Genistein Improves Skin Flap Viability in Rats: A Preliminary In Vivo and In Vitro Investigation.

Selective estrogen receptor modulators (SERMs) have been developed to achieve beneficial effects of estrogens while minimizing their side effects. In this context, we decided to evaluate the protective effect of genistein, a natural SERM, on skin flap viability in rats and in a series of in vitro experiments on endothelial cells (migration, proliferation, antioxidant properties, and gene expression profiling following genistein treatment). Our results showed that administration of genistein increased skin flap viability, but importantly, the difference is only significant when treatment is started 3 days prior the flap surgery. Based on our in vitro experiments, it may be hypothesized that the underlying mechanism may rather by mediated by increasing SOD activity and Bcl-2 expression. The gene expression profiling further revealed 9 up-regulated genes (angiogenesis/inflammation promoting: CTGF, CXCL5, IL-6, ITGB3, MMP-14, and VEGF-A; angiogenesis inhibiting: COL18A1, TIMP-2, and TIMP-3). In conclusion, we observed a protective effect of genistein on skin flap viability which could be potentially applied in plastic surgery to women undergoing a reconstructive and/or plastic intervention. Nevertheless, further research is needed to explain the exact underlying mechanism and to find the optimal treatment protocol.

Artichoke Leaf Extract Inhibits AKR1B1 and Reduces NF-κB Activity in Human Leukemic Cells.

The human intracellular enzyme AKR1B1 belongs to the aldo-keto reductase superfamily. The AKR1B1-catalyzed reduction of aldehydes is part of the intracellular inflammatory pathway leading to the activation of NF-κB and the expression of pro-inflammatory genes. The present study is aimed at determining the inhibition of AKR1B1 brought about by an extract of artichoke leaves (bracts), and the effects of this extract and three participating compounds on the expression of AKR1B1, COX-2, and MMP-2 proteins in THP-1 cells. It seeks to identify the ability of the test substances to modulate the lipopolysaccharide (LPS)-induced activation of NF-κB in cells and the intracellular oxidant effect of test substances after incubation with LPS. Low concentrations of the extract inhibit the enzyme AKR1B1. After stimulation by LPS, the extract attenuated the activity of NF-κB in THP-1 cells, but no changes in the expression of AKR1B1 were recorded. The extract diminished the expression of the inflammation-related enzymes COX-2 and MMP-2, probably by inhibiting the activity of NF-κB. The extract significantly diminished the intracellular reactive oxygen species after a brief LPS incubation, which may also have reduced intracellular inflammation. The diminished activity of NF-κB in the cells could be linked to the inhibition of the activity of AKR1B1. Copyright © 2017 John Wiley & Sons, Ltd.

Molecular Interactions and Implications of Aldose Reductase Inhibition by PGA1 and Clinically Used Prostaglandins.

Aldose reductase (AKR1B1) is a critical drug target because of its involvement in diabetic complications, inflammation, and tumorigenesis. However, to date, development of clinically useful inhibitors has been largely unsuccessful. Cyclopentenone prostaglandins (cyPGs) are reactive lipid mediators that bind covalently to proteins and exert anti-inflammatory and antiproliferative effects in numerous settings. By pursuing targets for modification by cyPGs we have found that the cyPG PGA1 binds to and inactivates AKR1B1. A PGA1-AKR1B1 adduct was observed, both by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and by SDS-PAGE using biotinylated PGA1 (PGA1-B). Insight into the molecular interactions between AKR1B1 and PGA1 was advanced by molecular modeling. This anticipated the addition of PGA1 to active site Cys298 and the potential reversibility of the adduct, which was supported experimentally. Indeed, loss of biotin label from the AKR1B1-PGA1-B adduct was favored by glutathione, indicating a retro-Michael reaction, which unveils new implications of cyPG-protein interaction. PGA1 elicited only marginal inhibition of aldehyde reductase (AKR1A1), considered responsible for the severe adverse effects of many AKR1B1 inhibitors. Interestingly, other prostaglandins (PGs) inhibited the enzyme, including non-electrophilic PGE1 and PGE2, currently used in clinical practice. Moreover, both PGA1 and PGE1 reduced the formation of sorbitol in an ex-vivo model of diabetic cataract to an extent comparable to that attained by the known AKR inhibitor epalrestat. Taken together, these results highlight the role of PGs as AKR1B1 inhibitors and the interest in PG-related molecules as leads for the development of novel pharmacological tools.

2-Chloro-1,4-naphthoquinone derivative of quercetin as an inhibitor of aldose reductase and anti-inflammatory agent.

The ability of flavonoids to affect multiple key pathways of glucose toxicity, as well as to attenuate inflammation has been well documented. In this study, the inhibition of rat lens aldose reductase by 3,7-di-hydroxy-2-[4-(2-chloro-1,4-naphthoquinone-3-yloxy)-3-hydroxy-phenyl]-5-hydroxy-chromen-4-one (compound 1), was studied in greater detail in comparison with the parent quercetin (compound 2). The inhibition activity of 1, characterized by IC50 in low micromolar range, surpassed that of 2. Selectivity in relation to the closely related rat kidney aldehyde reductase was evaluated. At organ level in isolated rat lenses incubated in the presence of high glucose, compound 1 significantly inhibited accumulation of sorbitol in a concentration-dependent manner, which indicated that 1 was readily taken up by the eye lens cells and interfered with cytosolic aldose reductase. In addition, compound 1 provided macroscopic protection of colonic mucosa in experimental colitis in rats. At pharmacologically active concentrations, compound 1 and one of its potential metabolite 2-chloro-3-hydroxy-[1,4]-naphthoquinone (compound 3) did not affect osmotic fragility of red blood cells.

Protection or cytotoxicity mediated by a novel quinonoid-polyphenol compound?

Many natural and synthetic quinones and naphthoquinones possess a variety of beneficial pharmacological properties. In plants, the cytotoxic properties of quinones serve in their defensive roles against invading bacteria, fungi and parasites. In this regard many quinones as well as polyphenols, exerting generally toxicity at high dosages, are able to induce favorable hormetic responses at a low dosage. The novel chloronaphthoquinone derivative of quercetin (CHNQ) showed a profound cytotoxicity followed by enhancement of intracellular generation of oxidants in human neonatal B-HNF-3 fibroblasts. Its synthetic precursors, quercetin and 2-chloro-3-hydroxy-[1,4]naphthoquinone, failed to induce these effects, and paradoxically, only CHNQ at a low concentration provided partial protection of the cells against oxidative challenge. Thus, the novel quinonoid-polyphenol CHNQ might have a merit in the search for new prospective agents in prevention and management of ageing and ageing-related pathologies.

(2-Benzyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indol-8-yl)-acetic acid: an aldose reductase inhibitor and antioxidant of zwitterionic nature.

Novel carboxymethylated pyridoindoles, characterized by antioxidant activity combined with the ability to inhibit aldose reductase, represent an example of a multitarget approach to the treatment of diabetic complications - severe diabetes-related health disorders of multifunctional nature. One of the novel carboxymethylated pyridoindoles, (2-benzyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indol-8-yl)-acetic acid (compound 1), was found to inhibit aldose reductase with the IC(50) value 18.2 ± 1.2 µM. Owing to aldose reductase pharmacophore requirements for an acidic proton, most aldose reductase inhibitors contain an acetic acid moiety, ionized at physiological pH, resulting in poor bioavailability of the drugs. The presence of a basicity center at the tertiary nitrogen of the carboxymethylated pyridoindoles, in addition to the acidic carboxylic function, predisposes these compounds to form double-charged zwitterionic species. The zwitterionic nature of compound 1 may remarkably affect its pH-lipophilicity profile allowing for increased membrane penetration in the pH region around its isoelectric point, which lies close to the physiological pH 7.4. In the first part of this study, the presence of zwitterionic species was experimentally proved by the concentration-dependent effect of sodium 1-hexanesulphonate on the distribution profile of compound 1. Then a series of experiments was performed in the cellular system of isolated erythrocytes in vitro. Isolated rat erythrocytes exposed to peroxyl radicals, generated in the solution by decomposition of the hydrophilic azoinitiator AAPH or intracellularly by decay of lipophilic t-BuOOH, underwent progressive hemolysis. The onset of the hemolysis was shifted from the starting zero point by the time interval assigned as a lag period. In the presence of compound 1 the lag period was significantly prolonged. Finally, under conditions of a short-term experiment in STZ-diabetic rats in vivo, increase in sorbitol levels in erythrocytes was recorded. Compound 1 administered in the dose 50mg/kg/day (i.g.) significantly decreased the sorbitol level in the erythrocytes. To conclude, the physico-chemical proof of the zwitterionic nature of compound 1 was established and the results obtained in isolated red blood cells indicated good cellular availability of the compound. In addition, in diabetic rats, sorbitol accumulation in red blood cells was significantly inhibited by compound 1 administered intra-gastrically, suggesting its ready uptake into the central compartment. The zwitterionic principle thus may have significant consequences for increased bioavailability of drugs bearing an acidic function.

[5-(Benzyloxy)-1H-indol-1-yl]acetic acid, an aldose reductase inhibitor and PPARγ ligand.

Based on overlapping structural requirements for both efficient aldose reductase inhibitors and PPAR ligands, [5-(benzyloxy)-1H-indol-1-yl]acetic acid (compound 1) was assessed for inhibition of aldose reductase and ability to interfere with PPARγ. Aldose reductase inhibition by 1 was characterized by IC50 in submicromolar and low micromolar range, for rat and human enzyme, respectively. Selectivity in relation to the closely related rat kidney aldehyde reductase was characterized by approx. factor 50. At organ level in isolated rat lenses, compound 1 significantly inhibited accumulation of sorbitol in a concentration-dependent manner. To identify crucial interactions within the enzyme binding site, molecular docking simulations were performed. Based on luciferase reporter assays, compound 1 was found to act as a ligand for PPARγ, yet with rather low activity. On balance, compound 1 is suggested as a promising lead-like scaffold for agents with the potential to interfere with multiple targets in diabetes.

Antioxidant action of the hexahydropyridoindole SMe1EC2 in the cellular system of isolated red blood cells in vitro.

OBJECTIVES: The subject of this study was the hexahydropyridoindole compound SMe1EC2 with reported antioxidant and neuroprotective effects and low toxicity. In this study, the antioxidant action of SMe1EC2 was investigated in a greater detail in the system of isolated rat erythrocytes. METHODS: First, the compound was subjected to the DPPH test. Second, the overall antioxidant action of the compound was studied in the cellular system of isolated rat erythrocytes oxidatively stressed by free radicals derived from either the hydrophilic azoinitiator AAPH or the lipophilic t-BuOOH, and compared with reference antioxidants. RESULTS: The DPPH test revealed significant antiradical activity of SMe1EC2 comparable with that of the standard trolox. In the cellular system, SMe1EC2 protected red blood cells against free radical-initiated hemolysis. The overall antioxidant efficacy of SMe1EC2 relative to the reference antioxidant stobadine was strongly affected by the lipophilicity of the initiating free radical species. CONCLUSIONS: The results proved high antiradical efficacy of SMe1EC2. In the system of t-BuOOH/isolated erythrocytes, a model cellular system of endogenously generated peroxyl radicals, SMe1EC2 significantly exceeded the parent stobadine in its antioxidant action. Considering the reported results of preclinical studies of SMe1EC2 showing its profound neuroprotective effects and low toxicity, the compound represents an example of a potential pharmacologically practicable antioxidant drug.

Screening for antiradical efficiency of 21 semi-synthetic derivatives of quercetin in a DPPH assay.

The group of 21 novel semi-synthetic derivatives of quercetin was screened for the antiradical efficiency in a DPPH assay. The initial fast absorbance decrease of DPPH, corresponding to the transfer of the most labile H atoms, was followed by a much slower absorbance decline representing the residual antiradical activity of the antioxidant degradation products. Initial velocity of DPPH decolorization determined for the first 75-s interval was used as a marker of the antiradical activity. Application of the kinetic parameter allowed good discrimination between the polyphenolic compounds studied. The most efficient chloronaphthoquinone derivative (compound Ia) was characterized by antiradical activity higher than that of quercetin and comparable with that of trolox. Under the experimental conditions used, one molecule of Ia was found to quench 2.6±0.1 DPPH radicals.

Aldose reductase inhibitory activity and antioxidant capacity of pomegranate extracts.

The pomegranate, Punica granatum L., has been the subject of current interest as a medicinal agent with wide-ranging therapeutic indications. In the present study, pomegranate ethanolic seed and hull extracts were tested, in comparison with a commercial sample, for the inhibition of aldose reductase, an enzyme involved in the etiology of diabetic complications. In vitro inhibition of rat lens aldose reductase was determined by a conventional method. Pomegranate ethanolic hull extract and commercial pomegranate hull extract exhibited similar aldose reductase inhibitory activity characterized by IC(50) values ranging from 3 to 33.3 µg/ml. They were more effective than pomegranate ethanolic seed extract with IC(50) ranging from 33.3 to 333 µg/ml. Antioxidant action of the novel compounds was documented in a DPPH test and in a liposomal membrane model, oxidatively stressed by peroxyl radicals. All the plant extracts showed considerable antioxidant potential in the DPPH assay. Pomegranate ethanolic hull extract and commercial pomegranate hull extract executed similar protective effects on peroxidatively damaged liposomal membranes characterized by 10