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The binding interactions of six ligands, neutral and monocationic asymmetric monomethine cyanine dyes comprising benzoselenazolyl moiety with duplex DNA and RNA and G-quadruplex structures were evaluated using fluorescence, UV/Vis (thermal melting) and circular dichroism (CD) spectroscopy. The main objective was to assess the impact of different substituents (methyl vs. sulfopropyl vs. thiopropyl/thioethyl) on the nitrogen atom of the benzothiazolyl chromophore on various nucleic acid structures. The monomethine cyanine dyes with methyl substituents showed a 100-fold selectivity for G-quadruplex versus duplex DNA. Study results indicate that cyanines bind with G-quadruplex via end π-π stacking interactions and possible additional interactions with nucleobases/phosphate backbone of grooves or loop bases. Cyanine with thioethyl substituent distinguishes duplex DNA and RNA and G-quadruplex structures by distinctly varying ICD signals. Furthermore, cell viability assay reveals the submicromolar activity of cyanines with methyl substituents against all tested human cancer cell lines. Confocal microscopy analysis shows preferential accumulation of cyanines with sulfopropyl and thioethyl substituents in mitochondria and indicates localization of cyanines with methyl in nucleus, particularly nucleolus. This confirms the potential of examined cyanines as theranostic agents, possessing both fluorescent properties and cell viability inhibitory effect.

Immortelle, as rich source of chlorogenic acid and the phloroglucinol alpha-pyrone compound arzanol, possesses anti-inflammatory and antioxidant properties, affects cell regeneration, and has positive effect on many skin conditions. Hemp oil, characterized by a favorable omega-6 to omega-3 ratio, as well as an abundance of essential fatty acids and vitamin E, participates in immunoregulation and also act as an anti-inflammatory. In the present study, we examined the effect on the skin of creams and emulgels with immortelle extract and hemp oil, by comparing them to placebo samples and a non-treated control. A long-term in vivo study of biophysical skin characteristics, which lasted for 30 days, was conducted on 25 healthy human volunteers. Measured parameters were electrical capacitance of the stratum corneum, trans-epidermal water loss (TEWL), and skin pH and erythema index. Further, a sensory study was carried out in which the panelists had to choose descriptive terms for sensory attributes in questionnaire. The results showed that application of all preparations led to increase of skin hydration and TEWL reduction, while the skin was not irritated, and its normal pH was not disrupted. This study also showed importance of the carrier. Not only were emulgels described by panelists as preparations with better sensory properties, there was a significant difference between the skin hydration effect of emulgel with immortelle extract and hemp oil compared to the placebo emulgel, which was not the case with creams. Such findings indicated enhanced delivery of herbal active substances from emulgel compared to the cream.

Supercritical CO2 extraction was applied on fenugreek (Trigonella foenum-graecum L., Fabaceae) seeds with the aim to define optimal process conditions to obtain the maximal extract yields content of steroidal sapogenins. Central composite rotatable design (CCRD) combined with response surface methodology (RSM) was applied to determine optimal process conditions defined by the influence and interaction of pressure, temperature and time of extraction through consumption of SC CO2. Optimization experiments revealed that the pressure of 24.73 MPa, the temperature of 38.°C and the consumption of SC CO2 of 19.24 g/gDM were the best process conditions enabling the maximal yield of extract and gain of the highest content of sapogenins. The optimal values of pressure and temperature defined SC CO2 density of 885.47 kg/m3, which provided the maximal yield of the extract with the highest content of steroidal sapogenins. The achieved yield of extract at these conditions was 0.073g/gDM, with 0.774 mg/gDM diosgenin, 0.477 mg/gDM protodioscin, 0.713 mg/gDM sarsapogenin and 0.205 mg/gDM oleanolic and ursolic acid with a significant quantity of 2.475 mg/gDM in the obtained extract. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. III45017]

Bilberry represents a valuable source of antioxidant substances responsible for its application for the treatment of different conditions (such as inflammation, cardiovascular disease, cancer, diabetes, and different age-related diseases) associated with increased oxidative stress. As oxidative stress might cause skin impairments, we aim to evaluate a topical preparation containing bilberry leaves extract and bilberry seeds oil, obtained as a byproduct of the food industry. To obtain the extracts, the conventional maceration technique for leaves, and supercritical carbon dioxide extraction for seeds were employed. The chemical profile of both actives was achieved by HPLC and GC methods, revealing the presence of phenolic acids (chlorogenic being the most abundant), flavonoids (isoquercetin in the highest amount), and resveratrol in leaves extract, while in seeds oil the essential ω-3 and ω-6 fatty acids were determined in favorable ratio, almost being 1. Antioxidant potential of the wild bilberry extract and seed oil was evaluated using in vitro DPPH and FRAP assays. Finally, effects of the oil-in-water creams with mentioned wild bilberry isolates on the skin were investigated in an in vivo study conducted on healthy human volunteers, revealing the significant beneficial effects when topically applied.

Background This study provides an insight into the impact of ultrasound-assisted extraction with water as solvent (UAEW) and extraction by supercritical carbon dioxide (SC-CO2 ) with 5% EtOH on antioxidant and enzyme inhibitory activity in regard to the chemical profile of the edible and medicinal mushroom, Pleurotus pulmonarius. Results Extraction efficiency was between 0.36% and 63.32%, depending on the extraction technique. The main compounds in the extracts were fatty acids. Supercritical CO2 extraction with co-solvent was the most suitable method for obtaining extracts that were rich in ergosterol content, reaching a value of 40.1 mg g-1 . The UAEW of crude mushroom powder ensured the highest yield, as well as the extracts with best antioxidative activity. The measurements of enzyme inhibitory activity revealed that all types of investigated extracts exhibited only tyrosinase and amylase inhibition at a significant level. Conclusion Based on our results, the extraction methods significantly affected the chemical profile and bioactivity of P. pulmonarius. © 2020 Society of Chemical Industry.

An experimental investigation on essential oil of a Montenegrin Helichrysum italicum (Roth) G. Don fil. (Asteraceae) is reported. The essential oil was analysed in both liquid and vapour phases. Gas chromatography-mass spectrometry analysis of the liquid oil showed the predominance of sesquiterpenes with β-eudesmene (21.65%) and β-bisabolene (19.90%) as the major ones. Monoterpene fraction was mainly represented by α-pinene (16.90%) and neryl acetate (10.66%). Head-space technique revealed the vapour phase enriched of monoterpenes with α-pinene (78.76%) predominance. The essential oil was tested against methicillin-sensitive Staphylococcus aureus (ATCC 29213), Escherichia coli (ATCC 25922), Candida albicans (ATCC 14053) and the clinical strains of methicillin-resistant S. aureus, carbapenem-resistant Klebsiella pneumoniae, carbapenem-resistant Acinetobacter baumannii and carbapenem-resistant Pseudomonas aeruginosa. Interesting fungicidal/bactericidal potency against C. albicans and carbapenem-resistant A. baumannii was revealed at concentration of 5% v/v.

Supercritical CO2 impregnation process was used for the first time to fabricate biodegradable gelatin-chitosan (G/Ch) films containing clove oil (CO) for potential use in active food packaging. All the impregnations were carried out at moderately low temperature (40°C). Aiming to maximize CO loading in the films with acceptable morphological, structural and thermal properties, CO2 pressure, impregnation time and G:Ch mass ratio were varied. Gelatin fraction in the films of 25-50 wt.%, scCO2 pressures of 10-20 MPa and impregnation time of 2-6 h impregnation favoured the CO loading. Processing of the film with equal gelatin to chitosan mass ratio (G/Ch50:50) at 10 MPa for 2 h yielded sufficiently high loading (56 mg CO/gfilm) without an adverse effect on morphological properties. The G/Ch50:50 film was therefore chosen for structural and thermal analyses. ATR-FTIR analysis confirmed successful CO incorporation into the G/Ch50:50 and its interaction with the film. Plasticizing effect of scCO2 and CO on the film was evidenced by DSC. Incorporation of 56 mg CO/gfilm into the G/Ch50:50 didn''t affect thermal stability of the film. Beside environmental benefits, supercritical impregnation process enables fast fabrication of G/Ch bio-composite films containing CO, thermally stable to 110°C, which is suitable for most of food packaging applications. [Projects of the Serbian Ministry of Education, Science and Technological Development, Grant no. III 45017 and Grant no. III45007]

Supercritical CO2 extraction of Helichrysum italicum and impregnation of starch xerogels with the extract by using an integrated scCO2 extraction and impregnation process were performed at 350 bar and 40?C in order to produce biomaterials for possible oral intake of the extract. Xerogels produced by air-drying of acetogels and alcogels were used as carriers in the supercritical impregnation process. The effect of ethanol as a co-solvent, contact time, plant material/carrier mass ratio and xerogel preparation on the impregnation loading was studied. The highest impregnation loading (1.26?0.22%) was achieved after 5 h impregnation of the xerogel obtained from alcogel using pure scCO2 and plant material/ /carrier mass ratio of 10. Chemical analysis of the extracts showed that the addition of ethanol as co-solvent had a positive effect on scCO2 selectivity to terpene fraction and total flavonoids, while it lowered the total phenolic content. Despite the difference in chemical composition, both extracts expressed similar antioxidant activity according to the DPPH and FRAP methods. The integrated process was shown to be a feasible method for isolation and incorporation of bioactive components of H. italicum into starch xerogels.

Supercritical fluid extraction as an environmentally friendly technology was applied to isolate biologically active extracts from celery and parsley fruits for potential applications in the food industry. The extractions were performed under mild temperature conditions of 39.85 &deg, C and at pressures of 10 and 30 MPa. The extracts were analyzed regarding their chemical composition, antibacterial activity, and cytotoxic effect. Sedanolide was the dominant component of the celery fruit extracts, comprising more than 70% of the obtained fraction, while the content of apiole in the parsley fruit SC CO2 extracts exceeded 85%. The celery fruit extracts showed strong and moderately strong antibacterial activity against tested Staphylococcus aureus, Bacillus (B.) cereus, B. subtilis, B. circulans, Listeria (L.) greyi, L. seeligeri and L. welshimeri, with minimal inhibitory concentration (MIC) values between 160 and 640 &micro, g/mL, and weak activity against the selected Salmonella isolates with a MIC of 2560 &micro, g/mL. The parsley extract obtained at 10 MPa showed strong and moderately strong antibacterial effects against Bacillus strains with obtained MICs of 160&ndash, 640 &micro, g/mL, and weak activity against Staphylococcus, Listeria, and Salmonella with a MIC of 2560 &micro, g/mL. Cytotoxicity investigation showed that the extracts with proven antibacterial activity had no cytotoxic effect on rabbit kidney cells at concentrations of up to 640 &micro, g/mL.

Usnic acid is one of the most investigated lichen secondary metabolites, with several proven biological properties with potential medical relevance. However, its unfavorable physico-chemical properties, as well as observed hepatotoxicity, have discouraged wide-range utilization of usnic acid as a promising therapeutic agent. In accordance with the growing research interest in the development of nanotechnology, especially in the arena of preparations based on natural sources of medicinal compounds, usnic acid incorporated into nano- and microsized colloidal carriers has been a subject of a large number of publications. Therefore, this review discusses the overall results of the studies dealing with usnic acid encapsulated into lipid-based, polymeric and nonorganic micro- and/or nanocarriers, as potential drug delivery systems for this natural compound, in an attempt to introduce its usage as a potential antitumor, antimicrobial, wound-healing, antioxidative and anti-inflammatory drug.

In recent years, naturally derived tyrosinase inhibitors and antioxidants have become very important, since the synthetic ones are related with several side effects. As wild fruits are rich source of bioactive compounds, this study focused on the examination of Prunus spinosa L. (blackthorn) fruit extracts, widely distributed in our country. However, blackthorn extracts are not enough investigated, and there are only a few published works. Extraction efficiency of P. spinosa ultrasonic fruit extracts obtained by using different solvents (methanol, 70% (v/v) ethanol, 45% (w/w) propylene glycol and water) was evaluated, regarding polyphenolic profile, tyrosinase inhibitory and antioxidant activity. Results suggested that extracts with 45% propylene glycol and water were particularly rich in polyphenolic compounds (especially phenols, flavonoids and anthocyanins). The highest extraction yield was for 45% propylene glycol extract. This extract showed higher tyrosinase inhibitory and antioxidant activity compared to others, while the methanolic extracts had the highest chelating capacity. Therefore, P. spinosa fruit extracts, especially extracts obtained by ultrasonic extraction with 45% propylene glycol, have a great potential to be incorporated in some pharmaceutical formulations for obtaining tyrosinase inhibitory and antioxidant effects. ********* In press - Online First. Article has been peer reviewed, accepted for publication and published online without pagination. It will receive pagination when the issue will be ready for publishing as a complete number (Volume 47, Issue 3, 2019). The article is searchable and citable by Digital Object Identifier (DOI). DOI link will become active after the article will be included in the complete issue. *********

Helichrysum italicum (Roth) G. Don fil., Asteraceae, possesses numerous secondary plant metabolites with a wide range of biological activities. Yet, data on the potential of supercritical fluid extraction (SFE) in their isolation are scarce. This study provides analyses of the chemical profiles of extracts obtained by SFE with or without ethanol as a cosolvent using GC-FID, GC-MS, HPLC, and UHPLC-MS techniques. Among the compounds with proven biological activity identified, the presence of arzanol was confirmed. In the next step, the integrated process of supercritical fluid extraction and impregnation was applied to deliver active compounds to cotton gauze and polypropylene fabric for possible topical applications. The analytical procedures results showed a considerable affinity of both textile materials for incorporating active components present in the H. italicum. The study indicated the high-pressure techniques applied as very efficient in the isolation of bioactive components from H. italicum and their adsorption on selected carriers. Supplementary material: [https://cherry.chem.bg.ac.rs/handle/123456789/4418]

peer-reviewed, Due to a high content of rutin (2-10%), dried buckwheat leaf and flower (DBLF) formulations were shown to be efficient in the treatment of vascular diseases. In order to find a cost effective way for the extraction of antioxidants, the effects of ethanol/water ratio and temperature on the extraction efficiency of phenolic compounds and the mechanisms of antioxidant action of the extracts were tested. Extraction with ethanol/water mixture (80:20, v/v) for 24 h at room temperature, after the mixture was just brought to boil was demonstrated to be an efficient and cheap way for obtaining a high yield of rutin (49.94±0.623 mg/g DBLF). The most abundant phenolic compounds in DBLF extracts were rutin and chlorogenic acid. Flavonoids, especially rutin, were shown to be the most responsible for the antioxidant activity in all investigated lipid model systems, acting as free radical scavengers, electron-donating substances and chelators of iron ions. In β-carotene bleaching tests, the extracts with the highest activity were as efficient as BHT (butylated hydroxytoluole). Regarding the results of antihemolytic and Schaal oven tests, the extracts demonstrated remarkable ability to inhibit the oxidative destruction of erythrocytes and to prolong the beginning of the oxidation process in sunflower oil., This work is a part of the National Project (TR-31029) financially supported by the Ministry of Education, Science and Technological Development, Republic of Serbia.

Objective The aim of the study was in vitro and in vivo characterization of cosmetic cream with 6% of standardized wild apple fruit extract, stabilized by conventional non-ionic emulsifier-CEW, in order to determine the influence of emulsifiers (conventional vs. biodegradable) on the characteristics of creams and their effects on the skin. Methods Organoleptic and physico-chemical (pH values and electrical conductivity) analysis was performed, determination of fruit acids-FAs content (using HPLC analysis) and estimation of its antioxidant activity-AA (using DPPH test) during 180 days. In vivo study included following examinations: screening of safety profile (after creams application under occlusion during 24 h at human skin); skin moisturizing potential, transepidermal water loss-TEWL, skin pH after 28 days of cream application and hypopigmentation efficacy 7 days of cream application at artificially induced skin hyperpigmentation. Results Investigated cosmetic cream-CEW showed satisfactory organoleptic, physico-chemical characteristics, stability, FAs content (0.13%) and AA (19.25 ± 0.67 %RSC) after preparation, which remained unchanged over the study period. In vivo investigation revealed absence of skin irritation after CEW's application under occlusion. An increase of skin moisturization (after 14 days ΔEC was 18.52 ± 11.51 and after 28 days of applications 16.52 ± 9.36) during 28 day-study, with unchanged TEWL and skin pH values was shown. Decrease of melanin index was revealed, too (after 7 days ΔMI was -31.40 ± 16.50). Conclusion Cosmetic cream stabilized by conventional emulsifier showed better antioxidant potential and weaker moisturizing and hypopigmentation effects related to the cream with same composition but stabilized by biodegradable emulsifiers. Based on all mentioned above, investigated cosmetic cream might be considered for potential use as modern, stable, safe and efficient cosmetic product in the prevention and/or treatment of oxidative stress-related skin changes and/or damages, for moisturization of dry, even irritated skin as well as for lightening of hyperpigmented skin. RESUME: OBJECTIF Le but de l'etude etait la caracterisation in vitro et in vivo d'une creme cosmetique contenant 6 % d'extrait normalise de pomme sauvage, stabilisee par un emulsifiant non ionique conventionnel-CEW, afin de determiner l'influence des emulsifiants (conventionnels vs biodegradables) sur les caracteristiques des cremes et les effets sur la peau. METHODES: Des analyses organoleptiques et physico-chimiques (pH et conductivite electrique) ont ete effectuees, la determination de la teneur en acides de fruits et en acides gras polyinsatures (par CLHP), et l'estimation de sa teneur en activite antioxydante (AA) (a l'aide du test DPPH) pendant 180 jours. L'etude in vivo comprenait les examens suivants : depistage du profil d'innocuite (apres application de cremes sous occlusion pendant 24 h sur la peau humaine); potentiel d'hydratation de la peau, perte d'eau transepidermique (PETE), pH de la peau apres 28 jours d'application de la creme et efficacite de l'hypopigmentation apres 7 jours d'application de la creme sur une hyperpigmentation de la peau induite artificiellement. RESULTATS: La creme cosmetique-CEW etudiee a montre des caracteristiques organoleptiques, physico-chimiques, une stabilite, une teneur en AG (0,13 %) et en AA (19,25 ± 0,67 % RSC) satisfaisantes apres preparation, qui sont demeurees inchangees au cours de la periode de l'etude. L'etude in vivo a revele l'absence d'irritation cutanee apres l'application de CEW sous occlusion. Une augmentation de l'hydratation de la peau (apres 14 jours de DEC etait de 18,52 ± 11,51 et apres 28 jours d'applications de 16,52 ± 9,36) pendant l'etude de 28 jours, avec une PETE et des valeurs de pH cutane inchangees, a ete demontree. Une diminution de l'indice de melanine a ete mise en evidence egalement (apres 7 jours, ΔMI etait de 31,40 ± 16,50). Conclusion La creme cosmetique stabilisee par un emulsifiant conventionnel a montre un meilleur potentiel antioxydant et des effets d'hydratation et d'hypopigmentation plus faibles par rapport a la creme de meme composition mais stabilisee par des emulsifiants biodegradables. D'apres les donnees precedentes, la creme cosmetique etudiee pourrait etre consideree comme un produit cosmetique moderne, stable, sur et efficace dans la prevention et/ou le traitement des changements et/ou dommages cutanes lies au stress oxydatif, pour l'hydratation de la peau seche, voire irritee, ainsi que pour eclaircir la peau hyperpigmentee.

Menopause is characterized by deep metabolic disturbances, including decreased insulin sensitivity, adiposity, and changes in lipid profiles. Estrogen replacement therapy can partially reverse these changes, and while it is safe in most healthy postmenopausal women, there are still existing concerns regarding an increased risk for breast and endometrial cancer as well as a risk for cardiovascular and thromboembolic disease. Therefore, certain natural compounds with positive metabolic effects may be considered as a possible alternative or adjunctive treatment in patients not willing to take estrogens or patients with contraindications for estrogens. The aim of this study was to investigate the influence of Sideritis scardica (mountain tea) extract on metabolic disturbances induced by ovariectomy in rats. The study included 24 rats divided into three groups: ovariectomized rats treated with 200 mg/kg S. scardica extract for 24 weeks (n = 8), ovariectomized non-treated (n = 8), and Sham-operated (n = 8) rats. Food intake, weight gain, body composition, fasting glucose levels, response to oral glucose challenge, liver glycogen content, catalase activity, thiol groups, and malondialdehyde concentrations as well as AMP-activated protein kinase activity in liver cells were studied. Ovariectomized rats treated with S. scardica extract had lower blood triglycerides, reduced fasting glucose levels, as well lower glucose peaks after oral glucose challenge, increased liver glycogen content, and significantly higher catalase activity and thiol group concentration than non-treated ovariectomized rats. The ability of S. scardica extract to attenuate metabolic disturbances associated with ovariectomy was associated with the activation of AMP-activated protein kinase in liver cells.

Paper description: Bilberry ( Vaccinium myrtillus L ., Ericaceae) and its extracts are used in traditional medicine. While research has focused on the health benefits of plant flavonoids, the element content is of potentialy great importance. This study provides information on which part of the bilberry (roots, stems, leaves or fruits) accumulates which elements and on how the metals taken up from the soil might be translocated from the roots to the above-ground parts of the plant. This study provides new findings on element distribution in bilberry and gives insight into the effect of the method of bilberry extract preparation on the element content. Abstract: Bilberry ( Vaccinium myrtillus L., Ericaceae) is a rich source of different mineral elements that are important as essential trace elements, or that can be potentially toxic, depending on their concentration. The contents of sixteen elements: the major (Al, Ca, Fe, K and Mg) and trace elements (As, Ba, Cd, Co, Cu, Cr, Mn, Ni, Pb, Sr and Zn) in roots, stems, leaves and fruits of bilberry and the corresponding soil substrate were determined in order to investigate element mobility and availability in soil. Soil was subjected to sequential extraction procedure as proposed by the Community Bureau of Reference in order to fractionate acid-soluble/exchangeable, reducible, oxidizable and residual fractions. Analysis of water and ethanol extracts of bilberry aerial parts revealed a significant transfer of elements during the extraction procedure, with corresponding extraction coefficient values of up to 95.12%. Results showed that bilberry fruits could serve as a good dietary source of essential elements for humans, especially Mn and Cu. https://doi.org/10.2298/ABS181004056B Received: October 4, 2018; Revised: November 28, 2018; Accepted: November 30, 2018; Published online: December 14, 2018 How to cite this article: Brasanac-Vukanovic S, Tadic VM, Blagojevic N, Vukasinovic-Pesic V, Đurđic S, Stankovic M, Mutic J. Element accumulation capacity of Vaccinium myrtillus from Montenegro: Comparison of element contents in water and ethanol extracts of bilberry plant parts. Arch Biol Sci. 2019;71(1):145-57.

Old man’s beard (Usnea barbata) is one of the most investigated lichens which biological properties (such as antimicrobial, anti-inflammatory and cytotoxic activities) have been proven in several scien- tific studies. Stated properties of old man’s beard have been attributed mainly to its major secondary metabolite - a dibenzofuran derivative, usnic acid. The aim of the current study was to examine cyto- toxic activity of the quantified supercritical CO2 extract and pure usnic acid in parallel against L929 fibrosarcoma cells. Our results indicated the tested extract to be more toxic towards the investigated cell line compared to usnic acid per se, despite the high amount of this compound (81.41% (w/w)) revealed in the extract using HPLC. Such result may be connected to the unidentified compounds present in the supercritical CO2 extract that could be responsible for apoptosis and oxidative stress in the exerted cytotoxicity of the investigated extract, but not usnic acid. Our study supports further in- vestigations of supercritical CO2 extract of U. barbata as a prospective therapeutic agent with potential relevance in the treatment of sarcoma.

The aim of this study was to establish correlation of chemical composition and antioxidant activity of bilberry plants from Montenegro. Total phenolic, tannin, flavonoid, procyanidin and anthocyanin contents were determined in fruits and leaves extracts using spectrophotometric methods, while the measurements of metal content was carried out in an Inductively Coupled Atomic Emission Spectrometer. Qualitative and quantitative analyses of major phenolics were achieved by HPLC. In the investigated extracts, the most abundant phenolic was chlorogenic acid, followed by protocatechuic acid, while resveratrol, isoquercetin, quecetin and hyperoside were also present in significant quantities. Antioxidant potential was evaluated using two in vitro assays&mdash, FRAP and DPPH&mdash, being in the accordance with the cyclic voltammetry tests, performed as well. The results revealed that all the investigated extracts were rich in phenolic and essential mineral constituents, with significant antioxidant activity, depending on the polyphenolic and mineral contents, which was confirmed by principal component analysis.

The objective of this work was to investigate and profoundly characterize low-energy nanoemulsions as multifunctional carriers, with slight reference to dermal administration. An evidence-based approach was offered for deepening the knowledge on their formation via spontaneous emulsification. Curcumin, a compound of natural origin, potentially powerful therapeutic, was chosen as a model API. Due to curcumin's demanding properties (instability, poor solubility, low permeability), its potentials remain unreached. Low-energy nanoemulsions were considered carriers capable of overcoming imposed obstacles. Formulation consisting of Polysorbate 80 and soybean lecithin as stabilizers (9:1, 10%), medium-chain triglycerides as the oil phase (10%) and ultrapure water was selected for curcumin incorporation in 3 different concentrations (1, 2 and 3 mg/mL). Physicochemical stability was demonstrated during 3 months of monitoring (mean droplet size: 111.3-146.8 nm; PDI LT 0.2; pH: 4.73-5.73). Curcumin's release from developed vehicles followed Higuchi's kinetics. DPPH (IC50 = 0.1187 mg/ mL) and FRAP (1.19 +/- 0.02 mmol/g) assays confirmed that curcumin acts as a potent antioxidant through different mechanisms, with no alterations after incorporation in the formulation. High biocompatibility in line with antigenotoxic activity of curcumin-loaded formulations (protective and reparative) was estimated through Comet assay. A multidisciplinary approach is needed to fully characterize developed systems, directing them to more concrete application possibilities. This is the peer-reviewed version of the article: I. Nikolic, D. Jasmin Lunter, D. Randjelovic, et. al., Curcumin-loaded low-energy nanoemulsions as a prototype of multifunctional vehicles for different administration routes: physicochemical and in vitro peculiarities important for dermal application, International, Journal of Pharmaceutics, 2018, 550, 1-2, 333-346, DOI: [https://doi.org/10.1016/j.ijpharm.2018.08.060] The published version: [https://cer.ihtm.bg.ac.rs/handle/123456789/2425]

Objective The aim of this study was in vitro and in vivo characterization of cosmetic cream with 6% of wild apple fruit water extract, containing 3.5% of alpha-hydroxyacids-AHAs and polyphenolic compounds, stabilized by biodegradable alkyl-polyglucoside emulsifiers. Methods In vitro characterization of cream included organoleptic and physico-chemical (pH values and electrical conductivity) analysis, antioxidant activity-AA estimation (using DPPH test) during 180 days of storage at 22±2°C and determination of cosmetic active substances content-AHAs (using HPLC analysis). In vivo estimation of skin irritation potential after creams application under occlusion during 24h was investigated employing the biophysical methods on 12 healthy volunteers; hydration efficiency, transepidermal water loss-TEWL, skin pH, erythema-EI and melanin index-MI on 10 healthy volunteers after 28 days of cream application and bleaching efficiency on 10 healthy volunteers after 7 days of cream application after artificially induced skin hyperpigmentation. Results Investigated cosmetic cream was pale beige color, odorless, semi-solid consistency and homogeneous, with pH values of 6.53±0.14, electrical conductivity above 50 μS/cm and AA of 24.96%RSC after preparation and these characteristics were stable during investigated period. In vivo measurements revealed absence of skin irritation after cream application under occlusion, which was part of it's safety profile. Increase of skin hydration after 14 days of cream application was 17.28±12.23 and after 28 days 21.19±7.59. In addition, in performed experiment TEWL and skin pH values during cream application remained unchanged. Cream application after artificial hyperpigmentation induced decrease of MI (∆MI after 7 days was -45.30±18.55) CONCLUSION: Formulated cosmetic cream with 6% of standardized wild apple fruit water extract and stabilized by biodegradable alkyl-polyglucoside emulsifiers demonstrated good stability, acceptable level of in vitro antioxidant activity, absence of skin irritation after cream application under occlusion and positive effects of cream on human skin after application (hydrating and bleaching effects). All mentioned properties make the cream suitable for possible usage as cosmetic product for preventing the skin damages caused by oxidative stress, for moisturizing the dry skin and bleaching of skin hyperpigmentation. This article is protected by copyright. All rights reserved.

The aim of the present work was to examine if hydrogen peroxide treatment of sugar beet fibre that aimed at improving its physicochemical properties would impair its antioxidant potential. Three different sugar beet fibres were obtained from sugar beet - non-treated fibre (NTF) from sugar beet cossettes extracted with sulphurous acid, treated fibre (TF) from NTF treated with hydrogen peroxide in alkaline solution and commercially available Fibrex(®) . The antioxidant activity of extractable and non-extractable fibre fractions in ethanol/water mixture (80:20, v/v) of three fibre samples was estimated. Non-extractable fractions obtained after alkaline treatment of investigated fibres were much higher in phenolic compounds and possessed higher antioxidant potential than extractable fractions. Ferulic acid was proven to be the dominant phenolic acid. Regarding both extractable and non-extractable fractions, Fibrex(®) had the highest antioxidant activity in chemical tests, while NTF was superior in comparison with TF. Based on the results of Caco-2 cells-based test, all non-extractable fractions possessed potential for reactive oxygen species inhibition. Regarding the extractable fractions, only the TF manifested this effect.Copyright © 2016 John Wiley & Sons, Ltd.

Wild apple fruit (Malus sylvestris (L.) Mill., Rosaceae) is a good source of polyphenolic compounds which have a therapeutic effects on many diseases caused by reactive oxygen species and oxidative stress (e.g. cardiovascular or degenerative diseases, atherosclerosis, diabetes, osteoporosis, cancer, dermatitis, phototoxity). The aim of our study was in vitro characterization of extracts of wild apple fruit originated from Serbia, obtained by different extraction methods and solvents, in order to examine their potential use as the source of substances with antioxidant properties in the production of nutraceuticals and cosmeceuticals. Extracts were made by maceration, percolation, Soxhlet and ultrasonic extraction using polar solvents: 70% ethanol, 45% and 80% propylene–glycol, and distilled water, and non-polar solvents: sunflower and olive oil. The extracts were characterized by determination of total content of phenols (TPC) by Folin–Ciocalteu assay and expressed as gallic acid equivalents (GAE), flavonoids (TFC) by Markham's method with rutin as standard (RE), tannins (TT) by vanillin assay and expressed as catechin equivalents (CE) and percentage content of anthocyanins (TA) by Eur.Ph.6.0. Besides, the antioxidant activity of the investigated extracts was estimated by three assays: DPPH (1,1-diphenyl-2-picryl-hydrazyl) test, FRAP (Ferric Reducing Antioxidant Power) test and test with linoleic acid, and expressed as %RSC (Radical Scavening Capacity), FRAP value (mM Fe2+) and %AOA (AntiOxidant Activity), respectively. The evidence of correlation between antioxidant properties and polyphenolic compounds content has been shown to be very good. The content of polyphenolic compounds and antioxidant activity depended on used extraction method and solvents. Namely, TPC in extracts ranged from 172.91–1556.99 mg GAE/100 g dry weight (d.w.) (dry plant material), TFC from 3.97 to 182.22 mg RE/100 g d.w., TT from 72.73 to 8872.73 mg CE/100 g d.w., and TA from 0.70 to 14.63%. %RSC ranged from 8.12 to 78.43 %RSC, FRAP value from 2.13 to 7.65 mM Fe2+ and %AOA from 57.73% to 95.32 %AOA. The study provided the data that the extracts obtained by ultrasonic extraction using ethanol and distilled water as solvents contained the highest amount of polyphenolic compounds and demonstrated the best antioxidant activity, as well. The results of our study indicated that wild apple fruit might be taken into consideration as a source of antioxidant substances for food, dermocosmetic and cosmetic industry.

This study discusses the possibility of supercritical solvent impregnation of polypropylene and polyamide fabrics as well as cellulose acetate in the form of beads and films with pyrethrum extract in order to fabricate functionalized materials with repellent properties. Impregnation experiments were performed at temperature of 40 degrees C and pressures of 10 and 20 MPa in supercritical carbon dioxide. The time of impregnation was varied from 0.5 to 18 h. Loadings of pyrethrum extract into polypropylene fabrics and cellulose acetate films higher than 30% were attainable at 10 MPa. In the case of polyamide fabrics and cellulose acetate beads, the pressure of 20 MPa was needed for obtaining the loadings higher than 11%. Pyrethrum extract release study revealed different capabilities of the tested materials for the repellent release, implying a variety of their possible applications. The impregnated solids were characterized by SEM, DSC, FTIR, Mercury intrusion porosimetry analyses and HPLC analyses. This the peer-reviewed version of the article: Pajnik J. et al., Journal of Supercritical Fluids, 2018, 136, 70-81, doi: [https://doi.org/10.1016/j.supflu.2018.02.014] Published version: [https://cer.ihtm.bg.ac.rs/handle/123456789/2298]

The antioxidant and antibacterial properties of Greek oregano extracts obtained by fractional supercritical fluid extraction (SFE) with carbon dioxide were investigated and compared with the properties of essential oil obtained by hydrodistillation. According to DPPH., hydroxyl radical and superoxide anion radical scavenging activity assays, the supercritical extracts expressed stronger antioxidant activity comparing to the essential oil. The most effective was the supercritical extract obtained by fractional extraction at 30 MPa and 100 degrees C after the volatile fraction had been extracted at lower pressure. At the same time this extract showed strong antibacterial activity against staphylococci, including MASA strain, but did not affect Escherichia coli of normal intestinal flora. The essential oil obtained by hydrodistillation showed stronger antibacterial activity against E. coli, Salmonella and Klebsiella pneumoniae, comparing to the supercritical extracts but at the same affected the normal gut flora.

Ethnopharmacological relevance Alchemilla vulgaris is an important remedy in European folk medicine, known for its astringent and anti-inflammatory properties; it is traditionally used to heal gynecological and gastrointestinal diseases. Despite its folkloric use in wound healing, there is a lack of scientific data to support this therapeutic application. Aim of the study To analyze the wound healing potential of different solvent A. vulgaris extracts per se and after incorporation into hydrogels as topical vehicles, using two complementary methods - in vitro wound healing assay with L929 fibroblasts and in vivo assessment of skin barrier repair potential. Besides scientific justification of the traditional usage, we aimed to ephasize the importance of a proper vehicle for herbal extracts. The wound healing activity has been connected to the chemical profile of the investigated extracts, their antioxidative properties, but also to pH of the investigated gels and their mechanical characteristics. Materials and methods Antioxidant activity of investigated extracts was estimated using both 2,2-diphenyl-1-picrylhydrazyl and β-carotene/linoleic acid models. Chemical profile was achieved applying spectrophotometric and HPLC methods. In vitro scratch assay with L929 fibroblasts, and in vivo study of skin barrier repair potential of hydrogels with A. vulgaris extracts on human skin employing biophysical measurements, were performed in order to confirm the wound healing potential of A. vulgaris. Texture analysis of the gels was performed alongside the pH measurements. Results All tested extracts and gels accelerated the wound healing process while the effect of ethanolic extract on migration of fibroblasts was the most pronounced. The highest extent of wound closure was also observed for the ethanolic extract. The most favorable effect on in vitro wound healing was observed for gel with propyleneglycolic extract. Results of in vivo study were in line with in vitro findings. Healing potential may be attributed to phenolic compounds found in A. vulgaris extracts, low pH of the gels, and the satisfying antioxidant activity of the extracts. Parameters obtained by textural analysis indicated satisfying mechanical properties of the gels, relevant to topical application. Conclusion Our study offers pharmacological evidence on the folkloric use of A. vulgaris in wound treatment, particularly after incorporation into hydrogel, and underlines an importance of a proper vehicle for incorporation of herbal extracts intended for topical treatment.

There is a growing research interest on investigation of appropriate alternatives of natural origin to synthetic additives used in food products. The aim of this study was to investigate correlation of chemical composition of the best known biomarker plants from the locality of Vrujci Spa, Serbia with their antioxidant activity in vitro, in terms of their potential usage as feedstock materials in the food industry. Total phenolic (TP), total tannins (TT) and total flavonoid (TF) contents were determined using spectrophotometric methods. Qualitative and quantitative analyses of major phenolics by high-performance liquid chromatography (HPLC) were also used. Major phenolic acids identified in analyzed species were chlorogenic, caffeic and ferulic acid, while predominant flavonoids were flavonols (hyperoside, rutin, isoquercitrin, quercetin) and, in lesser extent flavones (luteolin, apigenin and their 7-O-glucosides). Vitexin 2-O″-rhamnoside was found only in Crataegus species. Antioxidant capacity was estimated by the following methods: 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) test and ferric reducing antioxidant power (FRAP). Majority of investigated plants had high levels of phenolics and exhibited noteworthy antioxidant activity. Moreover, a positive linear correlation was found between TP and TT with antioxidant capacities regarding both methods used. The plants showing the highest antioxidant activities were Veronica officinalis, Mentha pulegium and Fragaria vesca. Hypericum perforatum demonstrated by far the most potent antioxidant activity, as well as TP and TT content. Our results indicate that, in addition to their traditional usage in folk medicine, these plants represent a valuable source of natural antioxidants, and thus may be considered as great potential for the food industry, representing possible alternatives to synthetic additives.

Helichrysum italicum presents a valuable source of natural bioactive compounds. In this work, a literature review of terpenes, phenolic compounds, and other less common phytochemicals from H. italicum with regard to application of different separation methods is presented. Data including extraction/separation methods and experimental conditions applied, obtained yields, number of identified compounds, content of different compound groups, and analytical techniques applied are shown as corresponding tables. Numerous biological activities of both isolates and individual compounds are emphasized. In addition, the data reported are discussed, and the directions for further investigations are proposed.

Sideritis scardica Griseb., Lamiaceae (ironwort, mountain tea), an endemic plant of the Balkan Peninsula, has been used in traditional medicine in the treatment of antimicrobial infections, gastrointestinal complaints, inflammation and rheumatic disorders. This study reports a comparison between conventional (hydrodistillation HD and solvent extraction SE) and alternative (supercritical carbon dioxide SC CO2) extraction methods regarding the qualitative and quantitative composition of the obtained extracts as analyzed by GC and GC-MS techniques and their anitimicrobial activity. Different types of extracts were tested, the essential oil EO obtained by HD, EO-CO2 and AO-CO2 obtained by SC CO2 at different preasures 10 and 30 MPa, at 40 degrees C, respectively, and the fractions A, B, C and D obtained by successive solvent extraction (SE) A: ethanol, B: diethyl ether, C: ethyl acetate and D: n-butanol). While EO was characterized by the presence of the high percentage of oxygenated monoterpenes and sesquiterpenes (30.01 and 25.54%, respectively), the rest of the investigated samples were the most abundant in fatty acids and their esters and diterpenes (from 16.72 to 71.07% for fatty acids and their esters, and from 23.30 to 72.76%, for diterpenes). Microbial susceptibility tests revealed the strong to moderate activity of all investigated extracts against the tested microorganisms (MIC from 40 to 2,560 mu g/mL). Although differences in the chemical compositions determined by GC and GC-MS analysis were established, the displayed antimicrobial activity was similar for the all investigated extracts.

Sideritis scardica Griseb. (ironwort, mountain tea), an endemic plant of the Balkan Peninsula, has been used in traditional medicine in the treatment of gastrointestinal complaints, inflammation, and rheumatic disorders. This study aimed to evaluate its gastroprotective and anti-inflammatory activities. Besides, continuously increasing interest in assessing the role of the plant active constituents preventing the risk of cancer was a reason to make a detailed examination of the investigated ethanol, diethyl ether, ethyl acetate, and N-butanol extracts regarding cytotoxicity. Oral administration of the investigated extracts caused a dose-dependent anti-inflammatory effect in a model of carrageenan-induced rat paw edema. Gastroprotective activity of the extracts was investigated using an ethanol-induced acute stress ulcer in rats. The cytotoxic activity of plant extracts was assessed on PBMC, B16, and HL-60 cells and compared to the cytotoxicity of phenolic compounds identified in extracts. Apoptotic and necrotic cell death were analyzed by double staining with fluoresceinisothiocyanate (FITC)-conjugated annexin V and PI. The developed HPLC method enabled qualitative fingerprint analysis of phenolic compounds in the investigated extracts. Compared to the effect of the positive control, the anti-inflammatory drug indomethacine (4 mg/kg), which produced a 50 % decrease in inflammation, diethyl ether and N-butanol extracts exhibited about the same effect in doses of 200 and 100 mg/kg (53.6 and 48.7 %; 48.4 and 49.9 %, respectively). All investigated extracts produced dose-dependent gastroprotective activity with the efficacy comparable to that of the reference drug ranitidine. The diethyl ether extract showed significant dose-dependent cytotoxicity on B16 cells and HL-60 cells, decreasing cell growth to 51.3 % and 77.5 % of control, respectively, when used at 100 µg/mL. It seems that phenolic compounds (apigenin, luteolin, and their corresponding glycosides) are responsible for the diethyl ether extract cytotoxic effect. It also appears that induction of oxidative stress might be involved in its cytotoxicity, since B16 and HL-60 cells increased their ROS production in response to treatment with diethyl ether extract. Neither of the tested extracts nor any phenolic compounds showed significant cytotoxic effect to human PBMC. These results demonstrated the potent anti-inflammatory and gastroprotective activities, as well as the promising cytotoxicity.

Oleum Hyperici, the oil extract of St. Johns Wort (SJW), is one of the oldest folk remedies, traditionally used in the topical treatment of wounds, bruises, ulcers, cuts, burns, hemorrhoids and also as an antiseptic. Considering the advantageous characteristics of emulsion applications, in the present study we have formulated three O/W creams containing 15% (w/v) of SJW oil extract as an active ingredient. The aim was to estimate dermatological application of the prepared creams for the abovementioned indications. The extracts were prepared according to the prescriptions from traditional medicine, however with different vegetable oils used as an extractant, namely: Olive, palm and sunflower oil. The investigated O/W creams demonstrated significant antiinflammatory effects in an in vivo double-blind randomized study, using a sodium lauryl sulphate test. Both skin parameters assessed in the study (electrical capacitance and erythema index), were restored to the baseline value after a seven-day treatment with the tested creams. Almost all investigated SJW oil extracts and corresponding creams displayed the same antimicrobial activity against the most of the investigated microorganisms with obtained minimal inhibitory concentrations values of 1,280 mu g/mL, 2,560 mu g/mL or >2,560 mu g/mL.

Two oil-in-water formulations, containing equal amounts of apigenin-enriched chamomile flower extracts, for potential use as topical antiinflammatory agents, were prepared and their physicochemical properties evaluated. A pilot clinical study was then carried out to assess patient acceptability and efficacy. The creams were either non-liposomal or liposomal. The liposomal formulations were more viscous, thus producing superior release characteristics in vitro. The clinical study also showed that the liposomal creams were, as antiinflammatory agents, slightly more effective in vivo than the non-liposomal formulations. These results suggest that there is scope for the further development of even more effective and safer alternatives to corticosteroids.

From a botanical perspective, the large number of species of the genus Sideritis coupled with the tendency to hybridise between the different species has prompted an accurate study designed to clarify all the controversial points in the botanical classification of this genus. The past phytochemical studies of the Sideritis species have been performed with different extracts from the aerial parts, essential oils and isolated compounds such as diterpenoids, flavonoids or phenylpropanoid glycosides. These investigations have justified the traditional uses for these plants and provided direction for new pharmacological research. In recent years, other compounds such as iridoids, coumarins, lignans and phenylpropanoid glycosides have also been isolated and identified. The results have shown that essential oils act as good antimicrobial agents against both Gram-positive and Gram-negative bacteria and against the Candida albicans fungus. Diterpenoids have shown antimicrobial, anti-inflammatory and antifeedant activity, and flavonoids are active as anti-ulcerous, anti-inflammatory and antioxidant agents. Future research should focus on the pharmacological activity of these isolated compounds to find new active principles and identify their mechanisms of action. In addition, it would be interesting to investigate new pharmacological activities apart from those used in traditional medicine; recent studies have shown that diterpenes and some diterpene derivatives act as effective anti-HIV and antiproliferative agents.

Three Hypericum perforatum L., Hypericaceae (St. John’s Wort) oil extracts (HPE) were prepared according to the prescriptions from traditional medicine – fresh flowering tops were macerated in three different vegetable oils: sunflower (E1), olive (E2) and palm oil (E3) for 40 days, exposed to the sunlight. The aim of the study was to investigate the gastroprotective activity of the obtained extracts in respect to their quercetin content. HPLC analysis confirmed the presence of quercetin in all of the investigated HPEs, but in different amounts: 15.1, 5.8 and 21.7 μg/mL in E1, E2, and E3, respectively. Gastroprotective activity was evaluated using cold-restraint stress (CRS) induced rat gastric mucosa lesions test. All of the HPEs showed gastroprotective activity, which was close to that achieved by the one of the most studied anti-ulcer flavonoids, quercetin [percentages of inhibition of ulcer index (UI) were 35, 62 and 40 % in E1, E2 and E3, respectively]. Contrary to the lowest quercetin content, HPE prepared with olive oil (E2) offered the highest protection against gastric damaging action of CRS. It may be assumed that this is due to other constituents of E2, which probably play an additional role in complex gastroprotective activity.

Some medicinal features of olive leaf have been known for centuries. It has been traditionally used as an antimicrobial and to prevent and treat diabetes mellitus and heart disease. Whether olive leaf, a natural antioxidant, influences the gastric defense mechanism and exhibits gastroprotection against experimentally-induced gastric lesions remains unknown. In this study, the content of total phenols, total flavonoids and tannins in olive leaf extract (OLE) were determined. Seven phenolic compounds were identified and quantified (oleuropein, caffeic acid, luteolin, luteolin-7-O-glucoside, apigenin-7-O-glucoside, quercetin, and chryseriol). Furthermore, the protective activity of the OLE in gastric mucosal injury induced by a corrosive concentration of ethanol was investigated. In relation to the control group, pretreatment with OLE (40, 80 and 120 mg kg-1) significantly (p < 0.001) attenuated the gastric lesions induced by absolute ethanol. The protective effect of the OLE was similar to that obtained with a reference drug, ranitidine. The results obtained indicate that OLE possesses significant gastroprotective activity, and that the presence of compounds with antioxidative properties would probably explain this effect.

Supercritical CO2 (SC-CO2) extraction of fenugreek seeds (Trigonella foenum-graecum L, Fabaceae) was analyzed using different pressure and temperature (16.6-33.4 MPa and, 30-50 degrees C), which corresponded to SC-CO2 density between 779 and 957 kg/m(3). The yield of extract as well as its composition were determined and analyzed for different solvent consumption defined as mass of used CO2 for extraction per mass of dry fenugreek seeds. A central composite design (CCD) combined with response surface methodology (RSM) was used to study the best extraction conditions which determined that 27.0 MPa, 42.6 degrees C, and 24.6 gCO(2)/g(dm), were the optimal conditions for isolation of total extract. The calculated yield of extract at these conditions defined by SC-CO2 density of 890 kg/m3 could be 2.57% based on fenugreek seed. Moreover, the best extraction conditions for obtaining extract which contains the maximal amount of sterols, or maximal amount of vitamin E or vitamin D were also analyzed. These RSM optimizations showed that at slightly lower pressure (22.8-24.8 MPa), temperature (39.9-42.0 degrees C) and consumption of SC-CO2 (19.0-19.7 gCO(2)/g(dm)) is followed by smaller amount of isolated extract but with maximum content of sterols (16.28%), or vitamin E or vitamin D (1.44% and 0.71%, respectively) at these conditions. Calculation of molar solubility of total extract, and apparent solubility of sterols, vitamin E and D in SC-CO2 was performed at different pressure and temperature. They were correlated to density of SC-CO2, temperature at which extractions were performed and Hildebrand solubility parameter of solvent and solubility parameter of compounds which are the main constituents of extract. These calculations also show that the highest molar solubility of total extract and apparent molar solubility of its constituents could be obtained using optimal density of SC-CO2 (830-890 kg/m(3)) defined by corresponding values of temperature and pressure (310-320 K and 20-25 MPa).

There is a worldwide ongoing investigation for novel natural constituents with cytotoxic and antioxidant properties. The aim of this study was to investigate chemical profile and stated biological activities of the supercritical CO2 extract (SCE) of old man's beard compared to the extracts obtained using the conventional techniques (Soxhlet extracts and macerate). The most abundant compound identified was usnic acid, which content was inversely proportional to the polarity of the solvent used and was the highest in the SCE, which was the sample revealing the highest cytotoxic activity in tested tumor cell lines (B16 mouse melanoma and C6 rat glioma), with lower IC50 values compared to pure usnic acid. Further investigations suggested both SCE and usnic acid to induce apoptosis and/ or autophagy in B16 and C6, indicating higher cytotoxicity of SCE to be related to the higher degree of ROS production. A good correlation of usnic acid content in the extracts and their antioxidant capacity was established, extricating SCE as the most active one. Presented results support further investigations of SCE of old man's beard as a prospective therapeutic agent with potential relevance in the treatment of cancer and/ or in oxidative stress-mediated conditions.

Topical treatment of skin infections is often limited by drawbacks related to both antimicrobial agents and their vehicles. In addition, considering the growing promotion of natural therapeutic products, our objective was to develop and evaluate naturally-based emulsion system, as prospective topical formulation for skin infections-treatment. Therefore, alkyl polyglucoside surfactants were used for stabilization of a vehicle serving as potential carrier for supercritical CO2-extract of Usnea barbata, lichen with well-documented antimicrobial activity, incorporated using two protocols and three concentrations. Comprehensive physicochemical characterization suggested possible involvement of extract's particles in stabilization of the investigated system. Raman spectral imaging served as the key method in disclosing extract's particles potential to participate in the microstructure of the tested emulsion system via three mechanisms: (1) particle-particle aggregation, (2) adsorption at the oil-water interface and (3) hydrophobic particle-surfactant interactions. Stated extract-vehicle interaction proved to be correlated to the preparation procedure and extract concentration on one hand and to affect the physicochemical and biopharmaceutical features of investigated system, on the other hand. Thereafter, formulation with the best preliminary stability and liberation profile was selected for further efficiency and in vivo skin irritation potential evaluation, implying pertinent in vitro antimicrobial activity against G+ bacteria and overall satisfying preliminary safety profile.

Chaerophyllum coloratum is an endemic plant for the Western Balkans. Despite of its common occurrence in the Sub-Mediterranean part of Dinaric countries, knowledge about its structure and biologically active compounds is rather poor. The plant material was collected in Montenegro. The structure was studied on paraffin sections, as well as on native sections stained with standard reagents for histochemical analysis. Chemical analyses of the essential oils from its leaves, flowers, roots, stems and fruits were performed. The total phenolic, flavonoid and tannin contents in different plant organs were determined, as well, in order to give an insight in the pattern of secondary metabolites distribution. Although it was shown that morphologically and ecologically Chaerophyllum coloratum differed significantly from other species of the genus native in the flora of the Western Balkans, it shared common structural features of the genus. In a comparative analysis of 33 anatomical characters of C coloratum and six previously studied Chaerophyllum species, C coloratum clustered with Chaerophyllum aureum. In addition it was closely linked with two other species of Chrysocarpum section: Chaerophyllum bulbosum and Chaerophyllum aromaticum. Histochemical and chemical analysis of secondary metabolites showed the presence of essential oil in all studied plant organs, but differing both in composition and content of individual compounds. The essential oil from the roots is characterized by a high content of monoterpene myrcene (72.18%). In the case of stem monoterpens beta-(E)-ocimene (33.59%), beta-(Z)-ocimene (20.43%) and terpinolene (10.77%) were predominant compounds, while the principal constituents in the oil of leaves were identified as spathulenol (10.19%), p-cymene-8-ol (9.45%) and p-cymene (7.6%). Monoterpenes terpinolene (17.08%), p-(Z)-(E)-ocimene (6.96%) were identified as the main constituents of the flower oil, while in the fruits, the constituents with the highest amount were caryophyllene oxide (6.93%), (Z)-beta-farnesene (5.92%), (E)-pinocarveol (5.56%) and myrtenol (5.25%).

Analysis of yield and composition profile of Melissa officinalis (Lemon balm) extract obtained by two-step CO2 extraction at high pressure was analyzed. The first or essential oil fraction, collected at 10 MPa and 40 degrees C, contained mostly the high volatile and aromatic compounds. The second fraction was consenquently extracted at 30 MPa using different temperature: 25 degrees C (higher pressure than critical pressure but at temperature below the critical; HPCP-CO2), 40 degrees C or 100 degrees C (supercritical CO2; SC-CO2). Obtained yield of essential oil fraction was 0.45% at 10 MPa, while depending on applied extraction temperature (HPCP-CO2 or SC-CO2), the yields of the second fractions collected at 30 MPa varied from 0.44% to 0.94%. Within the study, the content of active substances in obtained extracts was analyzed, too. For comparison with CO2 extracts (HPCP or SC) the yield and characterization of essential oil obtained by hydrodistillation was accomplished, as well. A significant difference was noted not only between the essential oil isolated by hydrodistillation and SC-CO2 extraction (40 degrees C, 10 MPa) but also between total extracts obtained by HPCP-CO2 and SC-CO2 extraction. The pseudo kinetic model was applied to define SC-CO2 extraction of Lemon balm to describe the relation between the yield of extraction and CO2 consumption. Antioxidant activity of obtained extracts was determined by free radical scavenging capacity (RSC) and significant correlation of antioxidant capacity and composition profile between obtained extracts was observed.

Ethanol extracts of the aerial parts of six Hypericum species, H. barbatum, H. androsaemum, H. richerii, H. hirsutum, H. perforatum and H. perforatum cultivated on mountain Tara were tested for antiinflammatory activity in comparison with indomethacin (IND) using the carrageenan-induced rat paw edema test. It was found that all examined extracts produced antiinflammatory activity, particularly those from H. hirsutum, and both wild and cultivated H. perforatum. A dose-dependent antiinflammatory effect was especially pronounced in extracts showing some lower antiinflammatory activity. There was no correlation between the amount of hypericin in the extracts and their antiinflammatory activity.

Extracts obtained from hops (Humulus lupulus L., Cannabaceae) by supercritical fluid extraction (SFE), SFE followed by isomerization, as well as by conventional technique, were investigated for their chemical composition and antibacterial activity against selected foodborne pathogens and microorganisms capable to cause the food spoilage. The antibacterial activity of the extracts was compared with the antibacterial activity of xanthohumol, compound known for its broad pharmacological properties, isolated from the raw material remained after the SFE. Xanthohumol (XH, 96%) proved to posses the most prominent activity against all the tested strains, with the MIC values ranged between 2.5 and 20 μg mL-1. Supercritical hops extract and potassium isomerized supercritical hops extract showed strong antibacterial activity against the tested strains as well. Escherichia coli was not affected by the extracts, meaning that their oral admission would not cause the same problem as antibiotic application in intestinal flora. The chemical composition of the investigated hops extracts was analysed by GC-MS. Contents of α-acids, β-acids, iso-α-acids and xanthohumol in the samples were determined by HPLC.

For the first time, an ultrasound-assisted extraction was used to investigate isolation of anthocyanin-containing extracts from the blackberry cultivar “Cacanska Bestrna” widely grown in Serbia. Sonication time and temperature influence on the yield, active substances content and antioxidant activity of the extracts isolated from blackberry fruit puree was discussed. Ultrasound-assisted extraction with acidified ethanol at room and moderately high temperature (40 °C) enabled rapid isolation (15–30 min) of anthocyanin-containing blackberry extracts with the high yields (5.3–6.3%). Both increase of the sonication temperature and time within studied ranges had positive effect on the extraction yield. The HPLC analysis revealed the presence of the anthocyanins in the investigated samples. After hydrolysis of all the samples, aglycone–cyanidin was detected in the samples. Determined cyanidin content in the obtained blackberry extracts (0.7–1.0%) was up to 20 times higher than in the blackberry juice. Cyanidin content was positively affected by sonication time and temperature increase. The antioxidant properties of the extracts were analyzed by the ferric reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity assays. Positive effect of increase of the sonication time and/or temperature on the scavenging activity of the extracts against free DPPH radicals and thus lower IC50 values was demonstrated. Strong linear correlation of DPPH radical scavenging capacities of the extracts with content of cyanidin and tannins was established. FRAP values significantly correlated with total tannin content in the extracts. The analyses of composition and antioxidant properties in this study revealed great potential of blackberry ultrasonic extracts of the cultivar “Cacanska Bestrna” for further use in food and pharmaceutical industries.

Sideritis scardica (mountain tea) is an endemic plant on the Balkan Peninsula traditionally used for treating different conditions, mainly of inflammatory nature. This study was aimed to examine the cytotoxic activity of different S. scardica extracts against the rat glioma C6 line and rat astrocytes in primary culture. The obtained data revealed that diethyl ether (extract 2) and ethyl acetate (extract 3) extracts of S. scardica exerted a cytotoxic effect on C6 rat glioma cells. Diethyl ether extract induced an increase in reactive oxygen species production, leading to apoptotic and autophagic cell death. Ethyl acetate extract induced G 2 M cell cycle arrest and autophagy. None of the tested extracts was cytotoxic to rat astrocytes in primary culture. Cytotoxic effects of S. scardica extracts were, at least in part, mediated by their flavonoid constituents apigenin and luteolin that, when applied alone, induced cell cycle arrest, apoptosis, and autophagy.

SUMMARY In this study we investigated the effects of four emollients (cetearyl octanoate, decyl oleate, C12-15 alkyl benzoate and olive oil) incorporated at a concentration of 10% on the structural properties of the cosmetic oil/water (o/w) creams (creams K1-K4, respectively) as well as the in vivo effects of those creams on pH and hydration level of the healthy volunteer’s skin after a single application. Creams’ structure was investigated throughout polarization microscopy. Anisotropic structures were found, i.e. stabilization of creams’ structure was performed by forming lamellar gel-crystalline and liquid-crystalline phase, different for different creams, which was probably caused by the presence of different emollients. The effects of creams (K1-K4) on skin hydration level and pH (30, 90, 150 and 300min after single application) were examined using Multi Probe Adapter (MPA ® 9, Courage& Khazaka Electronic, Germany), with appropriate sonde, Corneometer ® CM 825 for measuring capacitance as a measure of skin moisture level, while the skin pH was measured using Skin-pH-Meter, PH 900 sonde.The results indicate that the presence of the different emollients in o/w creams (10%) led to the creams'different effects on the skin hydration level. Cream with decyl oleate leads to a good initial hydration effect and probably rapid release of large amounts of water from the cream. Creams containing both C12-C15 alkyl benzoate or olive oil had a weaker initial effect on the skin moisture, showing good occlusive properties. A cream containing 10% cetearyl octanoate showed the lowest skin hydration potential.