Safety evaluation of aqueous extract from Eugenia uniflora leaves: Acute and subacute toxicity and genotoxicity in vivo assays.

ETHNOPHARMACOLOGICAL RELEVANCE: Eugenia uniflora Linn (Myrtaceae) is the native species of Brazil. The leaves of this species are used in folk medicine to treat different inflammatory and gastrointestinal disorders. However, research on the safety of using E. uniflora leaves has been poorly explored. AIM OF THE STUDY: This approach aims to investigate the phytochemical composition as well as the acute, subacute toxicity, and in vivo genotoxic profile of the aqueous extract of E. uniflora leaves. MATERIALS AND METHODS: The chemical composition of E. uniflora leaf extract was determined by Fingerprint by High-Performance Thin Layer and High-Performance Liquid Chromatography. The acute toxicity in vivo was evaluated for 14 days after the administration of E. uniflora leaves extract (2000 mg/kg). For the evaluation of subacute toxicity, mice were daily treated for 28 days with E. uniflora extract (250, 500, or 1000 mg/kg). Signs of behavioral toxicity and biochemical and hematological alterations, including the multiple organ toxicities were investigated. In addition, the micronucleus assay was used to evaluate the in vivo genotoxicity of the leaves extract in murine erythrocytes. RESULTS: The phytochemical analysis showed the majority presence of phenolic compounds (gallic acid, ellagic acid, and myricitrin). Single or repeated doses of the aqueous extract of E. uniflora leaves did not reveal any signs of in vivo toxicity. Daily doses of the extract for 28 days induced a slight reduction in cholesterol and triglyceride levels. Furthermore, E. uniflora leaves extract (1000-2000 mg/kg) showed no genetic damage in the micronucleus assay, indicating the absence of genotoxicity of the herbal species. CONCLUSION: The aqueous extract of E. uniflora leaves showed a predominance of phenolic compounds, with non-toxic and non-genotoxic action in vivo. This approach sheds light on the chemical composition of the leaves of E. uniflora and suggests a high margin of safety in the popular use of the leaves of this plant species.

Anti-inflammatory, antinociceptive effects and involvement of opioid receptors in the antinociceptive activity of Eugenia uniflora leaves obtained with water, ethanol, and propylene glycol mixture.

ETHNOPHARMACOLOGICAL RELEVANCE: Eugenia uniflora (Myrtaceae) is a species native to Brazil and has a traditional use in the treatment of inflammation. AIM OF THE STUDY: To evaluate the anti-inflammatory and antinociceptive effects, and the involvement of opioid receptors in the antinociceptive activity of extract and fractions from Eugenia uniflora leaves. MATERIALS AND METHODS: TLC and HPLC were used to characterize the spray-dried extract (SDE) and fractions. In the in vivo assays, Swiss (Mus musculus) mice were used. Carrageenan-induced hind-paw edema and carrageenan-induced peritonitis models were used to determine the anti-inflammatory effect of the extract (50, 100, or 200 mg/kg). Acetic acid-induced writhing, tail-flick, and formalin tests were used to determine the antinociceptive effect of the extract (50, 100, or 200 mg/kg). The aqueous (AqF) and ethyl acetate (EAF) fractions (6.25, 12.5, and 25 mg/kg) were then combined with naloxone to evaluate the involvement of opioid receptors in the antinociceptive activity. RESULTS: In this work, the TLC and HPLC analysis evidenced the enrichment of EAF, which higher concentration of gallic acid (5.29 ± 0.0004 %w/w), and ellagic acid (1.28 ± 0.0002 %w/w) and mainly myricitrin (8.64 ± 0.0002 %w/w). The extract decreased the number of total leukocytes and neutrophils in the peritoneal cavity (p < 0.05), at doses of 100 and 200 mg/kg and showed significant inhibition in the increase of paw edema volume (p < 0.05). The treatment per oral route (doses of 50, 100, and 200 mg/kg) significantly reduced the nociceptive response in acetic acid-induced abdominal writhing (p < 0.05). The effect of the extract on the tail-flick test showed a significant increase in latency time of animals treated at doses of 200 and 100 mg/kg (p < 0.05). The extract and ethyl acetate fraction reduced the nociceptive effect in both phases of formalin at all tested doses. The naloxone reversed the antinociceptive effect of EAF, suggesting that opioid receptors are involved in mediating the antinociceptive activity of EAF of E. uniflora in the formalin test. CONCLUSION: The current study demonstrates the anti-inflammatory and analgesic activities of water: ethanol: propylene glycol spray-dried extract from E. uniflora leaves using in vivo pharmacological models in mice. Our findings suggest that spray-dried extract and ethyl acetate fraction exhibit peripheral and central antinociceptive activity with the involvement of opioid receptors that may be related to the presence of flavonoids, mainly myricitrin.

Streptomyces hygroscopicus UFPEDA 3370: A valuable source of the potent cytotoxic agent nigericin and its evaluation against human colorectal cancer cells.

Streptomyces hygroscopicus UFPEDA 3370 was fermented in submerged cultivation and the biomass extract was partitioned, obtaining a fraction purified named EB1. After purification of EB1 fraction, nigericin free acid was obtained and identified. Nigericin presented cytotoxic activity against several cancer cell lines, being most active against HL-60 (human leukemia) and HCT-116 (human colon carcinoma) cell lines, presenting IC50 and (IS) values: 0.0014 µM, (30.0) and 0.0138 µM (3.0), respectively. On HCT-116, nigericin caused apoptosis and autophagy. In this study, nigericin was also screened both in vitro and in silico against a panel of cancer-related kinases. Nigericin was able to inhibit both JAK3 and GSK-3ß kinases in vitro and its binding affinities were mapped through the intermolecular interactions with each target in silico.

Anti-staphylococcal activity of Syagrus coronata essential oil: Biofilm eradication and in vivo action on Galleria mellonela infection model.

In this study, essential oil extracted from Syagrus coronata seeds (SCEO) was evaluated for antibacterial and antibiofilm activities against Staphylococcus aureus; in addition, Galleria mellonella model was used as an in vivo infection model. SCEO was mainly composed by fatty acids (89.79%) and sesquiterpenes (8.5%). The major components were octanoic acid, dodecanoic acid, decanoic acid and γ-eudesmol. SCEO showed bactericidal activity (minimal bactericidal concentration from 312 to 1250 µg/mL) against all tested S. aureus clinical isolates, which showed distinct biofilm-forming and multiple drug resistance phenotypes. SCEO weakly reduced biomass but remarkably decreased cell viability in pre-formed biofilms of S. aureus isolate UFPEDA-02 (ATCC-6538). Electron microscopy analysis showed that SCEO treatments decreased the number of bacterial cells (causing structural alterations) and lead to loss of the roughness in the multiple layers of the three-dimensional biofilm structure. In addition, overproduction of exopolymeric matrix was observed. SCEO at 31.2 mg/kg improved the survival of G. mellonela larvae inoculated with UFPEDA-02 isolate and reduced the bacterial load in hemolymph and melanization. In conclusion, SCEO is an antibacterial agent against S. aureus strains with different resistance phenotypes and able to disturb biofilm architecture. Our results show SCEO as a potential candidate to drug development.

Crataeva tapia bark lectin (CrataBL) is a chemoattractant for endothelial cells that targets heparan sulfate and promotes in vitro angiogenesis.

Formation of new blood vessels from preexisting ones, a process known as angiogenesis, is one of the limiting steps for success in treatment of ischemic disorders. Therefore, efforts to understanding and characterize new agents capable to stimulate neovascularization are a worldwide need. Crataeva tapia bark lectin (CrataBL) has been shown to have chemoattractant properties for endothelial cells through the stimulation of migration and invasiveness of human umbilical vein endothelial cells (HUVEC) because it is a positively charged protein with high affinity to glycosaminoglycan. In addition, CrataBL increased the production of chondroitin and heparan sulfate in endothelial cells. These findings orchestrated specific adhesion on collagen I and phosphorylation of tyrosine kinase receptors, represented by vascular endothelial growth factor receptor-2 (VEGFR-2) and fibroblast growth factor receptor (FGFR), whose downstream pathways trigger the angiogenic cascade increasing cell viability, cytoskeleton rearrangement, cell motility, and tube formation. Moreover, CrataBL inhibited the activity of matrix metalloproteases type 2 (MMP-2), a protein related to tissue remodeling. Likewise, CrataBL improved wound healing and increased the number of follicular structures in lesioned areas produced in the dorsum-cervical region of C57BL/6 mice. These outcomes altogether indicate that CrataBL is a pro-angiogenic and healing agent.

Evaluation of antitumor activity and toxicity of Schinus terebinthifolia leaf extract and lectin (SteLL) in sarcoma 180-bearing mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Schinus terebinthifolia Raddi is a plant broadly used in folk medicine and the use of its leaf extract as an antitumor agent has been reported. AIM OF THE STUDY: To evaluate the antitumor potential and the toxicity of saline extract (SE) and lectin (SteLL) from S. terebinthifolia leaves in sarcoma 180-bearing mice. MATERIALS AND METHODS: Cytotoxicity to sarcoma 180 cells was tested in vitro, and antitumor assay was performed using Swiss female mice. The treatments (0.15 M NaCl, negative control; methotrexate 1.5 mg/kg, positive control; SE 100 mg/kg; SteLL 1 and 5 mg/kg) by intraperitoneal injections started on the 8th day after tumor inoculation and lasted 7 days. It was analyzed: tumor weight; number and gauge of tumor vessels; hematological and biochemical parameters; histopathological changes; and occurrence of micronuclei in bone marrow cells. RESULTS: SE and SteLL showed IC50 values (concentrations that reduced cell viability to 50%) of 301.65 and 8.30 µg/mL, respectively. The lectin was able to induce apoptosis. Treatments with the extract and lectin caused a 57.6-73.6% reduction in tumor weight, which was not significantly different from the reduction in the methotrexate group. Tumors of animals treated with SteLL at 5 mg/kg showed reduced number of secondary vessels while the gauge was lower in all treated groups. In the groups treated with SteLL, tumors showed reduced and slightly vascularized parenchyma, with necrosis in the center and at the periphery. No alterations in the blood levels of urea, creatine, and glucose were detected while serum AST level was moderately increased in the SE group. Histopathological analysis revealed vacuolization and steatosis in the liver of animals treated with the extract and lectin. In addition, the treatments with SE and SteLL resulted in the reduction of filtration space and alterations in tubular architecture in kidneys. In respect to hematological parameters, it was only detected increase in the number of monocytes in SE group. The extract and lectin did not induce the formation of micronuclei in the bone marrow cells. CONCLUSIONS: SE and SteLL had antitumor effect against sarcoma 180 without inducing hematological changes and genotoxic effects in mice; however, some degree of hepatic and renal toxicity was observed, suggesting the evaluation of drug delivery strategies in the future.

CasuL: A new lectin isolated from Calliandra surinamensis leaf pinnulae with cytotoxicity to cancer cells, antimicrobial activity and antibiofilm effect.

This work describes the isolation of a lectin (CasuL) from the leaf pinnulae of Calliandra surinamensis and the evaluation of its cytotoxic, antimicrobial and antibiofilm properties. Proteins from pinnulae extract were precipitated with ammonium sulphate (60% saturation) and submitted to Sephadex G-75 chromatography, which yielded isolated CasuL (purification factor: 113). Native CasuL is an acidic protein (pI 5.82) with a relative molecular mass of 48kDa. This lectin is also an oligomeric protein composed of three subunits and mass spectrometry revealed similarities with a Sorghum bicolor protein. CasuL did not undergo unfolding when heated but changes in conformation and hemagglutinating activity were detected at basic pH. CasuL did not reduce the viability of human peripheral blood mononuclear cells but was toxic to leukemic K562 cells (IC50 67.04±5.78µg/mL) and breast cancer T47D cells (IC50: 58.75±2.5µg/mL). CasuL (6.25-800µg/mL) only showed bacteriostatic effect but was able to reduce biofilm formation by Staphylococcus saprophyticcus and Staphylococcus aureus (non-resistant and oxacillin-resistant isolates). CasuL showed antifungal activity against Candida krusei causing alterations in cell morphology and damage to cell wall. In conclusion, the pinnulae of C. surinamensis leaves contain a thermo-stable lectin with biotechnological potential as cytotoxic, antibiofilm, and antifungal agent.