Inhibition of LP (%) in Fe2+/ascorbate system of induction by essential oil of balm leaves and BHT (as a positive control) in the TBA assay.
Lipid peroxidation is used as a marker of cellular oxidative stress and contributes to the oxidative damage that occurs as a result of xenobiotics metabolism, inflammatory processes, ischemia, reperfusion injuries and chronic diseases such as atherosclerosis and cancer [1,2].
Cell membrane lipids (phospholipids, glycolipids and cholesterol) are the most common substrates of oxidative attack. Once initiated reaction autocatalytic continues, it has pro-gradient flow, and the ultimate consequence is the structural-functional changes of the substrate. Lipid peroxidation is one of the best studied processes of cell damage under conditions of oxidative stress [3-5]. In 1960s Hochstein et al.  found that the initiation of lipid peroxidation require the presence of iron ions. From that moment the mechanism of lipid peroxidation process has been studied in many
Since lipid peroxidation causes oxidative damage to cell membranes and all other systems that contain lipids, in investigation of total antioxidative activity of plant extracts it is necessary to investigate their effects on lipid peroxidation. However, the impact of various natural products (isolated compounds and extracts) on the intensity of lipid peroxidation is studied in a number of substrate (linoleic acid, liposomes, various fatty oils, liver homogenates or hepatocytes isolated from it). Some substrates (liposomes and linolenic acids) are used more frequently than others mainly because of the simpler ways of performing the method. Also, due to the complex composition, examining the process of lipid peroxidation in fatty oils and liver homogenates makes research more difficult.
2. Liposomes as a model system
Liposomes are microscopic structure consisting of the one or more lipid bilayer enclosing the same number of water compartments. First, they were produced in Great Britain in 1961 by Alex D. Bangham while he was studying blood clotting. It was discovered that when phospholipids were combined with water they immediately formed a sphere. This is due to the fact that one end of each molecule is water soluble, while the oposite end is water insoluble. Water-soluble medications added to the water were trapped inside the aggregation of the hydrophobic ends; fat-soluble medications were incorporated into phospholipids layer and then – an important delivery system was born! Generally, such a structure formed polar lipids (such as phospholipids) . Liposomes could be characterized as particles, similar to the structure and composition of cell membrane (Figure 1.). They occur in nature and could be artificially prepared .
The behaviour of liposomes in physical and biological systems is governed by the factors such as physical size, membrane permeability, percent entrapped solutes, chemical composition (estimation of phospholipids, phospholipids oxidation, and analysis of cholesterol), and quantity and purity of the starting material. Therefore, liposomes are characterized for physical attributes: shape, size, and its distribution; percentage drug capture; entrapped volume; lameliarity; percentage drug release. Based on the structure and size, we distinguish between different types of liposomes: Multilamellar Vesicles (MLV, size >0.5μm), Oligolamellar Vesicles (OLV, size 0.1-1μm), Unilamellar Vesicles (UV, all size ranges), Multivesicular Vesicle (MVV/MV, size >1μm). Unilamellar Vesicles are further divided into Small Unilamellar Vesicles (SUV, size 20-50nm), Medium Unilamellar Vesicles (MUV, size 50-100nm), Large Unilamellar Vesicles (LUV, size >100nm) and Giant Unilamellar Vesicles (GUV) (Figure 2.).
Based on composition and applications, liposomes are divided into conventional liposomes (CL), fusogenic liposomes, pH sensitive liposomes, cationic liposomes, long circulatory (stealth) liposomes (LCL) and immuno-liposomes . It is very difficult to measure dirrectly the phospholipid concentration, since dried lipids can often contain considerable quantities of residual solvent. Because of that, the method most widely used is an indirect one in which the phosphate content of the sample is first measured. The phospholipid concentration is measured using two methods - Bartlett and Stewart. In the Bartlett method the phospholipid phosphorous in the sample is first hydrolyzed to inorganic phosphate. This is converted to phospho-molybdic acid by the addition of ammonium molybdate and phospho-molybdic acid is quantitatively reduced to a blue colored compound by amino-naphthyl-sulfonic acid. The intensity of the blue color is measured spectrophotometrically and is compared with the curve of standards to give phosphorous and hence phospholipid content. This method is very sensitive. The problem is that test is easily upset by trace contamination with inorganic phosphate. In the other test, Stewart test, the phospholipid forms a complex with ammonium ferrothiocyanate in organic solution. The advantage of this method is that the presence of inorganic phosphate does not interfere with the test.
Until recently, liposomes are used as inert particles, carries of active principles, mostly for cosmetic purposes . Today liposomes are used as very useful models, reagents and tools in various scientific disciplines, including biophysics (properties of cell membranes and channels), chemistry (catalysis, energy conversion, photosynthesis), biochemistry (the function of membrane proteins) and biology (excretion, cellular functions, transports and signaling, the transfer of genes and their functions). Liposomal formulation of several active molecules are currently in pre-clinical and clinical trials in different fields, with promising results. Two of the key problems in drug therapy (biodistribution throught the body and targeting to specific receptors) can be overcome by using liposomal formulations – liposomes protect encapsulated molecules from degradation and can passively target tissues or organs that have a discontinuous endothelium, such as liver, spleen, and bone marrow . Comercial use of liposome was based on their colloidal, chemical and surface and microcapsuled proporties. These products include dosage formes of drugs (anti-cancer and antifugal agents, vaccines), cosmetic formulation (skin care products, shampoos), diagnostic products, a variety of applications in the food chemistry, as well as oral nutrient transport (liposomal vitamins, minerals and plants extracts for oral use). Liposome stability is an importrant aspect that must be met to be able to apply. By selecting the optimal value and size, pH and ionic strenght and the addition of complexing agents, liquid liposomical formulations could be stable for years.
Liposomal models have helped us to better understand the structure and dynamics of natural biomembrane systems. The concepts of structure and function of biomembranes, such as membrane fluidity, phase transition, the movement of lipids and proteins, triggering prosesses that affect metal ions or pH, have been very developed in this way. Modulatind effect of internal molecules (such as cholesterol) and insight into the mechanisms of membrane permeabillity for non-electrolytes and ions, are obtained by testing the model membranes. Liposomes that contains proteins as a components of membrane (reconstructes liposomes) were used in testing lipid-protein interactions in biological membranes, in examining the activities of active components such as membrane ionophores, anesthetics and divalent cations and mechanisms of antybody-antigen interactions .
Liposomes are very good models because they show the selectivity of the membrane to ions, osmotic swelling and response of range to agents that speed up or slow down the loss of ions and molecules from the particles in a way that at least, qualitatively mimic their activity in the natural membrane systems. Liposomes have also been successfully applied to „exclude the role“ of membranes lipids and other components in biomembranes interact with the physical or chemical agents. Nevertheless, liposomal systems are useful because they allow the manipulation of membrane lipid composition, pH, temperature, content of different compounds in a limited way and provide the ability to determine the individual effect of the investigation product .
An example of the advantages of liposome in investigation of lipid peroxidation is that the influence of free radicals can be explored in the absence of chemical systems that produce free radicals, which may affect the test reaction. It is also possible to control the chemical composition of the liposome. This is particularly useful for the determination of lipid peroxidation induced by different systems for the generation of free radicals, and monitoring the overall effect of the combined system, or synergistic effects of combined systems that may arise. In addition to this it is possible to determine the antioxidant activity of tested compounds and determine which system works the best, by simple monitoring of lipid peroxidation. The tests used to determine the power of antioxidants to exert suppression of lipid peroxidation based on an assessment of the strength of oxidation of lipid substrate in the presence or absence of potential antioxidant molecules of plant extracts. There are four different strategies for assessing antioxidant capacity of molecules to the lipid substrate. They include the determination of oxygen consumed, the loss of substrate and formation of primary and secondary oxidation products . The first method for determining the degree of lipid peroxidation, which includes the determination of oxygen consumed, based on following of initiation phase and its extension in the absence of antioxidants. The second method is based on measuring the loss of substrate in systems such as samples of food or biological samples and is very complicated, because they are full of potential oxiable substrates that are difficult to identify and characterize. The third method is based on monitoring the formation of primary oxidation products. It is a method that is well adapted to study such complex model systems and often involves the spectrophotometric determination of hydroperoxide, the dominant primary products of lipid peroxidation. Monitoring of secondary products of oxidation is the most commonly used method for the study of lipid model systems and lipid isolated from their natural environment. Both the
Despite all these advantage, liposomal systems remain different from the natural cellular systems. For this reason, regardless of the results obtained by testing the liposomes, they could not be reproduced on the natural membrane system, but they can provide useful information.
Therefore, the liposomes are still mostly used as a model system of biological membrane for testing the LP, especially when testing extracts and essential oils from plants on the intensity of LP. These studies are important because free radical oxidation of lipid components of food is a major strategic problem of food producers. The degree of oxidation of fatty acids and their esters in foods depends on the chemical structure of fatty acids, food processing technology, the temperature at which food is stored or prepared for eating and the presence of antioxidants. Synthetic antioxidants are widely used in many foods to retard undesirable changes as a result of oxidation. Chemicals, like tert-butyl-4-hydroxyanisole (BHA) and tert-butyl hydroxytoluene (BHT), can be used as antimicrobial and antioxidants agents. However, the use of some of these chemicals is restricted in several countries, as they may be dangerous to human health . Therefore, the search for new natural antioxidant sources has been greatly intensified. For this reason, there is a growing interest in the studies of natural additives as potential antioxidants. The antioxidant properties of many herbs and spices are reported to be effective in retarding the process of lipid peroxidation in oils and fatty foods and have gained the interest of many research groups. A number of studies on the antioxidant activities of various aromatic plants have been reported over the last 20 years [18,19]. Their aroma is associated with essential oils, complex mixtures of volatile compounds, dominated by mono- and sesquiterpenes. It is known that essential oils exhibit significant biological and pharmacological activities such as anti-inflammatory, antimicrobial, spasmolytic, stimulant effect on the CNS and the like. New research shows that they possess significant antitumor activity , and act as inhibitors of growth of breast tumors . It was confirmed that essential oils of some aromatic plants possess a high antioxidant potential . Widely used in the food industry to improve the flavor of foods.
In addition to essential oils, aromatic plants and characterized by the presence of plant phenolic compounds, primarily phenylpropanoids and coumarins which are proven to have multiple pharmacological activities. Studies of these secondary biomolecules have become intensified when some commercial synthetic antioxidants found to be expressed toxic, mutagenic and carcinogenic activities . In addition, it was found that excessive production of oxygen radicals in the body initiates oxidation and degradation of polyunsaturated fatty acids. It is known that free radicals attack the highly unsaturated fatty acid of membrane system and induce lipid peroxidation, which is a key process in many pathological conditions, and one of the reactions caused by oxidative stress. Particularly vulnerable are the biological membrane lipids in the spinal cord and brain because they contain high oxiable polyunsaturated fatty acids. These features facilitate the formation of oxygen radicals involved in the processes of aging, Alzheimer's and Parkinson's disease, ischemic damage, arthritis, myocardial infarction, arteriosclerosis and cancer. Phenolic antioxidants "stop" oxygen free radicals and free radicals formed from the substrate by giving hydrogen atom or an electron. Some flavonoids have strong inhibitory effect on lipid peroxidation processes. This action is based on their ability to chelate transition metal ions, thereby preventing the formation of radicals (initiators of LP), caught radicals initiators of LP (ROS), scavenge lipid-alkoxyl and lipid-peroxyl radicals and regenerate α-tocopherol by reduction of α-tocopheryl radicals. Flavonoids have the following characteristics: 3 ', 4'-dihydroxy group in ring B, or 4-keto and 3-hydroxy group in C ring, or 4'-keto group in C ring and 5-hydroxy group in A ring have the metal chelated properties (Figure 3.).
Different metals have different binding affinity of the flavonoids . Thus, for example, iron has the highest binding affinity for 3-OH group of ring C, then catechol group ring B and at the end of 5-OH group of ring A, while the copper ions bind to the first ring catechol group B . Solubility of flavonoids in the lipid phase and the ability to penetrate the lipid membrane is small, since flavonoids in nature are mostly in the form of polar glycosides. Numerous tests of the inhibitory effects of flavonoids on lipid peroxidation were carried out on models of cell membranes. Based on these studies, it is assumed that quercetin and other flavonoids probably located on the surface membrane could easily capture radicals from the aqueous phase and thus prevent the initiation of LP. Thus located, flavonoids faster capture radicals initiators LP than α-tocopherol, which is located within phospholipid bilayer and that the switch is a typical chain reaction. Prevention of initial attacks radicals from the aqueous phase to membrane phospholipids is essential in the antioxidant protection of biomembranes because free radicals are constantly generated in the aqueous phase of cellular and sub cellular structure [25,26].
In the present chapter, lipid peroxidation in a liposomal system was initiated by Fe2+-ascorbic acid system and the effects of four different Lamiaceae species (
3. Lamiaceae (Labiatae) family
The Lamiaceae family is one of the largest and most distinctive families of flowering plants, with about 220 genera and almost 4000 species worldwide . Lamiaceae are best known for the essential oils common to many members of the family . The family was established by De Jussieu in 1789 as the order Labiatae. This was the original family name, so given because the flowers typically have petals fused into an upper lip and a lower lip, the flower thus having an open mouth. Although this is still considered an acceptable alternative name, most botanists now use the name "Lamiaceae" in referring to this family. The main centre of diversity is the Mediterranean region to central Asia. Members are found in tropical and temperature regions . All Lamiaceae are aromatic plants. The essential oil contains mainly monoterpenes, sesquiterpenes and phenylpropanoid compounds. Also, the plant species of Lamiaceae have been shown as rich sources of phenolic compounds mostly flavonoids and phenolic acids.
3.1. Balm (
Balm is characterized by the presence of the other important plant phenolic substances such as phenolic acids (caffeic, rosmarinic and chlorogenic acid) (Figure 5.).
Also, balm leaf is characterized by the presence of pentacyclic triterpenes (ursolic, pomolic and oleanolic acid) (Figure 6.). The main biopharmacological effects shared by ursolic and oleanolic acid are anti-inflammatory, hepatoprotective, antitumor, and antioxidative [36-39].
Essential oil is present in all parts of the plant. The largest amount of oil obtained from aerial parts of plants, harvested in late summer. Balm leaves contain no more than 0.13% of essential oil which is of complex and variable composition. Among the more than 50 compounds identified to date, citronellal (dominantly the (
Beneficial effects of plants introduced by ancient Greeks and Romans.There is overlap with the use of plants in folk medicine and science. In relation to its complex composition it has multiple medicinal effects. Its herb has wide applications in the folk medicine. Due to the soothing action balm leaves enters into the composition of tea for calming, which is recommended for hysteria and neuralgia. Balm leaves mixed with bitter herbs are a great tool to enhance appetite. Various preparations containing extract or essential oil balm leaves are used as an addition to baths against rheumatism. In the folk medicine of Belarus alcoholic extract is drunk for stomach ulcer and duodenum, to calm the pain in the stomach, intestines, the liver, heart, and women’s diseases. Terpenes found in essential oil of balm leaves, have a relaxing and antiviral effects. Eugenol calms muscle spasms and destroy bacteria . It is also used as a carminative and sedative. Recent results indicate that the balm extract acts as depressants and have sedative effect on central nervous system of mice . In the folk medicine of central Italy inflorescences of this plant, called “Erba Lupa”, were used under infusion as antispasmodic, against insomnia and eyes inflammations [46,47].
Our research on balm was recently extended to the comprehensive
The protective effects on lipid peroxidation of balm extracts have been evaluated using the Fe2+/ascorbate system of induction, by the TBA-assay (Table 2.). In general, all of the examined extracts (except
It is known that quercetin, like many other flavonoids, prevents oxidation of LDL cholesterol, and its anti-inflammatory activity comes from inhibition of the enzyme lipooxigenase and inhibition of inflammatory mediators . Kaempferol acts synergistically with quercetin to reduce the proliferation of malignant cells, and treatments are a combination of quercetin and kaempherol efficient than their single use . It is, also, known that rutin has strong antioxidant effects, as well as a feature to built chelates with metal ions (e.g. iron) and reduces the Fenton reaction in which the resulting harmful oxygen radicals. It is supposed to stabilize vitamin C. If rutin is taken together with vitamin C, increases the activity of ascorbic acid . In addition, HPLC-DAD analysis showed that the aqueous extract, in large quantities, present phenolic acids (rosmarinic, chlorogenic and caffeic acid), which are known antioxidants. It was determined that rosmarinic acid has stronger antioxidant activity than vitamin E. Rosmarinic acid prevents cell damage caused by free radicals and reduce the risk of cancer and atherosclerosis. In contrast to the histamines, rosmarinic acid prevents activation of the immune system cells that cause swelling and fluid collection. Also, it is known that the caffeic acid by far surpassing other antioxidants because it reduces the production of α-toxin for more than 95% . Furthermore, it can be supposed that the reduction process of lipid peroxidation is caused, besides flavonoids, also by triterpenoids acids (especially ursolic, oleanolic, and pomolic acid) since non-polar extracts (Et2O and CHCl3) also exhibited high antioxidant potential . The
The represented antioxidant activity results show that extracts of examined plant species, especially EtOAc and H2O extracts are efficient in the protection of tissues and cells from oxidative stress. Anyway, according to variations in regard to antioxidant activity of tested by different
As compared with control, intensity of LP is statistically significant reduced during the treatment with ethylacetate and water extracts of balm leaves. The result derived by treatment with ethylacetate and water extracts is in according with amounts got
Similar results were obtained during examining the effects of extracts of bastard balm on LP in blood hemolysate in mice (Table 4.). Three extracts, CHCl3, EtOAc and H2O, induced a significant decrease of LP intensity, while Et2O and
The LP value showed a statistically insignificant increase with CCl4-treated animals compared with the untreated ones. A clear protective effect was seen in experimental animals administered H2O extract and CCl4 compared with untreated animals. Furthermore, EtOAc extract also significantly decreased the activity of LP, while Et2O, CHCl3 and
3.2. Horehound (
Marrubium genus includes about 40 species. Species of this genus growing in dry pastures, abandoned the places along the roads in central and southern Europe, but also in North Africa, in parts of Asia and the Americas. Horehound is a perennial plant with a rectangular stem, branched in the upper part. Rhizomes of this species are ligneous, leaves oblong, flowers grouped in loose inflorescence . A common plant blooms from July to September and harvested in that period. It has a bitter and pungent taste and smell. It is the drug of Herba
In previous phytochemical investigations on
Some species of
In our comprehensive study of chemical and biochemical investigation of
Also, our study showed that all of the examined essential oils express strong antioxidant activity and capability to reduce lipid peroxidation (Table 5.). The largest inhibitory activity was exhibited by essential oil from plant collected at Senta locality (No. 3.). Solution of all concentrations, except the most diluted (0.213 μg/mL), have exhibited a stronger protective effect (from 37.02 to 71.32% of inhibition of LP) than BHT (26.15%). The other two essential oils (from Rimski sanac and Novi Knezevac), at higher concentration (from 1.065 to 2.130 μg/mL), have also exhibited more intense protective effect than BHT .
The effect of crude MeOH extracts of
Obtained results can be related to the experiments in which the total amount of flavonoids was determined, which show that EtOAc and H2O extracts from Senta locality contains the largest amounts of total flavonoids, namely of luteolin, either being present as free or in the form of its glucosides. The suggested mechanism of flavonoid antioxidative action is as follows: the double bond in position 2, 3 is conjugated with C4-carbonyl group, and free OH groups (C5, C3 and C7) can form chelates with ions of d-elements. Once formed, complex with Fe2+ ion prevents formation of OH• radicals in Fenton’s reaction . Also, luteolin is thought to play an important role in the human body as an antioxidant, a free radical scavenger, an agent in the prevention of inflammation, a promoter of carbohydrate metabolism, and an immune system modulator. These characteristics of luteolin are also believed to play an important part in the prevention of cancer. Multiple research experiments describe luteolin as a biochemical agent that can dramatically reduce inflammation and the symptoms of septic shock . Furthermore, it is well known that some other flavonoids isolated from
3.3. Basil (
Basil is originally native to India and other tropical regions of Asia, having been cultivated there for more then 5.000 years. Ocimum genus includes about 150 species . There are many varieties of
The word market has several types of essential oils that differ in chemical structure, composition and fragrance. The dominant compounds of basil essential oil occur in two different biochemical pathways: phenylpropanoids (methyl chavicole, eugenol, methyl eugenol, and methyl cinnamate) through shicimic acid, and terpenoids (linalool and geraniol) through mevalonic acid. Based on chemical content, basils can be divided into four groups: European (French)
Among phenolic constituents flavonoids and their glucosides are dominant. The major flavonoids are: quercetin, kaempferol, apigenin, luteolin and rutin. Quercetin-3-O-diglucoside and kaempferol-3-O-β-rutinoside have been also identified. Beside, basil is rich in triterpenoid acids (ursolic and oleanolic), cinnamic acid (caffeic and rosmarinic), vitamin C and β-carotene, as well with calcium, copper, magnesium, sodium and potassium .
Basilici herba has been used in traditional and homeopathic medicine to treat number of diseases. Essential oil (
In our investigation, the examined essential oil expressed strong antioxidant activity (Table 7.). Solutions of all concentrations, except the most diluted (0.213 μg/mL), have exhibited a stronger protective effect (from 35.17 to 79.14% of inhibition of LP) than BHT (26.15%). The largest inhibitory activity was achieved by using the solution of the highest concentration. For the inhibition of LP, the most responsible compounds were the oxygenated phenolic monoterpens (methyl chavicole) and the mixture of mono- and sesquiterpene hydrocarbons. These findings are in correlation with the earlier published data on the antioxidant activities of the investigated essential oil and selected oil components [84,85].
The data presented in Table 8. show that the last three extracts of
3.4. Oregano (
Origanum is one of the most variable genera of Lamiaceae family. Originates from Europe, but is now cultivated throughout the world including USA, India and South America. This is an extremely variable species with several subspecies and named cultivars grown for ornamental, culinary and medicinal uses. Oregano is a bushy, semi-woody sub-shrub with upright or spreading stems and branches. Some varieties grow in mound like mats, spreading by underground stems (called rhizomes), and others with a more upright habit. The aromatic leaves are oval-shaped. Oregano will grow in a pH range between 6.0 (mildly acid) and 9.0 (strongly alkaline) with a preferred range between 6.0 and 8.0. The flowers are purple, 3–4 mm long, produced in erect spikes.
As the other three Lamiaceae species oregano is characterized by the presence of essential oil, flavonoids, phenolic acids (caffeic, chlorogenic and rosmarinic), triterpenoid acids (oleanolic and ursolic) and tannins. The oregano essential oil yield between 0.35-0.55% . According to Arnold et al. , the content of essential oil in
According to Duke , flavonoids are found in the leaves and whole plant, mostly as kaempferol, quercetin, apigenin, luteolin and rutin. Beside, oregano is rich in apigenin-7-O-β-D-glucoside and luteolin-7-O-β-D-glucuronide. In oregano flavanon naringenin and flavanon glucoside (naringin), have also been identified (Figure 12.).
Most of the healing properties are attributed to the essential oil and flavonoids. It has been widely used in agricultural, pharmaceutical and cosmetic industries as a culinary herb, flavoring substances in food products, alcoholic beverages and perfumery for its spicy fragrance . Regarding the nonvolatile components, the extracts of oregano have the most effective antioxidant activity among aromatic herbs . Oregano family, is widely known as possessing therapeutic properties (diaphoretic, carminative, antispasmodic, antiseptic, tonic) being used in traditional medicine systems in many countries. Different groups of researchers [96,97] studied oregano alcohol extracts. The antioxidant effect of the mentioned extracts is generally due to the presence of rosmarinic and caffeic acid .
Our tests showed that only concentrated solutions of essential oil exhibit a greater ability to inhibit LP in liposomes of synthetic antioxidant BHT. The antioxidant activities were dose dependent, but it is noticeable that the values obtained using two most concentrated solution of essential oils (1.598 and 2.130 μg/mL) are very close (49.58 and 51.13% of inhibition of LP). For the inhibition of LP, the most responsible compounds were the oxygenated phenolic monoterpens (thymol and carvacrol) and the mixture of mono- and sesquiterpene hydrocarbons .
The data presented in Table 10. show that the last three extracts of
It was found that excessive production of oxygen radicals in the body initiates oxidation and degradation of polyunsaturated fatty acids. It is known that free radicals attack the highly unsaturated fatty acid of membrane system and induce lipid peroxidation. Since lipid peroxidation causes oxidative damage to cell membranes and all other systems that contain lipids, in any investigation of total antioxidative activity of extracts and essential oils it is necessary to investigate their effects on lipid peroxidation. Some substrates (for example liposomes) are used more frequently than others, mainly because of the simplicity of the methods involved. In this way we get very useful information to direct future research. The results of our
This work was supported by the Ministry of Science and Environmental Protection of the Republic of Serbia (Project No. 172058) and by the Provincial Secretariat for Science and Technological Development, Autonomous Province of Vojvodina, Republic of Serbia.