Protease inhibitors characterisation by SDS-PAGE and MALDI-TOF from Alocasia macrorrhizos and their modulation of macrophage immune-inflammatory properties.

This study describes the extraction and identification by electrophoretic and spectrometric techniques of protease inhibitor from the medicinal plant Alocasia macrorrhizos as well as investigates their immunomodulatory properties and cell viability. The A. macrorrhizos tubers were subjected to protease inhibitor extractions and characterised using SDS-PAGE and MALDI-TOF. The protein extracts were assessed for activities trypsin inhibition stoichiometry, haemagglutinating, cell viability, NO and TNF-α production inhibition. Concerning the protease inhibitors analysis through SDS-PAGE, the results showed two bands with 11 and 24 kDa, and the MS analysis detected the ions more intense of m/z 4276.795 and 8563.361 in the roasted protein extract. The IC50 of trypsin inhibition was 0.119 and 0.302 mg L-1 in the roasted and crude tuber, respectively. The protease inhibitors extract from the roasted tubers showed a reduction in the production of NO and TNF-α at concentrations lower than 100 µg mL-1, without a reduction in cell viability.

Metabolic profiling by LC-DAD-MS, FTIR, NMR and CE-UV of polyphenols with potential against skin pigmentation disorder.

In traditional Brazilian medicine, tubers extracts from Alocasia macrorrhizos are widely used in the treatment of skin pigmentation disorder. However, studies that evaluate its benefits in the treatment of this disorder are non-existent. Thus, this work aims to investigate the bioactivity of A. macrorrhizos extracts in cell culture and murine model of Vitiligo and correlating with its phenolic profile. The metabolic profiling from the bioactive extracts was obtained by LC-DAD-MS, FTIR, NMR, and CE-UV. The murine model of Vitiligo was induced with 5% hydroquinone in C57BL/6 male mice, which were treated or not with 100 mg/kg of roasted tuber aqueous extract. In Vitiligo model assay was observed hair follicle repigmentation and reduction of the epidermal layer thickness at the histopathological level, in the animals treated with aqueous extract of roasted tubers. The present study provides new molecular insight and scientific evidence on the potential utility of the extract of A. macrorrhizos against Vitiligo.

Dereplication of Siparuna brasiliensis extract with in vivo anti-inflammatory activity.

Siparuna brasiliensis is a medicinal plant widely used by indigenous communities of the Amazon rainforest to treat inflammatory diseases and related pathologies. Considering its ethnopharmacological application, it constitutes an important source of biologically active molecules in the development of anti-inflammatory drugs. This study describes a dereplication methodology of the bioactive extract from S. brasiliensis leaves and the evaluation of the anti-inflammatory potential in an in vivo inflammatory model with mice of the BALB/c lineage and in vitro using cell lines, as well as determining the production of an inflammatory mediator. From their charge-to-mass ratios (m/z) and elemental composition obtained through Ultrahigh-resolution mass spectrometry analysis by ESI(-)-Orbitrap MS and chromatographic profile by RP-HPLC-PDA, it was possible to annotate polyphenols with anti-inflammatory properties classified as flavonoids and organic acids. The administration of the extract significantly inhibited carrageenan-induced paw edema and showed effects similar to those of drug dexamethasone without affecting cell viability.

Effect of Anadenanthera colubrina protease inhibitors as an antiinflamatory mediator.

This work aimed to obtain and characterize protease inhibitors from A. colubrina leaves and evaluate their potential as inflammatory mediator and cell viability. The protein extract was analyzed and characterized by SDS-PAGE, RP-HPLC-PDA, MALDI-TOF/MS and Zeta potential. Bioassays were conducted in order to evaluate the cell viability in RAW 264.7, in vitro (NO and TNF-α production inhibition) and in vivo anti-inflammatory potential, inhibition rate of trypsin and hemagglutination activity from protein extract. The results revealed the presence of bands at 14, 21 and 30 kDa in SDS-PAGE, the RP-HPLC-PDA analysis showed peaks at 12, 13, 28 and 40 minutes and MALDI-TOF/MS showed peaks with 3.4, 4.7, 5.6, 9.4 and 11.2 kDa. The protein extracts presented enzymatic activity inhibition of trypsin (IC50 59.2 µgmL-1), did not show any cytotoxicity to RAW264.7 cells, hemagglutination 8HU and insignificant reduction in NO and TNF-α production and reduced anti-inflammatory potential in vivo compared to dexamethasone.

Lipophilic amino alcohols reduces carrageenan-induced paw edema and anti-OVA DTH in BALB/c mice.

The inflammation process is a coordinated response of the organism related to immune response with release of pro-inflammatory substances, as nitric oxide, TNF-α and IL-1ß. In this work, a series of lipophilic amino alcohols were evaluated on RAW264.7 and primary macrophages for the modulation of nitric oxide and TNF-α. The most potent compounds were submitted to the treatment of BALB/c mice and evaluation of the carrageenan-induced paw edema and TNF-α and IL1-ß release in the paws and anti-OVA delayed type hypersensitivity reaction. RAW264.7 and primary macrophages were incubated in the presence of amino alcohols at different concentrations (1, 0.5, 0.05 and 0.005 µg mL(-1)). All tested compounds were not cytotoxic, however the inhibition of NO and TNF-α were observed only in RAW264.7 cultures. The NO production were reduced in 100% for all compounds, but only the compounds 4a and 4b expressively reduced the TNF-α release (67% and 92% respectively). On the carrageenan-induced paw edema, the compound 4b treatment showed reduction of edema, TNF-α and IL-1ß as efficient as dexamethasone treatment. Meanwhile, the compound 4a treatment showed only slight reduction of paw edema. In the anti-OVA DTH reaction, both compounds showed reduction in the paw edema as effective as dexamethasone. In function of the observed results in vitro and in the acute and anti-OVA inflammation of mice paw edema compound 4b showed promissory anti-inflammatory properties.

Synthesis and evaluation of cytotoxicity and inhibitory effect on nitric oxide production by J774A.1 macrophages of new anthraquinone derivatives.

Mitoxantrone is an anthracene-based anticancer agent whose efficacy in treating autoimmune diseases is believed to be due to cytotoxicity and inhibition of proliferation of cells. Several novel anthraquinone derivatives, analogs of mitoxantrone, were designed and synthesized. Lipophilic and functionalized mitoxantrone analogs were prepared by a simple methodology and the cytotoxicity and the inhibitory effect on nitric oxide release of these compounds were demonstrated in vitro on J774A.1 macrophages. Interestingly compounds 3, 4, 5, 6, 7, and 8 exhibited reduction in NO release (62.4%, 92.6%, 73.4%, 58.4%, 57.8% and 53.4%, respectively) in comparison to NG-n-methyl-arginine treated control, without cytotoxicity. In conclusion, anthraquinone derivatives were prepared in a good yield and showed promissory antiinflammatory properties.