Close relationship between high HbA1c levels and methemoglobin generation in human erythrocytes: The enhancement of oxidative stress in the process.

OBJECTIVE: This study aimed to investigate the effect of diabetic plasma on human red blood cells (RBCs) in order to highlight the amplification mechanisms of oxidative stress (OS) in relation to methemoglobin (metHb) production, a potential bio-indicator that could be related to diabetes disease. RESEARCH DESIGN AND METHODS: Normal RBCs were co-incubated with the diabetic plasma of 24 patients at different HbA1c levels, for 0, 24, and 48 h in order to assess cell turbidity and hemoglobin (Hb) stability. Hb and metHb production were quantified inside and outside RBCs. Malonaldehyde (MDA) level and cell morphology were concomitantly evaluated. RESULTS: The cell turbidity was significantly decreased in the group co-incubated with diabetic plasma at high HbA1c levels (0.074 ± 0.010 AU) compared to the control group (0.446 ± 0.019 AU). A significant decrease in intracellular Hb (0.390 ± 0.075 AU) and its stability (0.600 ± 0.001 AU) were revealed. Also, we found an important increase of metHb levels inside RBCs (0.186 ± 0.017 AU) and in its supernatant (0.086 ± 0.020 AU) after 48 h. Consequently, MDA absorbance increased significantly (0.320 ± 0.040 AU) in RBCs exposed to diabetic plasma with high HbA1c. CONCLUSION: These findings suggest that poor glycemic control in diabetes leads to metHb generation which is the main factor of the OS amplification.

Oxidative stress as an underlying mechanism of anticancer drugs cytotoxicity on human red blood cells' membrane.

The aim of this study is to investigate the direct in vitro effects of anticancer drugs on red blood cells (RBCs) and to explore the underlying mechanism, mainly by measuring RBCs oxidative stress (OS) status. After RBCs direct contact with fourteen (14) anticancer drugs, several parameters were assessed including: cellular turbidity, methemoglobin (metHb) generation, released Hb and Hb stability. Moreover, intracellular Hb, considered as new molecular target of anticancer drugs, was quantified inside RBCs. MDA level, the main biomarker of OS, was simultaneously measured. The cellular turbidity reveled severe (docetaxel "TXT", 0.03 ± 0.002), moderate (methotrexate "MTX", 0.49 ± 0.009), or none (5-fluorouracil "5-FU", 0.76 ± 0.029) membrane cytotoxicity (MC). An inverse relationship between cell concentration, released Hb and metHb content was obtained. High metHb generation, revealing intense OS, was also mostly expressed in paclitaxel "TXL" and etoposide "VP16". Further, epirubicin "EPI" and "TXT" induced important oxidation of membrane lipids with 0.32 ± 0.014 and 0.26 ± 0.004, respectively. Also, MTX (0.17 ± 0.006) and doxorubicin "DOX" (0.32 ± 0.034) affected significantly Hb stability by a direct contact with molecule. These findings demonstrated that anticancer drugs have the ability to induce membrane damages by the exacerbation of OS through membrane lipid peroxidation and Hb oxidation even inside RBCs.

High HbA1c levels affect motility parameters and overexpress oxidative stress of human mature spermatozoa.

The aim of this study is to investigate, by a validated in vitro model, the effect of diabetic plasma on ejaculated human spermatozoa. Plasma of 51 male diabetic patients (mean age 62.28 ± 9.28 years) was selected according to their HbA1c level: low HBA1c  ≤ 5% (31 mmol/mol), moderate HBA1c 6%-8% (42-64 mmol/mol) and high HBA1c  ≥ 10% (86 mmol/mol). The plasma was tested on eighteen normal semen samples by analysing gametes motility using a computer Sperm Class Analyzer® and their corresponding oxidative stress (OS) status using thiobarbituric acid-reactive substances assay. The results indicated that diabetic plasma affected all sperm motility parameters with high HbA1c showing the most important deleterious effects. Low gametes' straight-line velocity was observed in high HbA1c level, mainly after 20 min of co-incubation (8.78 ± 0.47 µm/s). Also, the highest lipid peroxidation (nmoles MDA/108 SPZ) was observed in high HbA1c values (0.92 ± 0.09), higher than those in spermatozoa treated with H2 O2 (0.85 ± 0.04). Conclusively, a direct impact of diabetic plasma on spermatozoa is revealed with overexpression of OS as the underlying mechanism. These findings suggested that it is strongly recommended to control clinically the glycaemic level and OS in diabetic patients for the maintenance of male fertility.

Inhibition of myeloperoxidase activity by the alkaloids of Peganum harmala L. (Zygophyllaceae).

ETHNOPHARMACOLOGICAL RELEVANCE: Seeds and aerial parts of Peganum harmala L. are widely used in Algeria as anti-inflammatory remedies. Evaluation of Peganum harmala total alkaloids extracts and pure ß-carboline compounds as an anti-inflammatory treatment by the inhibition of an enzyme key of inflammatory, myeloperoxidase (MPO) and HPLC quantification of the alkaloids from the different parts of plant. MATERIALS AND METHODS: MPO inhibition was tested using taurine chloramine test. The inhibition of LDL oxidation induced by MPO was carried out. The molecular docking analysis of Peganum harmala alkaloids on MPO was performed using the Glide XP docking protocol and scoring function and the redox potential of alkaloids was determined using an Epsilon potentiostat. The concentration of harmala alkaloids was determined using HPLC analysis. RESULTS: The HPLC profiling of the active total alkaloids indicates that ß-carboline e.g. harmine, harmaline, harmane, harmol and harmalol are major components. As ß-carbolines resemble tryptamine, of which derivatives are efficient inhibitors of MPO, the harmala alkaloids were tested for their activity on this enzyme. Total alkaloids of the seeds and of the aerial parts strongly inhibited MPO at 20µg/mL (97±5% and 43±4%, respectively) whereas, at the same concentration, those of the roots showed very low inhibition (15±6%). Harmine, harmaline and harmane demonstrated a significant inhibition of MPO at IC50 of 0.26, 0.08 and 0.72µM respectively. These alkaloids exerted a similar inhibition effects on MPO-induced LDL oxidation. Molecular docking analysis of Peganum harmala alkaloids on MPO showed that all active Peganum harmala alkaloids have a high affinity on the active site of MPO (predicted free energies of binding up to -3.1kcal/mol). Measurement of redox potentials versus the normal hydrogen electrode clearly differentiated (i) the high MPO inhibitory activity of harmine, harmaline and harmane (+1014, 1014 and 1003mV, respectively); and (ii) the low activity of harmalol and harmol (+629/778 and 532/644mV, respectively). A reverse phase HPLC method has been developed to determine simultaneously five alkaloids of Peganum harmala. Seeds contained all five ß-carboline derivatives with the main active alkaloids, harmaline and harmine, being up to 3.8% and 2.9%, respectively. Up to 3.2% of harmine was determined in the roots. The four ß-carboline derivatives, harmine, harmaline, harmane and harmalol were identified in the aerial parts. The highest inhibitory effect observed in seeds and the moderate effect of aerial parts could be explained by their harmine and harmaline content. In contrast, the very weak inhibition of the root extract, despite the presence of harmine, may tentatively be explained by the high concentration of harmol which can reduce Compound II of MPO to the native form. CONCLUSION: The inhibition of MPO by Peganum harmala ß-carboline alkaloids, herein reported for the first time, may explain the anti-inflammatory effect traditionally attributed to its herbal medicine.

Identification and quantification of the main active anticancer alkaloids from the root of Glaucium flavum.

Glaucium flavum is used in Algerian folk medicine to remove warts (benign tumors). Its local appellations are Cheqiq el-asfar and Qarn el-djedyane. We have recently reported the anti-tumoral activity of Glaucium flavum root alkaloid extract against human cancer cells, in vitro and in vivo. The principal identified alkaloid in the extract was protopine. This study aims to determine which component(s) of Glaucium flavum root extract might possess potent antitumor activity on human cancer cells. Quantitative estimation of Glaucium flavum alkaloids was realized by HPLC-DAD. Glaucium flavum effect on human normal and cancer cell viability was determined using WST-1 assay. Quantification of alkaloids in Glaucium flavum revealed that the dried root part contained 0.84% of protopine and 0.07% of bocconoline (w/w), while the dried aerial part contained only 0.08% of protopine, glaucine as the main alkaloid, and no bocconoline. In vitro evaluation of the growth inhibitory activity on breast cancer and normal cells demonstrated that purified protopine did not reproduce the full cytotoxic activity of the alkaloid root extract on cancer cell lines. On the other hand, bocconoline inhibited strongly the viability of cancer cells with an IC50 of 7.8 µM and only a low cytotoxic effect was observed against normal human cells. Our results showed for the first time that protopine is the major root alkaloid of Glaucium flavum. Finally, we are the first to demonstrate a specific anticancer effect of Glaucium flavum root extract against breast cancer cells, which can be attributed, at least in part, to bocconoline.

Revealing the anti-tumoral effect of Algerian Glaucium flavum roots against human cancer cells.

Glaucium flavum (G. flavum) is a plant from the Papaveraceae family native to Algeria where it is used in local traditional medicine to treat warts. G. flavum root crude alkaloid extract inhibited breast cancer cell proliferation and induced G2/M phase cycle arrest and apoptosis without affecting normal cells, which is a highly awaited feature of potential anti-cancer agents. G. flavum significantly reduced growth and vascularization of human glioma tumors on chicken chorioallantoic membrane (CAM) in vivo. The chromatographic profile of the dichloromethane extract of G. flavum root showed the presence of different constituents including the isoquinoline alkaloid protopine, as the major compound. We report for the first time that G. flavum extract may represent a new promising agent for cancer chemotherapy.