Echinodorus macrophyllus: Acute toxicological evaluation of hydroxycinnamoyl derivatives from SF1 subfractions.

ETHNOPHARMACOLOGICAL RELEVANCE: Echinodorus macrophyllus (Kunth.) Micheli (Alismataceae), known as chapéu-de-couro in Brazil, is popularly used to treat inflammatory diseases. We have previously demonstrated a significant reduction in the acute inflammation for the aqueous extract of E. macrophyllus (AEEm) and its ethanolic fraction (Fr20) and described that hydroxycinnamoyl derivatives present in SF1 (Fr20 subfraction) showed higher anti-inflammatory properties by mechanisms that include a reduction of TNF-α, IL-1ß, CKCL1/KC, LTB4, and PGE2 levels in exudate. AIM OF THE STUDY: This work describes the acute toxicological effect of SF1 subfraction on SW mice treated orally for five days in the air pouch model by evaluating the hematological and biochemical determinations on the blood samples; the relative organ weight and its histopathological analysis; the liver genotoxicity assessment and the activity of liver enzymes from xenobiotic metabolism. MATERIALS AND METHODS: Fr20 was earlier fractionated on the Sephadex LH-20 column, yielding mainly four subfractions, including SF1. The SF1 toxicity was evaluated in mice challenged with carrageenan on the air pouch inflammation model and orally treated for five days. The body weight was monitored daily, and the organs were weighed after the euthanasia. Hematological and biochemical determinations were carried out using specific commercial kits and following the protocols provided by the manufacturers. The organs were fixed, sectioned, processed for hematoxylin and eosin staining, and analyzed by light microscopy. Genotoxicity assessment was performed by the alkaline single-cell gel electrophoresis. Livers were processed for ethoxyresorufin-O-deethylase (EROD) and Glutathione S-transferase (GST) assays. RESULTS: SF1 exhibited low toxicity, as no significant discrepancy was observed in the relative weight of the body organs of mice. Moreover, the daily treatment with SF1 did not alter the number and percentage of red blood cells or hemoglobin concentration in the blood. The treatment with SF1 did not affect the creatinine concentration, but the 25 mg/kg dose reduced the plasma urea level and uric acid, suggesting its use in treating acute renal failure. The parameters analyzed did not present biochemical alterations indicative of liver disease. Regarding serum triglyceride and cholesterol levels, a significant decrease was detected in both parameters in mice treated with SF1. In addition, the histopathological analysis showed that inflammatory focus in the livers seemed more relevant in the control groups than in those treated. There were no significant changes in the renal or splenic tissues of animals treated with SF1. Treatment with SF1 also does not have a genotoxic effect on liver cells. CONCLUSION: Treatment with SF1 showed no toxicity in mice at doses equivalent to those recommended for humans, which provides evidence of the safety of the therapeutic use of this subfraction.

Echinodorus macrophyllus: Hydroxycinnamoyl derivatives reduces neutrophil migration through modulation of cytokines, chemokines, and prostaglandin in the air-pouch model.

ETHNOPHARMACOLOGICAL RELEVANCE: In Brazil, Echinodorus macrophyllus (Alismataceae), popularly known as chapéu-de-couro, is used to treat inflammatory diseases. Previous studies have shown a significant decrease in the acute inflammation for the aqueous extract of E. macrophyllus (AEEm) and its ethanolic fraction (Fr20). AIM OF THE STUDY: This work fractionated Fr20, identified the fraction and substances responsible for the in vivo anti-inflammatory property, and demonstrated important immunomodulatory mechanisms of action. MATERIALS AND METHODS: Fr20 was fractionated using Sephadex LH-20, and the most active fraction was chromatographically analyzed (HPLC-DAD and UPLC-ESI-TOF-MS). Leukotriene B4, Prostaglandin E2, and cytokines were determined by the enzyme-linked immunosorbent assay and in vivo acute inflammation by the air pouch model. RESULTS: The subfractions SF1, SF3, and mainly the SF4 decreased NO levels (p < 0.05). SF3 and SF4 showed high DPPH scavenger activity. SF1 was more effective than SF4 in reducing vasodilation, redness, and leukocyte migration into the 4-h air pouch. SF1 inhibited 90.5% (100 mg/kg) and SF4 54.0% (50 mg/kg), mainly affecting the number of neutrophils. SF1 and SF4 reduced the protein level in the exudate. SF1 was also more effective in inhibiting neutrophil migration in a transwell assay (46.3%) and reduced (86.1%) the Leukotriene B4 level in the exudate. After five days of treatment, some SF1 anti-inflammatory mechanisms were evaluated in the air pouch's 24 h exudate and tissue. Despite the high level of inflammation of the control group in this condition, SF1 confirmed the decrease in the protein level and neutrophils migration into the pouch. It decreased the number of bone marrow cells, indicating a systemic effect of SF1. SF1 also decreased TNF-α (87%), IL-1ß (77%), CKCL1/KC (71.3%), and PGE2 (97.8%) and increased IL-10 (74.1%) levels in the air pouch exudate. Phytochemical analysis of SF1 indicates mainly hydroxycinnamoyl derivatives. CONCLUSION: Hydroxycinnamoyl derivatives present in SF1 are related to the crucial anti-inflammatory mechanisms of E. macrophyllus, decreasing the levels of TNF-α, IL-1ß, CKCL1/KC, LTB4, and PGE2 on the exudate. These results explain the reduction of vasodilatation, erythema, and neutrophil migration into the air pouch model, confirming this plant's anti-inflammatory potential.

Flavonoid-enriched fraction from Echinodorus macrophyllus aqueous extract exhibits high in-vitro and in-vivo anti-inflammatory activity.

OBJECTIVES: Echinodorus macrophyllus (Kunth) Micheli (Alismataceae) is popularly used as an infusion to treat inflammatory diseases. This work fractionated the aqueous extract of E. macrophyllus (AEEm) to improve its anti-inflammatory effects. METHODS: Aqueous extract of E. macrophyllus was fractionated by Sephadex LH-20 and analysed by HPLC-DAD. Anti-inflammatory action was evaluated, in vivo, by air pouch model (total leucocyte, protein and leukotriene B4 (LTB4 )), and, in vitro, by neutrophil migration (transwell assay) and its Mac1 expression (flow cytometry), and RAW 264.7 nitric oxide (NO) production (Griess reaction). KEY FINDINGS: Fr20 reduced total leucocyte at 2.5 mg/kg (29.7%) while ethanolic extract of E. macrophyllus (EAEm) increased it (94.0%). Fr20 showed higher (P < 0.05) inhibition (89.8%) of LTB4 in exudate than EAEm (75.0%). Fr20 and EAEm decreased exudate protein and inflammatory infiltrate in pouch tissues, in-vitro neutrophil migration, and NO production. Otherwise, Fr40 did not reduce leucocytes and exudate protein (until 50 mg/kg) nor tissue inflammation, and increased in-vitro NO production. The inhibition of neutrophil migration by EAEm, but not Fr20, was dependent on reduced Mac-1 expression. CONCLUSIONS: The fractionation of AEEm provided a more potent anti-inflammatory fraction containing flavonoids (Fr20) that reduces the migration of neutrophils and LTB4 release, probably contributing to its mechanism of action.

Preclinical Studies Evaluating Subacute Toxicity and Therapeutic Efficacy of LQB-118 in Experimental Visceral Leishmaniasis.

Visceral leishmaniasis (VL) is the most severe form of leishmaniasis and is the second major cause of death by parasites, after malaria. The arsenal of drugs against leishmaniasis is small, and each has a disadvantage in terms of toxicity, efficacy, price, or treatment regimen. Our group has focused on studying new drug candidates as alternatives to current treatments. The pterocarpanquinone LQB-118 was designed and synthesized based on molecular hybridization, and it exhibited antiprotozoal and anti-leukemic cell line activities. Our previous work demonstrated that LQB-118 was an effective treatment for experimental cutaneous leishmaniasis. In this study, we observed that treatment with 10 mg/kg of body weight/day LQB-118 orally inhibited the development of hepatosplenomegaly with a 99% reduction in parasite load. An in vivo toxicological analysis showed no change in the clinical, biochemical, or hematological parameters. Histologically, all of the analyzed organs were normal, with the exception of the liver, where focal points of necrosis with leukocytic infiltration were observed at treatment doses 5 times higher than the therapeutic dose; however, these changes were not accompanied by an increase in transaminases. Our findings indicate that LQB-118 is effective at treating different clinical forms of leishmaniasis and presents no relevant signs of toxicity at therapeutic doses; thus, this framework is demonstrated suitable for developing promising drug candidates for the oral treatment of leishmaniasis.