Anti-Leishmania activity of condensed tannins isolated from Mimosa tenuiflora and β-1,3-1,6 glucan isolated from commercial nutraceuticals

Anti-Leishmania activity of condensed tannins isolated from Mimosa tenuiflora and β-1,3-1,6 glucan isolated from commercial nutraceuticals | 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 Research Article Anti-Leishmania activity of condensed tannins isolated from Mimosa tenuiflora and β-1,3-1,6 glucan isolated from commercial nutraceuticals Everton dos Santos Gomes, Fernanda da Silva, Rafael Francisco Rosalem, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7335507/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract This study evaluated the anti-Leishmania activity of condensed tannins from Mimosa tenuiflora and β -glucan (1,3 − 1,6) from commercial nutraceuticals to identify less toxic alternatives against leishmaniasis, a neglected disease affecting millions in tropical and subtropical regions. Current treatments rely on toxic drugs, motivating research on natural compounds with leishmanicidal activity. M. tenuiflora is known for antimicrobial, antioxidant, and anti-inflammatory properties, while β-glucans are immunomodulatory polysaccharides capable of activating macrophages and enhancing pathogen control. Condensed tannins showed activity against promastigotes (IC₅₀ = 24.56 µg/mL) but were ineffective against intracellular amastigotes, suggesting action on extracellular structures, possibly via membrane destabilization, nucleic acid alteration, or enzyme inhibition. β-glucan did not affect promastigotes but significantly reduced internalized amastigotes (IC₅₀ = 17.94 µg/mL). Cytotoxicity was low for both compounds (CC₅₀ >1,000 µg/mL), resulting in high selectivity indices: >40 for tannins (promastigotes) and > 55 for β-glucan (amastigotes).The anti-amastigote activity of β-glucan appears linked to immunomodulation via receptors such as Dectin-1 and TLR2 on macrophages, stimulating reactive oxygen species and proinflammatory cytokines production, which control intracellular parasites.In conclusion, condensed tannins demonstrated limited potential, acting only on promastigotes, while β-glucan was effective against amastigotes, the pathogenic form of leishmaniasis, with excellent cytotoxic safety. β-glucan (1,3–1,6) emerges as a promising candidate for novel therapeutic strategies, warranting further investigation of its immunological mechanisms and in vivo efficacy. Natural products Bioprospecting Leishmaniasis Drug development Figures Figure 1 Figure 2 Figure 3 INTRODUCTION Leishmaniasis is a complex of infectious and parasitic diseases caused by protozoa of the genus Leishmania and has a wide clinical spectrum depending on the species involved in the infection (WHO 2023). According to the WHO (2023), leishmaniasis can be divided into cutaneous and mucocutaneous leishmaniasis, characterized by the formation of dermal ulcers, and visceral leishmaniasis, the more severe clinical form of the disease that can lead to death if untreated. Considered a serious public health problem because of its wide geographical distribution, this form of anthropozoonosis is endemic to 90 countries, with a high prevalence in tropical and subtropical regions, where it affects millions of people (PAHO). Currently, chemotherapy is the most effective way to prevent diseases and mortality; however, even with the availability of different therapeutic options, there are still limitations (Ezatpour et al. 2015; Mendonça and Antônio 2020). The therapeutic arsenal available for the treatment of leishmaniasis involves five drugs, pentavalent antimonials, amphotericin B, pentamidine, paramomycin and miltefosine, which are associated with a series of side effects and restrictions due to their cardiotoxic, hepatotoxic, and nephrotoxic profiles, among others (Gervazoni et al. 2020; Silva et al. 2021; Garza-Tovar et al. 2020; Roatt et al. 2020; Ghorbani and Farhoudi 2018). Furthermore, prolonged treatment is painful because of its invasive administration in most cases, which can culminate in treatment failure (Chappuis et al. 2007; Croft and Coombs 2003). Given the existing limitations, natural compounds appear to be promising sources of new molecules with leishmanicidal potential (Shen and Hao 2020; Garza-Torvan et al. 2020; Gervazoni et al. 2020). Among the botanical species with therapeutic potential is Mimosa tenuiflora (Willd). Pois., popularly known as jurema-preta, is widely distributed in the semiarid region of Brazil and is traditionally used in folk medicine for the healing of wounds, skin infections and inflammatory processes (Bezerra et al. 2010; Hernandez et al. 2021). Previous phytochemical studies have shown that the bark of this species is rich in condensed tannins, which are known for their medicinal properties (Crepaldi et al. 2021; Santos et al. 2022; Borges et al. 2017; Azevêdo et al. 2013). Previous in vitro studies have shown that condensed tannins are classified as phenolic compounds and demonstrate promising activity against pathogens (Monteiro et al. 2005; Pizzi 2021). These molecules can inhibit the growth of microorganisms through multiple mechanisms, including changes in nucleic acids, nutrient deprivation and destabilization of organelles important for cellular metabolism (Ucella Filho et al. 2022; Pizzi 2021). Furthermore, these molecules have antioxidant, anti-inflammatory and antimicrobial effects (Sanches et al. 2005). Given its ability to act as a pathogenic agent, future studies should investigate its anti -Leishmania activity for possible therapeutic improvement. In addition to phenolic compounds, bioactive polysaccharides such as β - glucans (1,3 − 1,6) are also of interest in the therapeutic development of infectious diseases. β -Glucans can stimulate and activate macrophages and other cells of the innate immune system, resulting in an improved response against various pathogens (Castro; Calder; Roche 2021; Camilli; Tobouret; Quintin 2018). The adjuvant and modulatory effects of this molecule on the immune system suggest its potential in the development of new therapeutic strategies for leishmaniasis or the improvement of existing therapies. In view of the above, the present study aimed to evaluate the anti- Leishmania activity of condensed tannins isolated from the bark of M. tenuiflora and β -glucan (1.3–1.6), aiming to contribute to the development of innovative and less toxic therapeutic alternatives for the treatment of leishmaniasis. MATERIALS AND METHODS Compounds studied Condensed tannins isolated from the bark of Mimosa tenuiflora The condensed tannins used in the present study were extracted by the Semi-Arid Research Center (NUPEÁRIDO), municipality of Patos, Paraíba-PB, at the domains of the Federal University of Campina Grande (UFCG). The material was subsequently purified at the Laboratory of Forestry Products Technology (LTPF) of the UFCG and transferred to the Federal University of Rondonópolis, Faculty of Health Sciences, for studies of its biological activity. The entire procedure was performed according to the methods established by Paes et al. (2010) and Pereira et al. (2015). β-glucan (1.3-1.6) For the present study, a commercial nutraceutical rich in β-glucans (26 g) and amino acids, Bionutri AR-1®, was used. This product is obtained through a single fermentation process and is free of additives and preservatives, factors that classify it as a product of biotechnological innovation. Evaluation of anti -Leishmania activity in vitro Parasites For in vitro analysis, a standard strain of L. (L.) amazonensis (IFLA/BR/1967/PH8) was used. The parasites were previously isolated from BALB/c mice previously infected and maintained in Schneider® medium (Sigma‒Aldrich®) supplemented with 20% fetal bovine serum (FBS; Nova Biotecnologia) and 1% antibiotics (penicillin and streptomycin; Sigma‒Aldrich®), with repetitions every three days until the 20th serial passage. Preparation of compounds Five micrograms (0.0050 g) of condensed tannins isolated from the bark of M. tenuiflora were diluted in 100 μL of dimethyl sulfoxide (DMSO). The nutraceutical used to evaluate the biological activity of β -glucan (1.3–1.60 on L. (L.) amazonensis was a dilution of 5 μg (0.0050 g) in 100 μL of phosphate-buffered saline solution (PBS). This dilution was selected because of the properties of the compound. Analysis of anti -Leishmania activity Anti-promastigote assay In vitro susceptibility tests were performed on the promastigote forms of L. (L.) amazonensis in the log phase of growth in appropriate supplemented Schneider® medium (Sigma‒Aldrich®). Growth inhibition was assessed by cell viability after the addition of MTT (3-(4-5-dimethylthiazol-2-yl)-2,5-diphenyltetrasodium bromide). In 96-well plates, 100 μL of culture at a concentration of 10 6 parasites/mL (19.75 parasites/mL) and 100 μL of the compounds to be tested were added at concentrations of 50–3.12 μL/mL. The plates were subsequently incubated at 26 °C for 72 h (Bosquiroli et al. 2015). After 72 h of incubation, 5 mg/mL MTT was added to each well, after which the plates were stored again at 37 °C and 5% CO2 for 4 h, after which DMSO was added to dissolve the formazan crystals, after which the absorbance was measured with a spectrophotometer at a wavelength of 540 nm. Anti-amastigote test To evaluate the effects of the compounds against the amastigote forms, peritoneal macrophages from BALB/c mice were used, which were attached to circular coverslips at the bottom of 24-well plates supplemented with RPMI and infected with L. amazonensis promastigotes (IFLA)/BR/1967/PH8). Afterward, the plates were stored at 37 °C with 5% CO2 (Oliveira 2021). After 24 h, the compounds of interest were added at concentrations of 50–3.12 μL/mL for another 24 h. The coverslips were then fixed with Boiun's solution, stained with Giemsa and mounted on microscope slides with Entellan to evaluate the number of amastigotes within 200 macrophages under light microscopy. Each concentration of extract was tested in sextuplicate (Oliveira 2021). The inhibitory concentration was calculated using linear regression, and the log10 of each inhibitor concentration was plotted in relation to the percentage activity with the software GraphPad Prism 5 (Oliveira 2021). Cytotoxicity assay To evaluate the cytotoxicity of each compound, NIH/3T3 cells (ATCC CRL-1658 mouse fibroblast lineage) were grown in 96-well plates (1x10 4 cells in 100 μL per well) in RPMI 1640 medium supplemented with penicillin, streptomycin and 10% fetal bovine serum for 12 h at 37 °C and 5% CO2 (Oliveira 2021). After 12 h, the medium was replaced with fresh RPMI medium containing different concentrations of each compound (100 to 3.125 μL/mL) in quintuplicate, and the plates were incubated for 24 h. The viability/life control was determined by fibroblasts grown only in culture medium (Oliveira 2021). Subsequently, 10 μL of resazurin was added to each well, followed by incubation for 4 h at 37 °C and 5% CO2. After incubation, 80 μL of dimethyl sulfoxide was added to dissolve the formazan crystals, and the absorbance was estimated with a spectrophotometer at a wavelength of 570 nm (Oliveira 2021). The cytotoxic concentration (CC 50 ) was calculated by linear regression, and the log10 of each inhibitor concentration was plotted against the percentage activity with GraphPad Prism 5 (Oliveira 2021). Statistical analysis The results are expressed as the concentration of growth inhibition at 50% (IC 50 ) and were determined by nonparametric linear regression. One-way ANOVA was subsequently performed, followed by Tukey's post hoc test for comparisons between groups. The results were obtained using the statistical software GraphPad Prism 5.01®. The significance level was set at 5%. RESULTS Among the compounds evaluated, the condensed tannins isolated from the bark of Mimosa tenuiflora inhibited the growth of promastigote forms of Leishmania (L.) amazonensis , with a 50% inhibitory concentration (CI 50 ) of 24.56 µg (Fig. 1 -A). However, β-glucan (1.3–1.6) was not able to inhibit parasite growth in the same assay, with an IC50 >50 µg (Fig. 1 -B). In the investigation of the activity on intracellular forms, a change in the profile of the biological activity of the compounds was observed, where β -glucan (1.3–1.6) was able to reduce the number of amastigotes internalized in murine macrophages, with an IC50 of 17.94 µg (Fig. 2 -B), whereas tannins did not reduce the number of amastigotes per macrophage (Fig. 2 -A). In terms of the cytotoxicity profile on murine fibroblasts, both compounds showed low cytotoxicity to NIH/3T3 cells (Fig. 3 ). In view of the results, it was possible to estimate the selectivity index (SI) of the compounds tested, where the condensed tannin presented an IS > 40.65 for the promastigote form, which was more selective for the parasite than for the cell, while β -glucan (1.3–1.6) was more selective for the amastigote form than for the cell, with an SI > 55.74 (Table 1 ). Table 1 Anti- Leishmania activity on the promastigote and amastigote forms of L. amazonensis and selectivity index. Compost IC 50 (µg/ml) of L. amazonensis promastigotes CC 50 (µg/ml) Murine NIH/3T3 fibroblasts IS promastigote L. amazonensis IC 50 (µg/ml) L. amazonensis amastigotes IS amastigotes L. amazonensis Condensed tannin 24,59 > 1.000 > 40,65 > 50 - B-glucano (1,3 − 1,6) > 50 > 1.000 - 17,94 > 55,74 DISCUSSION The present study is a preclinical evaluation of different compounds against the evolutionary forms of L. amazonensis , as well as their toxicity to 3T3 lineage cells. The in vitro evaluation of molecules on the different evolutionary forms of the parasite is essential, since amastigotes are the intracellular form involved in human pathogenesis, and the efficiency and toxicity profile of the molecule can be evaluated for later scaling up for in vitro studies. The results of the present study revealed that the condensed tannins isolated from the bark of M. tenuiflora significantly affected the promastigote form but were ineffective against the amastigote form. These results show that tannins directly affect structures present in or more exposed to the extracellular form of the parasite, such as surface components or specific metabolic processes of the promastigote. The mechanisms underlying the inhibition of promastigote growth have not been elucidated, but they may be the same as those in other microorganisms, such as bacteria and fungi, where this molecule inhibits growth through changes in nucleic acids, nutrient deprivation and destabilization of important organelles. in cellular metabolism (Molino et al. 2019; Huang et al. 2024). Previous studies have shown that tannins can interact with membrane proteins, forming complexes and destabilizing their integrity, resulting in cell death by lysis (Ferreira and Evangelista 2021; Molino et al. 2019). In addition, these molecules can inhibit key enzymes involved in the metabolism of some microorganisms, preventing essential functions and slowing their growth. Since the inhibition mechanisms act on biological processes of organisms outside the intracellular environment, it is possible to explain the lack of inhibitory effects on the amastigote forms of L. amazonensis , showing that tannin has difficulty crossing the host cell membrane or maintaining intracellular activity, a fundamental characteristic for molecules with leishmanicidal activity. In view of the above, the condensed tannins isolated from the bark of M. tenuiflora are more selective for promastigote forms than for amastigote forms, a factor that discards it as a molecule of interest in the prospect of anti- Leishmania compounds. On the other hand, β -glucan (1.3–1.6) showed a distinct and potentially promising profile, as it showed significant inhibitory activity on the amastigote forms of L. amazonensis and zero toxicity on NIH/3T3 murine cells. Furthermore, β -glucan (1.3–1.6) was not able to inhibit the growth of the promastigote forms of the parasite. The profile observed in the present study suggests a selective action of the compound on the amastigote form, which is the pathogenic form in humans and other mammals The selectivity observed for amastigotes may be related to the immunomodulatory effect of β -glucan (1,3 − 1,6). Previous studies report that this polysaccharide acts as a modulator of the innate immune response, interacting with recognition pattern receptors, such as Dectin-1 and Toll-like receptors, on phagocytic cells (Zimara et al. 2018; Brown et al. 2003; Yadav and Schorey 2006). This interaction results in increased production of reactive oxygen species and proinflammatory cytokines, factors that may contribute to the control of amastigotes internalized by macrophages (Castro; Calder; Roche 2021; Stothers et al. 2021). Although no direct activity was observed for the promastigote forms, the ability of β -glucan (1.3–1.6) to reduce the number of parasites in infected macrophages is indirect. These results are corroborated by those of Patidar et al. (2020), who reported a reduction in the parasite load in murine peritoneal macrophages infected with Leishmania donovani and treated with β -glucan. In the same study, in silico evaluation revealed that the effect was due to the interaction between the β -glucan of Saccharomyces cerevisiae and Dectin-1 and TRL2 receptors, both of which are expressed by macrophages and dendritic cells, leading to the activation of different intracellular signaling pathways (IRAK-4, IKKα, IκB, LyN and SyK). The absence of significant cytotoxicity to NIH/3T3 cells corroborates the findings of previous studies, which demonstrated that β -glucans are safe and biocompatible compounds and present a low risk to mammalian cells (Chan; Chan; Sze 2009). The results obtained reveal a relevant profile for the development of alternative therapies, considering the current limitations in the treatment of leishmaniasis, which culminate in therapeutic failure (Vetvicka and Fernandez-Botran 2018). When the results concerning β-glucan (1.3–1.6) are considered, it is possible to discard its direct mechanism of action on the parasite, since the absence of activity on the promastigote forms of L. amazonensis; however, if necessary, further assays are needed to evaluate parameters such as phagocytosis, nitric oxide production and expression of macrophage activation markers to elucidate its mechanism of action on the amastigote forms. In conclusion, the results highlight the importance of screening molecules with anti- Leishmania activity and investigating their action profiles on different evolutionary forms. Given its activity on amastigotes and low cytotoxicity, the investigated β -glucan (1.3–1.6) showed a more adequate profile for further studies, making it necessary to characterize the mechanisms involved and evaluate its efficacy in complex biological systems. Declarations Acknowledgments I thank all the researchers involved in this study and the funding agencies. Authors' contribution All the authors of the present study contributed significantly to the design and preparation of the study. Ethical aspects To accomplish the present study, all the procedures presented are in accordance with the principles adopted by the Brazilian Society of Laboratory Animal Sciences (SBCAL). Support This research is funded by the National Research Development Council (CNPq), a public funding agency. Conflict of interest I declare that there are no conflicts of interest between the authors and the funding agency. 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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-7335507","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":501061781,"identity":"c883209f-d80a-4c5b-b058-abe629164e8c","order_by":0,"name":"Everton dos Santos Gomes","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAElEQVRIiWNgGAWjYPCDChsgwdh4gKBChIozaSAtDSRoYWw7jGYGFsAvkXzs84eKO/by7t2JD3+wnbdb234YaEuNTTQuLZIz0pJnHDjzLHHjmbObjXl4bidvO5MI1HIsLbcBhxaDM2eMGQ62HU4wnJG7TZpB4nay2QGgFsaGwzi12J85/xmkxR6oZfvPHwbnks3OP8SvxYC9hxmkhXG+RO42Bp6EA3ZmNwjYInG8zZjhzJnDiRt4zm6W5jmQnGB2A2hLAh6/8DczP2aoqDhsL9/eu/Hjz3929mbn0x8++FBjg1MLwoUHIHQiWGUCIeUgIA811J4YxaNgFIyCUTCyAAArWmp6PaDhOQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-1636-4840","institution":"FIOCRUZ Bahia: Instituto Goncalo Moniz","correspondingAuthor":true,"prefix":"","firstName":"Everton","middleName":"dos Santos","lastName":"Gomes","suffix":""},{"id":501061782,"identity":"569d577e-dd84-49f7-8917-55dfb4ea75f5","order_by":1,"name":"Fernanda da Silva","email":"","orcid":"","institution":"UFMS: Universidade Federal de Mato Grosso do Sul","correspondingAuthor":false,"prefix":"","firstName":"Fernanda","middleName":"da","lastName":"Silva","suffix":""},{"id":501061783,"identity":"0b7ad387-ce25-4b24-8db2-26c25fc36029","order_by":2,"name":"Rafael Francisco Rosalem","email":"","orcid":"","institution":"UFMS: Universidade Federal de Mato Grosso do Sul","correspondingAuthor":false,"prefix":"","firstName":"Rafael","middleName":"Francisco","lastName":"Rosalem","suffix":""},{"id":501061784,"identity":"b834fc74-7f70-4708-afd5-9b5ab7eba44d","order_by":3,"name":"Thalita Bachelli Riul","email":"","orcid":"","institution":"UFMS: Universidade Federal de Mato Grosso do Sul","correspondingAuthor":false,"prefix":"","firstName":"Thalita","middleName":"Bachelli","lastName":"Riul","suffix":""},{"id":501061785,"identity":"a7bec48b-eaa0-4728-b989-7b0a350273d3","order_by":4,"name":"Lara Nicolly Dias Santana","email":"","orcid":"","institution":"Universidade Federal de Rondonópolis: Universidade Federal de Rondonopolis","correspondingAuthor":false,"prefix":"","firstName":"Lara","middleName":"Nicolly Dias","lastName":"Santana","suffix":""},{"id":501061786,"identity":"e61d94de-120f-432d-8a24-4e5bc94c5507","order_by":5,"name":"Andreia Vieira Pereira","email":"","orcid":"","institution":"Universidade Estadual de Maringá: Universidade Estadual de Maringa","correspondingAuthor":false,"prefix":"","firstName":"Andreia","middleName":"Vieira","lastName":"Pereira","suffix":""},{"id":501061787,"identity":"34e8a5a9-ca76-434f-881e-6a9a418f624b","order_by":6,"name":"Carla Cardozo Pinto de Arruda","email":"","orcid":"","institution":"UFMS: Universidade Federal de Mato Grosso do Sul","correspondingAuthor":false,"prefix":"","firstName":"Carla","middleName":"Cardozo Pinto","lastName":"de Arruda","suffix":""},{"id":501061788,"identity":"4c67ee13-cad4-42f9-92ca-13688bf3b9d5","order_by":7,"name":"Marcelo Biondaro Gois","email":"","orcid":"","institution":"Universidade Federal de Rondonópolis: Universidade Federal de Rondonopolis","correspondingAuthor":false,"prefix":"","firstName":"Marcelo","middleName":"Biondaro","lastName":"Gois","suffix":""},{"id":501061789,"identity":"3aca15b4-6811-4cc8-ac68-c9381334c2fc","order_by":8,"name":"Ludiele Souza Castro","email":"","orcid":"","institution":"Universidade Federal de Rondonopolis","correspondingAuthor":false,"prefix":"","firstName":"Ludiele","middleName":"Souza","lastName":"Castro","suffix":""}],"badges":[],"createdAt":"2025-08-09 18:29:58","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7335507/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7335507/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89463454,"identity":"cddba918-8561-464e-bfa2-f089fee142ce","added_by":"auto","created_at":"2025-08-20 08:13:07","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":40216,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of anti\u003cem\u003e-Leishmania\u003c/em\u003eactivity in promastigotes\u003c/p\u003e\n\u003cp\u003eA) Activity of condensed tannins isolated from \u003cem\u003eM. tenuiflora\u003c/em\u003e on the promastigote form of \u003cem\u003eL. amazonensis\u003c/em\u003e; B) \u003cem\u003eβ\u003c/em\u003e-glucan (1.3–1.6) activity on the promastigote form of \u003cem\u003eL. amazonensis.\u003c/em\u003e ** and *** indicate p\u0026lt;0.01 and p\u0026lt;0.001, respectively (one-way ANOVA with Tukey's post hoc test). C = culture control with untreated promastigotes.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7335507/v1/b86a03061b452735dcfc66d2.jpg"},{"id":89463452,"identity":"1d4becfd-bf63-4eaa-8505-10dd88d4a560","added_by":"auto","created_at":"2025-08-20 08:13:07","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":31698,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of anti\u003cem\u003e-Leishmania\u003c/em\u003eactivity in intracellular amastigotes\u003c/p\u003e\n\u003cp\u003eA) Condensed tannins; B) \u003cem\u003eβ\u003c/em\u003e-glucan (1.3–1.6). *** indicates p\u0026lt;0.001 (one-way ANOVA followed by Tukey's post hoc test). C = control of infected macrophages without treatment.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7335507/v1/47545c5464922c017d58f9f3.jpg"},{"id":89465469,"identity":"0ad85a52-bc8c-43a8-9ddf-d0fe46060b75","added_by":"auto","created_at":"2025-08-20 08:29:07","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":40353,"visible":true,"origin":"","legend":"\u003cp\u003eCytotoxic activity on murine fibroblasts\u003c/p\u003e\n\u003cp\u003eA) Activity of condensed tannins isolated from \u003cem\u003eM. tenuiflora\u003c/em\u003e on murine fibroblasts; B) \u003cem\u003eβ\u003c/em\u003e-glucan (1.3–1.6) activity on murine fibroblasts. C = culture control with cells without treatment.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7335507/v1/c672ee5fde14019cdfaf45e0.jpg"},{"id":89465472,"identity":"2e79b7ab-c8b1-47d3-9d66-57fea633c3f5","added_by":"auto","created_at":"2025-08-20 08:29:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":812164,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7335507/v1/9bc4f4c6-e13f-4ca9-b5d1-c327031922f1.pdf"}],"financialInterests":"","formattedTitle":"Anti-Leishmania activity of condensed tannins isolated from Mimosa tenuiflora and β-1,3-1,6 glucan isolated from commercial nutraceuticals","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eLeishmaniasis is a complex of infectious and parasitic diseases caused by protozoa of the genus \u003cem\u003eLeishmania\u003c/em\u003e and has a wide clinical spectrum depending on the species involved in the infection (WHO 2023). According to the WHO (2023), leishmaniasis can be divided into cutaneous and mucocutaneous leishmaniasis, characterized by the formation of dermal ulcers, and visceral leishmaniasis, the more severe clinical form of the disease that can lead to death if untreated.\u003c/p\u003e\u003cp\u003eConsidered a serious public health problem because of its wide geographical distribution, this form of anthropozoonosis is endemic to 90 countries, with a high prevalence in tropical and subtropical regions, where it affects millions of people (PAHO).\u003c/p\u003e\u003cp\u003eCurrently, chemotherapy is the most effective way to prevent diseases and mortality; however, even with the availability of different therapeutic options, there are still limitations (Ezatpour et al. 2015; Mendon\u0026ccedil;a and Ant\u0026ocirc;nio 2020). The therapeutic arsenal available for the treatment of leishmaniasis involves five drugs, pentavalent antimonials, amphotericin B, pentamidine, paramomycin and miltefosine, which are associated with a series of side effects and restrictions due to their cardiotoxic, hepatotoxic, and nephrotoxic profiles, among others (Gervazoni et al. 2020; Silva et al. 2021; Garza-Tovar et al. 2020; Roatt et al. 2020; Ghorbani and Farhoudi 2018). Furthermore, prolonged treatment is painful because of its invasive administration in most cases, which can culminate in treatment failure (Chappuis et al. 2007; Croft and Coombs 2003).\u003c/p\u003e\u003cp\u003eGiven the existing limitations, natural compounds appear to be promising sources of new molecules with leishmanicidal potential (Shen and Hao 2020; Garza-Torvan et al. 2020; Gervazoni et al. 2020). Among the botanical species with therapeutic potential is \u003cem\u003eMimosa tenuiflora\u003c/em\u003e (Willd). Pois., popularly known as jurema-preta, is widely distributed in the semiarid region of Brazil and is traditionally used in folk medicine for the healing of wounds, skin infections and inflammatory processes (Bezerra et al. 2010; Hernandez et al. 2021). Previous phytochemical studies have shown that the bark of this species is rich in condensed tannins, which are known for their medicinal properties (Crepaldi et al. 2021; Santos et al. 2022; Borges et al. 2017; Azev\u0026ecirc;do et al. 2013).\u003c/p\u003e\u003cp\u003ePrevious \u003cem\u003ein vitro\u003c/em\u003e studies have shown that condensed tannins are classified as phenolic compounds and demonstrate promising activity against pathogens (Monteiro et al. 2005; Pizzi 2021). These molecules can inhibit the growth of microorganisms through multiple mechanisms, including changes in nucleic acids, nutrient deprivation and destabilization of organelles important for cellular metabolism (Ucella Filho et al. 2022; Pizzi 2021). Furthermore, these molecules have antioxidant, anti-inflammatory and antimicrobial effects (Sanches et al. 2005). Given its ability to act as a pathogenic agent, future studies should investigate its anti\u003cem\u003e-Leishmania\u003c/em\u003e activity for possible therapeutic improvement.\u003c/p\u003e\u003cp\u003eIn addition to phenolic compounds, bioactive polysaccharides such as \u003cem\u003eβ\u003c/em\u003e\u003cb\u003e-\u003c/b\u003eglucans (1,3\u0026thinsp;\u0026minus;\u0026thinsp;1,6) are also of interest in the therapeutic development of infectious diseases. \u003cem\u003eβ\u003c/em\u003e-Glucans can stimulate and activate macrophages and other cells of the innate immune system, resulting in an improved response against various pathogens (Castro; Calder; Roche 2021; Camilli; Tobouret; Quintin 2018). The adjuvant and modulatory effects of this molecule on the immune system suggest its potential in the development of new therapeutic strategies for leishmaniasis or the improvement of existing therapies.\u003c/p\u003e\u003cp\u003eIn view of the above, the present study aimed to evaluate the anti-\u003cem\u003eLeishmania\u003c/em\u003e activity of condensed tannins isolated from the bark of \u003cem\u003eM. tenuiflora\u003c/em\u003e and \u003cem\u003eβ\u003c/em\u003e-glucan (1.3\u0026ndash;1.6), aiming to contribute to the development of innovative and less toxic therapeutic alternatives for the treatment of leishmaniasis.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003eCompounds studied\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCondensed tannins isolated from the bark of \u003cem\u003eMimosa tenuiflora\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe condensed tannins used in the present study were extracted by the Semi-Arid Research Center (NUPEÁRIDO), municipality of Patos, Paraíba-PB, at the domains of the Federal University of Campina Grande (UFCG). The material was subsequently purified at the Laboratory of Forestry Products Technology (LTPF) of the UFCG and transferred to the Federal University of Rondonópolis, Faculty of Health Sciences, for studies of its biological activity. The entire procedure was performed according to the methods established by Paes et al. (2010) and Pereira et al. (2015).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eβ-glucan (1.3-1.6)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the present study, a commercial nutraceutical rich in β-glucans (26 g) and amino acids, Bionutri AR-1®, was used. This product is obtained through a single fermentation process and is free of additives and preservatives, factors that classify it as a product of biotechnological innovation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of anti\u003cem\u003e-Leishmania activity in vitro\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eParasites\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor \u003cem\u003ein vitro\u003c/em\u003e analysis, a standard strain of \u003cem\u003eL. (L.) amazonensis\u003c/em\u003e (IFLA/BR/1967/PH8) was used. The parasites were previously isolated from BALB/c mice previously infected and maintained in Schneider® medium (Sigma‒Aldrich®) supplemented with 20% fetal bovine serum (FBS; Nova Biotecnologia) and 1% antibiotics (penicillin and streptomycin; Sigma‒Aldrich®), with repetitions every three days until the 20th serial passage.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of compounds\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFive micrograms (0.0050 g) of condensed tannins isolated from the bark of \u003cem\u003eM. tenuiflora\u003c/em\u003e were diluted in 100 μL of dimethyl sulfoxide (DMSO). The nutraceutical used to evaluate the biological activity of \u003cem\u003eβ\u003c/em\u003e-glucan (1.3–1.60 on \u003cem\u003eL. (L.) amazonensis\u0026nbsp;\u003c/em\u003ewas a dilution of 5 μg (0.0050 g) in 100 μL of phosphate-buffered saline solution (PBS). This dilution was selected because of the properties of the compound.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of anti\u003cem\u003e-Leishmania\u003c/em\u003e activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnti-promastigote assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e susceptibility tests were performed on the promastigote forms of \u003cem\u003eL. (L.) amazonensis\u003c/em\u003e in the log phase of growth in appropriate supplemented Schneider® medium (Sigma‒Aldrich®). Growth inhibition was assessed by cell viability after the addition of MTT (3-(4-5-dimethylthiazol-2-yl)-2,5-diphenyltetrasodium bromide).\u003c/p\u003e\n\u003cp\u003eIn 96-well plates, 100 μL of culture at a concentration of 10 \u003csup\u003e6\u003c/sup\u003e parasites/mL (19.75 parasites/mL) and 100 μL of the compounds to be tested were added at concentrations of 50–3.12 μL/mL. The plates were subsequently incubated at 26 °C for 72 h (Bosquiroli et al. 2015).\u003c/p\u003e\n\u003cp\u003eAfter 72 h of incubation, 5 mg/mL MTT was added to each well, after which the plates were stored again at 37 °C and 5% CO2 for 4 h, after which DMSO was added to dissolve the formazan crystals, after which the absorbance was measured with a spectrophotometer at a wavelength of 540 nm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnti-amastigote test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the effects of the compounds against the amastigote forms, peritoneal macrophages from BALB/c mice were used, which were attached to circular coverslips at the bottom of 24-well plates supplemented with RPMI and infected with \u003cem\u003eL. amazonensis\u003c/em\u003e promastigotes (IFLA)/BR/1967/PH8). Afterward, the plates were stored at 37 °C with 5% CO2 (Oliveira 2021).\u003c/p\u003e\n\u003cp\u003eAfter 24 h, the compounds of interest were added at concentrations of 50–3.12 μL/mL for another 24 h. The coverslips were then fixed with Boiun's solution, stained with Giemsa and mounted on microscope slides with Entellan to evaluate the number of amastigotes within 200 macrophages under light microscopy. Each concentration of extract was tested in sextuplicate (Oliveira 2021).\u003c/p\u003e\n\u003cp\u003eThe inhibitory concentration was calculated using linear regression, and the log10 of each inhibitor concentration was plotted in relation to the percentage activity with the software GraphPad Prism 5 (Oliveira 2021).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eCytotoxicity assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the cytotoxicity of each compound, NIH/3T3 cells (ATCC CRL-1658 mouse fibroblast lineage) were grown in 96-well plates (1x10 \u003csup\u003e4\u003c/sup\u003e cells in 100 μL per well) in RPMI 1640 medium supplemented with penicillin, streptomycin and 10% fetal bovine serum for 12 h at 37 °C and 5% CO2 (Oliveira 2021).\u003c/p\u003e\n\u003cp\u003eAfter 12 h, the medium was replaced with fresh RPMI medium containing different concentrations of each compound (100 to 3.125 μL/mL) in quintuplicate, and the plates were incubated for 24 h. The viability/life control was determined by fibroblasts grown only in culture medium (Oliveira 2021).\u003c/p\u003e\n\u003cp\u003eSubsequently, 10 μL of resazurin was added to each well, followed by incubation for 4 h at 37 °C and 5% CO2. After incubation, 80 μL of dimethyl sulfoxide was added to dissolve the formazan crystals, and the absorbance was estimated with a spectrophotometer at a wavelength of 570 nm (Oliveira 2021).\u003c/p\u003e\n\u003cp\u003eThe cytotoxic concentration (CC \u003csub\u003e50\u003c/sub\u003e) was calculated by linear regression, and the log10 of each inhibitor concentration was plotted against the percentage activity with GraphPad Prism 5 (Oliveira 2021).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results are expressed as the concentration of growth inhibition at 50% (IC \u003csub\u003e50\u003c/sub\u003e) and were determined by nonparametric linear regression. One-way ANOVA was subsequently performed, followed by Tukey's post hoc test for comparisons between groups. The results were obtained using the statistical software GraphPad Prism 5.01®. The significance level was set at 5%.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003eAmong the compounds evaluated, the condensed tannins isolated from the bark of \u003cem\u003eMimosa tenuiflora\u003c/em\u003e inhibited the growth of promastigote forms of \u003cem\u003eLeishmania (L.) amazonensis\u003c/em\u003e, with a 50% inhibitory concentration (CI \u003csub\u003e50\u003c/sub\u003e) of 24.56 \u0026micro;g (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e-A). However, \u0026beta;-glucan (1.3\u0026ndash;1.6) was not able to inhibit parasite growth in the same assay, with \u003csub\u003ean IC50\u003c/sub\u003e \u0026gt;50 \u0026micro;g (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e-B).\u003c/p\u003e\n\u003cp\u003eIn the investigation of the activity on intracellular forms, a change in the profile of the biological activity of the compounds was observed, where \u003cem\u003e\u0026beta;\u003c/em\u003e-glucan (1.3\u0026ndash;1.6) was able to reduce the number of amastigotes internalized in murine macrophages, \u003csub\u003ewith an IC50\u003c/sub\u003e of 17.94 \u0026micro;g (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e-B), whereas tannins did not reduce the number of amastigotes per macrophage (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e-A).\u003c/p\u003e\n\u003cp\u003eIn terms of the cytotoxicity profile on murine fibroblasts, both compounds showed low cytotoxicity to NIH/3T3 cells (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). In view of the results, it was possible to estimate the selectivity index (SI) of the compounds tested, where the condensed tannin presented an IS\u0026thinsp;\u0026gt;\u0026thinsp;40.65 for the promastigote form, which was more selective for the parasite than for the cell, while \u003cem\u003e\u0026beta;\u003c/em\u003e-glucan (1.3\u0026ndash;1.6) was more selective for the amastigote form than for the cell, with an SI\u0026thinsp;\u0026gt;\u0026thinsp;55.74 (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab1\" border=\"1\" class=\"fr-table-selection-hover\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eAnti-\u003cem\u003eLeishmania\u003c/em\u003e activity on the promastigote and amastigote forms of \u003cem\u003eL. amazonensis\u003c/em\u003e and selectivity index.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCompost\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIC \u003csub\u003e50\u003c/sub\u003e (\u0026micro;g/ml) \u003cem\u003eof L. amazonensis\u003c/em\u003e promastigotes\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCC \u003csub\u003e50\u003c/sub\u003e (\u0026micro;g/ml) Murine NIH/3T3 fibroblasts\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIS promastigote \u003cem\u003eL. amazonensis\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIC \u003csub\u003e50\u003c/sub\u003e (\u0026micro;g/ml) \u003cem\u003eL. amazonensis\u003c/em\u003e amastigotes\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIS amastigotes \u003cem\u003eL. amazonensis\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eCondensed tannin\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e24,59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;40,65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eB-glucano (1,3\u0026thinsp;\u0026minus;\u0026thinsp;1,6)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17,94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;55,74\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe present study is a preclinical evaluation of different compounds against the evolutionary forms of \u003cem\u003eL. amazonensis\u003c/em\u003e, as well as their toxicity to 3T3 lineage cells. The \u003cem\u003ein vitro\u003c/em\u003e evaluation of molecules on the different evolutionary forms of the parasite is essential, since amastigotes are the intracellular form involved in human pathogenesis, and the efficiency and toxicity profile of the molecule can be evaluated for later scaling up for \u003cem\u003ein vitro\u003c/em\u003e studies.\u003c/p\u003e\u003cp\u003eThe results of the present study revealed that the condensed tannins isolated from the bark of \u003cem\u003eM. tenuiflora\u003c/em\u003e significantly affected the promastigote form but were ineffective against the amastigote form. These results show that tannins directly affect structures present in or more exposed to the extracellular form of the parasite, such as surface components or specific metabolic processes of the promastigote. The mechanisms underlying the inhibition of promastigote growth have not been elucidated, but they may be the same as those in other microorganisms, such as bacteria and fungi, where this molecule inhibits growth through changes in nucleic acids, nutrient deprivation and destabilization of important organelles. in cellular metabolism (Molino et al. 2019; Huang et al. 2024).\u003c/p\u003e\u003cp\u003ePrevious studies have shown that tannins can interact with membrane proteins, forming complexes and destabilizing their integrity, resulting in cell death by lysis (Ferreira and Evangelista 2021; Molino et al. 2019). In addition, these molecules can inhibit key enzymes involved in the metabolism of some microorganisms, preventing essential functions and slowing their growth. Since the inhibition mechanisms act on biological processes of organisms outside the intracellular environment, it is possible to explain the lack of inhibitory effects on the amastigote forms of \u003cem\u003eL. amazonensis\u003c/em\u003e, showing that tannin has difficulty crossing the host cell membrane or maintaining intracellular activity, a fundamental characteristic for molecules with leishmanicidal activity.\u003c/p\u003e\u003cp\u003eIn view of the above, the condensed tannins isolated from the bark of \u003cem\u003eM. tenuiflora\u003c/em\u003e are more selective for promastigote forms than for amastigote forms, a factor that discards it as a molecule of interest in the prospect of anti-\u003cem\u003eLeishmania\u003c/em\u003e compounds.\u003c/p\u003e\u003cp\u003eOn the other hand, \u003cem\u003eβ\u003c/em\u003e-glucan (1.3\u0026ndash;1.6) showed a distinct and potentially promising profile, as it showed significant inhibitory activity on the amastigote forms of \u003cem\u003eL. amazonensis\u003c/em\u003e and zero toxicity on NIH/3T3 murine cells. Furthermore, \u003cem\u003eβ\u003c/em\u003e-glucan (1.3\u0026ndash;1.6) was not able to inhibit the growth of the promastigote forms of the parasite. The profile observed in the present study suggests a selective action of the compound on the amastigote form, which is the pathogenic form in humans and other mammals\u003c/p\u003e\u003cp\u003eThe selectivity observed for amastigotes may be related to the immunomodulatory effect of \u003cem\u003eβ\u003c/em\u003e-glucan (1,3\u0026thinsp;\u0026minus;\u0026thinsp;1,6). Previous studies report that this polysaccharide acts as a modulator of the innate immune response, interacting with recognition pattern receptors, such as Dectin-1 and Toll-like receptors, on phagocytic cells (Zimara et al. 2018; Brown et al. 2003; Yadav and Schorey 2006). This interaction results in increased production of reactive oxygen species and proinflammatory cytokines, factors that may contribute to the control of amastigotes internalized by macrophages (Castro; Calder; Roche 2021; Stothers et al. 2021).\u003c/p\u003e\u003cp\u003eAlthough no direct activity was observed for the promastigote forms, the ability of \u003cem\u003eβ\u003c/em\u003e-glucan (1.3\u0026ndash;1.6) to reduce the number of parasites in infected macrophages is indirect. These results are corroborated by those of Patidar et al. (2020), who reported a reduction in the parasite load in murine peritoneal macrophages infected with \u003cem\u003eLeishmania donovani\u003c/em\u003e and treated with \u003cem\u003eβ\u003c/em\u003e-glucan. In the same study, \u003cem\u003ein silico\u003c/em\u003e evaluation revealed that the effect was due to the interaction between the \u003cem\u003eβ\u003c/em\u003e-glucan of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e and Dectin-1 and TRL2 receptors, both of which are expressed by macrophages and dendritic cells, leading to the activation of different intracellular signaling pathways (IRAK-4, IKKα, IκB, LyN and SyK).\u003c/p\u003e\u003cp\u003eThe absence of significant cytotoxicity to NIH/3T3 cells corroborates the findings of previous studies, which demonstrated that \u003cem\u003eβ\u003c/em\u003e-glucans are safe and biocompatible compounds and present a low risk to mammalian cells (Chan; Chan; Sze 2009). The results obtained reveal a relevant profile for the development of alternative therapies, considering the current limitations in the treatment of leishmaniasis, which culminate in therapeutic failure (Vetvicka and Fernandez-Botran 2018).\u003c/p\u003e\u003cp\u003eWhen the results concerning β-glucan (1.3\u0026ndash;1.6) are considered, it is possible to discard its direct mechanism of action on the parasite, since the absence of activity on the promastigote forms of \u003cem\u003eL. amazonensis;\u003c/em\u003e however, if necessary, further assays are needed to evaluate parameters such as phagocytosis, nitric oxide production and expression of macrophage activation markers to elucidate its mechanism of action on the amastigote forms.\u003c/p\u003e\u003cp\u003eIn conclusion, the results highlight the importance of screening molecules with anti-\u003cem\u003eLeishmania\u003c/em\u003e activity and investigating their action profiles on different evolutionary forms. Given its activity on amastigotes and low cytotoxicity, the investigated \u003cem\u003eβ\u003c/em\u003e-glucan (1.3\u0026ndash;1.6) showed a more adequate profile for further studies, making it necessary to characterize the mechanisms involved and evaluate its efficacy in complex biological systems.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eI thank all the researchers involved in this study and the funding agencies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors of the present study contributed significantly to the design and preparation of the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical aspects\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo accomplish the present study, all the procedures presented are in accordance with the principles adopted by the Brazilian Society of Laboratory Animal Sciences (SBCAL).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupport\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research is funded by the National Research Development Council (CNPq), a public funding agency.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eI declare that there are no conflicts of interest between the authors and the funding agency.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAZEVEDO TK et al (2017) Content of condensed tannins in the bark of jurema-preta (\u003cem\u003eMimosa tenuiflora\u003c/em\u003e) as a function of the phenophases. V. 24.\u003c/li\u003e\n\u003cli\u003eBEZERRA DAC et al (2009) Biological activity of jurema-preta (\u003cem\u003eMimosa tenuiflora\u003c/em\u003e) on \u003cem\u003eStaphylococcus aureus\u003c/em\u003e isolated from cases of bovine mastitis. Brazilian Journal of Pharmacognosy 19:814-817.\u003c/li\u003e\n\u003cli\u003eBORGES IV et al (2017) Identification of the antimicrobial fraction of the extract of \u003cem\u003eMimosa tenuiflora\u003c/em\u003e. Communicata Scientiae 8:1.\u003c/li\u003e\n\u003cli\u003eBOSQUIROLI LS et al (2015) \u003cem\u003eIn vitro\u003c/em\u003e anti-\u003cem\u003eLeishmania infantum\u003c/em\u003e activity of essential oil from \u003cem\u003ePiper angustifolium\u003c/em\u003e. Brazilian Journal of Pharmacognosy 25:124-128.\u003c/li\u003e\n\u003cli\u003eBROWN GD et al (2003) Dectin-1 mediates the biological effects of beta-glucans. 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Leishmaniasis: Cutaneous and mucosal leishmaniasis. Available in:\u003cu\u003ehttps://www.paho.org/pt/topicos/leishmaniose/leishmaniose-cutanea-e-mucosa\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003ePAHO \u0026ndash; Pan American Health Organization. Leishmaniasis. Available in:\u003cu\u003ehttps://www.paho.org/pt/topicos/leishmaniose\u003c/u\u003e.\u003c/li\u003e\n\u003cli\u003ePAES JB et al (2010) Tannic substances present in several parts of \u003cem\u003eAnadenathera colubrina\u003c/em\u003e (Vell.) Brenan. Var. cebil (Gris.) Alts). Tree. Scientia Forestalis 38:441-447.\u003c/li\u003e\n\u003cli\u003ePATIDAR A et al (2020) Barley beta-Glucan and Zymosan induce dectin-1 and toll-like receptor 2 colocalization and anti-leishmanial immune response in \u003cem\u003eLeishmania donovani\u003c/em\u003e-infected BALB/c mice. Experimental immunology 92.\u003c/li\u003e\n\u003cli\u003ePEREIRA AV et al (2025) Cashew bark tannins: antimicrobial activity. AGROTEC Magazine 36:121-127.\u003c/li\u003e\n\u003cli\u003ePIZZI A (2021) Tannins medical/pharmacological and related applications: A critical review. Sustainable Chemistry and Pharmacy.\u003c/li\u003e\n\u003cli\u003eROATT BM et al (2020) Recent advances and new strategies on leishmaniasis treatment. Applied Microbiology and Biotechnology 104:8965-8977.\u003c/li\u003e\n\u003cli\u003eSANCHES ACC et al (2005) Antioxidant and antifungal activities of extracts and condensed tannins from \u003cem\u003eStrypnodendron obovatum\u003c/em\u003e Benth. Brazilian Journal of Pharmaceutical Sciences 41.\u003c/li\u003e\n\u003cli\u003eSANTOS RF (2022) et al Antimicrobial properties of jurema-preta (\u003cem\u003eMimosa tenuifora\u003c/em\u003e (wild.) poir.) pear extracts 8.\u003c/li\u003e\n\u003cli\u003eSHEN Y, HAO X (2020) Natural products sciences: an integrative approach to the innovations of plant natural products. Sci China Life Sci 63.\u003c/li\u003e\n\u003cli\u003eSILVA VNS et al (2021) Considerations on leishmaniasis and the current scenario for developing new forms of treatment. J trop Pathol 50:255-264.\u003c/li\u003e\n\u003cli\u003eSTOTHERS CL et al (2021) \u003cem\u003e\u0026beta;\u003c/em\u003e-Glucan induces distinct and protective innate immune memory in differentiated macrophages. The Journal of Immunology 207:2785-2798.\u003c/li\u003e\n\u003cli\u003eVETVICKA V (2011) Glucan\u0026ndash;immunostimulant, adjuvant, potential drug. World Journal of Clinical Oncology 2:115\u0026ndash;119.\u003c/li\u003e\n\u003cli\u003eVETVICKA V, FRNANDEZ-BOTRAN R (2018) \u003cem\u003e\u0026beta;\u003c/em\u003e-Glucan and parasites. Helminthologia 55:177-184.\u003c/li\u003e\n\u003cli\u003eYADAV M, SCHOREY JS (2006) The \u003cem\u003e\u0026beta;\u003c/em\u003e-glucan receptor dectin-1 functions together with TLR2 to mediate macrophage activation by mycobacteria. Blood 1.\u003c/li\u003e\n\u003cli\u003eZIMARA M et al (2018) Dectin-1 positive dendritic cells expand after infection with \u003cem\u003eLeishmania major\u003c/em\u003e parasites and represent promising targets for vaccine development. Frontiers in Immunology 9.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-parasitic-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jopd","sideBox":"Learn more about [Journal of Parasitic Diseases](https://www.springer.com/journal/12639)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jopd/default.aspx","title":"Journal of Parasitic Diseases","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Natural products, Bioprospecting, Leishmaniasis, Drug development","lastPublishedDoi":"10.21203/rs.3.rs-7335507/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7335507/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study evaluated the anti-Leishmania activity of condensed tannins from \u003cem\u003eMimosa tenuiflora\u003c/em\u003e and \u003cem\u003eβ\u003c/em\u003e-glucan (1,3\u0026thinsp;\u0026minus;\u0026thinsp;1,6) from commercial nutraceuticals to identify less toxic alternatives against leishmaniasis, a neglected disease affecting millions in tropical and subtropical regions. Current treatments rely on toxic drugs, motivating research on natural compounds with leishmanicidal activity. \u003cem\u003eM. tenuiflora\u003c/em\u003e is known for antimicrobial, antioxidant, and anti-inflammatory properties, while β-glucans are immunomodulatory polysaccharides capable of activating macrophages and enhancing pathogen control. Condensed tannins showed activity against promastigotes (IC₅₀ = 24.56 \u0026micro;g/mL) but were ineffective against intracellular amastigotes, suggesting action on extracellular structures, possibly via membrane destabilization, nucleic acid alteration, or enzyme inhibition. β-glucan did not affect promastigotes but significantly reduced internalized amastigotes (IC₅₀ = 17.94 \u0026micro;g/mL). Cytotoxicity was low for both compounds (CC₅₀ \u0026gt;1,000 \u0026micro;g/mL), resulting in high selectivity indices: \u0026gt;40 for tannins (promastigotes) and \u0026gt;\u0026thinsp;55 for β-glucan (amastigotes).The anti-amastigote activity of β-glucan appears linked to immunomodulation via receptors such as Dectin-1 and TLR2 on macrophages, stimulating reactive oxygen species and proinflammatory cytokines production, which control intracellular parasites.In conclusion, condensed tannins demonstrated limited potential, acting only on promastigotes, while β-glucan was effective against amastigotes, the pathogenic form of leishmaniasis, with excellent cytotoxic safety. β-glucan (1,3\u0026ndash;1,6) emerges as a promising candidate for novel therapeutic strategies, warranting further investigation of its immunological mechanisms and in vivo efficacy.\u003c/p\u003e","manuscriptTitle":"Anti-Leishmania activity of condensed tannins isolated from Mimosa tenuiflora and β-1,3-1,6 glucan isolated from commercial nutraceuticals","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-20 08:13:02","doi":"10.21203/rs.3.rs-7335507/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-09-21T09:23:33+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-12T04:10:01+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Journal of Parasitic Diseases","date":"2025-08-12T12:15:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-12T07:44:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Parasitic Diseases","date":"2025-08-11T07:21:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-parasitic-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jopd","sideBox":"Learn more about [Journal of Parasitic Diseases](https://www.springer.com/journal/12639)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jopd/default.aspx","title":"Journal of Parasitic Diseases","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4ff902a5-5da2-4904-abaf-5ef061beda50","owner":[],"postedDate":"August 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-01-12T11:43:26+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-20 08:13:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7335507","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7335507","identity":"rs-7335507","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}