Antoaneta Zheleva

Sensitivity of alpha-amanitin to oxidation by a lactoperoxidase-hydrogen peroxide system.

Oxidation of alpha-amanitin - a potent hepatotoxin found in the mushroom Amanita phalloides - by a lactoperoxidase-hydrogen peroxide system was investigated by different techniques. (i). UV spectroscopy of the mixture after 24 h incubation reveals a significant decrease in the absorbance range characteristic of the putative reactive moiety of the toxin, the tryptathionine group. (ii). Formation of a new product was detected by Thin Layer Chromatography. (iii). In vivo experiments with non-inbred male albino mice showed a lowered toxicity of the modified toxin in comparison with that of the native one. Taking into account the latter results concerning the sensitivity of the toxin towards an oxidising system, the formation and reactivity of an alpha-amanitin derivative is discussed in the course of A. phalloides poisoning (inhibition of RNA polymerase type II and development of damaging radical species).

Radical scavenging and radiomodulatory effects of Psoralea corylifolia Linn. substantiated by in vitro assays and EPR spectroscopy.

The present study is the first report of the radiomodulatory effects of Psoralea corylifolia Linn. The extract (IBG-RA-26) prepared from P. corylifolia was chemically analysed by HPLC, LC-MS/MS and NMR. The total polyphenolic content of IBG-RA-26 was 0.287 mg/ ml of quercetin equivalents. IBG-RA-26 exhibited a dose-dependent increase in 2,2-diphenyl- 1-picrylhydrazyl (DPPH) radical scavenging activity. It exhibited comparable (> 50%) site-specific and non-site-specific hydroxyl radical scavenging activity in higher concentration ranges (500 - 1000 μg/ml), while at lower concentrations (5 - 50 μg/ml) it exhibited significantly (p < 0.05) higher non-site-specific scavenging ability compared to site-specific activity. Nitric oxide scavenging activity of IBG-RA-26 (5 - 1000 μg/ml) increased in a concentrationdependent manner, while maximum superoxide ion scavenging ability (79%) was observed at 50 μg/ml. The electron donation potential of IBG-RA-26 was found to be higher than that of ascorbic acid at lower concentrations (up to 5 μg/ml). Analysis of the ability of IBGRA- 26 to protect membranes against γ-radiation, utilizing an artificial membrane system (liposome), revealed a significant (p < 0.05) decrease in the formation of malondialdehyde (MDA) as a function of the concentration of IBG-RA-26. Radiation-induced lysis of human erythrocytes was monitored and efficacy of IBG-RA-26 was tested in the concentration range 25 - 1000 μg/ml, with significant protective efficacy observed in the range 25 - 50 μg/ml. IBG-RA-26 rendered significant (p < 0.05) protection against radiation (0.25 kGy)-induced DNA damage. EPR spectroscopy was used to investigate the DPPH radical scavenging capacity of IBG-RA-26. IBG-RA-26 exhibited a good DPPH radical scavenging capacity in a concentration-dependent manner. By direct EPR spectroscopy we have also demonstrated the possible formation of free radical species in a solution of IBG-RA-26. The wide spectrum of radioprotective and antioxidant properties exhibited by IBG-RA-26 indicate that P. corylifolia has potential as a radiomodulatory agent.

Electron paramagnetic resonance spectroscopy in radiation research: Current status and perspectives

Exposure to radiation leads to a number of health-related malfunctions. Ionizing radiation is more harmful than non-ionizing radiation, as it causes both direct and indirect effects. Irradiation with ionizing radiation results in free radical-induced oxidative stress. Free radical-mediated oxidative stress has been implicated in a plethora of diseased states, including cancer, arthritis, aging, Parkinsons disease, and so on. Electron Paramagnetic Resonance (EPR) spectroscopy has various applications to measure free radicals, in radiation research. Free radicals disintegrate immediately in aqueous environment. Free radicals can be detected indirectly by the EPR spin trapping technique in which these forms stabilize the radical adduct and produce characteristic EPR spectra for specific radicals. Ionizing radiation-induced free radicals in calcified tissues, for example, teeth, bone, and fingernail, can be detected directly by EPR spectroscopy, due to their extended stability. Various applications of EPR in radiation research studies are discussed in this review.

Preparation of a β-amanitin - concanavalin A conjugate of low toxicity

Zh. Zhelev, A.Zheleva and K. Halacheva. Preparation of a β-amanitin - concanavalin A conjugate of low toxicity. Toxicon25, 981 – 987, 1987.—Pure β-amanitin was isolated by combined adsorption chromatography of a crude methanol extract from Amanita phalloides on Sephasorb HP Ultrafine and Sephadex LH-20. The β-amanitin was coupled to concanavalin A by the carbodiimide method. The conjugate was purified by fractionation on a column of Sephadex G-75. The molar ratio β-amanitin to concanavalin A in this conjugate was 4.14, The purified conjugate was tested by thin-layer chromatography and showed characteristic (for amatoxins) bright purple staining but different mobility. The ultraviolet spectrum of the conjugate was different from the spectra of the native β-amanitin and the native concanavalin A. The toxicity of the conjugate was determined by in vivo toxicological studies and was four-fold lower than that of the native β-amanitin. These results suggest that this conjugate may be used for immunization procedures and for the production of amatoxin-specific antibodies.

Two glycine containing 2-chloroethylnitrosoureas—a comparative study on some physicochemical properties, in vivo antimelanomic effects and immunomodulatory properties

Physicochemical properties such as alkylating and carbamoylating activity and in vivo antimelanomic effects against B16 melanoma of the spin labeled (nitroxyl free radical containing) glycine nitrosourea (SLCNUgly) and its nonlabeled analogue (ChCNUgly), synthesized in our laboratory are studied and compared to those of antitumour drug 3-cyclohexyl-1-(2-chloroethyl)-1-nitrosourea (CCNU). We have demonstrated that introducing of glycine moiety in the nitrosourea structure in practice does not affect either alkylating or carbamoylating activity. On the other hand replacement of cyclohexyl moiety in ChCNUgly structure with nitroxyl free radical leads to a decrease in carbamoylating activity and an increase in alkylating activity. Compound ChCNUgly showed in vivo a higher antimelanomic activity against B16 melanoma in comparison with CCNU and SLCNUgly. It completely inhibited B16 melanoma growth (TGI=100%) at a dose 64.0 mg/kg. Moreover, we established that joint i.p. application in normal mice of SLCNUgly plus a new immunostimulator (C3bgp) formerly isolated in our laboratory led to a 75% restoration in immune function with respect to antibody production measured by Jerne hemolytic plaque assay. In contrast, no immunostimulation was found after joint application of C3bgp plus ChCNUgly or CCNU at the same experimental conditions. Based on these preliminary results, a possibility for developing of new combination immunochemotherapy schemes for treatment of human cancers is discussed.

Cytotoxicity on L1210 leukemic cells of beta-amanitin-concanavalin A and phallacidin-concanavalin A conjugates

The conjugates beta-amanitin-concanavalin A and phallacidin concanavalin A were tested for direct cytotoxicity on L1210 lymphocytic leukemia cells by a combined in vitro-in vivo bioassay. Both conjugates exerted strong direct cytotoxicity on the tumour cells.

Studies on Antioxidant Properties before and after UV- And Γ-Irradiation of Bulgarian Lavender Essential Oil Isolated from Lavandula Angostifolia Mill

ABSTRACT At present there is not enough data about different types of radiation on Bulgarian essential oils isolated from medicinal plants. Here we report for the first time our studies on the effects of UV-radiation (290 nm to 320 nm) and γ-radiation (doses of 5 Gy, 10 Gy, 20 Gy and 30 Gy) on Bulgarian lavender essential oil isolated from Lavandulla angustifolia Mill. A spectrophotometry method was used for evaluation of the changes in the reducing power of the Bulgarian lavender oil before and after radiation. The electron donation potential of non-irradiated oil was found to be lower (0.268 ± 0.0244) than those of both, UV-irradiated (0.336 ± 0.0121) and γ-irradiated, oil samples (0.427 ± 0.0251 at 5 Gy radiation, 0.341 ± 0.0371 at 10 Gy; 0.328 ± 0.0173 at 20 Gy). By direct electron paramagnetic resonance (EPR) spectroscopy, single almost symmetrical EPR signals with different values of the g-factor were registered in non-irradiated (g = 2.0047 ± 0.0002), UV-irradiated (g = 2.01050 ± 0.00005) and γ-irradiated oil samples (g = 2.0017 ± 0.0002), respectively. EPR spectroscopy was used for assessment of the radical scavenging capacity of the non-irradiated and UV- and γ-irradiated samples of the Bulgarian lavender oil towards the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH). Excellent DPPH radical scavenging capacities were found for UV- and 30 Gy-irradiated lavender oil samples. Based on these preliminary results, we consider that, after proper UV- or γ-radiation treatment, Bulgarian lavender oil might find application as a good radioprotector and antioxidant in cosmetics and medicine.

Epidermal barrier and oxidative stress parameters improve during in 311 nm narrow band UVB phototherapy of plaque type psoriasis

BACKGROUND Psoriasis is a multi-systemic inflammatory disease that results from dysregulation between epidermal keratinocyte homeostasis and both innate and acquired immunity. Epidermal barrier defect has been described in psoriatic lesions. Furthermore an imbalance between pro-oxidative stress and antioxidant defense mechanisms are known in psoriasis patients. AIM The aim of this study was to address the link between disease activity, epidermal barrier and systemic oxidative stress in the course of 311 nm narrow band ultraviolet B (NB-UVB) therapy of psoriasis. The dynamic of systemic oxidative stress parameters as well as local transepidermal water loss (TEWL) and stratum corneum hydration (SCH) was characterized before and after 311 nm NB-UVB therapy on the plaques of psoriasis vulgaris in comparison to untreated non-affected volar forearm sites of the same patients. MATERIAL AND METHODS 22 patients with plaque type psoriasis vulgaris and 25 gender- and age-matched healthy controls were enrolled. We assessed the psoriasis area and severity index (PASI) and the dermatology life quality index (DLQI) for monitoring disease activity, severity and self-perceived DLQI impact as patient related outcome parameter. We measured non-invasively TEWL (Tewameter TM 300) and SCH (Corneometer CM 825) and the end product of lipid peroxidation - malondialdehyde (MDA), Reactive oxygen species (ROS), ascorbyl radicals (Asc) and detoxifying activity of catalase (CAT) were measured in the peripheral blood with spectrophotometric and EPR spectroscopy methods. RESULTS Disease activity improved in all patients compared to baseline witnessed by significant decrease in PASI; (from 14.1 to 10.4; p < 0.0001) and DLQI (from 11.7 to 8.1; p < 0.0001). At baseline TEWL-values were significantly (p < 0.0001) higher on psoriatic plaques (16.8 g/h/m2) in comparison to uninvolved skin (5.3 g/h/m2); with a decrease at both sites after NB-UVB phototherapy. SCH was significantly lower at psoriatic plaque s (4.7AU) compared to uninvolved sskin (42.4AU) and increased after treatment (8.6AU) (p < 0.0001). Interestingly, SCH decrease slightly during therapy at uninvolved skin (40.6AU). ROS and Asc declined during therapy in parallel to a decrease in MDA. A mild decrease in the antioxidative enzyme CAT activity which did not reach the significance was observed. CONCLUSION The presented data is shows that a clinical improvement of psoriatic plaques under NB-UVB therapy, shown in with a decreased PASI and reflected by an increase in quality of life has beneficial effects on epidermal barrier function, SCH and improvement of systemic oxidative stress parameters (ROS, MDA and Asc). We assume that the general improvement in the oxidative stress parameters along with epidermal barrier parameters reflects mainly the improvement of disease activity which overwrites the possible negative pro-oxidative effects of the UV treatment.

Biological evaluation of new potential anticancer agent for tumour imaging and radiotherapy by two methods: 99mTc-radiolabelling and EPR spectroscopy

Recently, a new class of in vitro and ex vivo radiotracers/radioprotectors, the nitroxyl-labelled agent 1-ethyl-1-nitroso–3-[4-(2,2,6,6–tetramethylpiperidine-1-oxyl)]-urea (SLENU), has been discovered. Our previous investigations demonstrated that SLENU is a low-molecular-weight stable free radical which is freely membrane permeable, easily crosses the blood brain barrier and exhibited in/ex vivo the lowest general toxicity and higher anticancer activity against some experimental tumour models. Further investigation was aimed to develop a 99mTc-labelled SLENU (97%) as a chelator and evaluate its labelling efficiency and potential use as a tumour seeking agent and for early diagnosis. Tissue biodistribution of 99mTc-SLENU was determined in normal mice at 1, 2 and 24 h (n = 4/time interval, route of administration i.v.). The distribution data were compared using male albino non-inbred mice and electron paramagnetic resonance investigation. The imaging characteristics of 99mTc-SLENU conjugate examined in BALB/c mice grafted with Ehrlich Ascitis tumour in the thigh of hind leg demonstrated major accumulation of the radiotracer in the organs and tumour. Planar images and auto-radiograms confirmed that the tumours could be visualized clearly with 99mTc-SLENU. Blood kinetic study of radio-conjugate showed a bi-exponential pattern, as well as quick reduced duration in the blood circulation. This study establishes nitroxyls as a general class of new spin-labelled diagnostic markers that reduce the negative lateral effects of radiotherapy and drug damages, and are appropriate for tumour-localization.

Radical Scavenging Capacity of Seeds and Leaves Ethanol Extracts of Cynara Scolymus L.—A Comparative Study

ABSTRACT Artichoke with scientific name Cynara scolymus L. is an herbal medicinal plant widely used in traditional European medicine. This medicinal plant possesses many properties, including hepatoprotective ability and antioxidant effect. It has been reported that seeds and leaves of the artichoke contain different constituents such as caffeoylquinic acid derivatives, mono- and di- caffeoylquinic acids, bitter sesquiterpene such as cynatriol and cynaropicrin and flavonoids glycosides like apigenin, luteolin cynaroside, scolimoside, quercitine and others. The aim of the present study was by an Electron Paramagnetic Resonance (EPR) method and visible spectrophotometry to evaluate and compare the radical scavenging capacity towards 1,1 -diphenyl-2-picrylhydrazyl (DPPH) of ethanol extracts prepared from Cynara scolymus L. seeds and leaves. By both methods we have demonstrated that at any studied concentration of the seeds extract the percent of the scavenged DPPH radicals was considerably higher than that calculated for the leaves extract.