In vitro antioxidant and enzymatic approaches to evaluate neuroprotector potential of Blechnum extracts without cytotoxicity to human stem cells

Abstract

Background:Investigation of selected plant extracts on multi-targets related to neurodegeneration, such as monoamine oxidases (MAO), cholinesterase enzymes, and antioxidant activities (AOA) is a useful tool for identification of new scaffolds.Objective:This work investigated biological effects of three Blechnum methanol extracts from Brazil and chemical profile of the most active sample.Materials and Methods:AOA included scavenging of hydroxyl and nitric oxide radicals, also lipid peroxidation inhibition. Enzymatic modulation of Blechnum binervatum, Blechnum brasiliense, and Blechnum occidentale extracts on MAO and cholinesterases was conducted. Moreover, total phenol content was performed with all samples, and high-performance liquid chromatography-diode array detection mass spectrometry HPLC-DAD-MS analysis was carried out with B. brasiliense. Possible toxic effects were evaluated on Wistar rats polymorphonuclear cells (PMN) and human stem cells.Results:B. brasiliense extract presented the highest phenolic amount (9.25 g gallic acid equivalent/100 g extract) and lowest IC50 values (112.3 ± 2.61 and 176.1 ± 1.19 μg/mL) against hydroxyl radicals and on lipid peroxidation, respectively, showing strong AO effects. On nitric oxide assay and cholinesterase inhibition, all extracts were considered inactive. MAO-A selective action was evidenced, being B. brasiliense powerful against this enzyme (IC50: 72.7 μg/mL), followed by B. occidentale and B. binervatum (IC50: 130.85 and 165.2 μg/mL). No cytotoxic effects were observed on PMN and human stem cells treated with Blechnum extracts. HPLC-DAD-MS analysis of B. brasiliense allowed the identification of chlorogenic and rosmarinic acids.Conclusion:Our results especially highlight B. brasiliense, with pronounced phenols content and strong effects on selected targets related to neurodegeneration, being characterized as a natural safe source of bioactive hydroxycinnamic acids.SUMMARY Blechnum crude extracts showed high phenolic amounts and valuable IC50 values on targets related with neurodegenerative disordersBlechnum brasiliense was the most active sample, with strong radical scavenging and lipid peroxidation inhibition, also with monoamine oxidases: A selective modulationNo cytotoxic effects were observed on polymorphonuclear cells rat cells and human stem cells treated with Blechnum extractsHigh-performance liquid chromatography-diode array detection-mass spectrometry analysis of Blechnum brasiliense allowed the identification of hydroxycinnamic derivatives: Chlorogenic and rosmarinic acids. Abbreviations used: IC50: half maximal inhibitory concentration; MAO: monoamine oxidase; MAO-A: monoamine oxidase isoform A; MAO-B: monoamine oxidase isoform B; HO•: hydroxyl radical.

Identification of compounds from non-polar fractions of Blechnum spp and a multitarget approach involving enzymatic modulation and oxidative stress

“…On the other hand, as expected, Triton X (1%) displayed strong cell damage, evidenced by membrane injury observed in this methodology. Our group has already demonstrated the absence of toxic effects to this cell type when incubated with ferns crude extracts at 100 μg/ml and also with Blechnum crude extracts at 1 mg/ml, by the same method …”

“…Sodium hydroxide (2 m ) was added to stop reactions. Fluorescence readings at λ exc 320 nm/λ em 400 nm were carried out . Clorgyline and pargyline were used as positive control inhibitors of MAO‐A and MAO‐B, respectively.…”

“…Stem cells from human exfoliated deciduous teeth (SHED) were isolated, with previous approval by the Brazilian National Research Ethics Committee (Protocol number CAAE 36403514600005347). After cultivation, SHED characterization was performed by morphological analysis, differentiation assay in vitro (adipogenic, chondrogenic and osteogenic) and flow cytometry (FACSAria III BD ® , CA) and incubated with specific monoclonal antibodies (CD14, CD34, CD45, CD73, CD90, CD105 and HLADR) …”

“…The SHED were plated (7 × 10 3 cells/well) and, after reaching confluence, were treated with the HEX and DCM fractions at 100 and 500 μg/ml and dissolved in DMSO (2%), using Triton X‐100 (1%) as positive control. After 24 h, cell viability was determined by the reduction of 3‐(4,5 dimethylthiazole‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) . Cell membrane integrity was assessed in the culture supernatant by LDH assay, using a commercial kit (Labtest Diagnostica, Minas Gerais, Brazil) .…”

“…Plants of this group have been investigated for a wide range of biological activity, including anti‐inflammatory and antinociceptive, antioxidant, anticancer, antibacterial and wound healing effects . Recently, our group demonstrated the potential of crude methanol extracts and isolated compounds from southern Brazil Blechnum species on targets related to the aetiology of neurodegenerative diseases …”