Acute and sub-acute toxicity study of ethanol extract from Nectandra leucantha Nees & Mart. (Lauraceae) barks.

Nectandra leucantha has been used in traditional medicine. Several metabolites isolated from N. leucantha extracts displayed immunomodulatory, antileishmanial properties, but the determination of the toxicological profile in mammals has not previously been performed. In this study, the ethanol extract from N. leucantha barks (EENl) was characterized by HPLC/HRESIMS. To study acute toxicity, female mice received EENl in a single dose of 100, 300, 1000, or 2000 mg/kg bw. Later, sub-acute toxicity was introduced in female and male mice by oral gavage at 100, 500 or 1000 mg/kg bw for 28 consecutive days. Hematological and biochemical profiles from the blood as well as histological analysis from the liver and kidney were performed. The HPLC/HRESIMS analysis of the EENl revealed the presence of six neolignans chemically related to dehydrodieugenol B. In the oral acute and sub-chronic studies, EENl did not produce in all doses evaluated any alteration in behavior, biochemical, hematological, body weight gain and food intake or sudden death in Swiss mice. In addition, histopathological data did not reveal any disturbance in liver and kidney morphology after 28 days of EENl treatment. Our results indicate that EENl at dosage levels up to 2000 mg/kg bw is non-toxic and can be considered safe for mammals.

Structure-activity relationship study of cytotoxic neolignan derivatives using multivariate analysis and computation-aided drug design.

Dehydrodieugenol B and five related natural neolignans were isolated from the Brazilian plant species Nectandra leucantha. Three of these compounds were shown to be active against murine (B16F10) and human (A2058) melanoma cells but non-toxic to human fibroblasts (T75). These results stimulated the preparation of a series of 23 semi-synthetic derivatives in order to explore structure-activity relationships and study the biological potential of these derivatives against B16F10 and A2058 cell lines. These structurally-related neolignan derivatives were analyzed by multivariate statistics and machine learning, which indicated that the most important characteristics were related to their three-dimensional structure and, mainly, to the substituents on the neolignan skeleton. The results suggested that the presence of hydroxyl or alkoxyl groups at positions 3, 4 and 5 (with appropriate sidechains) promoted an increase in electropological and charge density, which seem to be important for biological activity against murine (B16F10) and human (A2058) melanoma cells.

Lapachol acetylglycosylation enhances its cytotoxic and pro-apoptotic activities in HL60 cells.

Lapachol is a plant-derived naphthoquinone that kills several types of cancer cells. Derivatives of this molecule may therefore prove to be useful chemotherapeutic agents. In this study, we explored whether glycosylation increases the cytotoxic potency of lapachol towards HL-60 human leukemia cells. Two beta-glycosides were synthesized and characterized: LA4A (lapachol-ß-glucoside) and LA4C (lapachol-N-acetylglucosamine-ß-glucoside). The sugar moieties of both novel molecules were per-acetylated to facilitate cellular uptake. The IC50 values (in µM) for LA4A (5.7) and LA4C (5.3) were lower than those for lapachol (25). LA4A and LA4C triggered typical signs of apoptosis, such as the exposure of phosphatidylserine on the outside of cells, chromatin condensation, DNA fragmentation and a decrease of the mitochondrial transmembrane potential (ΔΨm) prior to cell lysis. Moreover, DNA fragmentation triggered by the lapachol-glycosides was reduced by pre-treatment with the caspase inhibitor, z-VAD-fmk. While LA4A and LA4C activated caspases-3, -8 and -9, lapachol failed to activate these apoptotic proteases, even when used at high concentrations. Finally, the toxicity of lapachol and its derivatives was also tested on non-tumor cells. We used human peripheral neurons (PeriTox test) to evaluate the side effect potential of these compounds. LA4C was clearly less toxic than LA4A. We conclude that LA4C had the most favorable profile as drug candidate (high tumor cell toxicity, reduced neurotoxicity). In general, this study shows that the cytotoxicity of lapachol towards HL-60 can be enhanced by glycosylation, and that the therapeutic ratio may be modified by the type of sugar added.

A semi-synthetic neolignan derivative from dihydrodieugenol B selectively affects the bioenergetic system of Leishmania infantum and inhibits cell division.

Leishmaniasis is a neglected disease that affects more than 12 million people, with a limited therapy. Plant-derived natural products represent a useful source of anti-protozoan prototypes. In this work, four derivatives were prepared from neolignans isolated from the Brazilian plant Nectandra leucantha, and their effects against intracellular amastigotes of Leishmania (L.) infantum evaluated in vitro. IC50 values between 6 and 35 µM were observed and in silico predictions suggested good oral bioavailability, no PAINS similarities, and ADMET risks typical of lipophilic compounds. The most selective (SI > 32) compound was chosen for lethal action and immunomodulatory studies. This compound caused a transient depolarization of the plasma membrane potential and induced an imbalance of intracellular Ca2+, possibly resulting in a mitochondrial impairment and leading to a strong depolarization of the membrane potential and decrease of ATP levels. The derivative also interfered with the cell cycle of Leishmania, inducing a programmed cell death-like mechanism and affecting DNA replication. Further immunomodulatory studies demonstrated that the compound eliminates amastigotes via an independent activation of the host cell, with decrease levels of IL-10, TNF and MCP-1. Additionally, this derivative caused no hemolytic effects in murine erythrocytes and could be considered promising for future lead studies.

Genotoxic and cytotoxic effects of neolignans isolated from Nectandra leucantha (Lauraceae).

In this work was evaluated the cytotoxic activity of dehydrodieugenol B (1) and methyldehydrodieugenol B (2) isolated from Nectandra leucantha (Lauraceae) through cytokinesis-block micronucleus (CBMN) and Comet assay. Compounds 1 and 2 displayed in vitro toxicity against human melanoma cells (SKMEL-147) with IC50 values of 4.4 and 43.6 µg/mL, respectively. The interaction of these compounds with resistant human melanoma cell line SKMEL-29 was also investigated. Obtained results showed a concentration-response relationship for DNA damage (DI and D%) in SKMEL-29 cells for compounds 1 and 2 causing an increase in DNA damage on their lower concentrations. Concerning the cytokinesis-block micronucleus cytome (CBMNCyt) assay, all treatments demonstrated an increase in cytostatic and cytotoxic indexes. In micronucleus quantification, compound 1 displayed higher index in comparison to control. Regarding necrotic and apoptotic cells, treatments with SKMEL-29 cells demonstrated 100% of cell death induced by compounds 1 and 2 at 25 and 88 µg/mL, respectively. Additionally, it was observed that apoptosis is prevalent in SKMEL-147 cells treated with compound 1, while necrotic cells were observed in SKMEL-29 cells treated with both compounds. In conclusion, compounds 1 and 2 are suggested as promising cytotoxic agents against human melanoma resistant cells, emphasizing the potential use of these neolignans for the treatment of melanoma.

Chemical Structure and Localization of Levan, the Predominant Fructan Type in Underground Systems of Gomphrena marginata (Amaranthaceae).

Gomphrena marginata Seub. (Amaranthaceae) is an endemic species from Brazilian campos rupestres with a fructan accumulating underground reserve system. Analyses of high performance anion exchange chromatography (HPAEC-PAD) revealed the presence of the soluble carbohydrates glucose, fructose, sucrose, 1-kestose, 6-kestose, nystose and fructans with degree of polymerization (DP) up to approximately 40 fructose units. Data of 1H and 13C Nuclear Magnetic Resonance (NMR) spectroscopy, including Heteronuclear Single-Quantum Correlation (HSQC) and Heteronuclear Multiple-Bonds Correlation (HMBC) showed the presence of ß (2,6) linkages, characteristic of the linear molecule of levan-type fructan(2,6). These results confirmed previous studies suggesting that the reserve carbohydrate in the underground system of this species was levan-type fructans, similar to that of G. macrocephala. Structural analyses of the thickened underground system using light microscopy revealed a mixed origin system consisting mainly of a gemmiferous tuberous root with the upper region formed by short branched stems, both presenting vascular cylinders with unusual growth patterns. Fructan spherocrystals were visualized under polarized light and scanning electron microscopy (SEM) mostly in the cortex and vascular cylinder in both thickened stem and root. In addition to data reported in the literature concerning the occurrence of fructans in the Amaranthaceae, the results presented here suggest that fructans are a trait in this family while the levan-type fructan prevail in Gomphrena species.

Neolignans from leaves of Nectandra leucantha (Lauraceae) display in vitro antitrypanosomal activity via plasma membrane and mitochondrial damages.

Chagas disease is a neglected tropical disease, caused by the protozoan parasite Trypanosoma cruzi, which affects more than eight million people in Tropical and Subtropical countries especially in Latin America. Current treatment is limited to nifurtimox and benznidazole, both with reduced effectiveness and high toxicity. In this work, the n-hexane extract from leaves of Nectandra leucantha (Lauraceae) displayed in vitro antitrypanosomal activity against T. cruzi. Using several chromatographic steps, four related neolignans were isolated and chemically characterized as dehydrodieugenol B (1), 1-(8-propenyl)-3-[3'-methoxy-1'-(8-propenyl)-phenoxy]-4,5-dimethoxybenzene (2), 1-[(7S)-hydroxy-8-propenyl]-3-[3'-methoxy-1'-(8'-propenyl)-phenoxy]-4-hydroxy-5-methoxybenzene (3), and 1-[(7S)-hydroxy-8-propenyl]-3-[3'-methoxy-1'-(8'-propenyl)-phenoxy]-4,5-dimethoxybenzene (4). These compounds were tested against intracellular amastigotes and extracellular trypomastigotes of T. cruzi and for mammalian cytotoxicity. Neolignan 4 showed the higher selectivity index (SI) against trypomastigotes (>5) and amastigotes (>13) of T. cruzi. The investigation of the mechanism of action demonstrated that neolignan 4 caused substantial alteration of the plasma membrane permeability, together with mitochondrial dysfunctions in trypomastigote forms. In silico studies of pharmacokinetics and toxicity (ADMET) properties predicted that all compounds were non-mutagenic, non-carcinogenic, non-genotoxic, weak hERG blockers, with acceptable volume of distribution (1.66-3.32 L/kg), and low rodent oral toxicity (LD50 810-2200 mg/kg). Considering some clinical events of cerebral Chagas disease, the compounds also demonstrated favorable properties, such as blood-brain barrier penetration. Unfavorable properties were also predicted as high promiscuity for P450 isoforms, high plasma protein binding affinity (>91%), and moderate-to-low oral bioavailability. Finally, none of the isolated neolignans was predicted as interference compounds (PAINS). Considering the promising chemical and biological properties of the isolated neolignans, these compounds could be used as starting points to develop new lead compounds for Chagas disease.

Neolignans isolated from Nectandra leucantha induce apoptosis in melanoma cells by disturbance in mitochondrial integrity and redox homeostasis.

Six neolignans including three previously undescribed metabolites: 1-[(7R)-hydroxy-8-propenyl]-3-[3'-methoxy-1'-(8'-propenyl)-phenoxy]-4,5-dimethoxybenzene, 4-hydroxy-5-methoxy-3-[3'-methoxy-1'-(8'-propenyl)phenoxy]-1-(7-oxo-8-propenyl)benzene and 4,5-dimethoxy-3-[3'-methoxy-1'-(8'-propenyl)phenoxy]-1-(7-oxo-8-propenyl)benzene were isolated from twigs of Nectandra leucantha Nees & Mart (Lauraceae) using bioactivity-guided fractionation. Cytotoxic activity of isolated compounds was evaluated in vitro against cancer cell lines (SK BR-3, HCT, U87-MG, A2058, and B16F10), being dehydrodieugenol B and 4-hydroxy-5-methoxy-3-[3'-methoxy-1'-(8'-propenyl)phenoxy]-1-(7-oxo-8-propenyl)benzene the most active metabolites. These compounds displayed IC50 values of 78.8 ± 2.8 and 82.2 ± 3.5 µM, respectively, against murine melanoma. Different in vitro mechanism of induced cytotoxicity for this cell line is proposed for both compounds. Obtained results indicated a remarkable effect during the induction of morphological, biochemical and enzymatic features of apoptosis, such as disruption of mitochondrial membrane potential (ΔΨm), exposure of phosphatidylserine in the outer cell membrane, and genomic DNA condensation and fragmentation. Dehydrodieugenol B induced caspase-3 and PARP activation and 4-hydroxy-5-methoxy-3-[3'-methoxy-1'-(8'-propenyl)phenoxy]-1-(7-oxo-8-propenyl)benzene downregulated the levels of Bcl-2 protein. These effects were accompanied by increased levels of reactive oxygen species as a consequence of mitochondrial damage, followed by F-actin aggregation during the cell death process. Dehydrodieugenol B showed oxidative properties and both compounds, especially 4-hydroxy-5-methoxy-3-[3'-methoxy-1'-(8'-propenyl)phenoxy]-1-(7-oxo-8-propenyl)benzene, displayed potential to alkylate nucleophiles, suggesting an accessory mechanism of tumor-induced cytotoxicity by these metabolites.

Antitrypanosomal activity and evaluation of the mechanism of action of dehydrodieugenol isolated from Nectandra leucantha (Lauraceae) and its methylated derivative against Trypanosoma cruzi.

BACKGROUND: From a previous screening of Brazilian biodiversity for antiprotozoal activity, the hexane extract from leaves of Nectandra leucantha (Nees & Mart.) (Lauraceae) demonstrated activity against Trypanosoma cruzi. Chromatographic separation of this extract afforded bioactive dehydrodieugenol (1). Furthermore, methylated derivative 2 (dehydrodieugenol dimethyl ether) was prepared and also tested against T. cruzi. PURPOSE: To examine the therapeutical potential of compounds 1 and 2 against T. cruzi as well as to elucidate the mechanism of action of bioactive compound 1 against T. cruzi. METHODS/STUDY DESIGN: Crude hexane extract from leaves was subjected to chromatographic steps to afford bioactive compound 1. In order to analyze the effect of additional methyl group in the antiparasitic activity of 1, derivative 2 was prepared (both are no pan-assay interference compounds - PAINS). These compounds were evaluated in vitro against T. cruzi (trypomastigote and amastigote forms) and analyzed for the potential effect in host cells through the production of nitric oxide and reactive oxygen species. Finally, the plasma membrane effect of the most potent compound 1 was investigated in T. cruzi trypomastigotes. RESULTS: Compounds 1 and 2 displayed activity against amastigotes of T. cruzi. Although both compounds promoted activity against intracellular amastigotes, the production of nitric oxide and reactive oxygen species of host cells were unaltered, suggesting an antiparasitic activity other than host cell activation. Considering 1 the most effective compound against T. cruzi, the interference in the plasma membrane of the trypomastigotes was investigated using the fluorescent probe SYTOX® Green. After a short-term incubation, the fluidity and integrity of the plasma membrane was completely altered, suggesting it as a primary target for compound 1 in T. cruzi. CONCLUSION: Compounds 1 and 2 selectively eliminated the intracellular parasites without host cell activation and could be important scaffolds for the search of new hit compounds.

Immunomodulatory and Antileishmanial Activity of Phenylpropanoid Dimers Isolated from Nectandra leucantha.

Three phenylpropanoid dimers (1-3) including two new metabolites were isolated from the extract of the twigs of Nectandra leucantha using antileishmanial bioassay-guided fractionation. The in vitro antiparasitic activity of the isolated compounds against Leishmania donovani parasites and mammalian cytotoxicity and immunomodulatory effects were evaluated. Compounds 1-3 were effective against the intracellular amastigotes within macrophages, with IC50 values of 26.7, 17.8, and 101.9 µM, respectively. The mammalian cytotoxicity, given by the 50% cytotoxic concentration (CC50), was evaluated against peritoneal macrophages. Compounds 1 and 3 were not toxic up to 290 µM, whereas compound 2 demonstrated a CC50 value of 111.2 µM. Compounds 1-3 also suppressed production of disease exacerbatory cytokines IL-6 and IL-10 but had minimal effect on nitric oxide production in L. donovani-infected macrophages, indicating that antileishmanial activity of these compounds is mediated via an NO-independent mechanism. Therefore, these new natural products could represent promising scaffolds for drug design studies for leishmaniasis.