Remarkable rutin-rich Hypericum capitatum extract exhibits anti-inflammatory effects on turpentine oil-induced inflammation in rats

PubMed: 31664997Background: Natural extracts with beneficial biological activities are nowadays of high interest, in various treatment or prophylaxis. Hypericum capitatum has been known for its curative effects for centuries and its extracts have become of interest due to their distinct activity among other Hypericaceae members. In this study, further light is aimed to be shed on the secondary-metabolites composition of H. capitatum extracts, using chromatographic techniques and Electron paramagnetic resonance profiles in alkaline medium. Considering that no previous works explored the anti-inflammatory activity of H. capitatum, here, an in vivo study is also designed in order to evaluate this property by assessing the impact of one of H. capitatum extracts in ameliorating turpentine oil-induced inflammation on rats and to quantify their blood antioxidants level. Methods: Chromatographic techniques and Electron paramagnetic resonance spectroscopy were used in order to describe the chemical profile in different parts of the plant. The in vivo study on turpentine-oil induced inflammation in rats included three doses of H. capitatum extract expressed in rutin concentration. Oxidative stress was measured using total oxidative status, total antioxidant capacity, oxidative stress index, 3-nitrotyrosine, nitric oxide, malondialdehyde, superoxide dismutase, catalase and the inflammatory response was evaluated by performing a complete blood cells count and C reactive protein. Results: The extract was remarkably rich in rutin; however, other polyphenolic-like minor components appeared important in explaining the observed biological properties. The tested extract prevents the increase of inflammation-induced white blood cell count, number of neutrophils, and serum nitric oxide, and did so in a dose-dependent manner, similarly to the positive control - diclofenac. In addition, the same extract appeared to be a good alternative to diclofenac to restore total oxidative status, thiobarbituric active reactive species, total proteins and C reactive proteins. Moreover, antioxidant enzymes such as catalase, superoxide dismutase and total serum thiol concentration were significantly increased by the tested extract. Conclusions: Due to its powerful reservoir rich in rutin, H. capitatum extract depicted its in vivo antioxidant and anti-inflammatory effects indicating it to be a good alternative to conventional drugs for oxidative stress protection. © 2019 The Author(s).Ontario Ministry of Research, Innovation and Science, MRIS: PN-III-P1–1.1-PD-2016-0121This work was financially supported by Romanian Ministry of Research and Innovation, project PN-III-P1–1.1-PD-2016-0121. This financial support facilitated the purchase of several chemical reagents and special kits for analysis

Alternative fluorimetric-based method to detect and compare total S-nitrosothiols in plants

Nitric oxide (NO) is an important signaling molecule occurring in virtually all organisms, whose mechanism of action relies mainly on its interaction with proteins or peptides by nitrosylation, forming compounds such as S-nitrosothiols (SNO). The Saville reaction and the ozone-based chemiluminescence method are the main techniques used for nitrosylated protein quantification. While the Saville assay is not very sensitive, the ozone-based chemiluminescence is expensive and time-consuming. Here we propose a method in which the protein-bound NO groups are exposed to UV light, cleaving the bond and allowing the quantification of the derived NO molecules by diamino-rhodamine (DAR) dyes. The DAR-based method was shown to be specific in plant tissues by pre-treatment of the samples with reducing agents and parallel EPR analysis. Spike-and-recovery assays revealed 72% recovery after a GSNO spike. Moreover, the method was significantly more sensitive than the Saville reaction, and this increase in sensitivity was crucial for detecting the reduced levels of nitrosylated proteins in plant species other than Arabidopsis. The method presented here is a suitable alternative to compare plant samples, allowing simple and fast detection of nitrosylated proteins.Funding: This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) [grant numbers 2011/50637-0, 2013/18056-2 and 13/15108-1], Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) [grant numbers 442045/2014-0, and 309504/2014-7], Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) [grant number 99999.006262/2015-05] and CNCSIS/UEFISCDI [grant PN-II-RU-TE-2014-4-2555]. Research in FJC laboratory is supported by the ERDF-cofinanced grants from the Ministry of Economy and Competitiveness (AGL2015-65104-P) and Junta de Andalucía (group BIO192), Spain. MRR acknowledges an FPI contract (BES-2012-055904) from the Ministry of Economy and Competitiveness, Spain