Oxidative Stress and Human Health

Responsible structural features for cytotoxic and other kinds of activity of neoclerodane diterpenes from genus Scutellaria
Original Research Article
Pages 7 – 13
K. H. Nikolova, I. T. Stoykov, P. I. Bozov
Download PDF (1620 KB)
Abstract
Twelve natural neo-clerodane diterpenes, isolated from three Scutellaria (Labiatae) species, were tested for cytotoxicity on two cell lines, from human tumors of the lung designated as H1299 and normal cells from a navel string (HUVEC), using the MTT (3-/4,5-dimethylthiazol-2-yl/-2,5-diphenyltetrazolium bromide) method. The cytotoxic activity was evaluated as rate of IC50 (such concentration of the compound in μM by which half of the cells die). Three compounds, scutalpins A, E and F, exhibited moderate cytotoxic properties on both cell lines. Among all tested compounds the highest activity was detected for scutalpin A, with IC50 values of 21.35 and 23.9. Some significant aspects of the relationship structure-activity are discussed.

Rose oil isolated from oil-bearing Rosa damascena Mill. as a protectoragainst ionizing radiation-induced oxidative disorderst
Original Research Article
Pages 14 – 19
Y.D. Karamalakova, M.K. Adhikari, N.D. Kovacheva, V.A. Ivanov, G.D. Nikolova, V.G. Gadjeva
Download PDF (722 KB)
Abstract
Essential oils are derived from different natural plant materials such as leaves and flowers. They are commercially important and have been widely used in cosmetics, household products and medicines because of pharmacologically active components. Essential oil as antioxidant is able to prevent oxidative processes and to inhibit the oxidation reaction effect caused by radiation-induced oxygen/nitrogen free radicals. Ionizing radiation is a recognized method of maintaining the quality of aromatic herbs, spices and vegetables for a long time. The present study focused on identifying the radioprotective efficacy of rose oil against oxidative damage of molecules by ionizing radiation in in vitro models.

New mechanisms in preventive effect of ellagic acid on cognition in mice with Alzheimer’s disease type dementia
Original Research Article
Pages 20 – 24
L.P. Tancheva, A.B. Popatanasov, S.T. Dragomanova, E.R. Tzvetanova, S.M.Aleksandrova, L.G. Alova, M.O. Stefanova, R.E. Kalfin
Download PDF (781 KB)
Abstract
Antioxidant mechanisms in protective effects of some natural compounds on progression of Alzheimer’s disease (AD) were reported during last years. Our previous data revealed significant improving effect of a natural polyphenol Ellagic Acid (EA) on rodent cognitive functions. The goal of this study was to evaluate the effect of EA on cognition of mice with chemically induced dementia from AD type. This animal model was produced via Scopolamine treatment of male Albino mice and was verified by cognitive and biochemical methods. After 5-days treatment with EA both the changes in the cognitive functions of animals and biochemical correlates were evaluated. Significant preventive effect of EA on the processes of learning and memory (Step-through test) of dement animals was established. The high percent (50%) of memory prevention by EA was accompanied by significant antioxidant effect (decreased lipid peroxidation) and inhibited activity of acetylcholine esterase in the brains of EA-treated animals. An increase of dopamine uptake in the brains of EA-treated dement animals was also found. Our results reveal some of the complex mechanisms underlying the EA preventive effect on the cognition in mouse model of AD-dementia.

Interaction of natural thiols and catecholamines with reactive oxygen species
Original Research Article
Pages 25 – 29
K.M. Zinatullina, N. P. Khrameeva, O.T. Kasaikina , V.A.Kuzmin
Download PDF (487 KB)
Abstract
Natural thiols (TSH) cysteine, glutathione, and homocysteine, as well as catecholamines (CA) dopamine, noradrenaline and adrenaline are known as multifunctional biologically active compounds with antioxidant potential, i.e. bio-antioxidants, which play an important role in the regulation of the redox status and free radical formation – utilization in living organisms. The kinetic characteristics of interaction of TSH and CA with peroxyl radicals, RO2•, formed from the azo-initiator AAPH in aqueous solutions at 37°C by the method of competing reactions were determined. The kinetics of radical formation in the reactions of TSH with H2O2 was studied by the inhibitors method. The polymethine dye (A, pyridine salt of 3,3'-di-ϒ-sulphopropyl-9-methylthia-carbocyanine betaine) was used as a radical scavenger. CA demonstrated the highest antiradical activity (ki> 106(M·s)-1, whereas TSH possess moderate activity (ki 105(M·s)-1).

Oxidative stress and related diseases. Part 1: Bronchial asthma
Original Research Article
Pages 30 – 35
G.D. Nikolova, V. Ilieva, Y.D. Karamalakova, V. A. Ivanov, A. M. Zheleva, V. G. Gadjeva
Download PDF (674 KB)
Abstract
Oxidative stress (OS) plays a significant role in the pathogenesis of a number of human diseases such as ischemia/reperfusion injury, atherosclerosis, cancer, neurodegenerative diseases, allergy, etc. Bronchial asthma (BA) is a chronic inflammatory disease of the lungs, resulting in a restriction of airflow, hyperactivity, and airway remodeling. Clinical and experimental data in recent years show that increased OS and destructive effects of free radical oxidation can be an important cause of chronic pathological processes in the lungs. The aim of the present study was to investigate and compare the level of oxidative stress in blood of asthmatic patients that differ in the degree of disease control. In the study were included 30 patients with BA and 24 age-matched healthy volunteers. Patients were diagnosed with BA with allergic component longer than one year. For this purpose, the ROS and RNS products, the final products of lipid and protein were explored, the relationship between oxidative stress parameters and C-reactive protein (CRP) as a marker of inflammation degree was also studied. By using the EPR spin trapping technique, ongoing real time oxidative processes were confirmed in blood samples isolated from asthmatic patients differing in the degree of disease control. Moreover, positive correlation was found between the levels of studied OS biomarkers and CRP as a marker of inflammation degree. In BA patients, oxidative processes in real time were demonstrated. The results of correlation analysis confirmed that the development and maintenance of inflammatory processes in respiratory tract are associated with the oxidative and nitrosative stress.

Oxidative stress and related diseases. Part 2: Parkinson’ disease
Original Research Article
Pages 36 – 42
G.Nikolova, Y. Karamalakova, V. Mancheva, I. Manchev, A. Zheleva, V. Gadjeva
Download PDF (1027 KB)
Abstract
Parkinson’ disease is a progressive neurodegenerative movement disorder associated with a selective loss of neurons in the midbrain area called the substantia nigra pars compacta. Pharmacological treatment of PD traditionally has centered on administrering medications that restore dopaminergic influence in the basal ganglia. During the early stage of disease, Levodopa admistration can produce significant improvements in parkinsonian symptoms moreover, the effects of Levodopa, often diminish after only a few years of continuous use, and this drug often is ineffective in the advanced stages of PD. The aim of current research was to evaluate and compare oxidative status in blood of PD patients treated with Madopar, alone and with a combination of Madopar and vitamin C by several analytical techniques. To realize the aim were investigated: 1) levels of ROS products, ascorbate (Asc•) and NO• radicals as real time oxidative stress biomarkers using Electron paramagnetic resonance (EPR) spectroscopy; 2) the oxidative end products of lipids, proteins and DNA, levels of malondialdehyde (MDA) measured by visible spectrophotometry, protein carbonyl content (PCC) and also 8-hydroxy-2′-deoxyguanosine (8-OHdG) determined by ELISA; and 3) erythrocyte SOD activity by visible spectrophotometry in blood of PD patients.

Exhausted antioxidant defense in SSUV-exposed skin of hypothyroid rats
Original Research Article
Pages 43 – 47
G. S. Bocheva, T.E. Yordanova, M. L. Valcheva-Traykova, T. Traykov
Download PDF (489 KB)
Abstract
The decrease of the overall antioxidant defense due to solar simulated ultraviolet irradiation or hypothyroidism has been reported both in humans and animal models. Using a rat model, we aimed to investigate how the combination of UV radiation and hypothyroidism affects the antioxidant defense in the photo-exposed skin. The antioxidant resistance of the rat skin was characterized directly by the radical scavenging (RSA) activity toward stable 2,2-diphenyl-1-picryl-hydrazyl radical (DPPH), and indirectly by the formation of free radicals due to Fenton reaction-induced oxidative stress. Four groups of male Wistar albino rats, named C (controls), SSUV (irradiated), PTU (hypothyroid), and PTU+SSUV, were used in this experiment. Drug-induced hypothyroidism was developed by the addition of 0.01% (w/w) 6-n-propyl-2-thiouracil (PTU) for 5 weeks in the ad libitum consumed drinking water. Then SSUV and PTU+SSUV groups wereirradiated for 7 days. The results showed drastically lower antioxidant activity of the H-donors in PTU+SSUV skin thanthat in the skin of healthy controls. In PTU and SSUV groups a lower antioxidant activity than controls was found aswell, the decrease being in the order: C>PTUSSUV>>(PTU+SSUV). Free radical accumulation was many times higherin SSUV-treated euthyroid skin compared to non-irradiated skin of controls.The Fenton reaction in the PTU groupresulted in the formation of very few free radicals in the skin that might be related with the better RSA and slowed themetabolism of the hypothyroid rats. In conclusion, the combination of chronic sun exposure and hypothyroidism couldbe a risky and harmful factor leading to exhausted antioxidant defense and possible skin damage.

Effect of Melissa officinalis L. on the level of induced lipid peroxidation in mouse liver
Original Research Article
Pages 48 – 51
M.S. Chervenkov, T. A. Ivanova, E. N. Stoyanova, A.V. Alexandrova, E. R. Tzvetanova, L.P.Tancheva, A. P. Georgieva, E. K. Kistanova
Download PDF (379 KB)
Abstract
The aim of this study was to evaluate the effect of Melissa officinalis L. aqueous extract on the level of induced lipid peroxidation in mouse liver homogenate. Samples were prepared from homogenized BALB/c mice liver, and subsequently incubated with one of the following lipid peroxidation inducing agents: 0.5 mM H2O2; 0.1 mM FeCl3 +ascorbate or H2O2 +FeCl3+ascorbate (Fenton reaction), in the presence or absence of extract. M. officinalis aqueousextract was prepared by extraction with boiling deionized water in 1:10 ratio (w/v). In the experiments were used two-fold dilutions of the extract containing phenolics equivalent of 21.4 to 1.32 mg gallic acid following preliminarydetermination of the total phenolic content by Folin-Ciocalteu assay. The levels of lipid peroxidation in mouse liverhomogenate, caused by all of the oxidative agents were significantly reduced by all tested dilutions of the extract. M.officinalis aqueous extracts could be effective for protection of liver cells from induced lipid peroxidation.

Antioxidant mechanisms in neuroprotective action of lipoic acid on learning and memory of rats with experimental dementia
Original Research Article
Pages 52 –57
E. R. Tzvetanova, A.P. Georgieva, A.V. Alexandrova, L. P.Tancheva, M. I. Lazarova, S. T. Dragomanova, L. G. Alova, M. O. Stefanova, R. E. Kalfin
Download PDF (786 KB)
Abstract
Alzheimer's disease (AD) is one of the most common dementia affecting about 36 million people and without effective cure. Oxidative stress is one of many hypotheses for the AD mechanisms. Possible preventive AD effects of some antioxidants continue to be the object of clinic and experimental research. The aim of this study was to evaluate the antioxidant mechanism in the neuroprotective effect of lipoic acid (LA) on the cognitive functions in experimental dementia. Alzheimer's disease type dementia was produced via scopolamine treatment (Sco, 1 mg/kg i.p., 11 days) on male Wistar rats. Lipoic acid (LA, 30 mg/kg, i.p.) was applied for the same period. Learning and memory performance of the rats were evaluated using passive avoidance learning test (Step through test). At the 24th hour after the last treatment the brain frontal cortex, hippocampus, and striatum were isolated and homogenized. The homogenates were used for determination of malondialdehyde (MDA), total glutathione (tGSH), and activities of superoxide dismutase (SOD), glutathione peroxidase and catalase (CAT). The dementia model was verified by the cognitive tests used. In brain structures of the Sco-group increased MDA, and decreased tGSH levels, as well as activated antioxidant enzymes were observed. LA significantly improved cognitive functions and oxidative status damaged by Sco by increased tGSH level, restored CAT and SOD activities. Thus LA significantly protects memory impairments of dement animals due to its antioxidant capacity and could be used in prevention and therapy of AD.

Comparative analysis of real-time oxidative stress biomarkers measured in mussels (Mytilus galloprovincialis) and veined rapa whelks (Rapana venosa) in relation to two seasons - An electron paramagnetic resonance study
Original Research Article
Pages 58 – 63
G.Nikolova, Y. Karamalakova, A. Zheleva, D. Stratev, I. Vashin, G. Zhelyazkov, V.Gadjeva
Download PDF (474 KB)
Abstract
The aim of this study was to e lucidate the radical mechanisms for protection and survival of mussels (Mytilus galloprovincialis) and veined rapa whelks (Rapana venosa) during spring and summer season by following out levels of some real-time oxidative stress (OS) biomarkers. Thirty specimens of each species were analyzed by using electron paramagnetic resonance (EPR) spectroscopy. During spring, statistically higher levels of ROS products were found in R.venosa compared to M. galloprovincialis. During summer, statistically significant higher levels of ROS productswere found in both R. venosa and M. galloprovincialis, compared to the same groups during spring. NO radicals in R.venosa were higher, although not statistically significant than those in M. galloprovincialis during both spring andsummer periods. During summer, statistically significant higher levels of ascorbate radicals (Asc•) were found in bothR.venosa and M. galloprovincialis, compared to the same groups during spring. However, during the summer the levelsof ascorbate radicals measured in R. venosa were significantly higher compared to M. galloprovincialis. Our resultsshowed that changes in oxidative/antioxidant status may reflect the gradient of contamination confirming the rationaluse of biomarkers of oxidative stress in biomonitoring of contamination. R. venosa has effective biochemicalmechanisms of protection and survival, in particular a strong antioxidant system that provides this type of highadaptability and survival against oxidative stress.

Real time oxidative stress markers of patients with post-stroke depression: EPR study
Original Research Article
Pages 64 – 68
G. Nikolova, D. Komsiyska, Y. Karamalakova, Y. Petkov, V. Ivanov, T. Manolova, V.Gadjeva
Download PDF (651 KB)
Abstract
Depression is a common consequence of stroke. In the last few years, oxidative stress has been seen as one of the contributing factors in the pathogenesis of depression. Lately it has been discussed also as an accompanying factor in many chronic neurodegenerative pathologies, as well as in acute cerebrovascular disorders like stroke. The aim of our study is to investigate the role of oxidative stress in the etiopathogenesis of depressive disorders in post-stroke patients in order to optimize diagnostic, therapeutic and medical-social approaches. To achieve evaluating the level of oxidative stress in post-stroke depression we investigated the levels of ROS products, ascorbate (Asc•) and NO• radicals as real time oxidative stress biomarkers using electron paramagnetic resonance (EPR) spectroscopy.

A semiquinone glucoside derivative (SQGD) isolated from Bacillus sp. INM-1 as a provider of antioxidant protection to male mice against CCNU-induced oxidative toxicity
Original Research Article
Pages 69 – 73
Y.D. Karamalakova, A. M. Zheleva, R. Kumar, G. D. Nikolova, V. G. Gadjeva
Download PDF (416KB)
Abstract
Present investigation was focused on evaluation of a semiquinone glucoside derivative (SQGD) isolated from radioresistant bacterium Bacillus sp. INM-1 for its activity against CCNU- induced oxidative stress in healthy mice. Mice were divided into four groups, i.e., (I) untreated controls; (II) SQGD treated (40 mg/kg b. wt. i.p.) mice; (III) CCNU (40 mg/kg b.wt., i.p.); and (IV) SQGD (40 mg/kg b.wt., i.p.) administered 1 h prior to CCNU-administration (40 mg/kg b.wt., i.p.). Following treatment, liver homogenates and blood serum of the treated animals were subjected to ascorbate radical levels estimation and ROS production. Results indicated that SQGD+CCNU administration significantly (p< 0.05) reduced ascorbate radicals and ROS products in the liver and blood serum of mice as compared with CCNU-treated group. Reduction in oxidative disorders was observed in healthy mice which were treated with SQGD only, compared with controls. Further, the maximal concentration of free SQGD (a.u.) in the blood-flow was established at 30 min after i.p., and completely reduced after 240 min. The pharmacokinetic profile of free SQGD showed significant selective accumulation, mostly in liver and lungs (60 min), brain (90 min), followed by kidney, pancreas, spleen, blood and testicles. Thus, it can be concluded that SQGD treatment alone and in combination SQGD+CCNU neutralized oxidative toxicity caused by medicines not only by reducing lipid peroxidation but also by improving antioxidant status of organs and blood, and this effect may emphasize SQGD as a strong radical-scavenger and excellent natural protector.

Green extracts of grape seed oil - potential source of fatty acids and health benefits
Original Research Article
Pages 74 – 79
J.A.P. Coelho, M.P. Robalo, G.P. Naydenova, D.S. Yankov, R.P. Stateva
Download PDF (592 KB)
Abstract
Supercritical CO2 extraction of oil from grape seed samples obtained from a Portuguese industry without any previous treatment was carried out at temperatures from (313 to 333) K, pressures up to 40.0 MPa and different scCO2 flow rates The qualitative analysis of the crude oil was carried out by NMR. The fatty acids were analyzed by GC-FID with reference to the parameters in Annex I to European Commission Regulation. The results show similar content of triacylglycerols and diacylglycerols both in the n-hexane and scCO2 extracts, but the latter have higher content of polyunsaturated fatty acids and lower content of saturated fatty acids, and hence are more beneficial for human health and wellbeing.

Optimization of the extraction of natural antioxidants from avocado seeds
Original Research Article
Pages 80 – 84
S.S. Boyadzhieva, S.S. Georgieva, G. Angelov
Download PDF (699 KB)
Abstract
Food supplements of plant origin are widespread for dietary use. Most often they are in the form of extracts rich in natural antioxidants with high radical-scavenging action. Avocado (Persea americana) is a fruit widely cultivated throughout the world. There are many studies that attest to its health benefits. Avocado peels and seeds are considered as waste. However, there are indications that avocado seeds, which represent about 23 % of fruit weight, have even higher antioxidant activity than that of its flesh. So, the aim of this work is to study the influence of process parameters on the extraction of avocado seeds in order to determine the optimal conditions for obtaining extracts containing maximum amount of bioactive substances and having high antioxidant activity. An experimental approach is developed, which allows the optimal process parameters to be found by a reduced number of experiments. As a result, the following important process parameters are determined: selection of appropriate “green” solvent, which dissolves more antioxidant compounds; minimum solvent-to-solid ratio and minimum process duration necessary for complete extraction, which minimizes process costs; process temperature and ensures an acceptable compromise between higher solubility and thermal stability of the antioxidant compounds. The results obtained are useful for development of technological schemes for production of antioxidant extracts for use as functional supplements obtained from bio-wastes.

Characterization of polyphenol content and antioxidant capacity of spent coffee grounds
Original Research Article
Pages 85 – 89
S.Boyadzhieva, G. Angelov, S. Georgieva, D. Yankov
Download PDF (486 KB)
Abstract
In this study spent coffee grounds were investigated as a source of natural antioxidants. The polyphenolic content and antioxidant capacity of the extracts from fresh roasted coffee was determined and compared to those of spent coffee grounds (after a common espresso preparation). An optimized water-ethanol solvent was used. The extracts were analyzed for the total polyphenol content by Folin-Chiocalteau phenol reagent, by UV spectrophotometry for the content of chlorogenic acid. The antioxidant activity was investigated using the DPPH assay. It was found that the coffee remains still contain significant amount of polyphenols and show high antioxidant capacity. In our case study, spent coffee grounds (SCG) contain 17 mg polyphenols/g SCG, 15.8 mg chlorogenic acid/g SCG, and its antioxidant capacity was 86 mg DPPH/g SCG. The remaining polyphenolic content, chlorogenic acid and antioxidant capacity of spent coffee grounds represent 28%, 31% and 32.5%, respectively, as compared to untreated fresh coffee. In conclusion, spent coffee is far from being exhausted by a simple hot water extraction, and it can be considered as a rich, valuable and widely available source of useful natural bioactive substances with antioxidant activity.

Flavonoid content and antioxidant activity of Betonica bulgarica Degen et Neič
Original Research Article
Pages 90 – 97
M. T. Tzanova, N. H. Grozeva, M. A. Gerdzhikova, M. D. Argirova, D. H. Pavlov, S. R. Terzieva
Download PDF (752 KB)
Abstract
The Bulgarian endemic Betonica bulgarica Degen et Neič (syn. Stachys bulgarica Hayek) is a protected plant by the Biological Diversity Act and it is included in the Red Data Book of Bulgaria under the category “endangered“. The aim of this study was to determine the flavonoid content and antioxidant activity of different plant organs of this species (leaves, flowers, roots, stems and seeds), from four populations. Three flavonoids were found in significant amounts: rutin, quercetin and hispidulin. Rutin was in the largest quantity, followed by quercetin and hispidulin. The largest total flavonoid content was measured in leaves, followed by roots and flowers. The antioxidant activity of methanol extracts was tested by DPPH-method. The total polyphenol was also assayed. The correlation between flavonoid content and antioxidant activity of the studied plant organs was established.

In vivo evaluation of the antioxidant potential of dicaffeoylquinic acid isolated from Geigeria alata
Original Research Article
Pages 98 – 104
V.B. Vitcheva, R.L. Simeonova, M.S. Kondeva-Burdina, D. Zh. Zheleva-Dimitrova, V. I.Balabanova, S. Yagi, R. S. Gevrenova
Download PDF (623 KB)
Abstract
Geigeria alata (DC) Oliv. & Hiern. (Asteraceae) is an aromatic medicinal plant used in traditional Sudanese medicine, with a number of biological activities including suggested antidiabetic potential. The objective of this study was to evaluate the antioxidant properties of 3,5-dicaffeoylquinic acid (diCQA), the major compound isolated from Geigeria alata roots extract in an experimental model of streptozotocin-induced type 2 diabetes in male Wistar rats. Diabetes results in severe organ pathology which main pathophysiological mechanisms are related to oxidative stress, discerned by increased production of malondialdehyde (MDA) and disturbance in both non-enzymatic (GSH) and enzymatic (GPx, GR, GST) antioxidant defense. DiCQA (5 mg/kg/po) administered for 21 days to control and diabetic Wistar rats ameliorated the activity of the antioxidant enzymes and the levels of the cellular protector GSH, as well as reduced the production of MDA. It also exerts antidiabetic effect in diabetic rats. On the basis of these results, as well as knowing that formation of ROS is considered to be one of the mechanisms in the pathogenesis of diabetes we concluded that diCQA isolated from Geigeria alata possesses antioxidant properties which most probably determined its in vivo antidiabetic activity.

Evaluation of the antioxidant potential of defatted extract from Astragalus spruneri in spontaneously hypertensive rats (SHRs)
Original Research Article
Pages 105– 111
R. L. Simeonova, V. B. Vitcheva, M. S. Kondeva-Burdina, G. S. Popov A. M. Shkondrov, I. N. Krasteva, V. K. Manov
Download PDF (441 KB)
Abstract
The aim of this study was to evaluate the antioxidant potential of Astragalus spruneri (Fabaceae) in spontaneously hypertensive rats (SHR). Hypertension is a non-communicable disease and oxidative stress is regarded as one of the main pathophysiological mechanisms. Defatted extract of A. spruneri (EAS) was administered at a dose of 100 mg/kg bw (1/20 LD50) for 14 days. At the end of the treatment period the animals were euthanized and the activities of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) as well as the levels of non-enzyme cell protector reduced glutathione (GSH) were assessed in the brain, liver, kidney and spleen of SHR. In comparison to normotensive Wistar rats, in control, non-treated SHRs the GSH level and the activity of GPx were decreased in all organs, while the activity of CAT and SOD was decreased in brain, liver and kidney, and unchanged in spleen. Compared to the control SHRs A. spruneri exerted antioxidant activity, discerned by statistically significant increased activities of CAT and SOD in liver and kidney, of GPx and GSH in liver, kidney and spleen. It is worth to be noted that the extract did not exert any effect in the brain. This might be due to the fact that it cannot penetrate the blood brain barrier. Based on the results of our study we could conclude that the lyophilized extract of A. spruneri showed antioxidant potential in spontaneously hypertensive rats – a model of essential hypertension in humans.

Development of lipid damage of pumpkin seed oil stabilized with different antioxidants during long-term storage
Original Research Article
Pages 112 – 117
Zh.Y. Petkova, G.A. Antova, M.Y. Angelova-Romova
Download PDF (617 KB)
Abstract
Oxidative stability of pumpkin seed oil during long-term storage at different conditions was investigated. The examinations were carried out with pumpkin seed oil (as a control sample) and stabilized oil with different natural antioxidants such as caffeic acid, ethyl gallate and combination of both. Long-term storage for all samples was led for 6 months at room temperature (25°C) and in a dark place at 10°C. Indicators that define oxidative stability of the oils (acid and peroxide value, fatty acid composition and total tocopherol content) were monitored during the long-term storage. Significant increase in the peroxide value was detected in all samples during the whole period, especially in those that were kept at room temperature (25°C), while in acid value and fatty acid composition the deterioration was minor. Same tendency was observed in total tocopherol content where the latter decreased insignificantly in samples, stored at 10°C in a dark place. Generally, it could be considered that pumpkin seed oil was more stable when was stabilized with ethyl gallate and stored at 10°C.

Antioxidant and antimicrobial behaviour of alga Gracilaria gracilis extracts during hake (Merluccius merluccius) chilled storage
Original Research Article
Pages 118 – 124
R. G. Barbosa, M. Trigo, G. Dovale, A. Rodríguez, S. P. Aubourg
Download PDF (673 KB)
Abstract
The present research is a first attempt for the use of alga Gracilaria gracilis as a source of preservative compounds to be applied during the chilled storage of fish. For it, a combination of ethanolic and aqueous extracts of this edible seaweed were included in the icing medium employed for the chilling storage of hake (Merluccius merluccius). Chemical and sensory analyses related to quality loss were carried out in fish throughout 9-day storage. Three different alga concentrations were tested and compared to an icing control (traditional ice). As a result, an inhibitory effect (p<0.05) on lipid oxidation development (tertiary oxidation compounds) and microbial activity (trimethylamine formation) was observed in fish corresponding to batches including the two most concentrated alga conditions. However, a definite effect on lipid hydrolysis development (free fatty acids formation) could not be implied (p>0.05). Concerning sensory analysis, samples from the same two batches revealed a higher acceptance (p<0.05) than control; this difference was based on the evaluation of various descriptors such as skin, external odour, raw flesh odour and cooked flesh odour. On the basis of the presence of antioxidant and antimicrobial molecules, a profitable effect on
quality retention of chilled hake is concluded by including G. gracilis extracts in the icing system.

Optical characteristics and antioxidant activity of lingonberry (Vaccinium vitis-idaea) fruit juice
Original Research Article
Pages 125 – 130
Il. Milkova-Tomova, P. Radusheva, D. Buhalova, Kr. Nikolova, S. Krustev, T. Evtimov, I. Alexieva
Download PDF( 495 KB)
Abstract
This research is focused on juices of wild berries and more specifically on lingonberry juice. The kinetics of color parameters of the samples with inulin or lactulose during the storage is investigated. The content of anthocyanins, phenolic content, antioxidant activity and fluorescence spectra has been measured. The dependences between chemical parameters, color characteristics and ratio between intensity of emission and intensity of excitation for exciting wavelength 275 nm have been found. The dependence between antioxidant activity and total phenolic content also exists. Excitation in the UV region is suitable for distinguishing the phenolic content and antioxidant compounds. The most suitable wavelengths found to be 265 nm and 275 nm.

Preservative effect of jumbo squid (Dosidicus gigas) skin extract as glazing material during the frozen storage of Atlantic Chub mackerel (Scomber colias)
Original Research Article
Pages 131 – 137
J. M. Ezquerra-Brauer, M. Trigo, W. Torres-Arreola, S. P. Aubourg
Download PDF (568 KB)
Abstract
The present research was focused on the quality loss of Atlantic Chub mackerel (Scomber colias) during the frozen storage. Its basic objective was to investigate the effect of including a lipophilic extract of jumbo squid (Dosidicus gigas) skin (JSS) in the glazing system applied previously to the frozen storage. For it, two different concentrations of skin extracts were tested and compared with two control treatments (water glazing and non-glazing conditions). Quality changes were monitored for a 8-month frozen storage by sensory (odour and taste) and chemical (lipid hydrolysis and oxidation development) evaluation. An inhibitory effect (p<0.05) of skin extracts on lipid hydrolysis (free fatty acid formation) evolution was observed; furthermore, lower average values for lipid oxidation indices (peroxide and fluorescent compounds formation) were observed in fish samples corresponding to the highest JSS presence in the glazing system. Some sensory quality enhancement was evident in mackerel as a result of including JSS extracts in the glazing medium, especially for raw-flesh and cooked-flesh odours; as for chemical indices, an increasing effect was implied by increasing the skin extract presence in the glazing medium. Under the conditions tested in the present study, the JSS extract has shown promising antioxidant properties that could be applied to enhance the seafood quality during the commercialisation in frozen conditions. Further research would be necessary to optimise its use in the glazing system..

Antioxidant potential of high molecular weight polyphenol fraction from green tea
Original Research Article
Pages 138 – 143
M. A. Janiak, R. Amarowicz
Download PDF (814 KB)
Abstract
Green tea is one of the most popular beverages. Due to the high content of bioactive compounds it exhibits many health-promoting properties. Our study focused on analyzing the high molecular weight (HMW) fraction obtained from 13 green tea samples for their antioxidant capacity and chelating ability and comparing the results with those recorded for green tea extract (Ex) and its low molecular weight (LMW) fraction. HMW and LMW were obtained using Sephadex LH-20 column chromatography. Obtained fractions were characterized by total phenolic content, size-exclusion high performance liquid chromatography (SE-HPLC), 2,2-diphenyl-1-picrylhydrazyl radical (DPPH●) scavenging capacity, and ability to chelate Fe(II) ions. HMW exhibited the highest total phenolic content and scavenging activity. The results obtained for the chelating capacity showed that changes in UV-spectra after addition of ferric (II) chloride observed for HMW solutions were the most significant.

Antioxidant and antiproliferative activity of Juniperus L. species of Bulgarian and foreign origin and their anticancer metabolite identification
Original Research Article
Pages 144 – 150
D.I. Ivanova, A. N. Tashev, P. T. Nedialkov, Y. E. Ilieva, T. N. Atanassova, M. Olech, R.Nowak, G. Angelov, F. V. Tsvetanova, I. A. Iliev, Z. K. Kokanova-Nedialkova, G. H. Yotov
Download PDF (950 KB)
Abstract
The genus Juniperus L. (Cupressaceae) comprises more than 50 species in the world. Widely distributed junipers are evergreen plants that are easy for cultivation, produce a considerable amount of biomass all the year round and are rich of biologically active compounds, including antioxidants and anticancer substances. This study is pointed out at a systematic investigation of Bulgarian Juniperus species in comparison with foreign representatives with the aim to select these having high antiproliferative and antioxidant activity. In the group of studied plant extracts, the best antiproliferative activity in NB4 acute promyelocytic leukemia cells was determined for J. sabina L., J. virginiana L. and J. virginiana ‘Grey Owl’ extracts. The best antioxidant activity was exhibited by J. sibirica and J. excelsa leaves extracts in the group of analyzed species. While efficient antioxidant activity is desired for cancer prevention, efficient antiproliferative agents are required in the anticancer chemotherapy. Using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) we identified podophyllotoxin, a known anticancer drug precursor, in selected juniper extracts with antiproliferative activity. Identification of other plant metabolites (lignans, phenolic compounds etc.) is in progress. The identification of juniper extracts with efficient antiproliferative and antioxidant activity has potential application in clinical trials about combination therapy with other anticancer agents for treatment of resistance to conventional chemotherapy, disease relapse and reduction of the therapeutical doses of cytostatic drugs.

Activity-guided extraction optimization of highly efficient antioxidant plant species: study of Rhodiola rosea L. (Golden root)
Original Research Article
Pages 151 – 157
D.I. Ivanova, S. S. Boyadzieva, G. Angelov, P. T. Nedialkov, D. Nedeltcheva-Antonova,F.V. Tsvetanova
Download PDF (892KB)
Abstract
The species of genus Rhodiola L. (Crassulaceae) are mainly distributed in different cold regions of the Northern Hemisphere of the world. The Golden root (Rhodiola rosea L.) extract is known in the traditional medicine as adaptogen to treat fatigue, depression and stress-associated diseases. The Golden root extract was selected for a comprehensive study because it exhibited superior antioxidant properties in a group of other efficient antioxidant plant species studied here. Consequently, we aimed our study at an antioxidant activity – guided optimization of Rh. rosea extraction by selection of experimental conditions leading to maximum total polyphenolic content and antioxidant activity of the extract. Using a set of variable parameters (solvent composition, temperature, ratio solvent-to-raw material, process duration), it was suggested that extraction in 25% ethanol, at 50ºC, using solvent-to-solid ratio of 15 (v/w) and process duration of 3-5 min are optimal extraction conditions for obtaining of Golden root extracts with maximum total phenolic content (TPC) and antioxidant activity. Metabolite identification in the extract with the best antioxidant activity was performed by both HPLC and UHPLC/HRMS methods. It was found that Rh. rosea extract, obtained at optimal conditions, contained 2.29±0.05% of rosavin and 0.80±0.02% of salidroside. The Golden root extract, obtained in this study at optimal conditions regarding its antioxidant activity, has potential application in the production of high-quality plant extracts for prevention of cancer and oxidative-stress associated diseases, including cardio-, neuro- and hepato-degenerative disorders.

Comparative study of antioxidant potential of curcumin and its degradation products– vanillin, ferulic acid and dehydrozingerone
Original Research Article
Pages 158 – 163
A. K. Slavova-Kazakova, L. Koleva, V.D. Kancheva, G. Delogu
Download PDF (531 KB)
Abstract
Curcumin is one of the most intensively studied biologically active antioxidants during the last decade. Its chemical instability and rapid degradation at physiological conditions (pH ≈ 7) was stated as the most important limitation for its potential applications. Numerous approaches have been undertaken to overcome the problem with the bioavailability of curcumin, including the use of such adjuvants as piperine, liposomes, system of nanoparticles, phospholipid complexes and design of new structural analogs of curcumin. On the other hand, degradation of a compound does not necessarily lead to loss of its activity. Whether and how the degradation and oxidation pathways contribute to the biological and antioxidant activities of curcumin has also been discussed. The aim of this study was to compare the antioxidant activity of curcumin with those of its degradation products (ferulic acid, vanillin and dehydrozingerone), when added in binary and ternary mixtures. Lipid autoxidation was used for assessing the chain-breaking antioxidant efficiency and reactivity of the phenols. The results obtained showed much stronger activity of curcumin than that of all the individual compounds and their binary and triple mixtures.

Phenolics content and antioxidant activity of beverages on the Bulgarian market – wines, juices and compotes
Original Research Article
Pages 164 – 168
M.N. Todorova, M. G. Pasheva, Y. D. Kiselova-Kaneva, D. G. Ivanova, B. Tz. Galunska
Download PDF (640 KB)
Abstract
In wine, natural juices and compotes the taste is heavily influenced by the presence of phenolics. They also contribute to the antioxidant activity of fruits and processed foods from them and have health-protecting effect. The phenolic content and antioxidant activity are not included in the standard documentation for food labeling and control. The aim of the present work was to analyze and to compare the content of total phenolics and anthocyanins, as well as the antioxidant potential of alcoholic and non-alcoholic fruit drinks on the Bulgarian market. Commercial natural fruit juices, compotes, red, rosé and white wines on the Bulgarian market were used for testing total phenolics, anthocyanins and antioxidant activity. Among the tested alcoholic drinks the red wines revealed significantly higher polyphenolic (567±33 mg/L) and AC (97.9±40.7 mg/L) content vs rosé (323±84 mg/L, p<0.0001; 9.9+8.2 mg/L, p<0.01) and white (281±42 mg/L, p<0.0001; 0.2±0.1 mg/L, p<0.001) wines. In the group of the red wines, the highest polyphenolic content (625±13 mg/L) was detected in Merlot wine, and the lowest one in Syrah (534±20 mg/L). The wine Malbec was found to be the richest one in anthocyanins (156.6±1.5 mg/L), while in Mavrud the anthocyanins content was the lowest one (45.2±1.0 mg/L). The tested red wines showed high antioxidant activity, especially strong in Aronia wine (45.55±0.35 mM uric acid equivalent, UAE). In the tested non-alcoholic drinks, the highest polyphenolic content was found in Aronia juice (592±9 mg/L) and compote (556±62 mg/L). The red wines and compotes have been an element of traditional nutrition in Bulgaria and nowadays their input in the healthy diet is reassessed because of their high phenolics content and strong antioxidant potential.

Glycine-rich peptides from Cornu aspersum snail with antibacterial activity
Original Research Article
Pages 169 – 175
L. Velkova, A. Nissimova, A. Dolashki, E. Daskalova, P. Dolashka Y. Topalova
Download PDF (724 KB)
Abstract
Antimicrobial peptides are a unique and diverse group of molecules that have а great potential for use in new antimicrobial drugs, as many of them have a pronounced cytotoxicity to number of multi-drug resistant bacteria. We have been investigating different mucus extracts from the garden snail Cornu aspersum against the pathogen Gram-negative bacterial strain - Escherichia coli NBIMCC 878 and it has been found that the fraction below 10 kDa demonstrated strong antibacterial activity. Using tandem mass spectrometry we identified the primary structures of 9 novel antimicrobial peptides with molecular masses between 1000-3000 Da in this fraction. Most of them contain high level of glycine and leucine residues into the amino acid sequences.

Antioxidative screening of fractions from the mucus of garden snail Cornu aspersum
Original Research Article
Pages 176 – 183
N. Kostadinova, Y. Voynikov, A. Dolashki, E. Krumova, R. Abrashev, D. Kowalewski, S. Stevanovic, L. Velkova, R. Velikova, P. Dolashka
Download PDF (1113 KB)
Abstract
Antioxidative peptides have been utilized by various species to combat pathogenic microorganisms and stress. In the present study, the antioxidant potential of peptide fractions obtained from the mucus of the garden snail Cornu aspersum was evaluated. Bioactive compounds from the mucus of the garden snail were separated into four fractions with different molecular weight (MW): Fraction 1 (compounds with MW<5kDa), Fraction 2 (compounds with MW<10kDa), Fraction 3 (compounds with MW<20kDa) and Fraction 4 - in the region 10-30kDa. Three complementary test methods were employed for preliminary antioxidative screening, including measurement of the radical scavenging activity on the 1,1-Diphenyl-2-picrylhydrazyl free radical (DPPH), total antioxidant activity (ABTS method) and the inhibition of nitro blue tetrazolium (NBT) reduction by photochemically generated superoxide radicals (O2). The results demonstrated that the lower MW fraction of <5kDa exhibited better antioxidant potential compared to the others.

Kinetic parameters of the copigmentation effect of caffeic acid and strawberry anthocyanins
Original Research Article
Pages 184 – 189
I. J. Petrova, V. D. Gandova, P. N. Denev, K. M. Mihalev, V. T. Shikov
Download PDF (399 KB)
Abstract
The effect of temperature on the stability of the copigmentation complex of strawberry anthocyanin extract as pigment and caffeic acid as copigment was investigated. The system was studied with a high concentration of caffeic acid 1:20 to 1:100 molar ratio. Different temperatures and copigment concentrations were used for the investigated pigment:copigment interaction and kinetic parameters such as activation energy (Ea), z – factor and degradation rates (k) were calculated. According to the calculated results, at high temperatures (50oC) destruction of the complex was observed. Decreasing the temperature in the range of 20–30oC did not lead to restoration of the complex, indicating irreversibility of the copigmentation process.

Preparation of quercetin delivery systems on the basis of amino-modified KIL-2 mesoporous silica
Original Research Article
Pages 190 – 194
M. Popova, I. Trendafilova, I. Tsacheva, N. Georgieva, N. Koseva, A. Szegedi, J. Mihály, N. Novak-Tusar
Download PDF (555 KB)
Abstract
KIL-2 silica with textural mesoporosity was synthesized and modified with amino groups by post-synthesis method. Quercetin was successfully loaded on amino-modified KIL-2 by incipient wetness impregnation or solid-state method. Quercetin-loaded KIL-2NH2 particles were coated by a polyelectrolyte polymer complex containing κ-carrageenan-chitosan-κ-carrageenan. The parent, amino-modified and quercetin loaded samples were characterized by XRD, N2 physisorption, thermal gravimetric analysis and ATR-FT-IR spectroscopy. In-vitro release of quercetin from quercetin loaded formulations was studied in two acceptor media resembling physiological pH GIT (pH=1.2 and pH=6.8). The in-vitro release study showed slower quercetin release from polymer coated quercetin-loaded KIL-2NH2 samples prepared by both methods compared to the uncoated ones.

Structure and antibacterial activity of isolated peptides from the mucus of garden snail Cornu aspersum
Original Research Article
Pages 195 – 200
A.Dolashki, A. Nissimova, E. Daskalova, L. Velkova, Y. Topalova, P. Hristova, P. Traldi, W. Voelter, P. Dolashka
Download PDF (758 KB)
Abstract
The world provides a rich source of peptides with antimicrobial, antiviral and antitumor activity. Peptides and glycopeptides are an expanding group of structurally complex anti Gram positive antibacterial agents, which аrе used in human and veterinary medicine. Quite a series of proline-rich peptides, isolated from arthropods and molluscs, were considered to be promising candidates for the treatment of microbial infections and suppression of microbial resistance. In the present study, we report the primary structure and antimicrobial activity of peptides produced by the mucus of garden snail Cornu aspersum in comparison to similar peptides isolated from molluscs. Several peptides with molecular masses between 1 and 4 кDa measured by mass spectrometric analysis were identified in the mucus. Their amino acid sequences were determined by MS/MS analysis as is shown for peptide at m/z 1438.87 [M+H]+ (ML/INVAVNQ/KGEVKH). The fraction with peptides with molecular masses below 3 kDa exhibited antibacterial activity against Gram-negative Pseudomonas aeruginosa AP9 and Gram-positive Brevibacillus laterosporus BT271 bacteria and the inhibition effects of the peptides can be explained with the amino acid residues. The de novo sequence of six peptides revealed that most of them contain glycine, proline, tryptophan and valine which are typical for peptides with antimicrobial activity.

Effect of Pr(III) nitrate and Pr(III) complex on the accumulation of free radicals in rat blood serum
Original Research Article
Pages 201 – 206
C.Costanzo, L. T. Todorov, M. Valcheva-Traykova, I. Kostova
Download PDF (700 KB)
Abstract
The success of the cancer treatment depends on the good control over the oxidative stress (OS). Lanthanides and their compounds are promising anticancer agents due to the prooxidant activity of the Ln(III) cations. A complex formed by coordinatively bonding lanthanide ion with antioxidant ligand is promising strategy in the search of efficient anticancer medications. The 5-aminoorotic acid (HAOA) proved to be antioxidant at homeostatic pH. In this investigation, the effect of HAOA, Pr(III), and their complex, PrAOA, on the free radicals accumulation in rat blood serum was estimated. Pr(III) exhibited prooxidant properties. Below concentrations of 10-6 M both HAOA and PrAOA did not influence the OH-initiated OS in the serum. Above this concentration, both compounds were antioxidants, the complex being weaker than the ligand. It was proposed that the antioxidant effect of PrAOA resulted from both antioxidant properties of the ligands and prooxidant properties of Pr(III).

Radical scavenging activity toward 2,2-diphenyl-1-picrylhydrazyl and hydroxyl radicals of 5-aminoorotic acid and its Ga(III) complex
Original Research Article
Pages 207 – 212
L.T. Todorov, B. B. Chifchiev, M. L. Valcheva-Traykova, I. P. Kostova
Download PDF (841 KB)
Abstract
Along with anti-tumor activity, flexible control over oxidative stress (OS) levels is a desirable quality of any anticancer drug. Radicals scavenging activity (RSA) toward 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) is widely used to evaluate the ability to eliminate free radicals by donating hydrogen. 5-aminoorotic acid (HAOA) is known to have antioxidant properties and has been used as a ligand in lanthanide(III) complexes possessing anticancer activity in cell cultures. Ga(III) salts are known for their anticancer activity. Thus, the Ga(III) complex with HAOA, GaAOA, might be a promising anticancer agent with antioxidant properties that have not been explored so far. In the present work, the UV spectra and RSA of HAOA and GaAOA toward DPPH and OH were evaluated and discussed. The stereochemistry of HAOA and its Ga(III) complex was evaluated, and compared by means of IR, Raman IR and Raman spectral data. Two factors affected the UV spectra of the molecules: their arrangement (steric properties) and their interaction with the solvent. As far as the RSA was determined in absolute ethanol (for DPPH) and in water (for OH), the UV spectra of the molecules in water and ethanol were compared and discussed. The hypochromicity in the UV spectra of GaAOA, compared to the expected intensities, indicated an arrangement of the ligands that diminished the dipole moment. The RSA of HAOA and GaAOA towards both radicals was concentration-dependent. GaAOA, at the lowest concentration in ethanol, exhibits signs of dissociation, manifested in an anomalous RSA increase. That demonstrates the potential of GaAOAfor a controlled release of the antioxidant ligands.

Effects of orotic and 5-aminoorotic acids on the free radicals accumulation in rat blood serum
Original Research Article
Pages 213 – 217
L.Todorov, V.-M. B. Mladenova, M. Valcheva-Traykova, I. Kostova
Download PDF (556 KB)
Abstract
Orotic (HOA) and 5-aminoorotic (HAOA) acids are ligands in metal complexes with in vitro antioxidant and anticancer activities. Dietary orotic acid in vivo increases the free radicals formation in the liver by diminishing both activity and mRNA level of Cu,Zn-SOD. It seems that HOA may act as antioxidant by scavenging free radicals, but as prooxidant by diminishing the efficacy of Cu,Zn-SOD. The effect of orotic acid on the accumulation of free radicals (FRA) in the blood serum is still not assessed. In this investigation free radicals formation in rat blood serum was achieved by adding small amount of xanthine or Fe(II)/H2O2/ascorbate solution, thus superoxide or hydroxyl radical - induced free radicals formation was provoked. The effects of HOA and HAOA (within concentrations of 10-4 and 10-6 M)on the accumulation of free radicals in the blood serum were monitored using spectrophotometric method. FRAdecreased with increasing of concentration of both compounds was observed, the effect being stronger for HAOA, andfor the OH generating model system. The weaker antioxidant effect of HOA compared to HAOA on the free radicalsaccumulation in rat blood serum might be a result of negative influence on Cu,Zn-SOD, along with radicals scavengingactivity of the molecule. It was assumed that at concentrations below 0.1mM, the antioxidant effect of HOA and HAOAin the blood serum prevailed. More detailed investigations are under way.

Study on the cytoprotective and antioxidant in vitro activity of Pr(III) complex of 5-aminoorotic acid
Original Research Article
Pages 218 – 224
M. S. Kondeva-Burdina, A. V. Kasabova-Angelova, V. P. Atanasova, V. J. Tzankova, I. P. Kostova
Download PDF (550 KB)
Abstract
This study investigates the possible cytoprotective and antioxidant potential of Pr(III) complex of 5-aminoorotic acid (PrAOA), at a concentration of 100 μM, on sub-cellular (isolated rat liver microsomes) and cellular (isolated rat hepatocytes) level. The Pr(III) complex of 5-aminoorotic acid (PrAOA) was synthesized by reaction of the respective inorganic salt in amounts equal to ligand molar ratio of 1: 3. The newly synthesized complex was characterized by means of elemental analysis, FTIR and FTRaman spectroscopies. The effects of PrAOA were evaluated on two toxicity models: non-enzyme lipid peroxidation and tert-butyl hydroperoxide (t-BuOOH) and compared to those of 5-aminoorotic acid and quercetin. On isolated rat liver microsomes, in conditions of non-enzyme lipid peroxidation, PrAOA complex revealed good statistically significant antioxidant activity (decreasing malondialdehyde (MDA) production – marker for lipid peroxidation), closer to that of quercetin and stronger than that of 5-aminoorotic acid (AOA). On isolated rat hepatocytes, we determined the main parameters of the hepatocytes’ functional and metabolic status: cell viability (measured by trypan blue exclusion), levels of lactate dehydrogenase (LDH), reduced glutathione (GSH) and MDA. In t-BuOOH-induced oxidative stress, PrAOA complex showed statistically significant cytoprotective and antioxidant activities, closer to those of quercetin and stronger than those of AOA. The complex prevented the loss of cell viability and GSH depletion, decreased LDH leakage and MDA production. The stronger hepatoprotective and antioxidant activity of PrAOA than that of AOA on both in vitro toxicity models, might be due to the presence of Pr(III) ions in the complex.

Antioxidant effect of green synthesized silver nanoparticles on moderate local heat burn injuryx
Original Research Article
Pages 225 – 230
M.Valcheva-Traykova, G. Bocheva, S. Razmirov, D. Karashanova, T. Traykov
Download PDF (716 KB)
Abstract
By penetrating the damaged blood vessels walls, the free radicals formed in the heat burn wound field lead to oxidative stress-induced distant organs failure, latter being the most serious consequences of the burn injury. The oxidative stress (OS) may be successfully diminished by administering scavengers of free radicals along with substances preventing bacterial superposing. Silver is a well known highly efficient antimicrobial agent against antibiotic-resistant bacterial strains, with no registered allergic reactions. Recently silver nanoparticles on carriers (AgNPs) are preferred in wound treatment. In the present work, glycerol was used as a medium for AgNPs synthesis from AgNO3 and green tea extract. The resulting AgNPs suspension was applied on local moderate rat's heat burn wound for 1 to 5 days. The OS in the rat blood serum was monitored for 5 days. The topical treatment with green synthesized AgNPs resulted in a decreased OS level in the serum than those for the untreated burned animals.

Role of some styryl-heterocycles in the control of ochratoxin A biosynthesis
Original Research Article
Pages 231 – 237
R.Antonioletti, A. Viglianti, F. Massi, C. Sappino, A. Ricelli
Download PDF (705 KB)
Abstract
Ochratoxin A (OTA) is a mycotoxin whose dangers have been sufficient for many countries to regulate its presence in various foods. In different countries, the black Aspergilli group, in particular Aspergillus carbonarius, causes the highest OTA contamination in fruit. Here we describe the effects of different styryl-heterocyclic compounds on the prevention of OTA biosynthesis by A. carbonarius cultured in a conducive liquid medium. The most effective and long-lasting control of OTA biosynthesis was achieved with (E)-3,5-dimethoxy-(2-thienyl) styrene (10b) and (E)-3,4,5-trimethoxy-(3-thienyl) styrene (11d). In fungal cultures treated with these compounds at 50 ppm, OTA biosynthesis decreased by 65% and 90%, respectively, after 8 days of incubation. A lower reactivity enhances the inhibition of OTA biosynthesis, in particular long-term. This study underlines the greater effectiveness of the phenyl ring substitution model as compared with that of the thiophene substitution model. Natural compounds present in edible plants having a styryl-heterocyclic scaffold may be effective inhibitors of OTA biosynthesis.

Antioxidant activity of selected o-methoxyphenols and biphenols: theoretical and experimental studies
Original Research Article
Pages 238 – 246
L. Koleva, S. Angelova, M. A. Dettori, D. Fabbri, G. Delogu, V. D. Kancheva
Download PDF (1138 KB)
Abstract
A combination of theoretical and experimental approaches was applied to study and to explain the structure – antioxidant activity relationship for selected ortho-methoxyphenols (natural and natural-like phenols). The corresponding dimers (biphenols) possessing guaiаcyl moiety were handpicked in order to study the influence of the conformation and substituents in the aromatic ring on the antioxidant activity. Chain-breaking antioxidant activities of the compounds under study were determined from the kinetic curves of bulk lipid autoxidation. Full geometry optimization of neutral molecules and their corresponding phenoxyl radicals for all compounds under study were obtained by using DFT (B3LYP/6-31+G**) calculations. Good correlation between experimental and predicted activity was achieved which is helpful for the structure-activity relationship explanation.

Antioxidant activity of 3-hydroxyphenol, 2,2'-biphenol, 4,4'-biphenol and 2,2',6,6'- biphenyltetrol: theoretical and experimental studies
Original Research Article
Pages 247 – 256
L. Koleva, S. Angelova, M. A. Dettori, D. Fabbri, G. Delogu, V. D. Kancheva
Download PDF (796 KB)
Abstract
A set of selected phenolic compounds (phenol, 3-hydroxyphenol (resorcinol), 2,2'-biphenol, 4,4'-biphenol and 2,2',6,6'-biphenyltetrol) is designed in order to study the structure – antioxidant activity relationship for the compounds with one benzene ring and two C-C bridged benzene rings. The corresponding “dimeric” structures (biphenols and biphenyltetrol) of phenol and resorcinol are handpicked in order to study the influence of the number and mutual position of the substituents (OH group(s) in the aromatic ring) on the antioxidant activity. A combination of theoretical and experimental approaches is applied. Chain-breaking antioxidant activities of compounds under study are determined from the main kinetic parameters of bulk lipid autoxidation. Full geometry optimization of neutral molecules and their corresponding phenoxyl radicals for all compounds under study are obtained by using DFT (B3LYP/6-31+G**) calculations. Good correlation between experimental and predicted activity is achieved.

Mutual influence of lipid-antioxidant-surfactant in microheterogeneous systems
Original Research Article
Pages 254 – 259
O.T. Kasaikina
Download PDF (589 KB)
Abstract
This short review focuses specifically on the current understanding of the role of microheterogeneous reaction medium and surfactants in particular, in preventing and inhibiting lipid oxidation, and on the influence of surfactants on the behavior of known natural and synthetic antioxidants (AO). The mutual influence of the components and the conditions of occurrence of synergism and antagonism in a complex system (lipid – antioxidant – surfactant) are discussed as well.

Effects of pH and surfactant concentration on the local concentrations of antioxidants in binary oil-water mixtures and in oil-in-water emulsions
Original Research Article
Pages 260 – 267
J. Freiría-Gándara, C. Bravo-Díaz
Download PDF (851 KB)
Abstract
Antioxidants (AOs) react with radicals to minimize their harmful effects. The rates of their reactions depend on both the rate constant for the particular reaction and the concentrations of reactants at the reaction site. Thus, knowledge on their concentrations is very useful and desirable to interpret their reactivity (e.g., efficiency) because AOs with high rate constants may have low concentrations at the reaction site (i.e., their bioavailabity is low). Here we analyze the effects of surfactant concentration and acidity on the local concentrations of a model phenolic acid antioxidant in binary oil-water mixtures and, for the sake of comparisons, in corn oil-in-water emulsions. Local concentrations in emulsions can be up to 160 times higher than the stoichiometric concentrations, thus having a significant effect in AO efficiencies.

Expanding the antioxidant activity into higher temperatures – fullerene C60 conjugated with -tocopherol analogue as a hybrid antioxidant in saturated lipid systems
Original Research Article
Pages 268 – 274
R. Czochara, M. Grajda, J. Kusio, G. Litwinienko
Download PDF (480 KB)
Abstract
Oxidative stability of two fatty acids: stearic acid (STA) as a model of saturated lipid and linolenic acid (LNA) as model of polyunsaturated lipid containing C60 derivative with covalently bonded hydroxychromanyl moiety as analogue of -tocopherol (F-1) was monitored by differential scanning calorimetry. The overall Arrhenius kinetic parameters (activation energy Ea, pre-exponential factor Z, and rate constants k) of non-isothermal oxidative decomposition indicate that in saturated hydrocarbons the hybrid C60+hydroxychromane derivative is effective antioxidant acting at temperatures above 120°C, expressed as 9 kJ/mol increase of Ea and values of k twice smaller than for oxidation of noninhibited pure STA. However, experiments with LNA oxidized at temperatures above 80°C indicated that the hybrid derivative did not improve the oxidative stability of polyunsaturated lipids at higher temperatures (Ea, Z and k’s are almost the same as for oxidation of pure LNA). We suggest that C60 is able to inhibit STA autoxidation due to formation of thermally stable adducts with alkoxyl radicals whereas autoxidation LNA is mediated by peroxyl radicals that are not effectively scavenged by C60.

Effect of some membrane lipids on radical generation in the system acetylcholine-hydroperoxide
Original Research Article
Pages 275 – 279
N.V. Potapova, D.A. Krugovov, O.T. Kasaikina
Download PDF (580 KB)
Abstract
The influence of membrane lipids (0.001 - 0.1 mM) on the rate of -carotene oxidation initiated by the lipophilic azoinitiator AMVN and by microaggregates formed by acetylcholine (ACh) and tert-butyl hydroperoxide (ROOH) in n-decane (37 ° C) was studied. Phosphatidylcholine, sphingomyelin and cholesterol were investigated as lipid components. In homogeneous systems, all the additives slightly decrease the consumption rate of -carotene initiated by AMVN due to cross radical reactions. In the case of initiation by mixed microaggregates {mROOH…nACh} which generate peroxyl radicals RO2, a dose-dependent r etarding effect of phosphatidylcholine on the -carotene consumption rate was observed whereas cholesterol additives caused acceleration of -carotene oxidation. Sphingomyelin did not show any significant differences. It was found that the changes of radical initiation rate correlated with the changes of microaggregates sizes measured by dynamic light scattering.

Essentiality of dietary amino acids for antioxidative defense
Original Research Article
Pages 280 – 285
F. T. Ribarova
Download PDF (470 KB)
Abstract
The amino acids are not only building blocks for proteins synthesis. They also participate in many other biochemical processes and functions: cell signaling pathways, antioxidative reactions, acid-base balance, gene expression, regulation of immune function, and many others. The aim of this study was to present scientific data concerning the essentiality of dietary non-essential amino acids (NEAA) for antioxidative defense in the body, and to present data about their composition and quantity in Bulgarian foods. Information is provided on the content of amino acids with antioxidant activity (Cys,Glu, Gly, Меt and total content of S-amino acids) in 27 Bulgarian food products.The data were obtained by the classical method for analysis of amino acids – ion exchange chromatography with post-column derivatization. The importance of NEAA for the antioxidant defense of the body through their participation in glutathione, taurine and hydrogen sulfide synthesis is clarified. Their role in activating the antioxidant capacity of human serum albumin – an important endogenous antioxidant is outlined. Examples are listed showing effective implementation of antioxidant amino acids at thermal processing of dietary fats substituting the synthetic additives.The current data clearly showed that non-essential amino acids, especially those containing extra thiol, thioether, or extra amine group, have strong antioxidant activities. On the basis of the essentiality of dietary non-essential amino acids for the antioxidative defense of the human body, their broad participation in preventive and healthy diets is strongly recommended.

The flavonoids composition of Bulgarian foods – comparison with USDA database
Original Research Article
Pages 286– 292
S. P. Tsanova-Savova, S. K. Velikov, V. N. Petkov
Download PDF (670 KB)
Abstract
Flavonoids are polyphenolic antioxidants of plant origin, comprising more than 5000 individual compounds. Their protective role to human health is associated with reduced risk of development of series of degenerative diseases. Therefore the quantitative knowledge of the flavonoids composition is of crucial importance for elaboration of healthy nutrition diets. The aim of the study was to estimate differences between flavonoids content of Bulgarian foods and the USDA Flavonoids Database. For achieving the set aim, the Bulgarian Flavonoids Food Composition Database have been translated and transferred in Excel format, according the unified Access scheme of the USDA Database. The fast track and comparison of the data was made with automotive program written on Visual Basic for Application (VBA). The original data for representatives of 3 major classes of flavonoids in Bulgarian foods are presented. The data for the flavonols – myricetin, quercetin and kaempferol, flavan-3-ols – (+)-catechin and (-)-epicatechin and flavones – luteolin and apigenin were determined by validated HPLC methods. The food samples were collected according to a precise sampling plan and the origin of all foods was documented. In the current study data for 15 fruits, 30 vegetables and vegetable products and 3 leafy green condiments are reported. Our data are compared with the pool of results, summarized in USDA Database for the Flavonoids Content of Foods, where data for 506 foods are presented, covering 308 scientific literature papers. The current study revealed a number of topics whose further study would advance the development of nutritional databases.

Traditional Bulgarian food – source of antioxidant vitamins
Original Research Article
Pages 293 – 296
S. S. Paneva, D. S. Hristov, V. E. Vodenicharov, F. T. Ribarova
Download PDF (488 KB)
Abstract
Many studies have shown that traditional foods are related to good health and longevity of population. The aim of the current study was to provide data on the content and biological importance of antioxidant vitamins: vitamin A (as beta-carotene), vitamin E and vitamin C in some traditional Bulgarian foods. 15 traditional Bulgarian foods from plant origin are included in the present study. The antioxidant vitamins were determined using HPLC methods. A comparative analysis of the obtained results with information from European and American databases was carried out. The highest values of the studied vitamins were determined in vegetables - red peppers and tomatoes, and in fruits - cherries and peaches, which are traditional for Bulgarian diet. Enhancement of antioxidant defenses through dietary antioxidant vitamins would seem to provide a more reasonable and practical approach for reducing the level of oxidative stress. The data provided on antioxidant vitamins levels in the studied products are a scientific evidence that traditional foods are sources of antioxidants, which through the diet contribute to Bulgarians health and longevity, by enhancing the body antioxidant defenses.

The naturally derived “Stoletnik” dairy product as a model for nutraceutical traditional food
Original Research Article
Pages 297 – 301
Hr. Chomakov, I. Pantchev, G. Rakleova, Z. Nikolov, Zh. Dimitrov, M. Yordanova, A.Atanassov
Download PDF (494 KB)
Abstract
Current tendency for improving quality of live led, among all, to a demand for novel food products with beneficial effect on human health. The key requirement is that these products are either of natural origin or produced through organic farming and with as little processing as possible. Major sources for such products are the traditional regional recipes – ethnic foods. These recipes are an extract of a long-term experience (decades or even centuries) and reflect local biodiversity as well. Sporadic research on their composition and properties revealed significant beneficial effects on human health, sometimes in unexpected areas. These preliminary studies place the traditional foods as a promising new source for high quality healthy food products. In this paper we made an attempt to outline the beneficial properties of a traditional dairy product “Stoletnik” using the latest scientific knowledge in the field. Its traditional recipe was successfully converted into an industry-grade technology and was implemented in a small-scale production.

Phytochemical characteristics and in vitro antioxidant activity of fresh, dried and processed fruits of Cornelian cherries (Cornus mas L.)
Original Research Article
Pages 302 – 307
N. Tr. Petkova, M. H. Ognyanov
Download PDF (495 KB)
Abstract
In the current study, chemical and technological properties and antioxidant activity of fresh, dried and processed Cornelian cherry fruits were evaluated. Several fruit characteristics, such as total titratable acidity (TA), pH, total phenolic compounds, total monomeric anthocyanin (TMA), total antioxidant activity and sugar composition were studied. Average fruit mass and flesh ratio were as follows: for fresh fruits (1.53 g and 77.16%), for dry fruits (0.86 g and 67.61%) and for compôte (1.68 g and 63.15%), respectively. In fruits of Cornelian cherry dry matter varied from 18.7 to 81.4%, ash content from 0.5 to 2% and TA from 1.5 to 3.4%. The highest content of TMA - 32.1 mg cyd-3-glu/100 g fresh weight (fw), as well as of total phenolic compounds - 4.56 mg GAE/g fw was found in fresh fruits. Fresh Cornelian cherry demonstrated the highest antioxidant potential - 36.5 mM TE/g fw (DPPH assay) and 29.6 mM TE/g fw (FRAP assay). The sugar composition in all Cornelian cherry products mainly consisted of sucrose, glucose and fructose, as sucrose was in low levels from 0.15 to 0.30% fw. The polyuronic content was in the range from 1.1 to 1.3%. The current study demonstrated nutritional characteristics of Cornelian cherry fruits suitable for direct consumption and various forms as infusions or compôte. The analyzed products were evaluated as natural foods with high antioxidant activity and potential health benefits.

Different pathways of the para-O-H bond dissociation in di- and trihydroxyphenolic acids: a DFT investigation
Original Research Article
Pages 308 – 314
Zh. A. Velkov, L. P. Muteva, A. V. Tadjer
Download PDF (492 KB)
Abstract
Phenols may inhibit but can also enhance oxidative damage processes in biomolecules. This ambivalent behavior heavily depends on their chemical structure. Phenolic acids with two and three hydroxyl groups and a different length and degree of saturation of the side chain are selected for this investigation. The radical-scavenging activity of the compounds was assessed, as well as the role of the different structural features on it. The most appropriate mechanism for the para-O-H bond breaking in an aqueous medium is determined. A high level DFT investigation was performed using the B3LYP functional with the 6-311++G(d,p) orbital basis; the solvent effects were evaluated by PCM.

A preliminary study on radical scavenging abilities of two dihydroxy-coumarins by electron paramagnetic resonance (EPR) spectroscopy
Original Research Article
Pages 315 – 320
A.M. Zheleva, V.D. Kancheva, G.D. Nikolova, V.S.Parmar, V.G. Gadjeva
Download PDF (568 KB)
Abstract
In current research were evaluated and compared the radical scavenging abilities of two coumarins differing in positions of ortho-dihydroxyl groups in their aromatic rings. Scavenging abilities towards superoxide (·O2-) and hydroxyl (·OH) radical of 7,8-dihydroxy-4-methyl-2H-chromen-2-one (a1) and 6,7-dihydroxy-4-methyl-2H-chromen-2-one (b1) were studied. To realize the aim of the study proper Electron Paramagnetic Resonance (EPR) spin trapping spectroscopy was applied the only technique that allows scavenging, detecting and distinguishing of short live radicals such as ·O2- and ·OH species. In studied Fenton system was demonstrated higher inhibiting activity against •OH for a1 in comparison with b1, while in hypoxanthine/xanthine oxidase system b1 exhibited better inhibiting ability against •O2- generation.

Near-infrared spectroscopy as a tool for rapid estimation of the antioxidant capacity of red wines
Original Research Article
Pages 321 – 326
N. G. Hristova-Avakumova, L. A. Atanasova, S. L. Atanassova, T. V. Vangelova, V. A. Hadjimitova
Download PDF (435 KB)
Abstract
The present investigation evaluates the feasibility of using near-infrared (NIR) spectroscopy as an accurate, fast and non-destructive analytical tool for estimation of the antioxidant properties of red wines. The evaluation of the antioxidant properties was conducted using ABTS and DPPH assays for total antioxidant activity determination. NIR measurements were performed by NIRQuest 512 spectrophotometer in the region 900-1700 nm using transmittance mode. Partial least-square regression with internal cross-validation was used for calibration models development for determination of tested parameters and SIMCA for creation of model for classification on the base of the spectral data. All the investigated wines have demonstrated better antioxidant properties in the ABTS model system compared to the DPPH one. Although the observed activity highly varied, the wines containing varieties Syrah and Malbec presented better antioxidant effect in both model systems. The Merlot wines produced from the vineyard situated on the southern slopes of Sakar Mountain in the time interval of 2012 – 2016 denoted a tendency of decreasing SV50 values from year to year. In both model systems Merlot 2016 demonstrated the best antioxidant effect correlating to the lowest SV50 values. The determination of the antioxidant activity of the tested wines on the basis of their spectra in the NIR region revealed a high degree of accuracy of estimation. This indicates that NIR spectroscopy could be a promising technique in quantitative determination of antioxidant activity and building classification models for discrimination of wines according to their antioxidant properties.

Antioxidant properties of extracts from Daizo silkworm cocoons and relationship with near infrared spectra of intact cocoons
Original Research Article
Pages 327 – 331
L.A. Atanasova, N. G. Hristova-Avakumova, S.L. Atanassova, R. D. Ginin, M. V. Panayotov, V. A. Hadjimitova
Download PDF (341 KB)
Abstract
The aim of this study is to investigate antioxidant properties of water-soluble extracts from silkworm (Bombyx mori) cocoons, breed Daizo and their relation to near-infrared (NIR) spectra of intact cocoons. The process of extraction with water was carried out in two stages: an ultrasonic extraction for 30 min and subsequent incubation at 60°C for 1 h. The antioxidant activity (АОА) was determined after each stage by ABTS and DPPH test. It was found that the extract exhibits radical-scavenging properties in both model systems. The incubation at 60°C increases twice the yield of substances with antioxidant properties. The measurement of the standard sericin in the same conditions shows that the AOA of the obtained extracts is not only due to the sericin, but to the other extracted components, which contributes to the observed biological effect. Comparison between the AOA of the extracts with NIR spectra of intact cocoons shows a good correlation between these properties and spectral characteristics – obtained errors of estimation were low and correlation coefficients higher than 0.96.

4-Methoxy aroylhydrazones – promising agents protecting biologically relevant molecules from free radical damage
Original Research Article
Pages 332 – 337
N. G. Hristova-Avakumova, B. I. Nikolova-Mladenova, T. T. Traykov, V. A. Hadjimitova
Download PDF (613 KB)
Abstract
The concomitant toxicity of iron observed in many disorders associated with excess of this essential trace element is mainly due to its ability to promote and participate in free radical generation reactions and the subsequent oxidative damage. The dietary recommendations for some of the patients and the restriction for supplementation with essential antioxidants have led to the necessity of developing novel high-efficiency pharmacologically active substances with multipotent antioxidant properties. The present investigation aimed to determine the protection effect against oxidative stress of three structurally characterized derivatives of the active chelator salicylaldehyde benzoylhydrazone (SBH). The capability of the tested compounds 4-methoxysalicylaldehyde benzoylhydrazone (4mSBH), 4-methoxysalicylaldehyde-4-hydroxybenzoylhydrazone (4mShBH) and 4-methoxysalicylaldehyde isonicotinoylhydrazone (4mSIH)) to preserve biologically relevant molecules from oxidative damage was evaluated in in vitro spectrophotometric model systems with diverse mechanisms of free radical generation containing deoxyribose and egg yolk homogenate. Additional experiments have been performed in chemiluminescent systems containing different type of reactive oxygen species. The obtained results indicated that the incorporation of methoxy group at 4th position in the aldehyde part of the molecule ameliorates the evaluated properties. The extent of the observed improvement depends on the subsequent modifications in the hydrazide moiety. Greatest protection effect against the oxidative damage of the biologically important molecules was observed in the samples containing hydroxyl bearing compound (4mShBH). This hydrazone also demonstrated higher activity against OH● and O2●─ in the chemiluminescent model systems. Comparing the C50 values from the different model systems we suggest that the observed protection effect is associated with antioxidant activity different from Fe-chelation.

DPPH radical-scavenging activity of galantamine hydrobromide and pymadine alone and in combination
Original Research Article
Pages 338 – 344
D. D. Tsvetkova, St. A. Ivanova
Download PDF (930 KB)
Abstract
The overproduction of reactive oxygen species and the weakness of the antioxidant defense mechanisms in the human body are the main reasons for the oxidative stress, which underlies the development of neurodegenerative Alzheimer’s disease. Alkaloid Galantamine is nonselective acetylcholinesterase inhibitor with antioxidant activity. Pymadine is non-depolarizing potassium channel blocker having a synergistic effect with Galantamine on the symptomatic treatment of Alzheimer’s disease. The aim of the current study was the evaluation of the radical-scavenging activity (RSA) of Galantamine hydrobromide, Pymadine and the combination Galantamine hydrobromide/Pymadine towards 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical. The decrease in the absorbance of 0.05 mM methanol solution of DPPH at λ = 516 nm in presence of methanol solution of: 1 mM Butylhydroxytoluene (ВНТ) (standard); 1 mM ÷ 5 mM Galantamine hydrobromide; 1 mM ÷ 5 mM Pymadine and 5 mM Galantamine hydrobromide/5 mM Pymadine was monitored by spectrophotometry in equal intervals of 5 s for a total period of 30 min. The regression equations were used for calculation of the RSC50: RSA (Galantamine hydrobromide) = 3.419.e0.293.c, RSC50 (Galantamine hydrobromide) = 9.16 mM; RSA(Pymadine) = 0.460.e0.411.c, RSC50 (Pymadine) = 11.41 mM. RSA of the investigated compounds was compared with the effect of standard BHT and the relative radical-scavenging activity (RRSA) and relative decrease of radical-scavenging activity (RDRSA) were calculated. The experimental results showed that the combination of 5 mM Galantamine hydrobromide/5 mM Pymadine has a higher RSA (20.19 %), compared to 5 mM Galantamine hydrobromide (15.44 %) and 5 mM Pymadine (2.48 %) itself.