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Nowadays, climate changes induce deviations in the composition of honey varieties characteristic of a specific geographic region. Therefore, according to Codex Alimentarius for honey, the slight variations in values of determined parameters cannot be strictly regarded as a sign of its adulteration. Here, modified methods are presented for preparing honey samples for inductively coupled plasma-optical emission spectroscopy (ICP-OES) analysis and the high-performance liquid chromatography (HPLC) method to use them as fast and reliable methods in the food industry. ICP-OES analysis suggested that all analysed honey samples are natural, and HPLC-DAD (diode-arrray detection) analysis on the flavonoids showed that one sample is probably not natural. Invertase activity showed an overlap with this HPLC-DAD finding but suggested more samples as possibly adulterated, which can be neglected due to possible thermal treatment of those natural samples, causing a decrease in invertase activity. Therefore, results obtained from the analyses of investigated honey samples based on the mineral content, number, and quantity of flavonoids, together with the invertase activity, indicated that the combination of analyses could be a reliable tool for determining the quality of honey samples.

Background/Aim. Cyclamen hederifolium (C. hederifolium) Ait. belongs to the family Primulaceae, which includes 23 species of cyclamen, naturally distributed in the Central and Southern Europe, Western Asia and some parts of North Africa. This plant is considered highly poisonous and not suitable for human use. However, tuber extracts have been used in traditional medicine and homeopathy. The aim of this study was to investigate C. hederifolium growing naturally in Serbia for its metal content and biological activities (anti-oxidant, antibacterial, antifungal and cytotoxic activity). Methods. Content of metals was determined by atomic absorption spectrophotometric method. We used several different assays for assessment of antioxidant activity of both aqueous and ethanol extracts of C. hederifolium: 2.2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, 2,20-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging assay, ferric-reducing antioxidant power (FRAP) assay, total reducing power assay (TRP) and cupric reducing antioxidant capacity (CUPRAC) assay. The disk diffusion assay was used to investigate sensitivity of laboratory bacterial and fungal control strains against investigated extracts. Aqueous and ethanol extracts of C. hederifolium were examined on 4 different tumoral cell lines by in vitro MTT bioassay. Results. The presence of Mn, Ca, Mg, Fe, Zn, K, Cu was confirmed in aqueous and ethanol extract, as well as in whole tubers and soil, while Cr, Ni, Pb and Cd were not detected. Both aqueous and ethanol extract of C. hederifolium tubers showed antioxidant activity, that positively correlated to content of phenols and flavonoids in it. Aqueous extract was slightly superior in these terms than ethanol one. None of the tested extracts showed antimicrobial activity. Both investigated extracts showed cytotoxicity against four cancer cell lines 4T1, HCT116, CT26, LLC1, in the concentration range 15,625–2,000 μg/mL. Ethanol extract showed stronger cytotoxicity than aqueous ex-tract. Conclusion. Seven metals were identified in the C. hederifolium tubers, extracts and soil. Both extracts exhibited antioxidant and cytotoxic activity.

Three edible mushrooms of the Russulacea family (Lactarius deliciosus, Lactarius sanguifluus and Lactarius semisanguifluus), most frequently consumed in Serbia, were analyzed using the ICP-OES technique to evaluate the content of K, P, Ca, Mg, Na, Al, As, Cd and Pb, both in cap and stipe. Corresponding soils were analyzed, too. Based on the obtained values for the elemental composition of the mushrooms and the soil, bioaccumulation and translocation factors were calculated. All the examined mushrooms species were recognized as bioexclusors of analyzed toxic elements, but bioaccumulators of K, P and Ca. The studied mushrooms are good sources of macroelements. One portion of 300 g of fresh mushrooms had a significant contribution of K and P, exceeding 15 % of the recommended daily intake for the elements. On the contrary, mushrooms had a low potential to bioaccumulate toxic elements, and presented results indicated the regular consumption of wild edible mushrooms is safe for human health. Correlation analysis was applied to determine phosphorus’s influence on the elements’ content in the mushrooms and corresponding soils, demonstrating the most remarkable mushrooms' tendency to accumulate phosphorus.

Biperiden, an antiparkinsonian anticholinergic drug, has been found to inhibit enzymatic hydrolysis of butyrylthiocholine iodide, which is catalyzed by serum cholinesterase. By measuring the difference in the basic and inhibitory hydrolysis reaction rates, in the presence of biperiden as an inhibitor, it is possible to develop a kinetic method for its determination. Both systems, enzyme-substrate-chromogen and enzyme-substrate-chromogen-inhibitor, were characterized by biochemical parameters (KM = 0.326 – 0.330 mmol dm-3; Vmax = 40 - - 99 μmol dm-3 min-1), while inhibition was defined as non-competitive with the constant of inhibition Ki = 6.142 μmol dm-3. The reaction conditions have been optimized followed by determination of the calibration curve, the corresponding equation and the limits of detection and quantification yielding 3.84 and 12.80 nmol dm-3, respectively. Using the calibration chart, it is possible to determine biperiden in different samples in the concentration range of 0.035–35.940 μmol dm-3. Influence of a number of substances, found in the sample, on the reaction rate was also examined. The optimized method was applied for determination of biperiden in pharmaceutical preparations. Accuracy of the method was tested using the standard addition method. The proposed method has good sensitivity, selectivity, it is simple and fast, and above all easily accessible, and thus applicable in biochemical and pharmaceutical laboratories.

A study of medicinal flora of the Vidlič mountain situated in southeastern Serbia was conducted. The presence of 264 plant species, that are considered official or used in folk medicine is recorded. An overview of medicinal plants is given in a systematic order. For each herb species, main medicinal substances which enter in its chemical composition are listed. An overview of plant parts which are curative and which families contain the highest numbers of medicinal species, are presented. A special review of species that contain toxic substances, as well as species that have become so rare in their natural habitats by irrational exploitation in nature that they have been declared protected and strictly protected taxa in the Republic of Serbia is given.

Antioxidant and free radical scavenging activity of methanol, ethanol, ethyl acetate and chloroform extracts of aerial parts of Lamium purpureum L. was determined by DPPH, ABTS, FRAP and TRP assays. Contents of flavonoids and phenols were also investigated. The total phenolic content in the extracts, determined using Folin–Ciocalteu assay, ranged between 8.57 to 128.00 mg GAE/g d.e. while concentrations of flavonoids in the extracts varied from 24.20 to 39.80 mg QuE/g d.e. The highest phenolic content was found in methanol extract (128.00 mg GAE/g d.e.). The highest content of total flavonoids was identified in the methanol extract (39.80 mg QuE/g d.e.) and the lowest was in the chloroform (24.30 mg QuE/g d.e.). DPPH scavenging of the extracts was determined and obtained IC50 values ranged from 0.12 to 3.12 mg/mL of solution. The values of ABTS radical scavenging activity ranged from 0.35 to 1.80 mg AA/g. The highest ABTS antiradical activity was registered for methanol extract. The FRAP value was found within the range 0.08 to 1.04 μmol Fe/mg. The best radical scavenger was methanol (1.04 μmol Fe/mg). In reducing power assay different extracts of L. purpureum showed increasing of activity with increased concentration, and all extracts possessed substantial dose dependent antioxidant activity. The best reducing capacity was obtained with methanol extract of L. purpureum (0.0132 mg AA/mL). The results in this study confirmed that L. purpureum possesses moderate antioxidant properties.

This paper reports for the first time the effect of an acetone extract of Umbilicaria crustulosa on the micronucleus distribution of human lymphocytes, and on the cholinesterase activity and antioxidant activity by the cupric ion reducing antioxidant capacity (CUPRAC) method. Additionally, the total phenolic compounds (TPC) and the antioxidant properties were estimated via DPPH, ABTS and TRP assays. Moreover, the antibacterial activity against two Gram-positive and three Gram-negative bacteria were determined. Acetone extract of U. crustulosa at concentration of 1 and 2 μg mL-1 decreased a frequency of micronuclei (MN) by 10.8 and 16.8 %, respectively, acting more or slightly less than the synthetic protector amifostine (AMF, WR-2721, 11.4 %, at concentration of 1 μg mL-1). The tested extract did not inhibit cholinesterase activity nor did it exhibit activity toward the examined bacteria. The extract reduced the concentration of DPPH and ABTS radicals by 88.7 and 96.2 %, respectively. Values for total reducing power (TRP) and cupric reducing capacity (CUPRAC) were 0.6197±0.0166 μg ascorbic acid equivalents (AAE) per mg of dry extract, and 19.7641±1.6546 μg trolox equivalents (TE) per mg of dry extract, respectively. The total phenol content was 350.4188 ±14.587 μg gallic acid equivalents (GAE) per mg of dry extract. The results of the present study showed that U. crustulosa acetone extract is a promising candidate for in vivo experiments considering its antioxidant activity and protective effect on human lymphocytes. [Projekat Ministarstva nauke Republike Srbije, br. 172047]

The eggplant is a vegetable that has been used more in recent years thanks to the low calories versus high content of phenolic compounds. This study aimed to determine total phenolic, flavonoid, proanthocyanidin, and saponin content using spectrophotometric assays and antioxidant activity using 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH), 2,2'-azinobis(3-ethylbenzothiazol-6sulpho-nate (ABTS), total reducing power (TRP), ferric reducing antioxidant power (FRAP), and cupric reducing antioxidant capacity (CUPRAC) assassin peel and pulp of eggplant cultivars Robi, Tudela, Vernal, Aragon, and Rosa Bianca. The average total saponin content (TSC) in peel was 116.34 μg DSGE mg-1 DW (diosgenin equivalents per mg of extract's dry weight), whereas in pulp was slightly lower (107.86 μg DSGE mg-1 DW). A similar trend was observed for total phenolic content (TPC) (24.54 μg GAE mg-1 DW (gallic acid equivalents per mg of extract's dry weight) in peel and 15.88 μg GAE mg-1 DW in pulp); total proanthocyanidin (TP) (2.74 μg CE mg-1 DW (catechin equivalents per mg of extract's dry weight) in peel and 1.24 μg CE mg-1 DW in pulp) and total flavonoid content (TFC) (0,05 μg RE mg-1 DW (rutin equivalents per mg of extract's dry weight) in peel and 0.02 μg RE mg-1 DW in pulp). Principal component analysis (PCA) and cluster analysis (CA) methods were applied on the antioxidant activity (ABTS, DPPH, TRP, FRAP, and CUPRAC) and total bioactive compounds content (TSC, TPC, TP and TFC) parameters in order to reveal the relationships between analyzed samples. As a result of these approaches, analyzed samples were grouped into two groups. [ABSTRACT FROM AUTHOR]

The antioxidant activities of methanol and ethanol (10%, 30%, 50% and 96%) extracts of the aerial parts of Melittis melissophyllum L. were determined by DPP H, ABTS and FRAP assays. The content of flavonoids and phenols was also investigated. The total phenolic content in the extracts was determined using Folin-Ciocalteu assay; their amounts ranged between 63.5 and 111.7 mg GAeqv/g, while the concentrations of flavonoids were from 7.33 to 56.00 mg Queqv/g. IC50 values of the DPP H scavenging effect were from 0.109 to 0.664 mg/mL. The DPP H scavenging effect of the extracts was determined and the obtained IC50 values were from 0.109 to 0.664 mg/mL of solution. The values of ABTS radical activity were from 0.45 to 0.89 mg ascorbic acid/g. The FRAP value was within the range 0.160-0.382 mmol Fe/mg. The obtained values were analyzed by means of multivariate analysis, employing a hierarchical cluster analysis and between-groups linkage. The presented results confirmed that M. melissophyllum possesses good antioxidant properties and may serve as a promising source of natural antioxidants. [Projekat Ministarstva nauke Republike Srbije, br. 173029]

Primary role of peroxidase enzyme is to decompose endogenous hydrogen peroxide, when oxygen radical is being replaced by a less potent radical, which is its cosubstrates oxidized form. During this study, catalytic activity of horseradish peroxidase has been observed in the presence of antioxidants from vitamin group, such as C, E and A, i.e. their water-soluble forms. It was found that vitamin E showed no effect on the enzyme activity and fate of cosubstrate radicals from the group of benzidine derivatives. Vitamin C proceeds enzymatic reaction showing its antioxidative character, and absorbs electrons from radicals, bringing cosubstrate back to its relaxed state. On the other hand, vitamin A plays a role of uncompetitive peroxidase inhibitor, which is visible through decreasing initial rate of catalytic reaction, and is reflected as virtual decrease of enzyme concentration. Furthermore, it prolongs life of endogenous hydrogen peroxide, which could potentially lead to oxidative stress of cells. This inhibitory effect can be used in analytical purpose, for determination of retinol acetate content in a sample.

Propranolol, a widely used beta-blocker, inhibits the reaction of enzyme cholinesterase hydrolysis. Measurements of the hydrolysis rate differences between and inhibited reactions, allows the development of a kinetic method for its determination. Both systems, enzyme-substrate-chromogen and enzyme-substrate-chromogen-inhibitor, were characterized through biochemical kinetic parameters (KM = 0.326-0.330 mmol/L; Vmax = 40-42.99 μmol/Lmin), inhibition type was recognized as competitive, and inhibition constant, Ki, was determined to be 22.60 μmol/L. The detection and quantification limits were calculated as 0.004 and 0.0136 μmol/L, respectively. Accuracy and precision of proposed methods were tested. Proposed method is characterized with good sensitivity, selectivity, simplicity and rapidity, thus it is convenient for clinical applications.

In this study the content of chloroplast pigments (chlorophyll a, b, a+b, and carotenoids) in the leaves of Geranium macrorrhizum L., Doronicum columnae Ten., Aegopodium podagraria L. and Tussilago farfara L. from a beech forest that had undergone fire on Vidlič Mountain was determined. The same species of plants from a place that had not been exposed to fire were taken as controls. Chloroplast pigments were determined from acetone extracts of these plants spectrophotometrically. In the first year after the fire the content of chlorophyll a, b and a+b in Geranium macrorrhizum L. and Doronicum columnae Ten. was greater than in the plants not exposed to fire. The results were the opposite for Aegopodium podagraria L. and Tussilago farfara L. These differences can be attributed to the different physiology of the plants and consequently their different adaptation patterns. The carotenoid content was higher in the plant species at the fire site than in the area not exposed to fire.

Anew sensitive kinetic method has been developed for the determination of trace amounts of phenylhydrazine in the range of 1.08x10-7 to 1.08x10-6 g/cm3. The detection limit of this method is 0.008 μg/cm3, based on the 3Sb criterion. Themethod is based on the inhibitory effect of phenylhydrazine on the oxidation of Victoria Blue 4-R by KBrO3. The reaction was monitored spectrophotometrically at 596.3 nm. The method development includes the optimization of the reagent concentration and temperature. The kinetic parameters of the reaction are reported and a rate equation is suggested. The effects of certain foreign ions upon the reaction rate were determined for the assessment of the selectivity of the method. The new developed method was found to have fairly good selectivity, sensitivity, simplicity and rapidity.

A new reaction is suggested and a new method is elaborated for determination of micro amounts of Sn(II) based on its inhibiting effect on the oxidation of {4-bis[n-(dimethylamino) phenyl]methylene-2,5-cyclohexadiene-1-ilydene}dimethyl-ammonium chloride (crystal violet CV) by H2O2. The method sensibility is 0.4 μg/cm3. The probable relative error is 2.8-12.8 % for Sn(II) in the concentration interval of 3 to 0.8 μg/cm3. The kinetic equation for this process is given. The influence of some other ions on the reaction rate was tested. The method was applied to the determination of Sn(II) in a sample of microalloy.

In this study, the effect of various parameters such as solvent concentration (50 and 80%), extraction temperature (30, 40, and 50°C), and solvent/solid ratio (1:20, 1:25, and 1:30 g ml−1) on the extraction yield of apigenin-glycosides from parsley were investigated. The high-performance liquid chromatography analysis of parsley extracts confirms the presence of two apigenin-glycosides: apiin and malonyl-apiin. The kinetics of extraction was described using non-stationary diffusion model and Ponomarev's model. Models have a good fit to the experimental data (root mean square: RMS<±1.6%; standard deviation: SD<±1.5%; the coefficient of determination: R2 > 0.82). The effects of the process factors on the kinetic model parameters, equilibrium constant and Gibbs free energy change were assessed using the full factorial test plan 23. [ABSTRACT FROM AUTHOR]

The chemical composition of the essential oil and the volatiles obtained by static headspace (HS) of Inula oculus-christi L. is presented. The GC-MS analysis of the hydrodistilled oil resulted in the identification of 90 components, representing 92.7% of the oil. The most abundant compounds were: caryophyllene oxide (9.8%), trans-longipinocarveol (9.2%), eucalyptol (7.3%) and intermedeol (6.2%). The major constituent of I. oculus-christi L. HS volatiles was eucalyptol (87.4%). The antioxidant activity was evaluated by four different methods: 2,2-diphenyl-1-picryl-hydrazylhydrate free radical assay (DPPH), 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) method, total reducing power (TRP), ferric reducing antioxidant power (FRAP), and cupric reducing antioxidant capacity (CUPRAC). Total phenolic content in (TPC) examined oil was 177.95 µg GAE/mg oil. Radical scavenging potential of the oil was promising RSC-DPPH was 57.4% and RSC-ABTS was 82.7%. [ABSTRACT FROM AUTHOR]

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