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Thymus vulgaris L. (thyme), Origanum majorana L. (marjoram), and Origanum vulgare L. (oregano) were used to determine whether light modification (plants grown under nets with 40% shaded index or in un-shaded open field) could improve the quantity and quality of essential oils (EOs) and antioxidant activity. The yield of EOs of thyme, marjoram, and oregano obtained after 120 min of hydrodistillation was 2.32, 1.51, and 0.27 mL/100 g of plant material, respectively. At the same time under shading conditions plants synthetized more EOs (2.57, 1.68, and 0.32 mL/100 g of plant material). GC/MS and GC/FID analyses were applied for essential oils determinations. The main components of the thyme essential oil are thymol (8.05–9.35%); γ-terpinene (3.49–4.04%); p-cymene (2.80–3.60%) and caryophyllene oxide (1.54–2.15%). Marjoram main components were terpinene 4-ol (7.44–7.63%), γ-terpinene (2.82–2.86%) and linalool (2.04–2.65%) while oregano essential oil consisted of the following components: caryophyllene oxide (3.1–1.93%); germacrene D (1.17–2.0%) and (E)-caryophyllene (1.48–1.1%). The essential oil from thyme grown under shading (EC50 value after 20 min of incubation) have shown the highest antioxidant activity – 0.85 mg mL−1 in comparison to marjoram and oregano (shaded plants EC50 19.97 mg mL−1 and 7.02 mg mL−1 and unshaded, control plants EC50 54.01 mg mL−1 and 7.45 mg mL−1, respectively). The medicinal plants are a good source of natural antioxidants with potential application in the food and pharmaceutical industries. For production practice, it can be recommended to grow medicinal plants in shading conditions to achieve optimal quality parameters.

The aim of the presented work was to evaluate polar and non-polar extracts and fatty oil, isolated from pumpkin fruit (Cucurbita pepo L.), as a potential food supplements or raw material for cosmetic and pharmaceutical preparations. The extracts were isolated from the pumpkin peel, mesocarp and pulp and fatty oil from the seeds. The highest yield of total extractive matter and highest β-carotene content was determined in peel extracts at concentrations of 5.45 g/100gf.p.m and 136.22 μgg−1f.p.m, respectively. The presence of phenolic acids, flavonoids and carotenoids in polar and non-polar extracts was confirmed by UHPLC-DAD-ESI-MS and HPLC-DAD analysis, respectively. The yield of fatty oil in the seeds was 34.53%, while TLC and HPLC analysis showed the highest percentage of triglycerides (99.3%) and methyl esters (99.7%) in fatty oil and fatty acids methyl esters, respectively. Non-polar extracts of peel showed the highest antioxidant activity estimated by DPPH test (EC50 = 0.041 mgcm−3), fully comparable to activity of synthetic BHT antioxidant (EC50 = 0.021 mgcm−3). High linear correlation factor (0.9969) of EC50 values on β-carotene concentration suggests that β-carotene is the most responsible for significant antioxidant activity of non-polar extracts. Investigated isolates showed no antimicrobial activity against Lt. monocytogenes, St. aureus, E. coli, P. vulgaris, C. albicans, P. aeruginosa, B. cereus, K. pneumoniae and B. luteus under the examined conditions. Bearing in mind the results presented, the C. pepo isolates can be used as an alternative to synthetic antioxidants and a rich source of phytochemicals in food and pharmaceutical industry.

This work aimed to isolate the essential oil from the nutmeg (Myristica fragrans Houtt.) seed, to determine the qualitative and quantitative chemical composition of the obtained essential oil, as w...

Essential oil from rosemary (Rosmarinus officinalis L.) leaves was isolated by conventional hydrodistillation using hydromodulus of 1/15 m/v and by distillation after the plant material pretreatment with 10% v/v aqueous solution of natural deep eutectic solvent glyceline. Qualitative and quantitative composition of the oil was determined by GC/MS and GC/FID analyses, while its antioxidant activity was investigated by DPPH assay. The yield of the essential oil was higher after the pretreatment of the leaves. The distillation method affected the quantitative composition of essential oil, but not its qualitative composition. The essential oil from the pretreated leaves had a higher content of camphor, verbenone and borneol (33.3%, 12.2% and 8.3%, respectively) and better antioxidant activity than the oil obtained by classical hydrodistillation. The advantage of the pretreatment method is the effective improvement of the extraction efficiency of rosemary essential oil from rosemary leaves.

For quantitative analysis, contents of the selected three phenolic compounds and 17 minerals were determined in seven tea samples, i.e., infusions prepared on a traditional way from the dried aerial parts (herba) of St. John’s wort (Hypericum perforatum L.) found in the market, or collected from few locations in the Eastern and Southeastern Serbia. Four of them were commercially available in the markets: two in the bulk and two in filter bags. Phenolic and mineral contents were analyzed by high-performance liquid chromatography with diode array detector and inductively plasma optical emission spectrometry methods, respectively. The used instrument methods proved to be useful tools for analysis of St. John’s wort products. Main quantified phenolics (chlorogenic acid as phenolic acid, rutin and hyperoside as flavonols), as compounds with well-known biological activities, can contribute to a better quality of St. John’s wort teas from Serbia origin. Concentrations of the flavonols, rutin and hyperoside, in infusions samples were the highest, in the ranges of 13.1–79.6 mg/dm3 and 15.1–90.0 mg/dm3, respectively. Chlorogenic acid content was not much varied through the samples (2.6–3.6 mg/dm3). Among the macroelements, the potassium concentration (5868.9–10,608.5 μg/g) was the highest in all samples, and the concentration is in the order: K > P > Ca > Mg > Na. On the other hand, microelements concentrations follow the order: Zn > Mn > Cu. Among the analyzed toxic heavy metals, Cd was not detected in any of the samples, Pb detected only in two and Ba detected in small amounts in all samples.

The quality of the essential oil obtained from the wood verdure of fir from the territory of the Republic of Srpska was analyzed by examining the chemical composition, antioxidant activity and antimicrobial activity of the samples produced by the process of hydrodistillation in industrial production conditions. Two samples of fir essential oil were tested, depending on how long the plant material stood before the hydrodistillation process. The chemical composition of the essential oil was analyzed using GC/MS and GC/FID analysis. The antioxidant and antimicrobial activity of fir essential oil has been examined for its possible application as a natural antioxidant and antimicrobial agent. The obtained results prove the presence of 50 components in the first analyzed sample and 44 components in the second analyzed sample. The total components present (98.3% and 98.7%) were identified. The most common are monoterpene hydrocarbons, followed by sesquiterpene hydrocarbons and oxygen-containing monoterpenes. The antioxidant activity was investigated by the DPPH assay. Fir essential oil has shown the best antioxidant properties after 120 minutes of incubation with an EC50 value of 32.0 ± 0.03 mg/ml and 29.93 ± 0.08 mg/ml. Antimicrobial activity was investigated using a disk diffusion method. The results showed large zones of inhibition for both tested oil samples and for all eight bacterial strains analyzed (from 14.33 ± 0.58 mm to 16.67 ± 1.15 mm) as well as for the fungal strain. The chemical composition, antioxidant and antimicrobial action of fir essential oil obtained from plant material from the Republic of Srpska indicate significant phytomedicine potency.

The aim of the presented work was to investigate the potential of aqueous extract of cultivated strawberry (Fragaria x ananassa Duch.) leaves for stabilization of silver nanoparticles (AgNPs-E) synthesized at room (RT) and boiling temperature (BT). The synthesis and stability of AgNPs-E were monitored by UV-Vis spectroscopy confirming high stability of the AgNPs-E in the dark at room temperature. The Fourier-transform infrared spectra suggest that molecules containing oxygen and nitrogen functional groups (NH, (NH)C=O, CNO, C-O-C and OH) participate in the reduction and stabilization of formed nanoparticles. As determined by the DPPH test, AgNPs-E synthesized at RT exerted higher antioxidant activity as compared to AgNPs-E synthesized at BT (EC50 values of 0.025 and 0.039 mg cm-3, respectively). Also, the AgNPs-E synthesized at RT exerted higher antibacterial activity against Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Bacillus subtilis and Bacillus luteus. Examination of the AgNPs-E on HeLa and MDCK cell lines showed concentration-dependent and cell line specific effects on the cell viability as evaluated by the MTT test. The obtained results indicate that synthesized AgNPs-E can be used as a base material in production of pharmaceutical preparations for potential skin applications.

Both as food and spice, pepper is unavoidable on the tables in Serbia and many other countries. Because of its popularity, the contents of capsaicin, total phenols and total flavonoids, as well as the antioxidant activity of hot peppers extracts (Leskovac hot pepper, so called "džinka" and chili Habanero pepper) were examined in this paper. The extracts were obtained by Soxhlet extraction with 96% v/v ethanol. The content of capsaicin in dry extracts of the studied hot peppers was calculated by the HPLC method (13.038 mg/g d.e. for "džinka" and 76.516 mg/g d.e. for Habanero pepper, respectively). Total phenol and total flavonoids contents were examined by the UVVIS method. Leskovac "džinka" was characterized by both higher contents (47.17 mgGAE/g d.e. and 14.64 mgGAE/g d.e., respectively) in comparison to chili Habanero pepper (29.43 mgGAE/g d.e. and 0.25 mgGAE/g d.e., respectively). DPPH test was used for the antioxidant activity determination. The EC50 values obtained indicate a better antioxidant activity of Leskovac "džinka" (1.760 mg/ml) compared to chili Habanero pepper (6.016 mg/ml).

Allspice (Pimenta dioica (L.) Merr.) fruit was used for the essential oil and ethanolic extract isolation in the present study. The essential oil was obtained by Clevenger-type hydrodistillation from allspice fructus with hydromodule 1:10 m/v during 180 minutes, while the ethanolic extract was obtained by reflux extraction at the boiling temperature with solvomodule 1:10 m/v during 120 minutes. The qualitative and quantitative composition of the essential oil was determined by GC/MS and GC/FID methods, respectively. The total phenolic and total flavonoid content in the extract was determined according to the Folin-Ciocalteu and aluminium chloride methods, respectively the antioxidant activity of allspice fructus essential oil and the extract was determined spectrophotometrically by DPPH assay and the antimicrobial activity using the microdilution method on the following microorganisms: two types of bacteria (Bacillus cereus, Salmonella spp.) and one yeast (Candida albicans), pairs of reference strains and their isolates. The obtained results proved the presence of 20 components in the essential oil, mainly oxygenated monoterpenes. The extract and essential oil have shown the best antioxidant activities after 20 and 60 minutes of incubation with EC50 values of 0.011 mg/cm³ and 0.023 mg/cm³, respectively. Both the essential oil and the extract have shown an inhibitory effect on all investigated microorganisms. Allspice ethanolic extract showed a better inhibitory effect on B. cereus and S. enterica (isolate and ATCC strains) than the essential oil. On the other side, allspice essential oil showed a better inhibitory effect on C. albicans strains. The results obtained indicate that both the essential oil and the extract isolated from allspice fructus are a good source of natural antioxidants and antimicrobial agents with potential applications in food and pharmaceutical industries as a safer alternative to the synthetic additives.

The manipulation of light intensity by shade nets can lead to exchanges in the phytochemical quality and antioxidants ofsome herbs. This study aimed to determine whether shading by pearl nets (50% shade index) could improve the bioactive compounds in several medicinal herbs such as thyme (Thymus vulgaris L.), marjoram (Origanum majorana L.), oregano (Origanum vulgare L.), lemon balm (Melissa officinalis L.), and peppermint (Mentha piperita L.), and their correspondingherbal extracts during a four-week maceration process in ethanol. Oregano and thyme provided the highest yield of total extractive substances (TES) from both shaded and non-shaded plants. Among all studied herbs, the highest level of antioxidants, expressed as total phenolic content (TPC) was found in extracts from shaded plants of lemon balm. Herbal extracts produced from non-shaded thyme and marjoram had higher flavonoid contents compared to herbal extracts from shaded plants. Accumulation of the investigated secondary metabolites depends more on the specificity of a plant species rather than light intensity. This study was an initial step in the production of medicinal plants with an increased quantity of antioxidant and other bioactive compounds during the maceration process in extracts, aimed to be used as natural alcoholic productwith added value.

The purpose of the present study was to determine chemical composition and antioxidant activity of commercial peppermint (Aetheroleum menthae piperitae) and basil (Aetheroleum basilici) essential o...

The essential oil from birch (Betula pendula Roth.) buds was obtained by Clevenger-type hydrodistillation. Its qualitative and quantitative composition was determined by gas chromatography in combination with mass spectrometry (GC/MS) and flame ionization detection (GC/FID), respectively. Twenty-seven compounds, mainly sesquiterpene hydrocarbons (78.7%) and oxygenated sesquiterpenes (14.8%), were identified comprising 93.5% of total identified components in the essential oil. The most abundant compounds were germacrene D (21.7%) and -cadinene (17.0%). Antimicrobial activity of isolated essential oil against Gram-positive and Gram-negative bacteria as well as fungus Candida albicans was determined using the disc-diffusion method. The isolated essential oil has shown antimicrobial activity against the following microorganisms: Proteus vulgaris, Bacillus luteus and Klebsiella pneumoniae. Antioxidative activity of the essential oil was determined using the DPPH assay, after adding DPPH radical and after 20min, 30min and 60min incubation with radical. Essential oil showed antioxidant activity.

Synthesis of silver nanoparticles using plant extract of Fumaria officinalis L., (AgNPs-E), as a reducing and capping agent, is presented in this study. Formation of AgNPs-E is monitored by measuring surface plasmon resonance (SPR) absorption band in the UV-VIS range 200–800 nm. Based on UV-VIS spectra, the SPR band of AgNPs-E obtained by synthesis at the room and boiling temperatures appeared at 438 and 440 nm, respectively. After 15 days of equilibration, the SPR band was slightly shifted by 6 nm indicating a good stability of the formed complex at room temperature. The bands in the range 200–400 nm, originating from π−π* and n−π* transitions in the extract, disappeared in the spectra of AgNPs-E after reduction. Morphology, size and shape of formed AgNPs-E were investigated by scanning electron microscopy. The presence of both elemental silver and its crystalline structure (face centered cubic type) in AgNPs-E was confirmed by the energy dispersive X-ray spectroscopy (EDX) and X-ray diffraction (XRD) analyses. Particles size was determined by the XRD analysis: 20 ± 1 and 18 ± 1 nm at room (sample 1) and boiling temperature (sample 2), respectively. Fourier transform infrared spectroscopy (FTIR) assessments were carried out to identify biomolecules of F. officinalis responsible for the reduction and capping of the bioreduced silver nanoparticles and to predict their role in silver nanoparticles synthesis. FTIR spectroscopy indicated the activity of different functional groups during the synthesis by the changes in νC–OH, δOH, νC–O–C and (NH)C=O modes in the AgNPs-E spectrum relative to that of the extract. The AgNPs-E particles showed strong antibacterial activity against Staphylococcus aureus ATCC 25923, Bacillus cereus ATCC 11778, Bacillusluteus in haus strain, Bacillus subtilis ATTC 6633, Listeria monocytogenes ATCC 15313, Escherichia coli ATTC 25922, Pseudomonas aeruginosa ATTC 27853, Klebsiella pneumoniae ATTC 700603, Proteus vulgaris ATTC 8427, and antifungal activity against Candida albicans ATTC 2091.

This study reports for the first time the total extractive matter yield, total phenolics and total flavonoids content (TPC and TFC, respectively), chemical composition, and ability to scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals of the black mustard seed (BMS) extracts obtained by deep eutectic solvents (DESs). The following DESs: triethanolamine:glycerol (TEOA:G), triethanolamine:propylene glycol (TEOA:PEG), and choline chloride:urea (ChCl:U), as well as their mixtures with water and ethanol at a ratio of 1:4 (v/v), were used. Their extraction efficiencies were compared with the efficiency of ethanol that is most frequently used to extract phenolic compounds from BMS. The TPC and TFC of the obtained extracts were in the ranges of 19.9 +/- 0.1 to 32.2 +/- 0.2 mg gallic acid equivalents/g and 3.9 +/- 0.1 to 7.4 +/- 0.3 mg quercetin equivalents/g, respectively. The BMS extracts obtained with the pure ChCl:U and the mixtures of ChCl:U or TEOA:G with ethanol showed the best antioxidant activity (IC50 = 100 mg/mL). The obtained extracts contained syringic acid, ellagic acid, gallic acid, caffeic acid, caftaric acid, rutin, kaempferol, apigenin, taxifolin, hyperoside, and vitexin.

The quality of the essential oil obtained from white pine wood greenery from the territory of the Republic of Srpska was analyzed by examining the chemical composi- tion and antimicrobial activity of samples produced by the hydro-distillation process in industrial production conditions. The chemical composition of the essential oil was analyzed by GC/MS and GC/FID analysis. The antimicrobial activity of the ob- tained white pine oil was investigated for its possible use as an antimicrobial agent. The obtained results prove the presence of 37 components in the analyzed sam- ple, which represents 98.5% of the total components present. The most common are monoterpene hydrocarbons, followed by sesquiterpene hydrocarbons, much less represented are monoterpenes containing oxygen and aromatic compounds, while aliphatic esters are found only in traces. Antimicrobial activity was examined using the disk diffusion method. The results showed large zones of inhibition, both for the tested oil sample and for all eight analyzed bacterial strains (from 12.00 ± 0.00 to 17.33 ± 1.15mm), as well as for the fungal strain. The chemical composition and antimicrobial action of essential white pine oiled from plant material originating from the territory of the Republic of Srpska indicates its significant phytomedical potential.

The optimization of the headspace solid-phase microextraction in combination with the gas chromatography-mass spectrometry (HS-SPME-GC/MS) for the qualitative identification of the aroma compounds in fruit spirits (Williams, plum spirit, grape spirit , apricot spirit) was the subject of the presented paper. The SPME extraction conditions (temperature, time, and NaCl amount) have been varied in order to achieve the more efficient chromatographic separation of the gas mixtures, to efficiently salt-out a higher number of less volatile compounds, and to save the energy during the sample preparation. Optimal parameters were determined as: T = 40 °C; m NaCl = 2 g; dilution = 10 times. According to the results obtained, the analyzed samples of Serbian fruit spirits produced from different fruits have a comparable composition of volatile aroma compounds to whiskey. The detection and definition of specific compounds that are "fingerprint" of fruit spirits could be an efficient method of the product authentication developing.

The aim of this study was to isolate the essential oil from ripe and disintegrated caraway fruit by Clevenger-type hydrodistillation, to determine its qualitative and quantitative composition and to examine its antioxidant activity, as well as to determine the qualitative and quantitative chemical composition of the caraway volatile compounds extracted by HS-SPME technique. The essential oil from disintegrated caraway fruit was obtained by Clevenger-type hydrodistillation with hydromodulus 1:10 m/V during 180 minutes. Volatile compounds from caraway fruit were extracted by headspace-solid phase microextraction (HS-SPME) using 75 mm fiber coated with Carboxen®/Polydimethylsiloxane (PDMS). The qualitative and quantitative composition of the essential oil and volatile compounds was determined by gas chromatography coupled with mass spectrometry (GC-MS) and flame ionization detection (GC-FID). Twenty-seven different compounds were separated and identified in the essential oil. Twenty-six compounds were identified in the volatiles extracted by HS-SPME. The most abundant compounds in both examined samples were limonene and carvone. The content of carvone in the essential oil was 72%, while the limonene content was 25.6%. In the volatiles obtained by HS-SPME, the limonene was present with 72.1% and carvone with 24.6%. The difference in the chemical composition is due to the oxidation of limonene to carvone during the hydrodistillation. The antioxidative activity of the caraway essential oil was determined by using DPPH assay immediately after adding DPPH radical and after 20 and 40 min of incubation with the radical. The caraway fruit essential oil showed a satisfactory antioxidant activity. The oil incubated for 40 min (EC50=4.6 mg/cm³) showed the best antioxidant activity.

This study reports the yields, physicochemical properties, fatty acid compositions, and antioxidant activities of black mustard (Brassica nigraL.) seed oils extracted by cold pressing and the solvent extraction using different solvents (n-hexane, trichloroethylene, and a mixture of trichloroethylene and the triethanolamine:glycerol deep eutectic solvent (TEOA:G DES) with no or after pretreatment with the TEOA:G DES. Density (0.9791 +/- 0.0003-0.9851 +/- 0.0003 g/cm(3)), viscosity (121.15 +/- 1.62-123.32 +/- 0.10 mPas), boiling point (179-185 +/- 1 degrees C), refractive index (1.4692 +/- 0.0006-1.4758 +/- 0.0011), acid (2.08 +/- 0.19-3.22 +/- 0.15 mg KOH/g), iodine (99.2 +/- 1.6-105.4 +/- 1.5 g I-2/100 g), peroxide (1.68 +/- 0.01-1.82 +/- 0.03 mmol O-2/kg), and saponification values (165.1 +/- 2.7-169.4 +/- 2.3 mg KOH/g), fatty acid composition and antioxidant activity of the extracted oil were determined in order to evaluate its potential use as a foodstuff, a pharmaceutical or a feedstock for biodiesel production. Although the maceration of black mustard seeds with sole trichloroethylene yielded the highest oil yield of 31.10 +/- 0.42 g/100 g, it required almost twice the volume of trichloroethylene compared to the volume applied in the macerations after the pre-treatment with the TEOA:G DES and with the mixture of trichloroethylene/TEOA:G DES. The oil extracted by the extraction in the presence of TEOA:G DES has similar physicochemical properties but better antioxidant activity than the oil extracted by cold pressing.

Lemon balm, mint, and basil were used to determine whether light modification (plants grown under pearl shade nets with 40 % shaded index or in un-shaded open field) could improve the quantity and quality of essential oils (EOs) and antioxidant activity. The yield of EOs of lemon balm, mint and sweet basil obtained after 120 min of hydrodistillation was 0.18, 2.00, and 1.12 mL/100 g, respectively. At the same time under shading conditions plants obtained more levels of EOs (0.22, 2.23, and 1.32 mL/100 g). The main components of the lemon balm essential oil are geranial (6.84–7.78 %); neral (3.02–3.52 %) and piperitenone oxide (1.67–5.36 %). The main components of mint essential oil are piperitenone oxide (12–14 %) and 1,8 cineole (1.32–2.27 %), while basil essential oil consists of the following components: linalool (9.06–10.2 %); 1,8 cineole (1.26–8.06 %) and α-trans bergamotene (1.21–1.47 %). The essential oil obtained from basil leaves grown under shading has shown the highest antioxidant activity (EC50 value after 20 min of incubation = 0.68 mg/mL). Medicinal plants are a good source of natural antioxidants with potential application in the food and pharmaceutical industries. Medicinal plants can be recommended to grow in shading conditions for achieving optimal quality parameters.

Aronia (Aronia melanocarpa) leaves at the end of harvest season (in the leaves senescence vegetative stage), when it represents an agricultural waste material, have been extracted under different conditions in order to assess potential of this plant material in valuable bioactive compounds recovering. Response surface methodology was used in screening of the most important parameters effecting extraction (ethanol concentration, extraction time and solvomodule), as well as for mathematical modeling and process optimization. The optimal extraction conditions (ethanol concentration of 49.18%, extraction time of 75.26 min and solvomodule of 0.19 g/mL) that simultaneously maximize extractive matter yield and total phenolic content, minimizing the EC50 value, were proposed by desirability function approach. Finally, in further assessment of aronia agricultural waste as a potential source of bioactive compounds, the extract obtained under proposed optimal conditions was analyzed by UHPLC-MS, while the content of macro- and microelements was determined by ICP-OES technique.

Wild cyclamen tubers (Cyclamen purpurascens Mill.) (Kukavica mountain, south­east Serbia) was used as an extraction material in this study. The aqueous extract has been obtained by reflux extraction at the boiling temperature with hydromodulus 1:20 m/v during 180 minutes. The identification of bioactive components in the extract was performed by using UHPLC-DAD-HESI-MS analysis. The concentra­tions of macro- and microelements in the extract were determined by Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES). Isocyclamin and des­glucocyclamin I were identified in the obtained extract. Potassium was in the highest concentration - 10241.65 mg/kg of the plant material, while zinc was present in the highest concentration (11.57 mg/kg of plant material) among heavy metals. Presented results have shown that the obtained extract from wild cyclamen tubers is a potential source of triterpenoide saponin components isocyclamin and desglucocy­clamin I, as well as macro- and microelements.

Essential oils, as sources of natural products, represent an alternative to synthetic antioxidants and antimicrobial agents in food and pharmaceutical industry, alternative medicine and natural the...

The aim of this study was to determine the antimicrobial activity of essential oils obtained from sweet basil (Ocimum basilicum L. cv. ‘Genovese’) cultivated in the open field under different shading conditions (red, blue, and pearl nets with a shade index of 50 % and full sunlight exposure (control plants)), harvested at different times. The antimicrobial activity of basil essential oils (BEOs) obtained from all samples was determined for four microorganisms, while determinations for an additional five microorganisms included samples from non-shaded plants, plants grown under red and pearl nets, and second harvest of plants grown under blue net. Basil essential oil exhibited antimicrobial activity surpassing the activity of relevant commercial antibiotics regardless of growing conditions in the case of B. cereus, K. pneumoniae and C. albicans, while superior antimicrobial activity was exhibited in the case of essential oils from plants grown under blue nets in the case of S. aureus, E. coli and P. vulgaris. The influence of the application of colored shading nets was highly significant (p &lt, 0.01) in the cases of all analyzed microorganisms except C. albicans and P. aeruginosa, while the influence of harvest time was proven in the cases of all microorganisms except K. pneumoniae. ANOVA proved that antimicrobial activities are highly dependent on the methods of plant production, shading treatment, and harvest time. Obtained results are discussed in relation to previously determined composition and yield of essential oils from basil grown under shade nets and harvested in different periods.

Cranberry fruit (Vaccinium macrocarpon L., Ericaceae) is a valuable material for the production of juices and other fruit products, quite popular among consumers in Serbia. Different types of cranberry juice are produced in Serbia and other countries, but the chemical composition, as well as the antimicrobial and antioxidant activities of juices depend on both the used technology and the region in which cranberries are grown. Within this study, the screening of bioactive components found in cranberry juice from Serbia was performed by the UHPLC-DAD-MS/MS method, as well as the determination of its antibacterial and antioxidant activities. The juice was obtained by cold-pressing of ripe cranberry fruit grown in Serbia and enriched with sugar. Among the seventeen identified compounds, the most common were phenolic compounds and hexoses, followed by organic acids. The antibacterial activity of the juice was assessed by the disc diffusion method against Listeria monocytogenes, Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, Salmonella enteritidis, Klebsiella pneumoniae, and Pseudomonas aeruginosa. The DPPH test was used to determine the antioxidant effect. The cold-pressed cranberry juice showed higher antibacterial activity against Gram-positive bacteria. The obtained results will be useful in promoting further progress and development of cranberry cultivation and processing in Serbia.

Antioxidant activity of aqueous extracts from two cultivated strawberry leaves, Senga Sengana and Mount Everest, obtained by maceration, reflux extraction and ultrasonic extraction, was investigated. Antioxidant activity of extracts was assessed by DPPH, FRAP, FIC and H2O2 test; total phenolics and flavonoids were assessed by Folin–Ciocalteu and aluminium chloride methods, respectively. Extract obtained from Senga Sengana by reflux extraction, containing higher amounts of extractive matter, total phenolics and flavonoids, has shown higher antioxidant activity (EC50(DPPH) = 10.4 μg/mL; FRAP = 258.61 mgFe2+/g dry extract.; FIC activity = 62.6% in concentration of 2 mg/mL; H2O2 scavenging = 25.43% in concentration of 0.5 mg/mL) compared to extracts obtained by other extraction techniques. Rutin, coumaroyl-galloyl-glucopyranoside, hydroxyphenylethyl-coumaroyl-glucopyranoside, derivates of hydroxyphenylethyl-coumaroyl-glucopyranoside, chlorogenic acid and p-coumaroylhexose have been identified by UHPLC...

Dichloromethane soil samples extracts were prepared using Soxhlet extraction technique, and after clean-up step, gas chromatography-mass spectrometry analysis of 16 priority polycyclic aromatic hyd...

The subject of this study is to determine chemical composition, antioxidant and antimicrobial activity of essential oil obtained from chamomile flowers (Matricaria chamomilla L.). The essential oil...

The temperature- and pH-responsive poly(N-isopropylacrylamide-co-acrylic acid), p(NIPAM-co-AA), copolymer was synthesized by free radical polymerization and examined as a carrier for modified release of biochanin A. Biochanin A is a biologically active methoxylated isoflavone which exhibits estrogenic and other pharmacological activities. Due to its poor aqueous solubility and extensive first-pass metabolism, biochanin A has low bioavailability. The aim of this work was to incorporate biochanin A into the synthesized p(NIPAM-co-AA) copolymer and to examine its release at the body temperature and pH values that correspond to pH values of vaginal and rectal cavities. The amount of released biochanin A was monitored by the ultra-visible spectroscopy (UV-Vis) method. The structure of synthesized p(NIPAM-co-AA) copolymer and copolymer with incorporated biochanin A were characterized by using Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) methods. The content of residual monomers in the synthesized copolymer was analyzed by using the high-pressure liquid chromatography (HPLC) method. The swelling behavior of p(NIPAM-co-AA) copolymer was monitored in relation to the temperature and pH values of the surrounding medium. For modelling the process of p(NIPAM-co-AA) copolymer swelling, the full three-level factorial design was applied.

Five extraction techniques, maceration, reflux, Soxhlet, Tillepape, and ultrasonic extraction, were used to obtain the extractive matter from nettle leaves. The antioxidant activity of extracts was assessed by DPPH, FRAP, and H2O2 test, while the total phenolic and total flavonoid content was determined according to the Folin–Ciocalteu and aluminium chloride methods, respectively. Model Ponomarev and a non-stationary diffusion model through the plant material were used for modelling extraction process. The extract obtained by Soxhlet extraction, containing higher amounts of extractive matter as well as phenolic and flavonoid compounds, showed better antioxidant activity than those obtained by other extraction techniques.

The objectives of this study were to investigate the effects of coloured shade-nets (pearl, blue, or red all with a 50% shade index) compared to non-shaded plants on quality traits on the Discoa and ICE 40102 (green-leaf) and Eglantine (red-leaf) lettuce cultivars. Total chlorophyll content depended on the shading and lettuce genotype. The chlorophyll a and b contents were higher in shaded than unshaded plants. The cv. Eglantine had the highest total chlorophylls (637.03 μg∙g-1 F.M.) content. The highest carotenoid content was in leaves of cv. Discoa under pearl nets (208.89 μg∙g-1 F.M). The highest total phenols content (76.70±1.9 mg∙g-1 GAE D.M.) was in cv. Discoa under red shade; the highest flavonoids content was for cvs. Discoa (42.97) and Eglantine (42.91 mg∙g-1 RU D.M.) under blue shade. The blue and pearl shade nets resulted in slightly higher flavonoid contents in lettuce leaves compared to unshaded plants. The cv. Eglantine, under blue shade had the highest antioxidant capacity (EC50 - 0.197 mg∙mL-1). Red lettuce cv. Eglantine could be recommended for production due to its higher antioxidant properties. Generally blue shade can be used to retain antioxidant capacity.

Wild cyclamen tubers (Cyclamen purpurascens Mill.) (mountain Kukavica, Southeast Serbia) were used as material for extraction in this study. Aqueous extract was obtained by reflux extraction on boiling temperature with hydromodulus 1:20 m/v during 180 minutes. The total phenolic content was determined spectrophotometrically by the method of Folin-Ciocalteu, and the total flavonoids content by method with AlCl3. The antioxidant activity of extract was investigated spectrophotometrically by DPPH and ABTS test. Disc-diffusion method was used for antimicrobial activity investigation on the following pathogenic microorganisms: Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis, Pseudomonas aeruginosa and Klebsiella pneumoniae. The content of total phenols was 8.27 mg GAE/g dry extract while the total flavonoid content was 11.51 mg RE/g dry extract. The extract concentrations required to neutralize 50% of the initial concentration of DPPH radicals (EC50) after 20 minutes incubation and immediately after adding DPPH radical solution were 0.413 and 2.0 mg/ml, respectively, while concentrations of extract required to neutralize 50% of the initial ABTS radicals concentration is 0.743 mg/ml. The extract showed the highest antimicrobial activity on bacteria Staphylococcus aureus. The presented results indicate that cyclamen tubers extract is a potential source of natural antioxidants and antimicrobial agents.

The subject of this study was the examination of the chemical composition and antimicrobial activity of peppermint oil (Menthae piperitae L.), that was produced from the plant collected from the plantation in the city of Banja Luka. To examine the chemical composition of the Mentha piperita essential oil we used the GC-FID method. On it’s chromatogram it was clearly shown that the main components of the oil were menthole with the content of 43.66%, menthone 20.02%, iso-menthone 7.73%, following by 1,8-cineole with a percentage of 6.49% and menthil-acetate with 3.31%. The content of the previously mentioned components were all in accordance with the suggestions made by Ph.Eur 8.0. For the purpose of examination antimicrobial activity of the peppermint oil we used agar diffusion method. The results have shown that etheric oil of Menthae piperitae reflects very good antimicrobial activity on all bacterial cultures that we used in this study. The inhibiton zones moved from 12 mm for Pseudomonas aeruginosa to 37.66 mm for Staphylococcus aureus. Such an outcome of our study, and many others from all around the world, indicates the future potential use of peppermint oil in the treatment of bacterial infections, maybe even lowering the use of antibiotics.

The essential oils of plants have gained great interest in the pharmaceutical industry due to their biological activities. The objective of the present study was to evaluate the influence of fennel (Foeniculum vulgare Mill.) fruit milling on the essential oil yield, chemical composition and antimicrobial activity. The essential oil was obtained from disintegrated and non-disintegrated fruit by Clevenger-type hydrodistillation with hydromodulus 1:10 m/V during 180 min. The essential oil composition was analyzed using gas chromatography-mass spectrometry (GC–MS) technique combined with flame-chromatographic gas–chromatographic detection (GC-FID). Milling impacted the essential oil yield (3.6 ± 0.3 and 4.0 ± 0.2 g/100 g of plant material from non-disintegrated and disintegrated fruit, respectively), and quantitative, but not qualitative composition of essential oil. Among fourteen identified compounds, the most abundant in essential oil was trans-anethole (64.87 ± 0.07% and 69.89 ± 0.06% from non-disintegrated and disintegrated fruit, respectively). The antimicrobial activity of oil was evaluated using Staphylococcus aureus, Escherichia coli, Proteus vulgaris, Candida albicans, Pseudomonas aeruginosa, Bacillus subtilis, and Klebsiella pneumoniae. The essential oil isolated from disintegrated fruit showed higher antimicrobial activity than the essential oil from non-disintegrated fruit. Among the tested microorganisms, Candida albicans was the most sensitive to the essential oil, followed by Bacillus subtilis.

Light modification by colour shade net manipulation plays an important role in the synthesis of bioactive compounds and has been shown to affect quantity and quality of essential oils in basil. In the present study, basil was grown in the soil under net-house cover by pearl and red nets (50% shade index), or in unshaded condition (open field-control). Sweet basil (Ocumum basilicum L. cv. ‘Genovese’) were used to determine whether different times of harvest could improve yield, chemical composition of essential oils and antioxidant activity under light stress conditions. The lowest accumulation of essential oils was observed in the second harvest from unshaded, control plants (1.02 mL/100 g) while the highest oil accumulation was achieved in first harvest from red nets (3.23 mL/100 g). The main constituents found in the oil were linalool (46.7–53.9%), eugenol (9.7–20.9%), 1,8-cineole (8.7–15.3%), epi-α-cadinol (3.3–4.5%) and α-trans-bergamotene (2.2–3.4%). Light manipulation by color shade nets also increased antioxidant activity. Plants grown under blue shade nets from the second harvest are characterized by the highest eugenol content (20.9%) and highest antioxidant activity (efficient concentration - EC50, 0.003 mg mL−1). Direct sowing, high plant density, colour shade nets and successive harvests represents a new technology in basil production with high essential oil yield and quality.

In the last decade, agricultural producers in Serbia have been massively reoriented to aronia (Aronia) cultivation. Due to low and inadequately organized purchase, the accumulation of large amounts of aronia forced the producers to organize and start processing. The most common and most represented products of aronia in our market are cold pressed juice, pasteurized sweet syrup and liqueur. Processing can affect a decrease of the bioactive components concentration, as well as the reduction of beneficial effects on the human organism. The antioxidant activity of cold pressed aronia juice, pasteurized sweet aronia syrup and liqueur obtained in double processing of aronia (candied aronia) was studied using the DPPH (1.1-diphenyl- 2-picrylhydrazyl radical) test. The cold pressed juice has shown a higher antioxidant activity compared to the pasteurized sweet syrup. It was noticed that the antioxidant activity of liqueur from candied aronia was preserved but markedly decreased. So far, fresh aronia has mainly been used for the liqueur production, but candied aronia has been proved as an appropriate raw material, too. The liqueur obtained in that way could be applied in food and beverage industry as a substitute for synthetic antioxidants, food colors and aromas.

Earth smoke herb (Fumaria officinalis L.) was used as a source of extractive mat­ter in this study. The aqueous extract was obtained by pouring boiling water over the plant material (1:20 m/v) and letting it stay for 90 minutes at room temperature. The identification of bioactive compounds in the extract was performed using the UHPLC-DAD-ESI-MS analysis. The content of total phenols and total flavonoids was determined spectrophotometrically by Folin-Ciocalteu and AlCl3 methods, respectively. The antioxidant activity of the extract was also investigated spectropho­tometrically by DPPH and ABTS assays. A discdiffusion method was used in the antimicrobial activity investigation on the following pathogenic microorganisms: Candida albicans, Proteus vulgaris, Pseudomonas aeruginosa, Bacillus subtilis, Staphy­lococcus aureus, Escherichia coli and Klebsiella pneumoniae. Seven components were detected in the obtained extract, while caffeic and rosmarinic acid were identified. The main component in the extract was rosmarinic acid (16.7 g/100 g of dry extract). The content of total phenols was 77.67 mg GAE/g of the dry extract while the content of total flavonoids was 21.21 mg RE/g of the dry extract. The extract concentrations required to neutralize 50% of the initial DPPH radical concentration were 7.38 and 8.18 μg/cm3 after 30 and 20 min of incubation, respectively, as well as 48.6 μg/cm3 with no incubation. The extract concentration required to neutralize 50% of the initial ABTS radical concentration was 96.0 μg/cm3. The obtained extract has shown the highest antimicrobial activity against Candida albicans fungus. The presented results indicate that the aqueous earth smoke extract should be rightly considered as a potential source of natural antioxidants and antimicrobial agents.

In order to find the most efficient natural solvent for the extraction of quercetin, its solubility was tested in the selected deep eutectic solvents (DESs) such as choline chloride:urea (ChCl:U), choline chloride:glycerol (ChCl:G), citric acid:D-glucose (CA:Glc), citric acid:D-fructose (CA:Frc), lecithin:urea (Lec:U) and lecithin:glycerol (Lec:G), as well as in their aqueous solutions, in a temperature range of 6-75°C. Quercetin was most successfully dissolved in CA:Frc, followed by ChCl:U and ChCl:G, while CA:Glc was the least efficient. The lecithin-based DESs with urea and glycerol did not dissolve quercetin. The most optimal DES (ChCl:U) was employed in the extraction of quercetin from waste onion (Allium cepa L.) tunic and its efficiency was compared with those of methanol and ethanol. The largest amount of quercetin was extracted with ethanol, followed by methanol and ChCl:U, but quercetin extracted with this DES contained the least amount of impurities. Therefore, the ChCl:U DES was highly recommended for the selective extraction of quercetin.

This study investigates the biological activity of pure allantoin (PA) and aqueous extract of the comfrey (Symphytum officinale L.) root (AECR) standardized to the allantoin content. Cell viability and proliferation of epithelial (MDCK) and fibroblastic (L929) cell line were studied by using MTT test. Anti-irritant potential was determined by measuring electrical capacitance, erythema index (EI) and transepidermal water loss of artificially irritated skin of young healthy volunteers, 3 and 7 days after application of creams and gels with PA or AECR. Pure allantoin showed mild inhibitory effect on proliferation of both cell lines at concentrations 40 and 100 µg/ml, but more pronounced on MDCK cells. Aqueous extract of the comfrey root effect on cell proliferation in concentrations higher than 40 µg/ml was significantly stimulatory for L929 but inhibitory for MDCK cells. Pharmaceutical preparations that contained AECR showed better anti-irritant potential compared with PA. Creams showed better effect on hydration and EI compared with the gels that contained the same components. Our results indicate that the biological activity of the comfrey root extract cannot be attributed only to allantoin but is also likely the result of the interaction of different compounds present in AECR. Topical preparations that contain comfrey extract may have a great application in the treatment of skin irritation. Copyright © 2015 John Wiley & Sons, Ltd.

In this work the impact of four different techniques of Clevenger-type hydrodistillation (technique I–IV) on the yield, hydrodistillation kinetics and composition of the essential oils of Anethum graveolens L. seeds was investigated. The highest oil yield, after five consecutive hydrodistillation runs (3.74 ml/100 g of plant material), was achieved by the utilization of filtrated (from plant material) water used in the previous hydrodistillation run plus newly added water in the subsequent runs (technique III). The hydrodistillation of dill seeds took place in two stages: a rapid, early distillation of the oil followed by a much slower second phase. Two kinetics models were successfully used to interpret the hydrodistillation rate of the essential oil of dill. Independent of the technique used, the oil contained the same components but in differing amounts as inferred from detailed gas chromatography–mass spectroscopy (GC–MS) analyses. Carvone was found to be the major component in all obtained oils.

Strawberry is a member of the rose family (Rosaceae, subfamily Rosoideae, tribe Potentilleae) in the genus Fragaria. The cultivated varieties of commercial strawberries usually were designated as Fragaria ananassa. Root, leaf, flower and fruit have the healing properties. The strawberry leaves extract is used for blood cleaning, for treatment of oral inflammation, diarrhea, various gastro-intestinal inflammation, and hemorrhoids, as well as a diuretic. So far, many positive biological effects of strawberries (anticancer, antioxidant and anticoagulant effect) have been proven. The purpose of this study was to evaluate the antioxidant potential of ethanolic extract from cultivated strawberries (Fragariae folium, varieties Senga Sengana) by using different antioxidant assays (DPPH, FRAP, FIC, H2O2 and TBA-MDA). Ethanolic extract from strawberry leaves was obtained by reflux extraction at the boiling temperature. Total phenols and total flavonoids content was determined spectrophotometrically by the method of Folin-Ciocalteu and by method with AlCl3, respectively. In the extract was determined high content of total phenols, while the total flavonoid content is much lower. The concentrations of extract required to neutralize 50% of the initial concentration of DPPH radicals (EC50) after 20 minutes incubation and immediately after adding DPPH radical solution were 7,91 and 19,46 μgcm-3, respectively. Extract was achieved the maximum iron ions chelating ability (67.89%) at a concentration of 2 mgcm-3. Inhibition of lipid peroxidation of 70% was achieved by extract concentration of 0.03125 mgcm-3, while the maximum neutralization of H2O2 (30.47%) was achieved by extract concentration of 0.5 mgcm-3. FRAP value of the investigated extract is 284.51 mgFe/g of dry extract. Presented results of the antioxidant activity show that the obtained extract from the cultivated strawberry leaves is a potential source of natural antioxidants. [Projekat Ministarstva nauke Republike Srbije, br. TR 34012: Biljni i sintetski bioaktivni proizvodi novije generacije]

The effects of utilization of different color shade nets in lettuce production during the summer season were evaluated in Serbian climate conditions for pearl, blue, red and black shade nets in comparison to open field production. Applied shade nets (with shade index of 50%) significantly reduced solar irradiation (from 890 in open field to 400–560 Wm−2 under the shade nets) and photosynthetically active radiation (from 2020 in open field to less than 1000 μmol s−1 m−2 under the shade nets). Color shade nets affected both, properties of lettuce during the growing period and its morphological properties. Namely, the leaf area index was increased, lettuce heads were characterized with significantly higher marketable head weight and head diameter, the period from planting to bolting was significantly shorter, the outer leafs were characterized with more intensive green tone (–a*) and the lettuce leafs were softer and more tender for lettuce grown under the shade nets in comparison to open field production. In respect to lettuce composition, total chlorophyll content, contents of both chlorophyll a and chlorophyll b, as well as carotenoid content were significantly higher in shaded leaves of lettuce than in the control plants. Significantly higher (30.78 mg GAE g−1 d.m.) total phenol content was determined in plants grown under the pearl shade nets accompanied with significantly higher flavonoids content (14.28 mg RE g−1 d.m.) and significantly higher antioxidant properties (lower EC50 DPPH values) in comparison to all other nets. However, the antioxidant properties of control plants were at the same level as for the pearl net, in spite of lower phenols and flavonoids content.

The present paper deals with the fermentation of chopped, dried, woolly foxglove foliage, the extraction of secondary glycosides from fermented woolly foxglove foliage by the percolation method and the isolation and purification of digoxin. Optimal process conditions for fermenting the chopped, dried, woolly foxglove foliage, extracting of digoxin from the fermented woolly foxglove foliage by percolation, and further isolating and purifying of digoxin were defined. Under the optimum anaerobic conditions for fermentation of the chopped, dried woolly foxglove foliage at 37°C, the best yield of digoxin of 99-100% was achieved in 48 h. The optimal conditions for extraction of digoxin by percolation (plant particle size: 7 mm; height of foliage in the percolator: 30 cm; extracting solvent: 10%vol. ethanol- or methanol-water solution, volumetric percolate flow rate: 4 L/h and the percolate residence time in the percolator: 4h) ensured the digoxin extraction degree of 97%. Although ethanol is currently more expe...

The yield of extractive substances, antioxidant activity, as well as total phenolic and total flavonoid contents of aqueous-ethanolic extracts obtained from aerial parts of Galium mollugo L. by different extraction techniques (maceration, reflux and ultrasonic extraction) were reported. The antioxidant activity of extracts was tested by measuring their ability to scavenge a stable DPPH free radical, while the total phenolic and total flavonoid contents were determined according to the Folin-Ciocalteu procedure and a colorimetric method, respectively. The Duncan?s multiple range tests were used to evaluate if there were significant differences among yields of extractive substances, total phenolics, total flavonoids and EC50 values for the extracts obtained by different extraction techniques. The extracts obtained by the reflux extraction contained higher amounts of extractive substances, as well as phenolic and flavonoid compounds, and showed a better antioxidant activity than those obtained by the two other recovering techniques.

The photoselective netting concept was studied in a tomato ‘Vedeta’ cultivation in the south part of Serbia (Aleksinac) under high solar radiation, using four different colored shade-nets (pearl, red, blue and black) with different relative shading (40% and 50% PAR). Exposure to full sunlight was used as a control. Red and pearl nets with 40% shade significantly increased the total yield. Shading reduced the appearance of tomato cracking and eliminated sunscalds on tomato fruits and accordingly, increased the marketable tomato production by about 35% compared to non-shading conditions. Changing the light intensity by color shade nets affected the biosynthesis of lycopene and β-carotene in tomatoes. Thus, significantly higher lycopene content was observed in greenhouse tomato integrated with red shade netting technologies (64.9 μg g −1 ) than in field-grown tomatoes (48.1 μg g −1 ). By contrast, shaded fruits have lower content of β-carotene. The photo-selective, light-dispersive shade nets appear as interesting tools that can be further implemented within protected cultivation practices.

In this work was described poly(d,l-lactide) microwave synthesis using tin(II) 2-ethylhexanoate initiated ring-opening polymerization. Polymerization was performed at 100 °C with monomer to initiator molar ratio ([M]/[I]) of 5,000 in 30 min. The achieved number average molar mass of obtained polymers (determined by gel permeation chromatography) was 102,320 g/mol, with the polydispersion index, Q, 2.80. Structural characterization was performed by FT-IR spectroscopy followed characteristic bands. For applicative purposes the obtained polymer was purified during the procedure of microsphere preparation. Biodegradable microspheres prepared from poly(d,l-lactide) have been widely studied in recent years and have become well established controlled drug delivery systems. In this work microspheres were loaded with allyl thiosulfinate (allicin) and its transforments products (ajoene and vinyldithiine), as pharmacological active substances. The morphology of the microspheres was analyzed using a scanning electron microscope. Allicin was synthesized by acid oxidation of allyl disufide and purification of obtained products by liquid–liquid extraction with diethyl ether. Obtained allicin, purity 73%, was transformed using microwave in acetone solution, at solvent boiling temperature, for 5 min. For the quality and quantity analysis of allicin and its transformation process was used LC/MS chromatography. (E)- and (Z)-ajoene were detected at retention time 3.1 and 3.3 min, respectively, whence 3-vynil-4H-1,2-dithiine and 2-vynil-4H-1,3-dithiine were detected at 4.3 and 4.8 min, respectively. Retention time of allicin was 2.93 min, according to liquid chromatography results. HPLC method was used for assessment of pharmaceutical substances (alicine and alicine transforments) releasing from microspheres at room temperature in solutions with different pH (pH = 3 and pH = 8) for 24 h.