Inhibitory Activities","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe Euphorbiaceae family is a large group of higher plants that contains around 300 genera and 7,500 species. This family is primarily found in tropical regions (Vasas and Hohmann \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). One notable genus within this family is \u003cem\u003eExcoecaria\u003c/em\u003e, which comprises about 40 species in tropical Africa, Asia, and the Western Pacific (Karamsetty and Aluri \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn recent years, natural products derived from medicinal plants have attracted increasing attention as promising sources of therapeutic agents, particularly for cancer treatment and metabolic disorders (Rojas-Castano et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Manjia et al. 2024). \u003cem\u003eExcoecaria cochinchinensis\u003c/em\u003e Lour., belonging to the Euphorbiaceae family, is prevalent across Vietnam and was recognized as a traditional medicine plant to treat furuncles, pruritus, prolonged diarrhoea, urethrorrhagia, and dysentery (Giang et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Hieu et al. 2020). The phytochemical studies on \u003cem\u003eE. cochinchinensis\u003c/em\u003e have confirmed the presence of diverse secondary metabolites such as megastigmanes (Giang et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), diterpenoids (Yang et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), triterpenoids (Chen and Wang \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), lignans (Hieu et al. 2020; Le et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), flavonoids (Li 2006), and steroids (Wang et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eResearchers have focused on the biological activities of extracts from this plant. In particular, ethanol extract from the leaves of \u003cem\u003eE. cochinchinensis\u003c/em\u003e has been shown to inhibit the growth of MKN45 gastric cancer cells (Hung \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), while the methanol extract has demonstrated potential antibacterial activity (Nurhasana et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Additionally, water and ethanol extracts exhibited notable anti-inflammatory properties (Jiangcun et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Our previous studies reported a series of enzyme inhibitions of lignans and flavonoids from the ethyl acetate extract of this plant (Le et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The current work reports the isolation and biological activities of eleven known compounds (\u003cb\u003e1\u003c/b\u003e\u0026ndash;\u003cb\u003e11\u003c/b\u003e) from \u003cem\u003eE. cochinchinensis\u003c/em\u003e. Their structures were determined through comprehensive spectroscopic analysis and comparison with structures reported in the previous data. All the compounds were identified as triterpenoids, sterols, phenolic compounds, and dipeptides.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eThe aerial parts of \u003cem\u003eE. cochinchinensis\u003c/em\u003e were collected and identified as detailed in our previous report (Le et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The aerial parts of \u003cem\u003eE. cochinchinensis\u003c/em\u003e (20.0 kg) were air-dried and ground into powder, which was then extracted with methanol (50 L, three times) at room temperature. After evaporating the solvent under vacuum, the residues were combined and suspended in water. The mixture was then sequentially partitioned with \u003cem\u003en\u003c/em\u003e-hexane, a mixture of \u003cem\u003en\u003c/em\u003e-hexane and ethyl acetate (1:1), and ethyl acetate. This extraction process resulted in three fractions: H (70.0 g), HE (255.0 g), and EA (217.0 g). The HE extract (255.0 g) underwent silica gel column chromatography, eluted with the gradient solvent system of \u003cem\u003en\u003c/em\u003e-hexane:EtOAc (from 10:1 to 1:1), to afford six fractions (HE1\u0026ndash;HE6). Fraction HE2 (14.2 g) was further fractionated through silica gel column chromatography, using a solvent mixture of \u003cem\u003en\u003c/em\u003e-hexane:CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e:acetone (5:1:0.5), resulting in five subfractions (HE2.1\u0026ndash;HE2.5). Subfraction HE2.2 (8.9 g) was subsequently fractionated by a Sephadex LH-20 gel column with a solvent mixture of CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e and MeOH (1:1), yielding three subfractions (HE2.2.1\u0026ndash;HE2.2.3). Compounds \u003cb\u003e1\u003c/b\u003e (7.0 mg), \u003cb\u003e2\u003c/b\u003e (6.0 mg), \u003cb\u003e3\u003c/b\u003e (14.0 mg), and \u003cb\u003e7\u003c/b\u003e (7.5 mg) were isolated from HE2.2.2 (1.2 g) by employing silica gel chromatography with \u003cem\u003en\u003c/em\u003e-hexane:CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e:acetone (2:1:0.2). Subfraction HE3 (3.7 g) yielded compounds \u003cb\u003e4\u003c/b\u003e (4.5 mg), \u003cb\u003e5\u003c/b\u003e (10.5 mg), \u003cb\u003e6\u003c/b\u003e (2.0 mg), and \u003cb\u003e8\u003c/b\u003e (9.0 mg) \u003cem\u003evia\u003c/em\u003e successive silica gel separations using \u003cem\u003en\u003c/em\u003e-hexane:EtOAc (4:1) and \u003cem\u003en\u003c/em\u003e-hexane:CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e:acetone (2:1:0.5). From HE5 (4.1 g), compounds \u003cb\u003e9\u003c/b\u003e (2.0 mg), \u003cb\u003e10\u003c/b\u003e (10.0 mg), and \u003cb\u003e11\u003c/b\u003e (6.5 mg) were obtained using CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e:MeOH (4:0.1), followed by RP-C18 CC (MeOH:H\u003csub\u003e2\u003c/sub\u003eO 1:1, 2:1 and 4:1).\u003c/p\u003e\u003cp\u003eThe cytotoxic properties of the isolated compounds were assessed against MCF-7 and HeLa cancer cell lines utilizing the sulforhodamine B (SRB) assay (Monks et al. 1991) by established protocols (Tran et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The DPPH radical scavenging activity was evaluated to identify antioxidants among the isolated compounds, in alignment with the methodology presented by Kim et al. (Kim, Son, and Lee \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The inhibitory effect of the isolated compounds on \u003cem\u003eα\u003c/em\u003e-glucosidase activity was assessed following the procedure outlined by Duong et al. (Duong et al. 2024). The inhibition of tyrosinase activity was conducted according to the method of Chatatikun et al. (Chatatikun et al. 2023), using kojic acid as the positive control.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMolecular Docking\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMolecular docking was utilized to assess protein-ligand binding affinities and determine ligand-binding sites (Nguyen et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Trott and Olson \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), which provided an enhanced understanding of biological characteristics. The docking simulations were conducted using AutoDock Vina (Trott and Olson \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Since the crystallographic structure of \u003cem\u003eα\u003c/em\u003e-glucosidase of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e was unavailable, the 3D model designated AF-P38158-F1 (MAL32), was retrieved from AlphaFold Protein Structure Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://alphafold.ebi.ac.uk/entry/P38158\u003c/span\u003e\u003cspan address=\"https://alphafold.ebi.ac.uk/entry/P38158\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). This model was validated as the best representation of \u003cem\u003eS. cerevisiae\u003c/em\u003e-derived \u003cem\u003eα\u003c/em\u003e-glucosidase prior to the availability of its crystal structure (Tian et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Crystal structures of other target proteins were retrieved from the RCSB Protein Data Bank (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rcsb.org/pdb/\u003c/span\u003e\u003cspan address=\"https://www.rcsb.org/pdb/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). For cytotoxicity assessments, isolated compounds were evaluated against multiple cancer cell lines with associated protein structures: A549 (PDB ID: 4ZXT) (Shin et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and MCF-7 (PDB ID: 1H7K) (Andreoletti et al. 2001). The crystal structure of \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e R204Q HpxO complexed with FAD (PDB ID: 3RP8) (Hicks et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and mushroom tyrosinase structure (PDB ID: 2Y9X) (Ismaya et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) were also selected. Prior to molecular docking, each protein crystal structure was refined by removing water molecules, heteroatoms, and cofactors. The docking grid box was set to 23 \u0026Aring; \u0026times; 23 \u0026Aring; \u0026times; 23 \u0026Aring; for PDB IDs 2Y9X and 3RP8, and 26 \u0026Aring; \u0026times; 26 \u0026Aring; \u0026times; 26 \u0026Aring; for PDB IDs 1H7K, 4ZXT, and MAL32 (AlphaFold ID: AF-P38158-F1). The molecular docking outcomes were visualized using Biovia Discovery Studio Visualizer 2021 (Syst\u0026egrave;mes \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eThe chemical examination of the methanol extract from \u003cem\u003eE. cochinchinensis\u003c/em\u003e led to the isolation of eleven compounds. Their structures were established through NMR analysis and comparing with previous references, identified as cycloart-25-ene-3\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\beta\\:\\)\u003c/span\u003e\u003c/span\u003e,24-diol (\u003cb\u003e1\u003c/b\u003e) (Paula et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), euphorfistrine A (\u003cb\u003e2\u003c/b\u003e) (Wei et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), lupeol (\u003cb\u003e3\u003c/b\u003e) (Shwe et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), baccatin (\u003cb\u003e4\u003c/b\u003e) (Wang, Zhang, and Pan \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), ergosterol peroxide (\u003cb\u003e5\u003c/b\u003e) (Nowak et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), 7\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\beta\\:\\)\u003c/span\u003e\u003c/span\u003e-hydroxysitosterol (\u003cb\u003e6\u003c/b\u003e) (Li, Wang, and Li \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), 7\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\alpha\\:\\)\u003c/span\u003e\u003c/span\u003e-hydroxysitosterol (\u003cb\u003e7\u003c/b\u003e) (Li, Wang, and Li \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), 7-oxo-\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\beta\\:\\)\u003c/span\u003e\u003c/span\u003e-sitosterol (\u003cb\u003e8\u003c/b\u003e) (Pettit et al. 2000), gallic acid (\u003cb\u003e9\u003c/b\u003e) (Hern\u0026aacute;ndez-Garc\u0026iacute;a et al. 2019), emodin (\u003cb\u003e10\u003c/b\u003e) (Chu, Sun, and Liu \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), and aurantiamide acetate (\u003cb\u003e11\u003c/b\u003e) (Zhou et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Notably, all compounds, except for \u003cb\u003e3\u003c/b\u003e, \u003cb\u003e4\u003c/b\u003e, and \u003cb\u003e9\u003c/b\u003e, were isolated from \u003cem\u003eE. cochinchinensis\u003c/em\u003e for the first time.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAmong these isolated compounds, \u003cb\u003e1\u003c/b\u003e\u0026ndash;\u003cb\u003e8\u003c/b\u003e, and \u003cb\u003e10\u0026ndash;11\u003c/b\u003e, including triterpenoids (\u003cb\u003e1\u0026ndash;4\u003c/b\u003e), steroids (\u003cb\u003e5\u0026ndash;8\u003c/b\u003e), anthraquinone (\u003cb\u003e10\u003c/b\u003e), and dipeptide (\u003cb\u003e11\u003c/b\u003e) were selected for biological activity investigation on cytotoxicity against human cancer cell lines MCF-7, A549, and HeLa, antioxidant, and enzyme inhibition against \u003cem\u003eα\u003c/em\u003e-glucosidase and tyrosinase which were presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe cytotoxicity against MCF-7, A549, and HeLa cancer cell lines, antioxidant, and enzyme inhibition against \u003cem\u003eα\u003c/em\u003e-glucosidase and tyrosinase of the tested compounds.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eCompounds\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"6\" nameend=\"c7\" namest=\"c2\"\u003e\u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e (\u0026micro;M)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eCancer cell lines\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAntioxidant\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003eEnzyme inhibition\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMCF-7\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eA549\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHela\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eDPPH\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003eα\u003c/em\u003e-glucosidase\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eTyrosinase\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e95.39\u0026thinsp;\u0026plusmn;\u0026thinsp;3.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e61.08\u0026thinsp;\u0026plusmn;\u0026thinsp;3.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e69.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e182.26\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e67.72\u0026thinsp;\u0026plusmn;\u0026thinsp;3.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e59.80\u0026thinsp;\u0026plusmn;\u0026thinsp;3.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e41.29\u0026thinsp;\u0026plusmn;\u0026thinsp;1.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;300\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e92.01\u0026thinsp;\u0026plusmn;\u0026thinsp;4.35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e89.31\u0026thinsp;\u0026plusmn;\u0026thinsp;3.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e67.32\u0026thinsp;\u0026plusmn;\u0026thinsp;1.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;300\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e31.88\u0026thinsp;\u0026plusmn;\u0026thinsp;2.68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e45.17\u0026thinsp;\u0026plusmn;\u0026thinsp;3.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e60.68\u0026thinsp;\u0026plusmn;\u0026thinsp;3.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e163.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e155.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;300\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e54.50\u0026thinsp;\u0026plusmn;\u0026thinsp;2.89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;300\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e293.18\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e7\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e194.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;300\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e8\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e178.63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;300\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e10\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;300\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e11\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e195.75\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eEllipticine\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eAscorbic acid\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e82.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eAcarbose\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e168.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eKojic acid\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e251.28\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe cytotoxicity was evaluated using the sulforhodamine B assay, with ellipticine as the positive control. Compounds \u003cb\u003e1\u003c/b\u003e\u0026ndash;\u003cb\u003e5\u003c/b\u003e displayed moderate cytotoxic activity against these cell lines. Notably, compound \u003cb\u003e4\u003c/b\u003e exhibited the strongest cytotoxicity against the MCF-7 and A549 cell lines, with IC\u003csub\u003e50\u003c/sub\u003e values of 31.88\u0026thinsp;\u0026plusmn;\u0026thinsp;2.68 \u0026micro;M and 45.17\u0026thinsp;\u0026plusmn;\u0026thinsp;3.05 \u0026micro;M, respectively, followed by compounds \u003cb\u003e2\u003c/b\u003e and \u003cb\u003e3\u003c/b\u003e. Compound \u003cb\u003e2\u003c/b\u003e showed the most significant effect for the HeLa cell line, with an IC\u003csub\u003e50\u003c/sub\u003e value of 41.29\u0026thinsp;\u0026plusmn;\u0026thinsp;1.26 \u0026micro;M. In contrast, compounds \u003cb\u003e6\u003c/b\u003e\u0026ndash;\u003cb\u003e8\u003c/b\u003e, \u003cb\u003e10\u003c/b\u003e, and \u003cb\u003e11\u003c/b\u003e showed no inhibitory effects on all cancer cell lines. Regarding the antioxidants, compounds \u003cb\u003e1, 4\u003c/b\u003e, and \u003cb\u003e8\u003c/b\u003e displayed effectiveness in the DPPH assay with IC\u003csub\u003e50\u003c/sub\u003e values in the range of 69\u0026ndash;179 \u0026micro;M. Among them, \u003cb\u003e1\u003c/b\u003e revealed the most potent activity with an IC\u003csub\u003e50\u003c/sub\u003e value of 69.34 \u0026micro;M in comparison with the positive control (ascorbic acid, IC\u003csub\u003e50\u003c/sub\u003e 82.33 \u0026micro;M). Moreover, in the enzyme inhibition, compounds \u003cb\u003e4\u003c/b\u003e and \u003cb\u003e7\u003c/b\u003e (IC\u003csub\u003e50\u003c/sub\u003e value of 155.08 and 194.65 \u0026micro;M, respectively) showed good \u003cem\u003eα\u003c/em\u003e-glucosidase inhibition, while \u003cb\u003e1\u003c/b\u003e and \u003cb\u003e11\u003c/b\u003e (IC\u003csub\u003e50\u003c/sub\u003e value of 182.26 and 195.75 \u0026micro;M, respectively) revealed good tyrosinase inhibitory activity, in comparison with those of acarbose (IC\u003csub\u003e50\u003c/sub\u003e value of 168.0 \u0026micro;M) and kojic acid (IC\u003csub\u003e50\u003c/sub\u003e value of 251.28 \u0026micro;M), respectively. Furthermore, the above results also indicated that the triterpenoids (\u003cb\u003e1\u003c/b\u003e\u0026ndash;\u003cb\u003e4\u003c/b\u003e) displayed the most potent activities compared to those of steroids (\u003cb\u003e5\u0026ndash;8\u003c/b\u003e) and anthraquinone (\u003cb\u003e10\u003c/b\u003e). Among the isolated triterpenoids, the cycloartane-type triterpenoid (\u003cb\u003e1\u003c/b\u003e) showed more potential activity in the DPPH and tyrosinase assay, while nor-triterpene peroxide (\u003cb\u003e4\u003c/b\u003e) revealed more potential activity in the cytotoxicity against MCF-7 and A549 cancer cell lines and \u003cem\u003eα\u003c/em\u003e-glucosidase inhibition.\u003c/p\u003e\u003cp\u003eTo further understand the conformation of the most active compound for each bioassay in the target binding site of their proteins, the binding interactions of \u003cb\u003e1\u003c/b\u003e with the tyrosinase (2Y9X) and DPPH (3RP8) proteins and of \u003cb\u003e4\u003c/b\u003e with MCF-7 (1H7K), A549 (4ZXT), and \u003cem\u003eα\u003c/em\u003e-glucosidase (MAL32) proteins were studied using molecular docking simulation \u003cem\u003evia\u003c/em\u003e vina-docking.\u003c/p\u003e\u003cp\u003eIn the tyrosinase inhibition and antioxidant activities, compound \u003cb\u003e1\u003c/b\u003e exhibited one H-bonding to each protein as an H-donor from 3-OH to 2Y9X (with Asn B:81, 2.9 \u0026Aring;) and 3RP8 proteins (with Ser A:156, 2.2 \u0026Aring;) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), indicating the importance of the 3-OH moiety in the cycloartane triterpenoids to the respective activities. Moreover, the presence of carbon-carbon double bond moiety at C-25-C-26 led to the formation of H-\u003cem\u003eα\u003c/em\u003e (H-24 and H\u003csub\u003e3\u003c/sub\u003e-27), due to the hyper-conjugation effect, which can form the carbon-hydrogen bond (H-24 with Asn 260 of 2Y9X and with Tyr 216 of 3RP8) or pi-sigma bond (H\u003csub\u003e3\u003c/sub\u003e-27-His 263) and pi-alkyl bonds (H\u003csub\u003e3\u003c/sub\u003e-27-His 259 and H\u003csub\u003e3\u003c/sub\u003e-27-Val 283) of tyrosinase protein (2Y9X). Therefore, the presence of the C-25\u0026ndash;C-26 double bond in the cycloartane structure may significantly contribute to its tyrosinase inhibitory and antioxidant activities compared to those of a related 3-hydroxycycloartane (\u003cb\u003e2\u003c/b\u003e), lacking this feature.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAs to the cytotoxicity, compound \u003cb\u003e4\u003c/b\u003e interacted with the active site of the MCF-7 (1H7K) and A549 (4ZXT) proteins with affinity values of \u0026minus;\u0026thinsp;9.0 kcal/mol for each protein (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), explaining why this compound was the most active on the cytotoxicity against these cancer cell lines. The data was further supported by the formation of two hydrogen bonds of \u003cb\u003e4\u003c/b\u003e with Asp 339 (2.4 \u0026Aring;) and Ser 336 (3.1 \u0026Aring;), for 1H7K, and two with Ser 153 (2.5 \u0026Aring;) and Lys 114 (2.7 \u0026Aring;), for 4ZXT (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In two hydrogen bonds of \u003cb\u003e4\u003c/b\u003e with 1H7K or 4ZXT, one of these is H-donor (\u003cb\u003e4\u003c/b\u003e-Asp 339 or \u003cb\u003e4\u003c/b\u003e-Ser 153, respectively), and another is H-acceptor (\u003cb\u003e4\u003c/b\u003e-Ser 336 or \u003cb\u003e4\u003c/b\u003e-Lys 114, respectively), indicating that the highly oxygenated function could display more potential activity on the cytotoxicity. In addition, compound \u003cb\u003e4\u003c/b\u003e formed one hydrogen bond (with Phe 157, 2.7 \u0026Aring;) with MAL32 as an H-donor, indicating the significant role of the hydroxyl moiety on the A-ring of nor-triterpene peroxide in inhibiting the enzyme \u003cem\u003eα\u003c/em\u003e-glucosidase activity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe phytochemical analysis of the aerial parts of \u003cem\u003eE. cochinchinensis\u003c/em\u003e resulted in the isolation and identification of eleven compounds comprising triterpenoids, sterols, phenolic compounds, and a dipeptide. Notably, eight of these compounds were reported for the first time in this species. Their structures were determined through extensive spectroscopic analysis. Biological evaluations showed that the triterpenoids (compounds \u003cb\u003e1\u003c/b\u003e\u0026ndash;\u003cb\u003e4\u003c/b\u003e) exhibited moderate cytotoxicity against MCF-7 and HeLa cancer cell lines, with compound \u003cb\u003e4\u003c/b\u003e presenting the most potent cytotoxic activity. Additionally, compound \u003cb\u003e1\u003c/b\u003e displayed significant antioxidant and tyrosinase inhibitory activities, surpassing standard controls. Compounds \u003cb\u003e4\u003c/b\u003e and \u003cb\u003e7\u003c/b\u003e also illustrated considerable \u003cem\u003eα\u003c/em\u003e-glucosidase inhibition. Molecular docking studies confirmed these findings by highlighting the important interactions that contribute to the observed bioactivities.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe online version contains supplementary material available at\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge Nguyen Tat Thanh University, Ho Chi Minh City, Vietnam for supporting this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIsolation and purification: NVK, PDSN, VHGL; Biological tests: PTTM, BXH; Structure identification: NVK, LTTH, THNK; Molecular docking: HLTTT, TTN; Writing, review, and editing: NVK, PHVT, LLTH. All authors read and approved the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis research is funded by the Viet Nam Ministry of Education and Training under grant number B2024-SPS-06. Project title: \u0026ldquo;Khao sat thanh phan hoa hoc va nghien cuu hoat tinh chong oxy hoa, hoat tinh khang mot so dong te bao ung thu cua cay don la do (\u003cem\u003eExcoecaria cochinchinensis\u003c/em\u003e Lour.)\u0026rdquo;.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests. \u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAndreoletti P, Gambarelli S, Sainz G, Stojanoff V, White C, Desfonds G, Gagnon J, Gaillard J, Jouve HM (2001) Formation of a tyrosyl radical intermediate in \u003cem\u003eProteus mirabilis\u003c/em\u003e catalase by directed mutagenesis and consequences for nucleotide reactivity. 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J Trop Subtrop Bot 17:156\u0026ndash;159.\u003c/li\u003e\n\u003cli\u003eWei JC, Huang HH, Zhong NF, Gao YN, Liu XL, Long GQ, Hu GS, Wang AH, Jia JM (2021) Euphorfistrines A\u0026ndash;G, cytotoxic and AChE inhibiting triterpenoids from the roots of \u003cem\u003eEuphorbia fischeriana\u003c/em\u003e. Bioorg Chem 116:105395. https://doi.org/10.1016/j.bioorg.2021.105395\u003c/li\u003e\n\u003cli\u003eYang JH, Luo SD, Zhao JF, Wang YS, Huang R, Zhang HB, Li L (2005) Three new highly oxygenated diterpenoids from \u003cem\u003eExcoecaria cochinchinensis\u003c/em\u003e Lour. Helv Chim Acta 88:968\u0026ndash;973. https://doi.org/10.1002/hlca.200590090\u003c/li\u003e\n\u003cli\u003eZhou B, Yang Z, Feng Q, Liang X, Li J, Zanin M, Jiang Z, Zhong N (2017) Aurantiamide acetate from \u003cem\u003eBaphicacanthus cusia\u003c/em\u003e root exhibits anti-inflammatory and anti-viral effects via inhibition of the NF-\u0026kappa;B signaling pathway in Influenza A virus-infected cells. J Ethnopharmacol 199:60\u0026ndash;67. https://doi.org/10.1016/j.jep.2017.01.038\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"revista-brasileira-de-farmacognosia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbfa","sideBox":"Learn more about [Revista Brasileira de Farmacognosia](https://www.springer.com/journal/43450)","snPcode":"43450","submissionUrl":"https://www.editorialmanager.com/rbfa/default2.aspx","title":"Revista Brasileira de Farmacognosia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Excoecaria cochinchinensis. Cytotoxicity. α-Glucosidase inhibitory. Antioxidant. Antityrosinase","lastPublishedDoi":"10.21203/rs.3.rs-7268944/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7268944/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEleven known compounds, including four triterpenoids (\u003cb\u003e1\u003c/b\u003e\u0026ndash;\u003cb\u003e4\u003c/b\u003e), four sterols (\u003cb\u003e5\u003c/b\u003e\u0026ndash;\u003cb\u003e8\u003c/b\u003e), two phenolic compounds (\u003cb\u003e9\u003c/b\u003e\u0026ndash;\u003cb\u003e10\u003c/b\u003e), and a dipeptide (\u003cb\u003e11\u003c/b\u003e), were isolated from \u003cem\u003eExcoecaria cochinchinensis\u003c/em\u003e. Their structures were confirmed through spectroscopic analysis and compared with the existing literature. Eight compounds (\u003cb\u003e1\u003c/b\u003e, \u003cb\u003e2\u003c/b\u003e, \u003cb\u003e5\u003c/b\u003e\u0026ndash;\u003cb\u003e8\u003c/b\u003e, \u003cb\u003e10\u003c/b\u003e\u0026ndash;\u003cb\u003e11\u003c/b\u003e) have been identified in this species for the first time. The cytotoxicity of isolated compounds against MCF-7, A549, and HeLa cancer cell lines, antioxidant, \u003cem\u003eα-\u003c/em\u003eglucosidase, and tyrosinase inhibitory activities were evaluated. The results revealed moderate cytotoxic effects against MCF-7 cells (\u003cb\u003e2\u003c/b\u003e\u0026ndash;\u003cb\u003e4\u003c/b\u003e), A549 cells (\u003cb\u003e1\u003c/b\u003e\u0026ndash;\u003cb\u003e4\u003c/b\u003e), and HeLa cells (\u003cb\u003e1\u003c/b\u003e\u0026ndash;\u003cb\u003e5\u003c/b\u003e), while compounds \u003cb\u003e6\u003c/b\u003e\u0026ndash;\u003cb\u003e8\u003c/b\u003e, \u003cb\u003e10\u003c/b\u003e, and \u003cb\u003e11\u003c/b\u003e showed no activity in any of the cell lines tested. Compound \u003cb\u003e1\u003c/b\u003e exhibited more potent antioxidant activity and tyrosinase inhibitory properties than the positive control. Meanwhile, compound \u003cb\u003e4\u003c/b\u003e showed a significant inhibitory effect on the \u003cem\u003eα-\u003c/em\u003eglucosidase. In \u003cem\u003esilico\u003c/em\u003e study, the molecular docking simulation of compound \u003cb\u003e1\u003c/b\u003e to antioxidant (3RP8) and tyrosinase proteins (2Y9X), and of compound \u003cb\u003e4\u003c/b\u003e to MCF-7 (1H7K), A549 cells (4ZXT), and \u003cem\u003eα\u003c/em\u003e-glucosidase proteins (MAL32) were performed to calculate their binding affinities and to identify the ligand-binding sites.\u003c/p\u003e","manuscriptTitle":"Chemical Constituents of Excoecaria cochinchinensis Exhibiting Cytotoxic, Antioxidant, Antityrosinase, and α-Glucosidase Inhibitory Activities","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-11 19:08:51","doi":"10.21203/rs.3.rs-7268944/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-09-04T15:50:51+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-04T14:39:01+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Revista Brasileira de Farmacognosia","date":"2025-08-08T20:27:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-01T22:24:12+00:00","index":"","fulltext":""},{"type":"submitted","content":"Revista Brasileira de Farmacognosia","date":"2025-08-01T09:44:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"revista-brasileira-de-farmacognosia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbfa","sideBox":"Learn more about [Revista Brasileira de Farmacognosia](https://www.springer.com/journal/43450)","snPcode":"43450","submissionUrl":"https://www.editorialmanager.com/rbfa/default2.aspx","title":"Revista Brasileira de Farmacognosia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1f863c48-d165-45a5-9156-811eddef6c5b","owner":[],"postedDate":"September 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-22T16:01:16+00:00","versionOfRecord":{"articleIdentity":"rs-7268944","link":"https://doi.org/10.1007/s43450-025-00731-y","journal":{"identity":"revista-brasileira-de-farmacognosia","isVorOnly":false,"title":"Revista Brasileira de Farmacognosia"},"publishedOn":"2025-12-19 15:57:15","publishedOnDateReadable":"December 19th, 2025"},"versionCreatedAt":"2025-09-11 19:08:51","video":"","vorDoi":"10.1007/s43450-025-00731-y","vorDoiUrl":"https://doi.org/10.1007/s43450-025-00731-y","workflowStages":[]},"version":"v1","identity":"rs-7268944","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7268944","identity":"rs-7268944","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}