Deferiprone, an iron chelator, alleviates platelet hyperactivity in patients with ß-thalassaemia/HbE.

Background: Hyperfunctional platelets play important roles in thromboembolism in patients with ß-thalassaemia/ haemoglobin E (ß-thal/HbE). Our previous study revealed ex vivo inhibitory effects of deferiprone on normal platelets. Herein, we aimed to investigate the in vivo effects on platelets in patients with ß-thal/HbE. Methods: A prospective, self-controlled clinical study on 30 patients with ß-thal/HbE who had received therapeutic deferiprone (20.8-94.5 mg/kg/day) was conducted. The study included a 4-week washout period followed by 4 and 12 weeks of deferiprone treatment. Platelet aggregation was performed by a turbidimetric method. Levels of deferiprone and soluble platelet (sP)-selectin in serum were measured by high-performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA) kit, respectively. Results: The washout period significantly enhanced platelet hyperactivity both in patients who had undergone splenectomy and in those who had not. At 2 hours following the administration of a single dose of deferiprone, platelet sensitivity to ADP and arachidonic acid was significantly reduced. The inhibitory effects of deferiprone were gradually increased over the period of 4 and 12 weeks. Deferiprone also depressed sP-selectin levels, but the effect was stable over longer follow-up periods. Correlation analysis demonstrated the relationship between serum levels of deferiprone, sP-selectin, and platelet activities induced by ADP and arachidonic acid. Conclusion: We first demonstrated the in vivo antiplatelet effect and benefit of short-term treatment of deferiprone in patients with ß-thal/HbE. The impact on thrombotic outcomes deserves further study.

Streptomyces barringtoniae sp. nov., isolated from rhizosphere of plant with antioxidative potential.

A novel actinomycete strain, JA03T, belonging to the genus Streptomyces, was isolated from the rhizosphere of Barringtonia racemosa (L.) Spreng. It was characterized taxonomically using a polyphasic approach. It grew at 25-37 °C, at pH 5-10 and with 6 % (w/v) NaCl. It contained ll-diaminopimelic acid in the cell-wall peptidoglycan. Ribose and glucose were detected in its whole-cell hydrolysate. The predominant cellular fatty acids were iso-C16 : 0, anteiso-C15 : 0, C16 : 0, iso-C14 : 0 and iso-C15 : 0. Detected polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannosides, unidentified phospholipids and unidentified amino lipids. Based on the results of 16S rRNA gene sequence analyses, strain JA03T showed highest similarity to Streptomyces filipinensis NBRC 12860T (98.76 %), Streptomyces fodineus TW1S1T (98.69 %) and Streptomyces shenzhennensis 172115T (98.68 %). Strain JA03T has a genome size of 9 092 851 bp with DNA G+C content of 71.28 mol%. The average nucleotide identity (ANI)-blast and ANI-MUMmer values of strain JA03T and related type strains were 79.6-89.2 and 86.7-92.5 %, respectively, and the digital DNA-DNA hybridization values were 27.3-46.4 %. Ethyl acetate extract of JA03T exhibited total phenolic content (33.4±0.6 µg mg-1 gallic acid equivalent), ferric reducing power value (70.8±1.8 µg mg-1 ascorbic acid equivalent) and 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity (IC50=67.0±21.1 µg ml-1). Intracellular reactive oxygen species and NO production in RAW264.7 macrophage cells induced by H2O2 and lipopolysaccharide were inhibited with IC50 of 67.40 and 16.95 µg ml-1, respectively. Based on the taxonomic results, it has been concluded that strain JA03T represents a novel species of the genus Streptomyces for which the name Streptomyces barringtoniae sp. nov., is proposed. The type strain is JA03T (=LMG 32415T=TISTR 2999T).

Inhibitory Mechanisms of Lusianthridin on Human Platelet Aggregation.

Lusianthridin is a phenanthrene derivative isolated from Dendrobium venustum. Some phenanthrene compounds have antiplatelet aggregation activities via undefined pathways. This study aims to determine the inhibitory effects and potential mechanisms of lusianthridin on platelet aggregation. The results indicated that lusianthridin inhibited arachidonic acid, collagen, and adenosine diphosphate (ADP)-stimulated platelet aggregation (IC50 of 0.02 ± 0.001 mM, 0.14 ± 0.018 mM, and 0.22 ± 0.046 mM, respectively). Lusianthridin also increased the delaying time of arachidonic acid-stimulated and the lag time of collagen-stimulated and showed a more selective effect on the secondary wave of ADP-stimulated aggregations. Molecular docking studies revealed that lusianthridin bound to the entrance site of the cyclooxygenase-1 (COX-1) enzyme and probably the active region of the cyclooxygenase-2 (COX-2) enzyme. In addition, lusianthridin showed inhibitory effects on both COX-1 and COX-2 enzymatic activities (IC50 value of 10.81 ± 1.12 µM and 0.17 ± 1.62 µM, respectively). Furthermore, lusianthridin significantly inhibited ADP-induced suppression of cAMP formation in platelets at 0.4 mM concentration (p < 0.05). These findings suggested that possible mechanisms of lusianthridin on the antiplatelet effects might act via arachidonic acid-thromboxane and adenylate cyclase pathways.

Protective Effect of Lusianthridin on Hemin-Induced Low-Density Lipoprotein Oxidation.

Oxidation of low-density lipoprotein (LDL) plays a crucial role in the pathogenesis of atherosclerosis. Hemin (iron (III)-protoporphyrin IX) is a degradation product of hemoglobin that can be found in thalassemia patients. Hemin is a strong oxidant that can cause LDL oxidation and contributes to atherosclerosis in thalassemia patients. Lusianthridin from Dendrobium venustrum is a phenolic compound that possesses antioxidant activity. Hence, lusianthridin could be a promising compound to be used against hemin-induced oxidative stress. The major goal of this study is to evaluate the protective effect of lusianthridin on hemin-induced low-density lipoprotein oxidation (he-oxLDL). Here, various concentrations of lusianthridin (0.25, 0.5, 1, and 2 µM) were preincubated with LDL for 30 min, then 5 µM of hemin was added to initiate the oxidation, and oxidative parameters were measured at various times of incubation (0, 1, 3, 6, 12, 24 h). Lipid peroxidation of LDL was measured by thiobarbituric reactive substance (TBARs) assay and relative electrophoretic mobility (REM). The lipid composition of LDL was analyzed by using reverse-phase HPLC. Foam cell formation with he-oxLDL in RAW 264.7 macrophage cells was detected by Oil Red O staining. The results indicated that lusianthridin could inhibit TBARs formation, decrease REM, decrease oxidized lipid products, as well as preserve the level of cholesteryl arachidonate and cholesteryl linoleate. Moreover, He-oxLDL incubated with lusianthridin for 24 h can reduce the foam cell formation in RAW 264.7 macrophage cells. Taken together, lusianthridin could be a potential agent to be used to prevent atherosclerosis in thalassemia patients.

Antiplatelet activity of deferiprone through cyclooxygenase-1 inhibition.

Thalassemia patients are susceptible to both iron overload and thromboembolism. Deferiprone is an iron chelator that shows an antiplatelet activity and thus may alleviate platelet hyperactivation in thalassemia. Therefore, this study aimed to characterize the inhibitory effects and mechanisms of deferiprone on normal human platelets. The results illustrated that deferiprone inhibited platelet aggregation at the iron chelating concentrations (0.08-0.25 mmol/l). Deferiprone inhibited human platelet aggregation stimulated by arachidonic acid and ADP more potently than epinephrine and collagen, with the IC50 of 0.24 mmol/l and 0.25 mmol/l vs. 3.36 mmol/l and 3.73 mmol/l, respectively. Interestingly, deferiprone significantly inhibited COX-1 activity, with the IC50 of 0.33 mmol/l, and slightly increased cAMP level at the high concentration of 4 mmol/l. Moreover, the results from molecular docking showed that deferiprone interacted closely with key residues in the peroxidase active site of COX-1. These results suggested that deferiprone possessed antiplatelet activity mainly through the inhibition of COX-1 activity.

Kinetics and localisation of haemin-induced lipoprotein oxidation.

Haemin (iron (III)-protoporphyrin IX) is a degradation product of haemoglobin in circulating erythrocytes. Haemin may play a key oxidising agent for lipoprotein oxidation in patients with haemolytic anaemia. In this study, kinetic changes in chemical composition and target sites of haemin-induced LDL and HDL oxidation were investigated. Haemin initially induced the loss of α-tocopherol, followed by accumulation of lipid hydroperoxide (LP) and alteration of core lipid fluidity. The absence of LP in HDL was explained by the antioxidant activity of PON in addition to α-tocopherol. The target site of haemin was evaluated by ESR spin labelling with 5- and 16-doxyl steric acids. In the presence of t-BuOOH and haemin, ESR signal decay of the doxyl moiety demonstrated the initiation phase and the propagation phase of lipid peroxidation. The results of the lag time and the rate of signal decay indicated that haemin is located near the 16th carbon atom of the fatty acid chain in the phospholipid layer. The analyses of motion parameters, order parameter (S) of 5-DS and rotational correlation time (τ) of 16-DS, supported the observation that the lipid properties changed near the hydrophobic region rather than at the surface region of lipoproteins. Moreover, ESR spin labelling demonstrated that haemin molecules but not iron ions caused lipoprotein oxidation. In conclusion, haemin is a potent inducer of lipoprotein oxidation, and the target site for this oxidation is near the hydrophobic core of the lipoprotein leading to the loss of antioxidant activities and changes in lipid composition and physical properties.

Inhibitory and inductive effects of Phikud Navakot extract on human cytochrome P450.

Effects of the hydroethanolic extract of Phikud Navakot (PN), a Thai traditional remedy, on human cytochrome P450s (CYPs) were investigated in vitro. Selective substrates of CYPs were used to investigate the effects and kinetics of PN on CYP inhibition using human liver microsomes. Primary human hepatocytes were used to assess the inductive effects of PN on CYP enzyme activities and protein expressions. The results showed that PN inhibited the activities of CYP1A2, CYP2C9, CYP2D6, and CYP3A4 with half maximal inhibitory concentration (IC50) values of 13, 62, 67, and 88 µg/mL, respectively. Meanwhile, it had no effect on the activities of CYP2C19 and CYP2E1 (IC50 > 1 mg/mL). PN exhibited competitive inhibition of CYP1A2 (Ki = 34 µg/mL), mixed type inhibition of CYP2C9 and CYP2D6 (Ki = 80 and 12 µg/mL, respectively), and uncompetitive inhibition of CYP3A4 (Ki = 150 µg/mL). PN did not have an inductive effect on CYP1A2, CYP2C9, CYP2C19 and CYP3A4 in primary human hepatocytes, which is an advantageous characteristic of the extract. However the extract may cause herb-drug interactions via inhibition of CYP1A2, CYP2C9, CYP2D6 and CYP3A4, and precautions should be taken when PN is coadministered with drugs that are metabolized by these CYP enzymes.

Synergistic antioxidant action of Phikud Navakot ameliorates hydrogen peroxide-induced stress in human endothelial cells.

BACKGROUND: Phikud Navakot (PN), a combination of nine herbs, has been used traditionally in Thai medicinal formulas to relieve circulatory disorder. The present study aimed to compare the synergistic antioxidant efficacy and toxicity of the hydroethanolic and water extracts of PN at cellular level. METHODS: PN and its nine herbs were extracted with either 50% ethanol or water. All extracts were tested for in vitro antioxidant potential using standard antioxidant assays. Evaluation of cytotoxicity, genotoxicity, and intracellular reactive oxygen species were performed using human endothelial ECV304 cells. RESULTS: Antioxidant assays in cell-free systems showed that the hydroethanolic extract of PN scavenged superoxide, hydroxyl, nitric oxide radicals, and hydrogen peroxide more effectively than its water extract. Combination indices were calculated to show that the ingredients of the hydroethanolic extract acted synergistically to exhibit antioxidant activities against all tested radicals, whereas, in the case of water extract, this effect was observed only against 2,2-diphenyl-1-picrylhydrazyl, superoxide, and hydroxyl radicals. A cell-based assay also revealed that the hydroethanolic extract concentration-dependently attenuated hydrogen peroxide-induced stress more effectively than the water extract. At the antioxidant and cytotoxic concentrations of both extracts, no genotoxicity was found. CONCLUSION: Our findings demonstrate that the synergistic antioxidant action of PN ameliorates endothelial stress, which may provide some clues for understanding the traditional use of PN for the treatment of circulatory disorder. Additionally, the selection of a suitable solvent for the extraction of PN herbal combination is essential for maximal efficacy and safety.

Imperatorin sensitizes anoikis and inhibits anchorage-independent growth of lung cancer cells.

The anoikis-sensitization activity of imperatorin, an active furanocoumarin component of Angelica dahurica root, is reported herein for the first time. The present study demonstrated that the imperatorin treatment at sub-toxic concentrations enhanced human lung cancer H23 cell apoptosis after detachment. A Western blot analysis showed that imperatorin significantly enhanced the p53 protein level, which subsequently down-regulated Mcl-1 protein and up-regulated Bax, while it had a minimal effect on Bcl-2 expression. In addition, an anchorage-independent growth assay was performed to support the anti-metastasis potential of imperatorin. Consistent with anoikis assay, imperatorin exhibited a strong inhibitory effect on the anchorage-independent growth of the cells. Further, this study demonstrated that imperatorin sensitizes anoikis in other lung cancer cells, namely, H292 and A549. Because anoikis was shown to be a critical hindrance in preventing cancer cell metastasis, the knowledge regarding such an activity and an underlying mechanism may lead to the development of this compound for a cancer therapy.

Curcumin extract for prevention of type 2 diabetes.

OBJECTIVE: To assess the efficacy of curcumin in delaying development of type 2 diabetes mellitus (T2DM) in the prediabetic population. RESEARCH DESIGN AND METHODS: This randomized, double-blinded, placebo- controlled trial included subjects (n = 240) with criteria of prediabetes. All subjects were randomly assigned to receive either curcumin or placebo capsules for 9 months. To assess the T2DM progression after curcumin treatments and to determine the number of subjects progressing to T2DM, changes in ß-cell functions (homeostasis model assessment [HOMA]-ß, C-peptide, and proinsulin/insulin), insulin resistance (HOMA-IR), anti-inflammatory cytokine (adiponectin), and other parameters were monitored at the baseline and at 3-, 6-, and 9-month visits during the course of intervention. RESULTS: After 9 months of treatment, 16.4% of subjects in the placebo group were diagnosed with T2DM, whereas none were diagnosed with T2DM in the curcumin-treated group. In addition, the curcumin-treated group showed a better overall function of ß-cells, with higher HOMA-ß (61.58 vs. 48.72; P < 0.01) and lower C-peptide (1.7 vs. 2.17; P < 0.05). The curcumin-treated group showed a lower level of HOMA-IR (3.22 vs. 4.04; P < 0.001) and higher adiponectin (22.46 vs. 18.45; P < 0.05) when compared with the placebo group. CONCLUSIONS: A 9-month curcumin intervention in a prediabetic population significantly lowered the number of prediabetic individuals who eventually developed T2DM. In addition, the curcumin treatment appeared to improve overall function of ß-cells, with very minor adverse effects. Therefore, this study demonstrated that the curcumin intervention in a prediabetic population may be beneficial.

Lipid fluidity at different regions in LDL and HDL of beta-thalassemia/Hb E patients.

Atherosclerosis-related vascular complications in beta-thalassemia/hemoglobin E (beta-thal/Hb E) patients may result from iron induced oxidation of lipoproteins. To identify the specific site of oxidative damage, changes in lipid fluidity at different regions in LDL and HDL particle were investigated using two fluorescence probes and two ESR spin probes. The magnitude of increased lipid fluidity in thalassemic lipoproteins was dependent on the location of the probes. In hydrophobic region, the rotational correlation times for 16-doxyl stearic acid and DPH anisotropy were markedly changed in LDL and HDL of the patients. In the surface region, there was only a slight change in the order parameter (S) for 5-doxyl stearic acid and TMA-DPH anisotropy. Lipid fluidity at the core of LDL and HDL showed good correlation with oxidative stress markers, the ratio of CL/CO, and the level of alpha-tocopherol, suggesting that hydrophobic region of thalassemic lipoprotein was a target site for oxidative damage.

The reduction of cholesteryl linoleate in lipoproteins: an index of clinical severity in beta-thalassemia/Hb E.

BACKGROUND: Oxidative modification of lipoproteins has been reported in beta-thalassemia and has been suggested to relate to atherogenesis-risk. This study focused on the change in cholesteryl esters in plasma lipoproteins under oxidative stress resulting from iron overload in beta-thalassemia/hemoglobin E (beta-thal/Hb E) patients. METHODS: Markers of oxidative damage and cholesteryl esters (CEs) were measured in plasma and lipo-proteins from 30 beta-thal/Hb E patients and compared to those from 10 healthy volunteers. CEs in plasma, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) were separated and identified using HPLC. RESULTS: beta-Thal/Hb E patients presented iron overload, a precipitous decrease in alpha-tocopherol and increased lipid peroxidation (thiobarbituric acid-reactive substances; TBARs) in both plasma and lipoproteins. Cholesteryl linoleate, the most abundant CE in lipoproteins, showed a reduction of 70% in LDL, while other CEs showed a lower reduction (50%). An inverse relationship between the cholesteryl linoleate/cholesteryl oleate ratio (CL/CO) and the degree of clinical severity suggested that the CL/CO ratio is an index of damaged lipoproteins and could be used as a pathologic marker of underlying iron overload. Good correlation of non-transferrin-bound iron (NTBI) and TBARs (r=0.8, p<0.01) in LDL strongly supported the contention that iron overload is responsible for initiating the lipid peroxidation in beta-thal/Hb E. CONCLUSIONS: This study suggests that cholesteryl linoleate is the primary target of oxidative modification induced by NTBI in beta-thal/Hb E patients and that reduction in cholesteryl linoleate in lipoproteins could be used as a severity index for beta-thal/Hb E.

Preliminary study of the effect of vitamin E supplementation on the antioxidant status of hemoglobin-E carriers.

The antioxidant status of hemoglobin-E carriers was studied pre- and post-treatment with vitamin E for 3 months. Fourteen hemoglobin-E carriers (age = 21.36 +/- 1.08 years, BMI = 18.32 +/- 1.22 kg/m2) were treated with vitamin E 200 I.U. daily for 3 months. Fasting blood samples were collected and analyzed for erythrocyte superoxide dismutase activity, total antioxidant activity, hemoglobin concentration, hematocrit, MCV, Heinz body formation and osmotic fragility test. The blood parameters before and after vitamin E treatment were compared. The results showed that superoxide dismutase activity in the erythrocytes was significantly decreased, while total antioxidant activity in plasma, and the osmotic fragility of the erythrocytes, was significantly increased after vitamin E supplementation. However, hematocrit, MCV, and Heinz body formation did not change significantly. This demonstrated that vitamin E 200 IU could be used as a lipophilic antioxidant in red blood cells and could help increase the level of antioxidant in hemoglobin-E carriers.