Intra-specific biodiversity of Italian myrtle (Myrtus communis) through chemical markers profile and biological activities of leaf methanolic extracts.

Methanolic extracts of Myrtus communis leaves from two Italian regions (Calabria and Sardinia) were processed to determine the content of myrtenol, linalool and eucalyptol. Among the Calabrian and Sardinian myrtle samples, linalool and eucalyptol chemotypes were prevalent. The extracts were also tested for antioxidant, antibacterial and antifungal activities. Myrtle leaves samples were dried and extracted through maceration. Partition chromatography was adopted to separate myrtenol, linalool and eucalyptol fractions. Analyses were performed through GC and GC-MS. Some of the samples showed a good scavenger activity evidenced by DPPH radical scavenging assay and beta-carotene bleaching test. Antibacterial and antifungal activities were generally weak. The phytochemical and biological characterization of all the extracts were determined with an aim to characterize the intra-specific biodiversity of myrtle populations.

Influence of environmental factors on composition of volatile constituents and biological activity of Helichrysum italicum (Roth) Don (Asteraceae).

The biovariability of Helichrysum italicum (Roth) Don grown wild in Calabria and Sardinia (Italy) was reported. This species has been characterized through the detection, isolation and quantitative evaluation of chemical markers (alpha-terpinolene, trans-cariophyllene and neryl acetate) by GC and GC-MS. Antioxidant activity of the methanolic H. italicum extracts using DPPH and beta-carotene bleaching test showed that the Calabrian samples were more active than those from Sardinia. The antibacterial activity of all extracts evidenced the best performance on the Gram positive bacteria particularly on Micrococcus luteus. Moreover, antifungal activity of all extracts was also tested evidencing important results particularly on the phytopathogene fungus Pythium ultimum. In general, as regards the antifungal activity, the extracts from Sardinia were more active than those from Calabria. The phytochemical analysis and the biological activity data suggested a possible use of these plant matrices in alimentary, cosmetic and pharmaceutical fields.

Tocopherols, fatty acids and sterols in seeds of four Sardinian wild Euphorbia species.

Sardinian wild Euphorbia pithyusa, E. semiperfoliata, E. dendroides and E. characias seed oils were analyzed for their fatty acids, unsaponifiable and tocopherol content. Total tocopherol content showed a wide variability, ranging from 939 mg/kg in E. semiperfoliata seeds to its absence in E. characias. The results on tocopherol content were statistically correlated with both 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging test and the beta-carotene bleaching antioxidant test. All seeds were rich in linolenic acid, while no uncommon fatty acids were detected.

Wild Amaranthus caudatus seed oil, a nutraceutical resource from Ecuadorian flora.

Seed oil of wild Amaranthus caudatus from Ecuador was analyzed for determining the tocopherol, fatty acid, and sterol contents. The data obtained were compared with the analogous chemical profile of seed oil of Italian A. caudatus with the objective of evaluating the nutraceutical and alimentary potential of the Ecuadorian matrix. Supercritical fluid and ultrasound-enhanced extractions were performed on both matrices. Qualitative and quantitative determinations of tocopherols were performed by HPLC, whereas GC and GC-MS were used to determine the fatty acid composition and sterols, respectively. Supercritical fluid extraction at 400 atm was the most efficient extraction method in terms of both total yield extract and tocopherol yield. Seeds of Ecuadorian of A. caudatus contained higher levels of tocopherols than Italian samples, whereas the fatty acid composition and sterol content were similar. From the obtained results it can be suggested that seed oil of wild Ecuadorian A. caudatus can prove to be an effective nutraceutical and alimentary resource and a valid alternative to the European varieties.

Detection of xanthine oxidase activity products by EPR and HPLC in bronchoalveolar lavage fluid from patients with chronic obstructive pulmonary disease.

Xanthine oxidase (xanthine: oxygen oxidoreductase, EC 1.1.3.22), a molybdenum-containing hydroxylase that produces superoxide and uric acid from purine substrates and molecular oxygen, is involved in the oxidative stress underlying several human pathologies including lung diseases. An enzymatic activity similar to xanthine oxidase was previously reported in bronchoalveolar lavage fluid of patients with chronic obstructive pulmonary disease (COPD-BAL), by fluorometric analysis of DNA unwinding and cytochrome c reduction kinetics. Here we report the detection of xanthine oxidase activity products by electron paramagnetic resonance (EPR) in presence of the spin-trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and reversed-phase high-performance liquid chromatography (RP-HPLC) in COPD-BAL (n = 14, average age of patients 65 years, range 38-81) and BAL from healthy nonsmoker controls (n = 6, average age 64 years, range 44-73). Superoxide DMPO adducts were detected in COPD-BAL and in an in vitro system containing xanthine and xanthine oxidase (XA/XO), but not in BAL controls and when superoxide dismutase (SOD, 1000 I.U./ml) was added to COPD-BAL. The HPLC analyses after addition of xanthine showed production of uric acid in COPD-BAL and in the XA/XO system but not in BAL controls. These results support the involvement of xanthine oxidase in the mechanisms of superoxide production by BAL supernatant, which increases oxidative stress in chronic obstructive pulmonary disease.

Correlation between age and DNA damage detected by FADU in human peripheral blood lymphocytes.

Fluorometric analysis of DNA unwinding (FADU) is a fast and reliable method for detecting single strand DNA breaks as an index of DNA damage induced by clastogenic agents. A study of damage detected by FADU was conducted on DNA extracted from peripheral blood lymphocytes of 128 healthy nonsmoking regular donors (ranging in age from 19 to 67 years) and from 5 umbilical cord blood samples. DNA damage was measured as percentage of unwound DNA after alkalinization. Statistical analyses, both parametric (Pearson r correlation coefficient, b regression coefficient, ANOVA) and nonparametric (Kruskal-Wallis H test, Spearman rs rank correlation coefficient), support a significant correlation between age of donors and amount of DNA damage. The same results are found when adult donors are divided in four age classes and the ANOVA test performed among the mean percentages of unwound DNA of each class. Furthermore, donors of the same age belonging to different blood groups (A, B, AB and O) do not show any difference in DNA damage detected by FADU.

Xanthine oxidase activity in bronchoalveolar lavage fluid from patients with chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a serious respiratory pathology characterized by irreversible limitation of expiratory flow and includes chronic obstructive bronchitis, chronic airflow limitation, and emphysema. To determine whether xanthine oxidase activity increased in the airspaces of COPD patients, we examined bronchoalveolar lavage fluid (BAL) from COPD patients recruited during a 2-year clinical study. Filtered BAL supernatant from COPD patients and healthy nonsmoking controls was examined by fluorometric analysis of DNA unwinding (FADU) and spectrophotometric assays (cytochrome c reduction kinetics and uric acid kinetics). Compared to controls, filtered BAL supernatant of subjects with COPD exhibited a detectable clastogenic activity probably related to superoxide production. The method of BAL preparation as an acellular system strongly suggests that superoxide production may be due to xanthine oxidase activity.

Oxygen radical scavengers inhibit clastogenic activity induced by sonication of human serum.

Clastogenic factors (CF) are diffusible molecules that damage DNA. They are generated within biological media by a variety of physical and chemical stimuli. Their nature and mechanism of action remain largely unknown. Clastogenic activity can be experimentally generated by pulsed ultrasound treatment of human serum. To investigate whether oxygen radicals are involved in the clastogenic activity induced by sonication of human serum, we examined the effects on such clastogenic activity of different oxygen radical scavengers added to human serum before and after sonication. Human serum was sonicated for 50 min at 24 microW/cm2 by pulsed ultrasound. The clastogenic activity of sonicated human serum was examined in the presence or absence of oxygen radical scavengers by measuring the amount of DNA damage induced in autologous human lymphocytes, assessed with the fluorometric analysis of DNA unwinding (FADU). Sonication of human serum generated significant DNA damage in autologous lymphocytes (DNA unwinding averaged 31.79% +/- 2.1 after sonication vs. 12.82% +/- 2.6 in the controls, p < 0.005). Superoxide dismutase (SOD; 500 I.U./ml), catalase (500 I.U./ml), mannitol (50 mM), and glutathione (50 mM) completely prevented DNA damage when added before serum sonication, whereas only mannitol (86%) and glutathione (90%) almost completely inhibited DNA damage when added after sonication. SOD and catalase had only a partial inhibitory effect when added after sonication (49% and 63%, respectively). The prevention of DNA damage was also obtained by an association of subliminal amounts of glutathione (20 mM) and vitamin E (1 I.U./ml). These results suggest that the clastogenic activity generated by sonication of human serum is mediated by oxygen radicals.

In vitro production of free oxygen radicals induced by pulsed ultrasounds in whole blood exposed to diagnostical frequencies and intensities.

DNA alkalinization experiments on lymphocytes from sonicated whole blood and on in vitro cultured lymphocytes in presence of free radical scavengers (superoxide dismutase, catalase and mannitol) showed that lesions inflicted upon DNA by pulsed ultrasounds could be ascribed to production of free radicals (O2-, OH.) and H2O2, which could mediate the production of still unidentified organic radicals, likely to be responsible for DNA damage.