Scientific project No. БИН-7/08 for bilateral scientific collaboration between The Republic of Bulgaria and The Republic of India

Project title: Studies on Antioxidants against Oxidative Stress Induced by Drugs and Radiation

Project coordinator from Bulgaria:
DSc.Vesselina Georgieva Gadjeva
Professor and Head of Dept. Chemistry and Biochemistry, Medical Faculty, Trakia University, Stara Zagora, Bulgaria
Project coordinator from the partner country:
Dr. Rakesh Kumar Sharma
Scientist 'F' & Joint Director
Institute of Nuclear Medicine and Allied Sciences (INMAS), Delhi, INDIA

The main task of the project is creation of a new, mechanism-based approach for determination and prevention of oxidative stress induced by drugs and radiation. Free radicals have been implicated in a number of malignant diseases, thyroid autoimmune diseases, diabetes, rheumatoid arthritis, ets. Studies on such diseases are useful for evaluation of prognostic importance of the oxidative stress parameters on the therapeutic response to the corresponding chemotherapeutic schemes. Based on this rationale, we will find possibilities for decreasing the toxic effects of chemotherapeutic drugs and also mitigate the deleterious effects of ionizing radiation-induced damage with application of antioxidants (both synthetic and of natural origin). The parameters for evaluation of the protective effects of the antioxidant compounds will be standardized in in vitro and in vivo models. The project also aims to investigate the mode of action of the promising antioxidants at cellular/molecular level. The role of reactive oxygen/nitrogen species in (ROS/RNS) inducing oxidative damage will be studied and attempts made to discover novel antioxidant molecules that can be useful in the treatment of free radical - mediated human ailments.
Expected Results:
Drugs that induce oxidative stress particularly in different population of humans will be selected and evaluated for the studies. The effect of ionizing radiation will also be investigated in both in vitro/ex vivo and in vivo model systems.
The indigenous medicinal plants that are widely used in traditional systems of medicine will be collected from the different geographical regions of India and parameters for their extraction and fractionation standardized. Bio-activity guided fractionation will be performed and attempts will be made to characterize the bioactive molecule(s) using modern analytical techniques.
Synthetic drugs and the different extracts/fractions of natural antioxidants will be evaluated in vitro for their free radical protective ability and radioprotective efficacy will be studied using standardized protocoscavenging ability by EPR spectroscopy techniques. DNA protective ability, membrane protocols . Efforts will be made to isolate some novel antioxidants.
The protective effects of antioxidant(s) will also be evaluated at ex vivo and in vivo level in lower animal models by EPR spectroscopy techniques.
Mechanistic studies will be taken up to find out the exact mechanism of action at cellular/molecular level. EPR spectroscopy techniques will be used for evaluation and development of new strategies for prevention against the oxidative toxic effects using new antioxidants. Techniques like COMET assay, Western blotting, Flow cytometry, spectroflourimetry and spectrometry, Fluorescence microscopy etc. will be utilized.

DPPH Radical scavenging capacity of grude extracts and their fractions isolated from bactirium Bacillus SP
Antoaneta Zheleva¹, Raj Kumar², Rakesh Sharma ², Yanka Karamalakova¹, Veselina Gadjeva^1
1Department of Chemistry and Biochemistry, Medical Faculty, Trakia University, 6003 Stara Zagora, Bulgaria
²Institutes of Nuclear Medicine and Alliend Sciences (INMAS), Delhi-110054, India

Abstract
Stable free radical DPPH has been widely used to evaluate antioxidant activity of various plant extracts, food suppliments and many other pure natural and synthetic compounds.
In present EPR spectroscopy study we have reported our preliminary results in relation with DPPH scavenging capacity of several crude extracts and their fractions formerly isolated from bacterium Bacillus – sp. INM-I.
Aqueous solutions of the studied samples of IBG20, IBG21, IBG23, IBG24, IBG25 and IBG26 were added to DPPH (0.08 mM) ethanol solution and were incubated at room temperature for 10, 20 or 30 min in the dark. After incubation the corresponding mixture was transfer into a capillary and placed in the microwave cavity of EPR spectrometer (Bruker EMXmicro, Germany) and its EPR spectrum was recorded at room temperature. For all studied samples a considerable decrease in EPR signal intensity of DPPH was registered and compared with the control sample containing ethanol solution of DPPH, only.
EPR spectroscopic analysis demonstrated a significant decrease in the DPPH signal intensity with all the tested extracts and fractions in a range of their concentrations between 0.2% and 6.4%. Therefore, based on this preliminary study it can be concluded that all tested bacterial extracts and fractions manifest a good antioxidant activity at our experimental conditions.
Key wards: EPR, DPPH, antioxidant activity.

EPR spectra in powder and solution forms of crude extracts and fractions isolated from bacterium Bacillus sp
Raj Kumar², Veselina Gadjeva¹, , Rakesh Sharma², Yanka Karamalakova¹, Antoaneta Zheleva¹,
¹Department of Chemistry and Biochemistry, Medical Faculty, Trakia University, 6003 Stara Zagora, Bulgaria
²Institutes of Nuclear Medicine and Alliend Sciences (INMAS), Delhi-110054, India

Abstract
Formerly, crude extracts and their fractions were isolated from bacterium Bacillus – sp. and they exhibited high radioprotective effect against gamma radiation at in vivo models.
To evaluate the chemical nature of these extracts and fractions in powder and solution forms, EPR spectroscopic analysis was carried out. Capillaries filled with either the powders or solutions were placed in microwave cavity of EPR spectrometer (Bruker, EMXmicro, Germany) and their EPR spectra were recorded at room temperature.
A well expressed EPR signal (singlet) was registered with all extracts in powder form. Interestingly, similar EPR signals were also registered with all extracts and fractions in the solution form, as well. It was also found that UV irradiation did not affect the EPR signal intensity of the most potent radioprotective fraction IBG 21 in powder form.
Based on the results obtained, we assume that in vivo high protective effect against gamma radiation established for IBG 21 might be explained by its free radical properties demonstrated by the present EPR spectroscopy study.
Key wards: EPR spectroscopy, gamma radiation, radioprotective