Pulmonaria obscura and Pulmonaria officinalis Extracts as Mitigators of Peroxynitrite-Induced Oxidative Stress and Cyclooxygenase-2 Inhibitors-In Vitro and In Silico Studies.

The Pulmonaria species (lungwort) are edible plants and traditional remedies for different disorders of the respiratory system. Our work covers a comparative study on biological actions in human blood plasma and cyclooxygenase-2 (COX-2) -inhibitory properties of plant extracts (i.e., phenolic-rich fractions) originated from aerial parts of P. obscura Dumort. and P. officinalis L. Phytochemical profiling demonstrated the abundance of phenolic acids and their derivatives (over 80% of the isolated fractions). Danshensu conjugates with caffeic acid, i.e., rosmarinic, lithospermic, salvianolic, monardic, shimobashiric and yunnaneic acids were identified as predominant components. The examined extracts (1-100 µg/mL) partly prevented harmful effects of the peroxynitrite-induced oxidative stress in blood plasma (decreased oxidative damage to blood plasma components and improved its non-enzymatic antioxidant capacity). The cellular safety of the extracts was confirmed in experimental models of blood platelets and peripheral blood mononuclear cells. COX-2 inhibitor screening evidently suggested a stronger activity of P. officinalis (IC50 of 13.28 and 7.24 µg/mL, in reaction with synthetic chromogen and physiological substrate (arachidonic acid), respectively). In silico studies on interactions of main components of the Pulmonaria extracts with the COX-2 demonstrated the abilities of ten compounds to bind with the enzyme, including rosmarinic acid, menisdaurin, globoidnan A and salvianolic acid H.

Protective Role of Glutathione against Peroxynitrite-Mediated DNA Damage During Acute Inflammation.

Inflammation is an immune response to protect against various types of infections. When unchecked, acute inflammation can be life-threatening, as seen with the current coronavirus pandemic. Strong oxidants, such as peroxynitrite produced by immune cells, are major mediators of the inflammation-associated pathogenesis. Cellular thiols play important roles in mitigating inflammation-associated macromolecular damage including DNA. Herein, we have demonstrated a role of glutathione (GSH) and other thiols in neutralizing the effect of peroxynitrite-mediated DNA damage through stable GSH-DNA adduct formation. Our observation supports the use of thiol supplements as a potential therapeutic strategy against severe COVID-19 cases and a Phase II (NCT04374461) open-label clinical trial launched in early May 2020 by the Memorial Sloan Kettering Cancer Center.

Pine (Pinus sylvestris L.) bark proanthocyanidins affords prevention of peroxynitrite-induced l-tyrosine nitration, DNA damage and hydroxyl radical formation.

Peroxynitrite is known as a strong deleterious species that may readily trigger several geriatric diseases via injuring cellular constituents. Proanthocyanidins, a biological flavonoids constituent of Pinus sylvestris L. bark, has been attributed a large variety of pharmacological functions to its antioxidant potential. The results revealed that peroxynitrite could cause the generation of hydroxyl radical, the breakage of φX-174 plasmid DNA brand as well as the nitration of L-tyrosine. However, pine (Pinus sylvestris L.) bark proanthocyanidins extracts at low concentration range markedly inhibited the peroxynitrite -induced the formation of open circular DNA form (IC50 = 5.03±0.39 mg/mL). The 3-Nitro-L-tyrosine generated by the reaction of peroxynitrite with L-tyrosine was reduced by PBP (IC50 = 1.01±0.01 mg/mL). Besides, electron spin resonance spectroscopy data indicates that the intensive signal of dimethyl pyridine N-oxide hydroxyl radical adduct from peroxynitrite was reversed by pine bark proanthocyanidins extracts (IC50 =1.02±0.04 mg/mL). Moreover, the obtained data shows that PBP provides more efficient protection against peroxynitrite than that of ascorbic acid. Together, the present study suggests that pine bark proanthocyanidins could exert potent preventive activity against peroxynitrite -elicited cytotoxicity on the biomacromolecules, a study-worthy finding with pharmacological importance.

The extract from hop cones (Humulus lupulus) as a modulator of oxidative stress in blood platelets.

The plant Humulus lupulus is known as the raw material of the brewing industry. Hop cones, rich in polyphenolic compounds and acyl phloroglucides, are widely used to preserve beer and to give it a characteristic aroma and flavor. Hop cones have long been used for medicinal purposes. In particular, hop preparations were mainly recommended for the treatment of sleeping disorders. The antioxidative action of hop cones, however, is poorly understood. The aim of our present study was to investigate in vitro changes in human blood platelets induced by peroxynitrite (ONOO(-), the compound of particular importance for vascular thrombosis and inflammatory process) in the presence of hop cone extract (Humulus lupulus). The antioxidative action of the extract was also compared with the properties of a well-characterized antioxidative commercial monomeric polyphenol, resveratrol (3,4',5-trihydroxystilbene) in a model system in vitro. Various biomarkers of oxidative/nitrative stress, such as carbonyl groups, 3-nitrotyrosine and thiobarbituric acid reactive substances (TBARS) were estimated. The 3-nitrotyrosine formation and carbonyl group generation was assessed by the use of a competition ELISA test and ELISA test, respectively. Tested plant extract (12.5-50 µg/ml), like resveratrol, significantly inhibited protein carbonylation and nitration in the blood platelets treated with ONOO(-) (0.1 mM). The extract from hop cones, like resveratrol, also caused a distinct reduction of platelet lipid peroxidation induced by ONOO(-). The present results indicate that the hope cone extract has in vitro protective effects against ONOO(-), such as induced oxidative/nitrative damage to the human platelet proteins and lipids. However, in comparative studies the extract was not found to be a more effective antioxidant than the solution of pure resveratrol.

An in vitro examination of the antioxidant and anti-inflammatory properties of buckwheat honey.

OBJECTIVE: Hydroxyl radical and hypochlorite anion formed at the wound site from superoxide anion produced by activated polymorphonuclear neutrophils (PMNs) are considered important factors in impaired wound healing. Superoxide anion may also react with nitric oxide produced by macrophages to form peroxynitrite, a third strong oxidant that damages surrounding tissue. In order to select honey for use in wound-healing products, different samples were compared for their capacity to reduce levels of reactive oxygen species (ROS) in vitro. METHOD: Honey samples were tested in assays for inhibition of ROS production by activated human PMNs, antioxidant activity (scavenging of superoxide anion in a cell-free system) and inhibition of human complement (reducing levels of ROS by limiting formation of complement factors that attract and stimulate PMNs). For buckwheat honey (NewYork, US), moisture and free acid content were determined by refractive index measurement and potentiometric titration respectively. Honey constituents other than sugars were investigated by thin layer chromatography, using natural product reagent to detect phenolic compounds. Constituents with antioxidant properties were detected by spraying the chromatogram with DPPH. RESULTS: Although most honey samples were shown to be active, significant differences were observed, with the highly active honey exceeding the activities of samples with minor effects by factors of 4 to 30. Most pronounced activities were found for American buckwheat honey from the state of NewYork. Phenolic constituents of buckwheat honey were shown to have antioxidant activity. CONCLUSION: As buckwheat honey was most effective in reducing ROS levels, it was selected for use in wound-healing products. The major antioxidant properties in buckwheat honey derive from its phenolic constituents, which are present in relatively large amounts. Its phenolic compounds may also exert antibacterial activity, whereas its low pH and high free acid content may assist wound healing.

Antioxidant and antiaggregatory effects of an extract from Conyza canadensis on blood platelets in vitro.

The antioxidative activity of the polysaccharide extract from Conyza canadensis in blood platelets treated with peroxynitrite (ONOO-) was studied. Peroxynitrite as a strong biological oxidant has toxic effects on blood platelets and induces the oxidation of thiols, carbonylation and nitration of platelet proteins and lipid peroxidation. Therefore, the aim of our study was to assess if the natural extract from herbal plant, Conyza Canadensis, may protect platelet proteins against nitrative and oxidative damage induced by ONOO-. In our study we measured oxidative damage of platelet proteins induced by peroxynitrite and protectory effects of this extract by estimation of the level of carbonyl groups and nitrotyrosine (a marker of platelet protein nitration). We also used cytochrome c reduction method to test the ability of this extract to change O2-* generation in platelets. Moreover, we determined the effects of the extract on blood platelet aggregation induced by ADP. We observed that the extract from Conyza canadensis distinctly reduced oxidation and nitration of proteins in blood platelets treated with ONOO-(0.1mM) and O2-* production in these cells. The extract from Conyza canadensis also inhibited platelet aggregation. The ability of the extract to decrease O2-* generation in blood platelets supports the importance of free radicals in platelet functions, including aggregation process. The present study suggests that the natural polysaccharide extract from Conyza canadensis has antiaggregatory and antioxidative activities, and therefore may be beneficial in the prevention of peroxynitrite-related diseases, such as cardiovascular or inflammatory diseases.

Alveolar macrophage cytotoxicity for normal lung fibroblasts is mediated by nitric oxide release.

Nitric oxide (NO) released by activated alveolar macrophages (AM) can mediate effects on target cells and can also react with superoxide anion (O2-) to form peroxynitrite (PN), a highly cytotoxic product. The role of NO and PN in AM cytotoxicity for normal lung cells was investigated using co-cultures of rat lung fibroblasts (FB) and rat AM treated with lipopolysaccharide + interferon-gamma (LI). AM and FB, alone and in co-culture, were treated with LI for 5 days and cell viability measured. The culture media was analyzed for NO, TNF-alpha, O2-, and IL-1beta. A decreased FB viability was correlated with increased NO release by LI-activated AM. Pretreatment of co-cultures with the inducible NOS inhibitor L-NAME caused dose-related decreases in NO release by AM and increases in FB viability. Although TNF-alpha release was increased in co-cultures treated with LI, the viability of FB was not affected when cultures were treated with similar concentrations of TNF-alpha in the absence of AM. O2- could not be detected in the media and addition of superoxide dismutase (SOD) did not protect FB. These data suggest that neither O2- nor PN contributed to the loss of cell viability. Activated AM may kill normal rat lung FB through a NO-mediated pathway that does not involve PN.

Tyrosine 125 of alpha-synuclein plays a critical role for dimerization following nitrative stress.

alpha-Synuclein is a major component of Lewy bodies in Parkinson's disease, dementia with Lewy bodies, and glial cytoplasmic inclusions in multiple system atrophy. Increasing evidence suggests that the nitration of tyrosine residues in alpha-synuclein induced by oxidative injury is involved in the formation of inclusions characteristic to these synucleinopathies. Exposure of alpha-synuclein to peroxynitrite induces nitration of tyrosine residues, thereby forming alpha-synuclein oligomers. However, the contribution of tyrosine residues to either the nitration or the oligomerization is currently unknown. The present study used recombinant wild-type and mutant alpha-synuclein proteins to investigate the role of each alpha-synuclein tyrosine residue in the in vitro formation of alpha-synuclein oligomers under nitrative stress. Confocal microscopic analysis revealed that wild-type alpha-synuclein protein was able to accumulate and form an inclusion-like structure in the cytoplasm of living cells upon introduction by streptolysin O. Authentic peroxynitrite induced nitration of tyrosine residues in alpha-synuclein protein, as well as dimerization of alpha-synuclein. The formation of both SDS- and heat-stable dimers suggests cross-linking between nitrated tyrosine residues. Nonetheless, dimerization of alpha-synuclein proteins lacking tyrosine 125 was significantly decreased compared with alpha-synuclein proteins lacking tyrosine residues at positions 39, 133, or 136. Presumably, tyrosine 125 plays a critical role for alpha-synuclein dimerization under nitrative stress.

Prevention of peroxynitrite-induced renal injury through modulation of peroxynitrite production by the Chinese prescription Wen-Pi-Tang.

The effect of Wen-Pi-Tang extract on renal injury induced by peroxynitrite (ONOO-) production was investigated using rats subjected to intravenous lipopolysaccharide (LPS) injection and then renal ischemia followed by reperfusion. The plasma level of 3-nitrotyrosine, a marker of cytotoxic ONOO formation in vivo, was enhanced markedly in control rats subjected to LPS plus ischemia-reperfusion, but was significantly reduced by the oral administration of Wen-Pi-Tang extract, at doses of 62.5 and 125 mg/kg body weight/day, for 30 days prior to LPS plus ischemia-reperfusion. The activities of inducible nitric oxide synthase (iNOS) and xanthine oxidase (XOD) in renal tissue of control and Wen-Pi-Tang extract-treated rats did not change significantly, while those of the antioxidant enzymes, superoxide dismutase, catalase and glutathione peroxidase, were significantly increased by the administration of Wen-Pi-Tang extract, indicating that Wen-Pi-Tang improved the defense system by scavenging free radicals, not by directly inhibiting nitric oxide and superoxide production by iNOS and XOD. In addition, the levels of the hydroxylated products, m- and p-tyrosine, declined, whereas that of phenylalanine increased, after oral administration of Wen-Pi-Tang extract. Furthermore, the elevated plasma urea nitrogen and creatinine levels resulting from LPS plus ischemia-reperfusion process were significantly reduced by Wen-Pi-Tang extract, implying amelioration of renal impairment. The present study indicates that Wen-Pi-Tang extract contributes to the regulation of ONOO- formation and plays a beneficial role against ONOO(-) -induced oxidative injury and renal dysfunction in vivo.