In Vitro Effects of Charged and Zwitterionic Liposomes on Human Spermatozoa and Supplementation with Liposomes and Chlorogenic Acid during Sperm Freezing.

Semen handling and cryopreservation induce oxidative stress that should be minimized. In this study, human semen was supplemented during cryopreservation with formulations of handmade liposomes and chlorogenic acid (CGA), an antioxidant compound. Zwitterionic (ZL), anionic (AL), and cationic (CL) liposomes were synthesized and characterized. Three aliquots of swim-up-selected sperm were incubated with ZL, AL, and CL (1:10,000), respectively. The percentages of sperm with progressive motility, high mitochondrial membrane potential (MMP; JC-1), double-stranded DNA (dsDNA acridine orange), and acrosome integrity (Pisum sativum agglutinin) were assessed. Then, human semen was frozen using both 1:10,000 ZL and CGA as follows: freezing medium/empty ZL (EL), freezing medium/empty ZL/CGA in the medium (CGA + EL), freezing medium/CGA loaded ZL (CGA), freezing medium (CTR). The same sperm endpoints were evaluated. ZL were the most tolerated and used for semen cryopreservation protocols. All the supplemented samples showed better endpoints versus CTR (p < 0.001). In particular, spermatozoa from the CGA and CGA + EL A samples showed increased motility, dsDNA, and acrosome integrity versus CTR and EL (p < 0.001; motility EL vs. CGA + EL p < 0.05). ZL and CGA can improve post-thaw sperm quality, acting on both cold shock effect management and oxidative stress. These findings open new perspectives on human and animal reproduction.

Apelin is found in human sperm and testis and is raised in inflammatory pathological conditions.

Apelin/APJ receptor (R) is involved in many oxidative stress-induced pathological conditions. Since this system is not yet explored in male reproduction, we studied apelin/APJ-R in human semen and testis. Semen of 41 infertile patients with varicocele, genitourinary infections, unexplained infertility and 12 fertile men was analysed (WHO guidelines, 2021). Apelin was quantified by ELISA in seminal fluid and spermatozoa, interleukin (IL)-1ß in seminal fluid. Apelin/APJ-R were immunolocalized in spermatozoa and testis. Apelin was present in spermatozoa and its levels were negatively correlated with normal sperm morphology% (r = -0.857; p < 0.001), and positively with IL-1ß levels (r = 0.455; p < 0.001). Apelin and IL-1ß concentrations were increased in patients' samples with varicocele (apelin p < 0.01; IL-1ß p < 0.05) and infections (apelin p < 0.01; IL-1ß p < 0.001). By logistic regression analysis, apelin (OR 1.310; p = 0.011) and IL-1ß (OR 1.572; p = 0.005) were predictors of inflammatory diseases (varicocele, infections). Apelin and APJ-R immunofluorescence labels were weak in sperm tail of fertile men and intense along tail, cytoplasmic residues and post-acrosomal sheath of sperm from infertile men. In testis, apelin and APJ-R labels were evident in Leydig cells and weak inside the seminiferous tubule. Apelin/APJ-R system is present in human spermatozoa and testicular tissue and probably involved in human fertility.

Follicular Fluid Components in Reduced Ovarian Reserve, Endometriosis, and Idiopathic Infertility.

Follicular fluid (FF) molecules, and their increase or decrease, can contribute to appropriate follicular growth and oocyte maturation, thus being related to female infertility conditions. In this paper, we studied the changes and the relationships of some biochemical components, hormones, antioxidant enzymes, F2-Isoprostanes (F2-IsoPs), and resolvin (Rv) D1 in the FF of infertile women with different reproductive conditions such as endometriosis, reduced ovarian reserve, and idiopathic infertility during assisted reproductive techniques (ART). In the whole population, positive correlations between albumin (ALB)/iron (Fe), ALB/beta-2-microglobulin (B2MG), and F2-IsoPs/RvD1 were detected in the FF. In FF from aged women, increased levels of follicle stimulating hormone (FSH) and reduced anti-Müllerian hormone (AMH) levels were associated with a worse oocyte quality. The negative ART outcome was influenced by patient age and AMH, B2MG, and FSH levels. Moreover, the reduced ovarian reserve condition was characterised by a significant decrease in oocyte number and quality, AMH amount, and lactate dehydrogenase (LDH) activity, as well as by an increase in age and FSH levels. In the presence of endometriosis, high levels of MDA and RvD1 were detected in FF, with a decrease in luteinising hormone (LH). Finally, among the molecules examined, none characterised the condition of idiopathic infertility. These data could support the identification of new FF markers in different reproductive disorders, suggesting the need for personalised therapeutic approaches and optimised ART outcomes. In particular, the evaluation of resolvins and lipid mediators in FF could be a promising field of investigation with which to understand the entity of oxidative stress and inflammation in some female infertility conditions.

F4-Neuroprostane Effects on Human Sperm.

Swim-up selected human sperm were incubated with 7 ng F4-neuroprostanes (F4-NeuroPs) for 2 and 4 h. Sperm motility and membrane mitochondrial potential (MMP) were evaluated. The percentage of reacted acrosome was assessed by pisum sativum agglutinin (PSA). Chromatin integrity was detected using the acridine orange (AO) assay and localization of the ryanodine receptor was performed by immunofluorescence analysis. Sperm progressive motility (p = 0.02) and the percentage of sperm showing a strong MMP signal (p = 0.012) significantly increased after 2 h F4-NeuroP incubation compared to control samples. The AO assay did not show differences in the percentage of sperm with dsDNA between treated or control samples. Meanwhile, a significantly higher number of sperm with reacted acrosomes was highlighted by PSA localization after 4 h F4-NeuroP incubation. Finally, using an anti-ryanodine antibody, the immunofluorescence signal was differentially distributed at 2 and 4 h: a strong signal was evident in the midpiece and postacrosomal sheath (70% of sperm) at 2 h, whereas a dotted one appeared at 4 h (53% of sperm). A defined concentration of F4-NeuroPs in seminal fluid may induce sperm capacitation via channel ions present in sperm cells, representing an aid during in vitro sperm preparation that may increase the positive outcome of assisted fertilization.

Fatty Acid Oxidation and Pro-Resolving Lipid Mediators Are Related to Male Infertility.

Specialized pro-resolving lipid mediators regulate the resolution of acute inflammation. They are formed by enzymatic oxygenation of polyunsaturated fatty acids and are divided into families including lipoxins, resolvins, protectins, and maresins. Resolvin D1 (RvD1), produced by docosahexaenoic acid, exerts anti-inflammatory and pro-resolving activities. This research aimed to investigate the implication of seminal RvD1 in human infertility. Infertile patients (n° 67) were grouped based on pathological reproductive conditions as idiopathic infertility, varicocele, and leukocytospermia; the fourth group was composed of fertile men (n° 18). Sperm characteristics were evaluated by light microscopy (WHO guidelines) and by transmission electron microscopy (TEM). The seminal levels of RvD1 and F2-isoprostane (F2-IsoPs) were dosed. In twenty men (6 fertile men, 8 with varicocele, 6 with leukocytospermia) seminal phospholipase A2, iron, cholesterol, transferrin, estradiol, ferritin, testosterone, and sperm membrane fatty acids were detected. The results indicated that: (i) RvD1 amount was positively correlated with F2-IsoPs and reduced sperm quality; (ii) RvD1 levels were significantly higher in patients with leukocytospermia, varicocele, and idiopathic infertility compared to fertile men; (iii) RvD1 increased along with other markers of oxidative stress and inflammation as fatty acids content and clinical biomarkers. This study suggests a panel of inflammatory markers and lipid mediators for a diagnosis of inflammatory status and a subsequent appropriate therapeutic approach.

Do Seminal Isoprostanes Have a Role in Assisted Reproduction Outcome?

F2-isoprostanes (F2-IsoPs), stereoisomers of prostaglandin F2α generated by the free radical-induced oxidation of arachidonic acid, have been associated with different male infertility conditions. This study aimed to evaluate the role of seminal isoprostane levels and sperm characteristics in the reproductive outcome and embryo quality of 49 infertile couples. Semen analysis was performed following WHO guidelines. Sperm chromatin maturity was detected using an aniline blue (AB) assay, and DNA integrity was assessed using the acridine orange (AO) test. Seminal F2-IsoP levels were quantified by gas chromatography/negative ion chemical ionization tandem mass spectrometry (GC/NICI-MS/MS) analysis. Correlations among variables and their impact on in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) outcome were investigated. F2-IsoP levels are positively correlated with double-stranded DNA sperm (p < 0.001) and negatively correlated with mature sperm chromatin (p < 0.001). Patients with positive outcomes had an increased percentage of sperm with double-stranded DNA, as did patients producing high-quality embryo, who showed higher F2-IsoP levels compared to those detected in the low-quality embryo group. An intriguing relationship between a mild increase in F2-IsoP levels, DNA integrity, and embryo quality seems to indicate that the non-enzymatic oxidation of arachidonic acid can be also a marker of metabolic activity in human semen.

Tissue Antioxidant Status and Lipid Peroxidation Are Related to Dietary Intake of n-3 Polyunsaturated Acids: A Rabbit Model.

Polyunsaturated fatty acid (PUFA) metabolism and tissue distribution is modulated by the oxidation of these molecules. This research aimed to investigate the implication of dietary n-3 PUFA supplementation (precursor and long-chain PUFA) on the PUFA profile and oxidative status of the liver, testis, and brain of adult rabbit bucks. Twenty New Zealand White rabbit bucks were divided into four experimental groups (n = 5 per group) and were fed different diets for 110 days: control (CNT), standard diet containing 50 mg/kg alpha-tocopheryl acetate (vitamin E); CNT+, standard diet + 200 mg/kg vitamin E; FLAX, standard diet + 10% flaxseed + 200 mg/kg vitamin E; or FISH, standard diet + 3.5% fish oil + 200 mg/kg vitamin E. Antioxidants (enzymatic and non-enzymatic), oxidative status (malondialdehyde and isoprostanoids), and n-3 and n-6 PUFAs of tissues were analysed. A chain mechanism of oxidant/antioxidant molecules, which largely depended on the particular PUFA composition, was delineated in the different organs. The liver showed an oxidant/antioxidant profile and lipid pathways widely modulated by PUFA and vitamin E administration; on the other hand, the testis' oxidative profile rather than its lipid profile seemed to be particularly affected, an outcome opposite to that of the brain (modulation operated by dietary PUFA).

Redox imbalance induced by docetaxel in the neuroblastoma SH-SY5Y cells: a study of docetaxel-induced neuronal damage.

OBJECTIVES: In cancer survivors, chemotherapy-associated adverse neurological effects are described as side effects in non-targeted tissue. We investigated the role of redox-imbalance in neuronal damage by a relative low dose of Docetaxel (DTX). METHODS: The neuroblastoma cells (SH-SY5Y cells) were exposed to DTX at a dose of 1.25 nM for 6 h. Antioxidant defenses (i.e. ascorbic acid, glutathione, and catalase) and lipid oxidation products (i.e. F2-isoprostanes) were evaluated. To investigate cell ultrastructure and tubulin localisation, transmission electron microscopy (TEM) and immunofluorescence techniques were applied. RESULTS: In the SH-SY5Y cells, DTX induced a significant reduction of total glutathione (P < 0.001) and ascorbic acid (P < 0.05), and an increase in both total F2-Isoprostanes (P < 0.05) and catalase activity (P < 0.05), as compared to untreated cells. Additionally, TEM showed a significant increase in cells with apoptotic characteristics. Immunolocalisation of tubulin showed a compromised cytoskeletal organisation. DISCUSSION: The investigated sublethal dose of DTX, to which non-targeted cells may be exposed throughout the duration of chemotherapy treatment, induces a redox imbalance resulting in a specific modulation of the antioxidant response. This study provides new insights into DTX-induced cellular mechanisms useful for evaluating whether the concomitant use of antioxidants associated with chemotherapy mitigates chemotherapy side effects in cancer survivors.

Cytotoxic Effects of Cannabinoids on Human HT-29 Colorectal Adenocarcinoma Cells: Different Mechanisms of THC, CBD, and CB83.

In this study, we investigated the effects of exposition to IC50 dose for 24 h of a new synthetic cannabinoid (CB83) and of phytocannabinoids Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) on HT-29 colorectal carcinoma cells. Cell viability and proliferative activity evaluated using the MTT, lactate dehydrogenase (LDH), and CyQUANT assays showed that cell viability was significantly affected when CB83, THC, and CBD were administered to cells. The results obtained showed that the reduced glutathione/oxidized glutathione ratio was significantly reduced in the cells exposed to CBD and significantly increased in the cells treated with the CB83 when compared to the controls. CBD treatment causes a significant increase in malondialdehyde content. The catalase activity was significantly reduced in HT-29 cells after incubation with CB83, THC, and CBD. The activities of glutathione reductase and glutathione peroxidase were significantly increased in cells exposed to THC and significantly decreased in those treated with CBD. The ascorbic acid content was significantly reduced in cells exposed to CB83, THC, and CBD. The ultrastructural investigation by TEM highlighted a significantly increased percentage of cells apoptotic and necrotic after CB83 exposition. The Annexin V-Propidium Iodide assay showed a significantly increased percentage of cells apoptotic after CB83 exposition and necrotic cells after CBD and THC exposition. Our results proved that only CBD induced oxidative stress in HT-29 colorectal carcinoma cells via CB receptor-independent mechanisms and that CB83 caused a mainly CB2 receptor-mediated antiproliferative effect comparable to 5-Fuorouracil, which is still the mainstay drug in protocols for colorectal cancer.

Resistin in Human Seminal Plasma: Relationship with Lipid Peroxidation, CAT Activity, GSH/GSSG Ratio, and Semen Parameters.

Resistin is an adipokine involved in inflammation and able to induce the expression of other proinflammatory cytokines. It is known that, in human semen, resistin is correlated with inflammatory cytokines and sperm quality. The aim of this prospective study was to explore the potential relationship between resistin, lipid peroxidation (LPO), catalase (CAT) activity, and reduced and oxidized glutathione (GSH/GSSG) ratio in semen samples of infertile patients with leukocytospermia (no. 19), infertile patients with varicocele (no. 17), and fertile men (no. 17). Semen analysis was performed following the WHO guidelines, and sperm apoptosis and necrosis were evaluated with annexin V/propidium iodide assay. Seminal plasma samples were used to determine resistin levels by an immunological method, MDA concentration by a HPLC analysis with UV detection, GSH/GSSG ratio by an enzymatic method, CAT activity by a spectrophotometric method. The results showed that, in both groups of infertile patients, semen parameters were significantly reduced (P < 0.001) and sperm apoptosis and necrosis percentages were increased. Resistin levels were significantly higher in leukocytospermia and varicocele groups (P < 0.001 and P < 0.01, respectively) as well as MDA concentration (P < 0.001) compared to controls. The MDA level was also significantly increased in the leukocytospermia group versus the varicocele group (P < 0.05). The GSH/GSSG ratio was higher in fertile controls than the leukocytospermia group (P < 0.05) and the varicocele group (P < 0.001) and in the leukocytospermia group versus the varicocele group (P < 0.05). Both the leukocytospermia and varicocele groups showed increased values of CAT activities (P < 0.001) than controls. Briefly, the correlation between variables, calculated in the whole patient population, showed that resistin levels positively correlated with MDA levels, CAT activity, sperm apoptosis, and necrosis and negatively with sperm parameters and GSH/GSSG ratio. These results support an active role of resistin in an inflammatory process causing LPO, increase of CAT activity, and decrease of GSH/GSSG ratio in seminal plasma of infertile men vs. fertile controls.

Relationships between Ghrelin and Obestatin with MDA, Proinflammatory Cytokines, GSH/GSSG Ratio, Catalase Activity, and Semen Parameters in Infertile Patients with Leukocytospermia and Varicocele.

Ghrelin and obestatin are involved in many biological functions including reproduction. Growing evidences suggest that both peptides could exert protective and antioxidant activities. In this study, the relationships between ghrelin/obestatin, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), malondialdehyde (MDA), reduced glutathione (GSH), oxidized glutathione (GSSG), expressed as the GSH/GSSG ratio, catalase (CAT), and semen parameters in infertile patients with varicocele or leukocytospermia and controls were investigated. Fifty-six infertile patients (32 with leukocytospermia and 24 with varicocele) and 14 controls participated in this study. Semen analysis was performed following the WHO guidelines. Apoptotic and necrotic sperm were scored by annexin V/propidium iodide assay. Seminal plasma samples were used for the following determinations: ghrelin, obestatin, IL-6, and TNF-α were measured by an immunological method, GSH/GSSG by an enzymatic method, and CAT by spectrophotometric determination. With respect to controls, both the leukocytospermia and varicocele groups showed altered sperm parameters, significantly increased sperm apoptosis (P = 0.009 and P = 0.011, respectively), IL-6 (P = 0.0001 and P = 0.004, respectively), and TNF-α levels (P = 0.0001 and P = 0.002, respectively); both groups had significantly decreased levels of ghrelin (P = 0.0001), obestatin (P = 0.0001 and P = 0.006, respectively), and GSH/GSSG ratio (P = 0.003 and P = 0.0001, respectively). The MDA concentration was significantly increased in the leukocytospermia group vs. controls (P = 0.0001), in the varicocele group vs. controls (P = 0.011), and in the leukocytospermia group vs. the varicocele group (P = 0.008). CAT activity was augmented in both the leukocytospermia and varicocele groups (P = 0.0001)vs. controls. The results indicate that both ghrelin and obestatin may play a protective role in human semen and this effect is probably due to their antioxidant properties.

Nitric oxide, malondialdheyde and non-enzymatic antioxidants assessed in viable spermatozoa from selected infertile men.

There are growing evidences that the semen of infertile male population shows higher reactive oxygen species (ROS) levels concomitant with lower antioxidant capacity compared to those detected in semen of fertile population. The plasma membrane of the sperm cell, which has high levels of polyunsaturated fatty acids, renders it particularly sensitive to ROS. The aim of this study was to compare the sperm parameters (concentration, motility, morphology and vitality) and the levels of malondialdehyde (MDA), as marker of lipid peroxidation (LPO), nitric oxide (NO), ascorbic acid (AA), total (GSHt) and oxidized glutathione (GSSG) in viable sperm in a group of 38 infertile patients and in a group of 55 control subjects with unknown reproductive potential. The comparison between variables in infertile patients and controls revealed that the sperm quality was reduced in the infertile group, whereas the levels of NO, AA and GSH were significantly increased in viable spermatozoa from infertile men; however, the endogenous levels of MDA were similar in infertile and control groups. Based on our results, we could speculate that the rise of GSHt and AA levels in viable sperm of infertile group help partially to counteract the damaging effect of ROS and partly prevent a substantial LPO. The observation of the concomitant increase of NO and antioxidant indices in viable spermatozoa of infertile subjects is a novel finding and we think that these results can be useful since the viable sperm population is conceivably used in assisted reproductive technology.

Myocardial oxidative damage is induced by cardiac Fas-dependent and mitochondria-dependent apoptotic pathways in human cocaine-related overdose.

The aim of this study is to analyse cardiac specimens from human cocaine-related overdose, to verify the hypothesis that cardiac toxicity by acute exposure to high dosage of cocaine could be mediated by unbalanced myocardial oxidative stress, and to evaluate the apoptotic response. To address these issues, biochemical and immunohistological markers of oxidative/nitrosative stress were evaluated. We found that i-NOS, NOX2 and nitrotyrosine expression were significantly higher in the hearts of subjects who had died from high doses of cocaine, compared to the control group. Increase of these markers was associated with a dramatic increase in 8-OHdG, another marker of oxidative stress. A high number of TUNEL-positive apoptotic myocells was observed in the study group compared to the control group. The immunoexpression of TNF-α was significantly higher in the cocaine group compared to the control group. Furthermore, we detected a significantly stronger immunoresponse to anti-SMAC/DIABLO in our study group compared to control cases. Both cardiac Fas-dependent and mitochondria-dependent apoptotic pathways appeared to be activated to a greater extent in the cocaine group than in the control group. Our results highlight the central role of oxidative stress in cocaine toxicity. High levels of NOS can promote the oxidation process and lead to apoptosis.

Phase I trial of thymidylate synthase poly-epitope peptide (TSPP) vaccine in advanced cancer patients.

Thymidylate synthase (TS) poly-epitope peptide (TSPP) is a 27-mer peptide vaccine containing the amino acidic sequences of three epitopes with HLA-A2.1-binding motifs of TS, an enzyme overexpressed in cancer cells, which plays a crucial role in DNA repair and replication. Based on the results of preclinical studies, we designed a phase Ib trial (TSPP/VAC1) to investigate, in a dose escalation setting, the safety and the biological activity of TSPP vaccination alone (arm A) or in combination with GM-CSF and IL-2 (arm B) in cancer patients. Twenty-one pretreated metastatic cancer patients, with a good performance status (ECOG ≤ 1) and no severe organ failure or immunological disease, were enrolled in the study (12 in arm A, nine in arm B) between April 2011 and January 2012, with a median follow-up of 28 months. TSPP resulted safe, and its maximal tolerated dose was not achieved. No grade 4 toxicity was observed. The most common adverse events were grade 2 dermatological reactions to the vaccine injection, cough, rhinitis, fever, poly-arthralgia, gastro-enteric symptoms and, to a lesser extent, moderate hypertension and hypothyroidism. We detected a significant rise in auto-antibodies and TS-epitope-specific CTL precursors. Furthermore, TSPP showed antitumor activity in this group of pretreated patients; indeed, we recorded one partial response and seven disease stabilizations (SD) in arm A, and three SD in arm B. Taken together, our findings provide the framework for the evaluation of the TSPP anti-tumor activity in further disease-oriented clinical trials.

Hepatotoxicity of Teucrium chamaedrys L. decoction: role of difference in the harvesting area and preparation method.

AIM: Two recurrent cases of severe acute liver injury attributed to the use of a wild germander decoction, prepared with some variation in traditional method has been reported. The aim of the present study was to correlate the hepatotoxic effect observed in patients who consumed germander decoction with teucrin A levels. Antioxidant properties were analyzed to assess any possible differences between the decoction used traditionally by the family (without negative consequences) and the decoction taken by the patients. MATERIALS AND METHODS: Different types of germander decoctions were prepared in the laboratory by simulating the same conditions for preparing the decoction by the patients and their family members. The levels of teucrin A, the polyphenols and the antioxidant power were determined. One-way analysis of variance was used to test for differences between the groups. RESULTS AND CONCLUSIONS: The extract consumed by the patients had higher concentration of teucrin A, lower antioxidant activity and lower content of polyphenols compared with the traditional decoction, revealing an inverse relationship between teucrin A content and antioxidant capacity. These case reports emphasize that more information is needed on the safety and quality of these natural products.

Severe hypoglycemia associated with levofloxacin in Type 2 diabetic patients receiving polytherapy: two case reports.

OBJECTIVE: Although hypoglycemia is known to be associated with levofloxacin, patients are continually being hospitalized because of this adverse event. Here we have reported two further cases of severe hypoglycemia and discussed the possibility that the hypoglycemia is the result of interactions between levofloxacin and certain drugs, in order to alert physicians to be aware that geriatric patients, particularly those with Type 2 diabetes and in polytherapy, are at risk of showing this adverse reaction. CASES SUMMARY: A 91-year-old woman with Type 2 diabetes on metformin and glibenclamide, also under treatment with oral antihypertensive drugs, platelet antiaggregant, low-molecular- weight heparin and buprenorphine, was prescribed levofloxacin for a bacterial infection. Liver function and renal function parameters were within normal limits. After repeated administration of levofloxacin, her serum glycemic levels had decreased to 47 mg/dl and the patient was in a coma. After stopping levofloxacin her glycemia level returned to the normal value. A 61 year-old male, affected by tonsillar squamous cell carcinoma, with Type 2 diabetes on metformin and glibenclamide, under treatment with low-molecular-weight heparin for deep venous thrombosis, hydromorphone and undergoing nutritional support, was treated with levofloxacin for a bacterial infection. After 72 h the patient was unresponsive and his blood glucose levels were 38 mg/dl. After discontinuation of levofloxacin administration the patient was treated with glucose infusion and his glycemic values gradually returned to the normal range. DISCUSSION: The causality relationship between the levofloxacin and the hypoglycemia was established using Naranjo's ADR probability scale. These case reports confirm the literature data that serious hypoglycemia may develop due to the use of levofloxacin and appears to occur most frequently in elderly patients with Type 2 diabetes who are receiving oral hypoglycemic agents. We described the possible pharmacokinetic and pharmacodinamic mechanisms of the interaction between levofloxacin and the other drugs. CONCLUSIONS: We hypothesized that the concomitant use of several drugs and particularly levofloxacin in association with oral antidiabetic drugs, opioid analgesics and low-molecular-weight heparin could concur to cause this side effect. The safety and tolerability of this anti-infective agent should be revised urgently.

In vitro antioxidant activity of aged extracts of some Italian Allium species.

Antioxidant activity of fresh Allium sativum L. (garlic) is well known and is mainly due to unstable and irritating organosulphur compounds. Fresh garlic extracted over a prolonged period (up to 20 months) produces odourless aged garlic extract (AGE) containing stable and water soluble organosulphur compounds that prevent oxidative damage by scavenging free radicals. The aim of this study was to investigate the in vitro antioxidant activity of aged (up to 20 months) 15% hydroethanolic extracts of different parts (bulbs, bulblets, flower bulblets, flowers, and leaves) of three Allium spontaneous species which are endemic for Italian flora: Allium neapolitanum Cyr., Allium subhirsutum L., Allium roseum L. and to compare it with the in vitro antioxidant activity of aged 15% hydroethanolic extracts of bulbs and leaves of garlic. The antioxidant potential of aged extracts of all species has been evaluated using two different spectrophotometric assays: 2,2-diphenylpicrylhydrazyl (DPPH) test and the ferric reducing/antioxidant power (FRAP) assay. Furthermore the polyphenol content was determined. The aged extracts obtained from the leaves showed the best antioxidant activity, followed by flowers and then by bulbs in both used tests, while flower bulblets and bulblets exhibited lower results or no activity. The polyphenol content was generally directly correlated with antioxidant/antiradical activity. This study confirms the data obtained in previous researches, the wild-type species of Allium and in particular organs other than bulbs are more active and efficacious than garlic bulb. Surely leaves of these Allium spp. deserve special attention.

Cardiovascular receptor binding affinity of aqueous extracts from Allium species.

INTRODUCTION: The aims of this study were to evaluate whether the antihypertensive effect of garlic could to be associated to interactions with adrenergic and dopaminergic receptors involved in regulating blood pressure and to compare these data with those obtained from wild Allium species. METHODS: The aqueous extracts of bulbs or leaves of Allium sativum L. (garlic), Allium neapolitanum Cyr., Allium subhirsutum L., and Allium roseum L. were tested for their in vitro affinity for the adrenergic (alpha(1), alpha(2), beta(1) and beta(2)) and dopaminergic (D(1) and D(2)) receptors by radioligand binding assays. RESULTS: Interesting results were shown by bulbs extracts of A. neapolitanum and A. subhirsutum with higher affinities for the beta(2) receptors and by bulbs extract of A. roseum for D(2) receptors. CONCLUSIONS: The known antihypertensive activity of Allium sativum cannot be correlated with binding to receptors involved in blood pressure regulation. However, aqueous extracts of the wild-type species of Allium show much higher affinities, warranting further explorations.

Protective effect of Allium neapolitanum Cyr. versus Allium sativum L. on acute ethanol-induced oxidative stress in rat liver.

This study investigated the protective effect of Allium neapolitanum Cyr., a spontaneous species of the Italian flora, compared with garlic (Allium sativum L.) on liver injury induced by ethanol in rats. Male albino Wistar rats were orally treated with fresh Allium homogenates (leaves or bulbs, 250 mg/kg) daily for 5 days, whereas controls received vehicle only. At the end of the experimental 5-day period, the animals received an acute ethanol dose (6 mL/kg, i.p.) 2 hours before the last Allium administration and were sacrificed 6 hours after ethanol administration. The activities of catalase (CAT), superoxide dismutase (SOD), and glutathione reductase (GR) and the levels of malondialdehyde (MDA), ascorbic acid (AA), and reduced (GSH) and oxidized glutathione in liver tissue were determined. Administration of both Allium species for 5 days (leaves or bulbs) led to no statistical variation of nonenzymatic parameters versus the control group; otherwise Allium treatment caused an increase of GSH and AA levels compared with the ethanol group and a diminution of MDA levels, showing in addition that A. neapolitanum bulb had the best protective effect. Regarding to enzymatic parameters, GR and CAT activities were enhanced significantly compared with the ethanol group, whereas SOD activity showed a trend different from other parameters estimated. However, the treatment with both Allium species followed by acute ethanol administration reestablished the nonenzymatic parameters similar to control values and enhanced the activities of the enzymes measured. These results suggest that fresh Allium homogenates (leaves or bulbs) possess antioxidant properties and provide protection against ethanol-induced liver injury.

Evaluation of antioxidative properties of Allium species growing wild in Italy.

The genus Allium (Alliaceae) is an important dietary source of antioxidant phytochemical products. The antioxidant activity of some Allium species is well known but no information is available on the in vitro antioxidant activities of Italian Allium species growing wild. The aim of this study was to examine the in vitro antioxidant activity of aqueous extracts of different parts belonging to three Allium species growing wild, endemic to Italian flora: Allium neapolitanum Cyr., Allium subhirsutum L. and Allium roseum L., compared with the in vitro antioxidant activity of aqueous extracts of bulbs and leaves of the greatly studied garlic (Allium sativum L.). The antioxidant potential of extracts was evaluated using two different spectrophotometric assays: the DPPH test and FRAP assay. Furthermore the polyphenolic content was determined in all Allium species. The flowers of species growing wild showed the higher antioxidant power. Interesting results were shown even by the leaves, while the antioxidant capacity of the bulbs was lower. A correlation between the phenolic contents and the antioxidant activity was discovered. The differences in antioxidant capacity reflect the variability in the Allium species and the parts of the plant used and that the bulb of Allium sativum did not show higher antioxidant power.