Oxidative Hemolysis Research Articles

Dracaena draco L. fruit: Phytochemical and antioxidant activity assessment

The present study reports for the first time the metabolite profile and antioxidant activity of aqueous extract obtained from Dracaena draco L. fruit. Volatiles profile was determined by HS-SPME/GC-IT-MS, with 9 compounds being identified, distributed by several distinct chemical classes: 1 alcohol, 3 aldehydes, 2 carotenoid derivatives, and 3 terpenic compounds. Aldehydes constituted the most abundant class in this exotic berry, representing 59% of total identified volatile compounds. Phenolics profile was determined by HPLC/DAD and 5 constituents were identified: 5-O-caffeoylquinic, 3,5-O-dicaffeoylquinic, ferulic and sinapic acids, and quercetin-3-O-rutinoside. The major phenolic compound is quercetin-3-O-rutinoside, comprising 42% of the total phenolic content. Organic acids composition was also characterized, by HPLC-UV, and oxalic, citric, l-ascorbic, malic, quinic and shikimic acids were determined. The most abundant is quinic acid, representing 39% of the total organic acid content. The antioxidant potential of this matrix was assessed by (i) reducing power of Fe3+/ferricyanide complex, (ii) scavenging effect on DPPH free radicals, and (iii) ability to inhibit the 2,2´-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced oxidative hemolysis in human erythrocytes. Strawberry (Fragaria×ananassa Duch. cv. Camarosa) extract was used for comparison purposes. All assay models showed remarkable concentration dependent antioxidant activity, reducing power and radical scavenging efficiency for D. draco fruit, being invariably higher than that of strawberry extract. This is the first report showing that D. draco fruit is a promising new antioxidant agent.

Effect and its mechanism of Coptis chinensis on oxidative hemolysis of erythrocytes in mice induced by acetylphenyhydrazine

To investigate the effect of Coptis chinensis on oxidative hemolysis of erythrocytes in mice. Acetylphenyhydrazine (APH)-induced oxidative hemolysis of erythrocytes in mice were used. The contents of free hemoglobin of blood plasma, indirect bilirubin of serum, reticulocytes of blood, and malondialdehyde (MDA) of erythrocytes were measured. The activity of superoxide dismutase (SOD), reduced glutathione hormone (GSH), and glucose-6-phosp hate dehydrogenase (G-6-PD) of erythrocytes were also determined and the total-antioxygen capability (T-AOC) of blood was analyzed. The levels or amount of free hemoglobin of blood plasma, indirect bilirubin of serum, reticulocytes of blood and MDA of erythrocytes were higher in APH (0.03 g x kg(-1))-induced mice than normal mice. The activity or content of SOD, GSH and G-6-PD was lower in APH-induced mice than in normal mice. Primaquine (0.058 g x kg(-1)) could aggravated the degree of elevated hemolysis of erythrocytes in APH-induced mice. C. chinensis (0.6 g x kg(-1) could deprssed significantly the elevated levels of indirect bilirubin in serum. The levels of free hemoglobin of blood plasma, indirect bilirubin of serum, reticulocytes of blood, the production of SOD and GSH and T-AOC were also decressed by C. chinensis (0.6 g x kg(-1)). C. chinensis suppressed t he degree of hemolysis of erythrocytes in APH-induced mice due to the suppression of the production of lipid peroxidation and increasing of the activity of antioxidase of erythrocytes.

When pure is not so pure: Chloramine‐related hemolytic anemia in home hemodialysis patients

Worldwide, chloramines are used as the preferred disinfectant for city water supplies. Although they have distinct advantages compared with chlorine and are deemed harmless to the general population, hemodialysis (HD) patients are at risk from chloramine-induced hemolytic anemia. In recent years, this has been highlighted in regional dialysis units but not as frequently in the home HD group. We report on 2 home HD patients who succumbed to severe oxidative hemolysis due to high mains water chloramine concentrations. Both patients were extensively investigated for other cause of anemia before a definitive diagnosis was reached. Delays in diagnosing this uncommon condition can be costly in terms of significant morbidity and excessive usage of recombinant erythropoietin and blood transfusion. Prevention primarily involves enforcing strict water quality control and establishing regular communication with water supply boards and home HD patients. Double (inline) carbon filters should be installed in patient's homes as an effective means for removing high incoming chloramine concentrations.

Inhibition of oxidative hemolysis by quercetin, but not other antioxidants

We previously reported that lipid-soluble quercetin, not water-soluble dihydroquercetin, protects human red blood cells against oxidative damage. The objectives of this study were to determine if an antihemolytic effect could be produced by other lipid-soluble antioxidants and if anti-inflammatory activity played a role in antihemolysis by quercetin. This study compared three lipid-soluble polyphenols, muscadine, curcumin and quercetin, and three lipid- (α-tocopherol and α-tocotrienol) or water-soluble (ascorbic acid) vitamins. Among the tested polyphenols, muscadine was the most potent in inhibiting superoxide and 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH)-generated peroxyl radicals, whereas ascorbic acid was the most potent inhibitor of hydrogen peroxide. Activities of the polyphenols after lipid extractions showed that curcumin inhibited superoxide production to a greater extent than quercetin and muscadine. All blood cells were tested 20 min after incubation with the selected compounds. All the polyphenols caused inhibition of N-formyl- l-methionyl- l-leucyl- l-phenylalanine-induced neutrophil oxidative bursts. Quercetin, but not other polyphenols, significantly reduced AAPH-induced oxidative hemolysis. No significant effect on neutrophil oxidative burst or oxidative hemolysis was found with any of the tested vitamins. These results suggest that quercetin enhances the resistance of membrane to destruction by free radicals. This effect of quercetin is not directly mediated through antioxidative or anti-inflammatory actions. Antioxidant or anti-inflammatory potency may not be used as a simple criterion to select polyphenols for cell protection benefits.

Antioxidant activity of α-pyridoin and its derivatives: possible mechanism

alpha-Pyridoin (1, 1,2-di(2-pyridyl)-1,2-ethenediol) is a unique enediol antioxidant. To explore the detailed antioxidant mechanism of alpha-pyridoin, we synthesized alpha-pyridoin and its 5,5'- or 6,6'-bis-substituted derivatives (2-7) and compared their capacities to scavenge galvinoxyl radical (GO*) and protect human red blood cells (RBCs) from oxidative haemolysis. It was found that the compounds (5 and 6) with a methyl or methoxy group at the 5-position exhibit significantly higher GO*-scavenging and anti-haemolysis activities than other derivatives and vitamin C. Kinetic analysis of the GO*-scavenging reaction and the effect of added base on the reaction rate revealed that in ethyl acetate, the reaction occurs primarily by the direct hydrogen atom transfer (HAT mechanism). However, in ethanol that supports ionization, the kinetics of the process is mostly governed by sequential proton loss electron transfer (SPLET mechanism).

Dicranostigma leptopodum (maxim) fedde induced apoptosis in SMMC-7721 human hepatoma cells and inhibited tumor growth in mice

Dicranostigma Leptopodum (Maxim) Fedde (DL- F), which had been previously documented to suppress oxidative hemolysis of erythrocytes and enhance immune functions of murine peri- toneal macrophages, was investigated for its effect on anti-tumor activity. Of alkaloids extracted from DLF, five have been identified with employment of chromatographic analysis. An antiproliferative role of these alkaloids was determined on SMMC-7721 Human Hepatoma Ce- lls in an apoptosis-inducing manner, through MTT assaying, Trypan blue exclusion assaying and cytometric analysis of cell cycle distribution. To further examine their inhibitory effects on tumor progression, murine H22 cells were inoculated into Kunming mice to determine the role of these alkaloids of DLF in inhibiting tumor growth in the tumor-implanted mice. It was found that these alkaloids of DLF enhanced the tumor shrinkage effectively wherein its tumor inhibitory rate and immunohistochemistry stain- ing of the tumor were determined and profiled, respectively.

Enhanced Protective Effect of Ultrafine Particles of Red-Ginseng against Phenanthrene-induced Cell Damage

Phenanthrene, one of the polycyclic aromatic hydrocarbons, has been known to be toxic to the environment. In this investigation, the protective effect of red ginseng on phenanthrene-induced oxidative DNA damage was evaluated using Comet assay in A549 cells. Red ginseng's cytoprotective effect on phenanthrene-induced hemolysis was also investigated. This study's findings show that oxidative DNA damage and hemolysis were significantly prevented by red ginseng treatment. Notably, it was found that pulverizing red ginseng into ultra-fine particles even enhanced its protective effects against DNA damage and hemolysis. The results suggest that particle size reduction seems to effectively enhance red ginseng's pharmacological efficacies.

Powerful Protective Role of 3,4-Dihydroxyphenylethanol−Elenolic Acid Dialdehyde against Erythrocyte Oxidative-Induced Hemolysis

The present work studied and compared the capacity of four important olive oil polyphenolic compounds, oleuropein, hydroxytyrosol, and the oleuropein aglycones 3,4-dihydroxyphenylethanol-elenolic acid (3,4-DHPEA-EA) and 3,4-dihydroxyphenylethanol-elenolic acid dialdehyde (3,4-DHPEA-EDA), to protect red blood cells (RBCs) from oxidative hemolysis induced by the physiological initiator H2O2. The amount of hemolysis was evaluated spectrophotometrically. The compounds were also tested in the presence and absence of the naturally occurring antioxidant ascorbic acid. All compounds were revealed to significantly protect RBCs from oxidative hemolysis induced by H2O2 at 40 and 80 microM, with the order of activity being 3,4-DHPEA-EDA>3,4-DHPEA-EA>hydroxytyrosol=oleuropein. At 20, 10, and 5 microM, only 3,4-DHPEA-EDA showed a significant protection against the oxidative injury. In the presence of ascorbic acid at physiological concentration, the addition of individual compounds at 40 microM increased the stability of erythrocytes. The addition of phenolic compounds at 20 and 10 microM did not produce further protection when compared with the protection given by ascorbic acid alone, except for 3,4-DHPEA-EDA. This compound was shown to produce further protection even at 5 microM. In summary, 3,4-DHPEA-EDA plays an important protective role against reactive oxygen species-induced oxidative injury in RBCs, and this effect is more potent than the one evidenced by hydroxytyrosol or oleuropein.

High Prevalence of Dapsone-Induced Oxidant Hemolysis in North American Stem Cell Transplant Recipients without Glucose-6-Phosphate-Dehydrogenase Deficiency.

Abstract 3314Poster Board III-202 IntroductionDapsone (4-4'-diaminodiphenylsulfone) is commonly used for Pneumocystis jirovecii pneumonia (PCP) prophylaxis in immunocompromised patients. Oxidant hemolysis is a known complication of dapsone, but its frequency in adult patients who have undergone stem cell transplantation (SCT) for hematologic malignancies is not well established. We report a high prevalence of hemolytic anemia associated with dapsone use in SCT patients in the absence of glucose-6-phosphate dehydrogenase (G6PDH) deficiency. Patients and MethodsWe retrospectively analyzed laboratory data from 30 SCT (29 Caucasian and 1 African-American; 15 male and 15 female) patients receiving dapsone (100 mg orally daily) for PCP prophylaxis to determine the frequency of oxidant hemolysis. All patients had undergone allogeneic (n = 27) or autologous SCT (n = 3) for hematological malignancy or aplastic anemia. Peripheral blood (PB) smears were reviewed in a blinded fashion for the presence of morphologic features of oxidant hemolysis (multiple bite cells in each high power field), with PB smears from the same patient group while not on dapsone serving as controls. Additional demographic and laboratory data, including G6PDH enzyme activity screening test, hemoglobin, reticulocyte count, lactate dehydrogenase and bilirubin levels, were collected from chart review. Results29 patients had normal G6PDH activity (results were not available for 1 patient). 38 PB smears were obtained while on dapsone (some patients had more than one bone marrow evaluation). 30 (79%) PB smears from 26 (87%) patients had morphologic evidence of overt oxidant hemolysis. This was significantly higher than in the controls, in which 5/22 (23%) PB smears had morphologic features of oxidant hemolysis (p<0.001, Fisher's exact test). Hemoglobin levels were lower in the dapsone group, compared to the controls (10.0 vs. 12.5 g/dL, p=0.012). Mean LDH (507 IU) and direct bilirubin (1.6 g/dL) were also increased in the patients on dapsone. None of the patients had laboratory signs of hemolysis while not on dapsone. ConclusionsDapsone-induced oxidant hemolysis in SCT patients is 20-fold higher than the reported rate in HIV-infected patients receiving this agent. This high prevalence in patients without G6PDH deficiency suggests the coexistence of other sources of oxidant stress specific to this patient cohort. DisclosuresBredeson:Esai: Honoraria.

Human cancer cell antiproliferative and antioxidant activities of Juglans regia L.

Several studies suggest that regular consumption of nuts, mostly walnuts, may have beneficial effects against oxidative stress mediated diseases such as cardiovascular disease and cancer. Walnuts contain several phenolic compounds which are thought to contribute to their biological properties. The present study reports the total phenolic contents and antioxidant properties of methanolic and petroleum ether extracts obtained from walnut ( Juglans regia L.) seed, green husk and leaf. The total phenolic contents were determined by the Folin–Ciocalteu method and the antioxidant activities assessed by the ability to quench the stable free radical 2,2′-diphenyl-1-picrylhydrazyl (DPPH) and to inhibit the 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced oxidative hemolysis of human erythrocytes. Methanolic seed extract presented the highest total phenolic content (116 mg GAE/g of extract) and DPPH scavenging activity (EC 50 of 0.143 mg/mL), followed by leaf and green husk. In petroleum ether extracts, antioxidant action was much lower or absent. Under the oxidative action of AAPH, all methanolic extracts significantly protected the erythrocyte membrane from hemolysis in a time- and concentration-dependent manner, although leaf extract inhibitory efficiency was much stronger (IC 50 of 0.060 mg/mL) than that observed for green husks and seeds (IC 50 of 0.127 and 0.121 mg/mL, respectively). Walnut methanolic extracts were also assayed for their antiproliferative effectiveness using human renal cancer cell lines A-498 and 769-P and the colon cancer cell line Caco-2. All extracts showed concentration-dependent growth inhibition toward human kidney and colon cancer cells. Concerning A-498 renal cancer cells, all extracts exhibited similar growth inhibition activity (IC 50 values between 0.226 and 0.291 mg/mL), while for both 769-P renal and Caco-2 colon cancer cells, walnut leaf extract showed a higher antiproliferative efficiency (IC 50 values of 0.352 and 0.229 mg/mL, respectively) than green husk or seed extracts. The results obtained herein strongly indicate that walnut tree constitute an excellent source of effective natural antioxidants and chemopreventive agents.

Hemolytic anemia after zopiclone overdose

Introduction. Zopiclone is associated with methemoglobinemia. We report a case of zopiclone overdose manifested with another hematological complication, hemolysis. Case. A 46-year-old woman overdosed with 50–100 tablets of 7.5 mg zopiclone. Oxidative hemolysis was evident by anemia, bite cells, raised unconjugated bilirubin and lactate dehydrogenase, lowered haptoglobin, and reticulocytosis. Discussion. In zopiclone overdose, there may be oxidative stress rendering the development of not only methemoglobinemia but also hemolysis.

Biological activity of novel synthetic derivatives of carnosine.

Two novel derivatives of carnosine--(S)-trolox-L-carnosine (STC) and (R)-trolox-L-carnosine (RTC) are characterized in terms of their antioxidant and membrane-stabilizing activities as well as their resistance to serum carnosinase. STC and RTC were synthesized by N-acylation of L-carnosine with (S)- and (R)-trolox, respectively. STC and RTC were found to react more efficiently with 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) and protect serum lipoproteins from Fe(2+)-induced oxidation more successfully than carnosine and trolox. At the same time, STC, RTC and trolox suppressed oxidative hemolysis of red blood cells (RBC) less efficiently than carnosine taken in the same concentration. When oxidative stress was induced in suspension of cerebellum granule cells by their incubation with N-methyl-D-aspartate (NMDA), or hydrogen peroxide (H(2)O(2)), both STC and RTC more efficiently decreased accumulation of reactive oxygen species (ROS) than carnosine and trolox. Both STC and RTC were resistant toward hydrolytic degradation by human serum carnosinase. STC and RTC were concluded to demonstrate higher antioxidant capacity and better ability to prevent cerebellar neurons from ROS accumulation than their precursors, carnosine and trolox.

Antioxidant capacity of curcumin-directed analogues: Structure–activity relationship and influence of microenvironment

Curcumin is the active ingredient of turmeric powder with a variety of biological activities including antioxidative activity. In order to find more active antioxidants with curcumin as the lead compound we synthesised a series of enone analogues of curcumin. The present work studied and compared the capacity of curcumin-directed analogues to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH ) and protect human red blood cells (RBCs) from oxidative haemolysis. It was found that these compounds which bear o-diphenoxyl and o-dimethoxyphenoxyl groups exhibited significantly higher DPPH -scavenging and anti-haemolysis activities than those which bear no such groups. In contrast to curcumin analogues that retained the 7-carbon spacer, the compounds with a 5-carbon linker had lower activity. In the case of the latter, the introduction of a ring further decreased DPPH -scavenging activity. However, the introduction of a ring did increase anti-haemolysis activity, suggesting that the lipophilicity of these compounds might play an important role in the antioxidant activity.

Antioxidant property of a Thai traditional formula for longevity

Antioxidant activity of a Thai traditional formula for longevity was studied. The formula comprised six herbs, as follows: Albizia procera, Cyperus rotundus, Diospyros rhodocalyx, Piper nigrum, Tinospora crispa and Streblus asper. Each components and the formula were separately extracted by 95% ethanol and concentrated using a reduced-pressure evaporator. The antioxidant properties were detected by DPPH method using vitamin C and Trolox as reference standards. In vitro oxidative hemolysis of sheep red blood cells was used as a model to study the free radical-induced damage of biological membranes by AAPH. From the DPPH method, Albizia procera extract demontrated the most potent antioxidant properties (IC 50 44.34 µg/ml), while vitamin C and Trolox had IC 50 of 17.47 µg/ml and 22.75 µg/ml, respectively. From the AAPH hemolysis method, Albizia procera, the formula for longevity, Cyperus rotundus, Diospyros rhodocalyx, Piper nigrum, Tinospora crispa and Streblus asper extracts at a concentration of 5 mg/ml could prolong the time of 50% hemolysis from 78 min to 157, 142, 126, 114, 108, 101 and 100 min, respectively, while the time of 50% hemolysis of Trolox at a concentration of 0.5 mg/ml was 160 min. Phytochemical screening tests showed the presence of phenolic compounds, tannins and flavonoids in the formula Cyperus rotundus and Albizia procera; and phenolic compounds and flavonoids in Piper nigrum, Diospyros rhodocalyx and Streblus asper. Tinospora crispa extract contained only phenolic compounds. Keyword: Albizia procera, Antioxidant activities, Cyperus rotundus, Diospyros rhodocalyx, DPPH method, Piper nigrum, Streblus asper, Thai traditional formula for longevity, Tinospora crispa, 50% hemolysis

Unexpected glucose‐6‐phosphate dehydrogenase deficiency

A 50-year-old man with refractory intestinal T-cell lymphoma was started on combination chemotherapy (ifosfamide, methotrexate, l-asparaginase, etoposide, dexamethasone). Cotrimoxazole was commenced for pneumocystis prophylaxis after a normal glucose-6-phosphate dehydrogenase (G6PD) spot fluorescence screen [>4·5 iu/g haemoglobin (Hb)]. Initial tumour shrinkage was accompanied by renal impairment (creatinine 393 μmol/l, normal 67–109) and hyperuricaemia (1259 μmol/l, normal 260–530), and rasburicase (4·5 mg × 1 dose) was given for tumour lysis. Despite a dramatic fall in uric acid level (55 μmol/l), anaemia suddenly occurred (Hb fell from 113 g/l to 59 g/l), followed by hypotension and oliguria. Biochemical screening showed raised unconjugated bilirubin (41 μmol/l, normal <6) and lactate dehydrogenase (798 iu/l, normal 118–211). Intravascular haemolysis was confirmed by raised methaemalbumin (14·3 mg/l, normal <01·0) levels and haemoglobinuria. A peripheral blood film showed bite cells and abundant hemi-ghost cells (left), classical for oxidative haemolysis. The patient survived with transfusion, haemodialysis and supportive treatment, with complete renal recovery. A repeat G6PD enzyme assay on his stored blood sample showed a marginal G6PD level of 5·5 iu/g Hb, which lies within the range expected for female heterozygotes. His four brothers were all G6PD deficient (0·21–1·24 iu/g Hb). Using allele-specific polymerase chain reaction, a G6PD Kaiping allele (nt 1388 G→A) was detected in the patient and all siblings. Surprisingly the patient also carried a second normal G6PD allele, suggesting extra X chromosome material. On further questioning, the patient volunteered a history of azoospermia and bilateral absent vas deferens. There were no other abnormal features suggestive of Klinefelter syndrome. Karyotype analysis on stimulated peripheral blood lymphocytes and fluorescence in situ hybridization with an X whole chromosome painting probe (Oncor Inc., Gaithersburg, MD, USA) confirmed the presence of extra X chromosome material on the Y-chromosome (right, red arrow) in all 20 metaphases analysed: 46, XY.ish add(Y)(p11)(wcpX+). Hence the patient was an occult carrier of extra X chromosome material and a heterozygous carrier of G6PD deficiency. Deficiency of G6PD is the commonest red cell enzymopathy world-wide. It affects 4·8% of all Southern Chinese males, and is screened for at birth and before the commencement of oxidative medications. In female heterozygotes, random lyonization of the X chromosomes results in a mixture of normal and G6PD deficient red cells. The resultant average enzyme levels usually lie within the normal range. However female heterozygotes for G6PD deficiency can still suffer haemolysis as a result of skewed lyonization because of age, haemopoietic transplantation or clonal haemopoiesis. Without mandatory screening, this can cause unexpected severe haemolysis. Our case showed that a simple fluorescent screen might still be insufficient. Severe oxidative stresses because of a combination of drugs (cotrimoxazole and rasburicase) could still cause en masse destruction of the G6PD deficient subpopulation, resulting in life-threatening intravascular haemolysis. Hence, a quantitative enzyme level assay is a better option in areas endemic for G6PD deficiency.

Effects of olive oil polyphenols on erythrocyte oxidative damage

Many studies have investigated the protective effects of oleuropein and hydroxytyrosol against cell injury, but few have investigated the protective effects of oleuropein aglycones 3,4-dihydroxyphenylethanol-elenolic acid (3,4-DHPEA-EA) and 3,4-dihydroxyphenylethanol-elenolic acid dialdehyde (3,4-DHPEA-EDA). The present work studied and compared the capacity of these four compounds, found at high concentrations in olive oil, to protect red blood cells (RBCs) from oxidative injury. The in vitro oxidative stress of RBCs was induced by the water-soluble radical initiator 2,2'-azo-bis(2-amidinopropane) dihydrochloride. RBC changes were evaluated either by optical microscopy or by the amount of hemolysis. All compounds were shown to significantly protect RBCs from oxidative damage in a dose-dependent manner. The order of activity at 20 microM was: 3,4-DHPEA-EDA > hydroxytyrosol > oleuropein > 3,4-DHPEA-EA. Even at 3 microM, 3,4-DHPEA-EDA and hydroxytyrosol still had an important protective activity. However, deleterious morphological RBC changes were much more evident in the presence of hydroxytyrosol than with 3,4-DHPEA-EDA. For the first time it was demonstrated that 3,4-DHPEA-EDA, one of most important olive oil polyphenols, may play a noteworthy protective role against ROS-induced oxidative injury in human cells since lower doses of this compound were needed to protect RBCs in vitro from oxidative mediated hemolysis.

Protective effect of quince (Cydonia oblonga Miller) fruit against oxidative hemolysis of human erythrocytes

The aim of this study was to determine the phenolic content and evaluate the antioxidant activity of quince (Cydonia oblonga) fruit. For this purpose, fruits were separated into pulps, peels and seeds and methanolic extracts were prepared. The phenolic profiles were determined by HPLC/UV and antioxidant properties were studied for their ability to quench the stable free radical 2,2′-diphenyl-1-picrylhydrazyl (DPPH) and to inhibit the 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced oxidative hemolysis of human erythrocytes.The main phenolic compounds were 5-O-caffeoylquinic acid for pulp and peel (57% and 29%, respectively) and stellarin-2 for seed (18%). Total phenolics content was 2.5, 6.3 and 0.4g/kg of methanolic extract for pulp, peel and seed, respectively. Pulp and peel extracts showed similar DPPH free radical scavenging activities (EC50 of 0.6 and 0.8mg/ml, respectively), while seed extract presented much lower antioxidant potential (EC50 of 12.2mg/ml). Under the oxidative action of AAPH, pulp and peel extracts showed significant protection of the erythrocyte membrane from hemolysis, in a time- and concentration-dependent manner. Seed extracts by themselves induced extensive hemolysis. These results indicate higher antioxidant activity for certain parts of quince fruit, namely pulp and peel, that may therefore represent accessible sources of natural antioxidants with potential application in nutritional/pharmaceutical fields, as preventive or therapeutic agents in diseases in which free radicals are implicated.

Evaluation of free radical-scavenging and antihemolytic activities of quince ( Cydonia oblonga) leaf: A comparative study with green tea ( Camellia sinensis)

This study aimed to determine the phenolic profile and to investigate the antioxidant potential of quince ( Cydonia oblonga) leaf, comparing it with green tea ( Camellia sinensis). For these purposes, methanolic extracts were prepared and phenolics content of quince leaf was determined by HPLC/UV. The antioxidant properties were assessed by Folin–Ciocalteu reducing capacity assay and by the ability to quench the stable free radical 2,2′-diphenyl-1-picrylhydrazyl (DPPH) and to inhibit the 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced oxidative hemolysis of human erythrocytes. 5- O-Caffeoylquinic acid was found to be the major phenolic compound in quince leaf extract. Quince leaf exhibited a significantly higher reducing power than green tea (mean value of 227.8 ± 34.9 and 112.5 ± 1.5 g/kg dry leaf, respectively). Quince leaf extracts showed similar DPPH radical-scavenging activities (EC 50 mean value of 21.6 ± 3.5 μg/ml) but significantly lower than that presented by green tea extract (EC 50 mean value of 12.7 ± 0.1 μg/ml). Under the oxidative action of AAPH, quince leaf methanolic extract significantly protected the erythrocyte membrane from hemolysis in a similar manner to that found for green tea (IC 50 mean value of 30.7 ± 6.7 and 24.3 ± 9.6 μg/ml, respectively, P > 0.05). These results point that quince leaf may have application as preventive or therapeutic agent in diseases in which free radicals are involved.

Oxidative haemolysis due to ‘poppers’

A 38-year-old man was admitted to hospital with several days of malaise and dyspnoea. He was on no prescribed medications, and had previously been well apart from an episode of gangrenous cholecystitis due to mixed gallstones three years previously. Full blood examination showed haemoglobin 110 g/l, white cell count 12AE9 · 10/l with neutrophil count 9AE2 · 10/l, and platelet count 266 · 10/l. The blood film (left) demonstrated marked polychromasia with features of oxidative haemolysis and hyposplenism. Supravital staining with methyl-violet demonstrated numerous Heinz bodies (top right). Other indicators of haemolysis included a reticulocytosis of 241 · 10/l, lactate dehydrogenase 374 IU/l, bilirubin 54 lmol/l with undetectable haptoglobin and a negative direct antiglobulin test. On further enquiry, the patient denied fava bean ingestion but disclosed heavy recreational use of inhalational amyl nitrite. Avoidance of further nitrite exposure resulted in resolution of haemolysis, and the patient was provided with counselling on drug use. Laboratory testing in the convalescent phase did not detect evidence of glucose-6-phosphate dehydrogenase deficiency or an underlying haemoglobinopathy. Nitrite inhalants, such as amyl nitrite, are highly volatile yellow liquids that cause rapid vasodilation and hence were once used as anti-anginals. Nitrites efficiently oxidize haemoglobin to methaemoglobin and may also increase the proteolytic susceptibility of haemoglobin leading to globin chain denaturation and Heinz body formation. The recreational use of alkyl nitrites, colloquially known as ‘poppers’ (bottom right), should be considered in apparently idiopathic episodes of oxidative haemolysis. Significant complications may occur even in patients with intact red cell reduction pathways.

Protective effect of saponins from Argania spinosa against free radical-induced oxidative haemolysis

Saponins from Argania spinosa at a non-haemolytic concentration diminish by 53.2% erythrocyte haemolysis induced by free radicals. 2 mM aspirin and acetaminophen diminish by 75% and 68% , respectively, erythrocyte haemolysis induced by free radicals, while 0.3 microM vitamin E shows no significant antioxidant activity. Interestingly, a combination of 1 mg/l of A. spinosa saponins and vitamin E at 0.3 microM resulted in a 68% level of protection against free radical-induced erythrocyte haemolysis, which may suggest that A. spinosa saponins enhance the antioxidant effect of vitamin E. In contrast, no synergic effect was observed for acetaminophen (2 mM) when in combination with vitamin E (0.3 microM). These results demonstrate the antioxidant properties of saponins from A. spinosa and their ability to potentate the antioxidant effect of vitamin E.