Phytochemical Analysis, Biological Activities, and Molecular Docking Studies of Root Extracts from Paeonia Species in Serbia.

Without being aware of their chemical composition, many cultures have used herbaceous peony roots for medicinal purposes. Modern phytopreparations intended for use in human therapy require specific knowledge about the chemistry of peony roots and their biological activities. In this study, ethanol-water extracts were prepared by maceration and microwave- and ultrasound-assisted extractions (MAE and UAE, respectively) in order to obtain bioactive molecules from the roots of Paeonia tenuifolia L., Paeonia peregrina Mill., and Paeonia officinalis L. wild growing in Serbia. Chemical characterization; polyphenol and flavonoid content; antioxidant, multianti-enzymatic, and antibacterial activities of extracts; and in vitro gastrointestinal digestion (GID) of hot water extracts were performed. The strongest anti-cholinesterase activity was observed in PT extracts. The highest anti-ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) radical potential was observed in PP extracts, whereas against DPPH (2,2-diphenyl-1-picrylhydrazyl radicals), the best results were achieved with PO extracts. Regarding antibacterial activity, extracts were strongly potent against Bacillus cereus. A molecular docking simulation was conducted to gather insights into the binding affinity and interactions of polyphenols and other Paeonia-specific molecules in the active sites of tested enzymes. In vitro GID of Paeonia teas showed a different recovery and behavior of the individual bioactives, with an increased recovery of methyl gallate and digallate and a decreased recovery of paeoniflorin and its derivatives. PT (Gulenovci) and PP (Pirot) extracts obtained by UAE and M were more efficient in the majority of the bioactivity assays. This study represents an initial step toward the possible application of Paeonia root extracts in pharmacy, medicine, and food technologies.

Chemical Profile and Skin-Beneficial Activities of the Petal Extracts of Paeonia tenuifolia L. from Serbia.

Without being aware of its chemical makeup, many ancient societies have used Steppe peony in their traditional medicine. Given that modern phytopreparation intended for use on human skin requires, above all, knowledge of its chemical composition, the goal of this study was to make a screening of the composition of aqueous and methanolic extracts of the petals of P. tenuifolia L. and to examine them for various skin-beneficial properties. The extracts were prepared by maceration, ultrasound-assisted, and microwave-assisted extraction procedures. The chemical profiling was conducted by the use of UHPLC-LTQ-OrbiTrap MS and UHPLC/MS, and spectrophotometric methods for the determination of total polyphenol and total flavonoid contents. The biological activities entailed antioxidant ABTS, DPPH, CUPRAC (Cupric Ion Reducing Antioxidant Capacity), and FRAP (Ferric Reducing Antioxidant Power) assays, antimicrobial (antibacterial and antifungal) and antibiofilm activities, cytotoxicity, wound healing potential, as well as the adhesion and invasion of Staphylococcus lugdunensis. The results showed that the petals are rich in phenolic acids and flavonoids, which are commonly associated with numerous biological activities. The aqueous extracts were more efficient in the majority of the bioactivity assays then the methanolic ones, whereas the optimal extraction method varied between the assays. This study is the first step towards the safe use of the aqueous extracts of P. tenuifolia petals for therapeutic skin treatments.

The Structuring of Sage (Salvia officinalis L.) Extract-Incorporating Edible Zein-Based Materials with Antioxidant and Antibacterial Functionality by Solvent Casting versus Electrospinning.

In this study, in order to develop zein-based, edible, functional food-contact materials in different forms incorporating sage extract (10, 20, and 30%), solvent casting and electrospinning were employed. The study aimed to assess the effects of the applied techniques and the extract's incorporation on the materials' properties. The solvent casting generated continuous and compact films, where the extract's incorporation provided more homogenous surfaces. The electrospinning resulted in non-woven mats composed of ribbon-like fibers in the range of 1.275-1.829 µm, while the extract's incorporation provided thinner and branched fibers. The results indicated the compatibility between the materials' constituents, and efficient and homogenous extract incorporation within the zein matrices, with more probable interactions occurring during the solvent casting. All of the formulations had a high dry matter content, whereas the mats and the formulations incorporating the extract had higher solubility and swelling in water. The films and mats presented similar DPPH• and ABTS•+ radical scavenging abilities, while the influence on Staphylococcus aureus and Salmonella enterica subsp. enterica serovar Typhimurium bacteria, and the growth inhibition, were complex. The antioxidant and antibacterial activity of the materials were more potent after the extract's incorporation. Overall, the results highlight the potential of the developed edible materials for use as food-contact materials with active/bioactive functionality.

Freeze vs. Spray Drying for Dry Wild Thyme (Thymus serpyllum L.) Extract Formulations: The Impact of Gelatin as a Coating Material.

Freeze drying was compared with spray drying regarding feasibility to process wild thyme drugs in order to obtain dry formulations at laboratory scale starting from liquid extracts produced by different extraction methods: maceration and heat-, ultrasound-, and microwave-assisted extractions. Higher total powder yield (based on the dry weight prior to extraction) was achieved by freeze than spray drying and lower loss of total polyphenol content (TPC) and total flavonoid content (TFC) due to the drying process. Gelatin as a coating agent (5% w/w) provided better TPC recovery by 70% in case of lyophilization and higher total powder yield in case of spray drying by diminishing material deposition on the wall of the drying chamber. The resulting gelatin-free and gelatin-containing powders carried polyphenols in amount ~190 and 53-75 mg gallic acid equivalents GAE/g of powder, respectively. Microwave-assisted extract formulation was distinguished from the others by a higher content of polyphenols, proteins and sugars, higher bulk density and lower solubility. The type of the drying process mainly affected the position of the gelatin-derived -OH and amide bands in FTIR spectra. Spray-dried formulations compared to freeze-dried expressed higher thermal stability as confirmed by differential scanning calorimetry analysis and a higher diffusion coefficient; the last feature can be associated with the lower specific surface area of irregularly shaped freeze-dried particles (151-223 µm) compared to small microspheres (~8 µm) in spray-dried powder.

Design of biopolymer carriers enriched with natural emulsifiers for improved controlled release of thyme essential oil.

This work aims to characterize a novel system for thyme essential oil delivery based on the combination of natural emulsifiers (soy protein and soy lecithin) and alginate, produced using the extrusion technique. The formulations are optimized concerning alginate and soy protein concentrations (both 1 to 1.5 wt.%), and consequently lecithin amount, in order to achieve spherical beads in the range 2.0 to 2.3 mm and 1.2 to 1.4 mm, wet and dry, respectively. Fourier-transform infrared analysis was performed, proving that there are interactions between all components. Lecithin-soy protein synergistic combination improved entrapment efficiency of total polyphenols (for nearly 12%) and decreased thymol release in a simulated gastric solution for nearly 35%, in comparison with beads without lecithin. The addition of lecithin enhances the thermal properties of the polysaccharide-protein systems at 50 °C after 3 hr of heating. The mechanical stability of the biopolymer carriers is improved with lecithin addition and the elastic modulus varied from 80.06 to 123.7 kPa, depending on the formulation. Alginate/soy protein/lecithin are effective carriers for the encapsulation, protection, and controlled release of thyme essential oil. PRACTICAL APPLICATION: There is unfortunately growing human resistance to antibiotics. This work offers a novel system for effective protection and controlled release of thyme essential oil in the small intestine. The mechanical and thermal properties of the carrier were estimated as they indicate how the beads will be able to resist stress during their incorporation into food (i.e. cookies-mixing, baking). The proposed approach offers ''green advantage'' as arises from all-natural materials.

Native bovine hemoglobin reduces differentiation capacity of mesenchymal stromal cells in vitro.

We have tested in vitro effects of hemoglobin from bovine slaughterhouse blood (BHb) on stromal cells of mesodermal origin, with an aim to explore its use as a component of cell culture media. Human peripheral blood mesenchymal stromal cells (PB-MSCs) and three mouse cell lines (ATDC5, MC3T3-E1 and 3T3-L1) were employed to study BHb effects on their growth and migration. The cells multilineage differentiation capacity in the presence of BHb was evaluated after induced differentiation, by histochemical staining and by RT-PCR analysis of the expression of genes specific for chondrogenic, adipogenic and osteogenic lineages. The effects of BHb on the cell proliferation and motility were dependent on both, cell type and BHb concentration (0.1 µM, 1 µM and 10 µM). In the lowest concentration (0.1 µM) BHb showed the least prominent effect on the cell proliferation and migration. In this concentration BHb reduced the differentiation capacity of all tested cells and its effect was dependent of composition of induction medium and the culture period. Obtained data suggest that BHb has the potential to be used as a component of cell culture media through maintaining proliferation and reducing differentiation capacity of mesenchymal stromal cells.

Characterisation of peppermint (Mentha piperita L.) essential oil encapsulates.

Aim: The aim was to choose the optimal encapsulation system and to incorporate encapsulates loaded with essential oil into the ice cream as a model food product. Methods: Ca-alginate beads were produced by electrostatic extrusion process. Gelatine/alginate coacervates were processed with coacervation. Carnauba wax microparticles were produced using melt dispersion process. Morphological properties, chemical, and thermal stabilities of encapsulates were tested by SEM, FTIR spectral, and thermogravimetric analysis. Results: Alginate provided sufficient emulsion stability over 1 h. Ca-alginate showed higher encapsulation efficiency (EE) (98.4 ± 4.3%) compared to carnauba wax (94.2 ± 7.8%) and gelatine/alginate coacervates (13.2 ± 1.2%). The presence of essential oil in all three types of encapsulates confirmed with FTIR. The encapsulation process ensured controlled release and thermal stability of the oil. Conclusions: Ca-alginate matrix as the most suitable for peppermint essential oil encapsulation. The sensory analysis showed that ice cream incorporating encapsulates is a promising system for the consumption of health beneficial peppermint essential oil.

Adipoinductive effect of extracellular matrix involves cytoskeleton changes and SIRT1 activity in adipose tissue stem/stromal cells.

Adipose tissue (AT) homeostasis and expansion are dependent on complex crosstalk between resident adipose stromal/stem cells (ASCs) and AT extracellular matrix (ECM). Although adipose tissue ECM (atECM) is one of the key players in the stem cell niche, data on bidirectional interaction of ASCs and atECM are still scarce. Here, we investigated how atECM guides ASCs' differentiation. atECM altered shape and cytoskeleton organization of ASCs without changing their proliferation, ß-galactosidase activity and adhesion. Cytoskeleton modifications occurred due to fostered parallel organization of F-actin and elevated expression of Vimentin in ASCs. After seven-day cultivation, atECM impaired osteogenesis of ASCs, simultaneously decreasing expression of Runx2. In addition, atECM accelerated early adipogenesis concomitantly with altered Vimentin organization in ASCs, slightly increasing PPARγ, while elevated Adiponectin and Vimentin mRNA expression. Early adipogenesis triggered by atECM was followed by upregulated mitochondrial activity and Sirtuin 1 (SIRT1) expression in ASCs. Proadipogenic events induced by atECM were mediated by SIRT1, indicating the supportive role of atECM in adipogenesis-related metabolic state of ASCs. These results provide a closer look at the effects of atECM on ASC physiology and may support the advancement of engineering design in soft tissue reconstruction and fundamental research of AT.

Alginate/soy protein system for essential oil encapsulation with intestinal delivery.

Preparation of alginate-soy protein isolate (AL/SPI) complex beads containing essential oil of thyme was carried out by emulsification of thyme oil in aqueous sodium alginate solution blended with SPI solution, followed by atomization via electrostatic extrusion and gelification with calcium ions. The process parameters were optimized by variation of the alginate (1-2.5 wt.%) and SPI (0-1.5 wt.%) concentrations. Dry alginate-SPI particles exhibited wrinkle surface while shape distortion of hydrogel beads occurred with ≥1.5 wt.% alginate concentration, whereas SPI induced reduction of the particle size. Encapsulation efficiency of 72-80 % based on total polyphenols was achieved. In SGF the samples exhibited oil release of 42-55 % (due to matrix shrinkage and proteins degradation by pepsin activity), while the rest was delivered in SIF within 2.5 h simultaneously with swelling and degradation of the matrix.

Production of egg white protein hydrolysates with improved antioxidant capacity in a continuous enzymatic membrane reactor: optimization of operating parameters by statistical design.

This study focuses on the influence of operating conditions on Alcalase-catalyzed egg white protein hydrolysis performed in a continuously stirred tank reactor coupled with ultrafiltration module (10 kDa). The permeate flow rate did not significantly affect the degree of hydrolysis (DH), but a significant increase in process productivity was apparent above flow rate of 1.9 cm3 min-1. By contrast, an increase in enzyme/substrate (E/S) ratio provided an increase in DH, but a negative correlation was observed between E/S ratio and productivity. The relationship between operating conditions and antioxidant properties of the hydrolysates, measured by three methods, was studied using Box-Behnken experimental design and response surface methodology. The statistical analysis showed that each variable (impeller speed, E/S ratio, and permeate flow rate) had a significant effect on the antioxidant capacity of all tested systems. Nevertheless, obtained response functions revealed that antioxidative activity measured by DPPH, ABTS and FRAP methods were affected differently by the same operating conditions. High impeller speeds and low permeate flow rates favor ABTS while high impeller speeds and high permeate flow rates had a positive effect on the DPPH scavenging activity. On the other hand, the best results obtained with FRAP method were achieved under moderate operating conditions. The integration of the reaction and ultrafiltration membrane separation in a continuous manner appears to be a right approach to improve and intensify the enzymatic process, enabling the production of peptides with desired antioxidant activity.

Antioxidant edible films based on chitosan and starch containing polyphenols from thyme extracts.

The aim of this study was to analyse the antioxidant activity of different polymeric matrices based on chitosan and starch, incorporating a thyme extract (TE) rich in polyphenols. TE provided the films with remarkable antioxidant activity. When mixed with chitosan, the polyphenols interacted with the polymer chains, acting as crosslinkers and enhancing the tensile behaviour of films. The opposite effect was observed when incorporated into the starch matrix. All the films became darker, more reddish and less transparent when TE was incorporated. These colour changes were more marked in starch matrices, which suggests that TE compounds were poorly encapsulated. The use of chitosan-based matrices carrying TE polyphenols is recommended as a means of obtaining antioxidant films, on the basis of their tensile response and greater antioxidant activity, which could be associated with the development of polyphenol-chitosan interactions, contributing to a better protection of the functionality of polyphenols during film formation and conditioning.

Upregulation of Heme Oxygenase-1 in Response to Wild Thyme Treatment Protects against Hypertension and Oxidative Stress.

High blood pressure is the most powerful contributor to the cardiovascular morbidity and mortality, and inverse correlation between consumption of polyphenol-rich foods or beverages and incidence of cardiovascular diseases gains more importance. Reactive oxygen species plays an important role in the development of hypertension. We found that wild thyme (a spice plant, rich in polyphenolic compounds) induced a significant decrease of blood pressure and vascular resistance in hypertensive rats. The inverse correlation between vascular resistance and plasma heme oxygenase-1 suggests that endogenous vasodilator carbon monoxide generated by heme oxidation could account for this normalization of blood pressure. Next product of heme oxidation, bilirubin (a chain-breaking antioxidant that acts as a lipid peroxyl radical scavenger), becomes significantly increased after wild thyme treatment and induces the reduction of plasma lipid peroxidation in hypertensive, but not in normotensive rats. The obtained results promote wild thyme as useful supplement for cardiovascular interventions.

Chitosan crosslinked microparticles with encapsulated polyphenols: Water sorption and release properties.

Chitosan-glutaraldehyde microparticles were produced by emulsion crosslinking method to be used as drug delivery system for polyphenols from Thymus serpyllum L. aqueous extract. The effect of preparation conditions, chitosan concentration (1.5-3% w/v), and glutaraldehyde/chitosan (GA/Ch) mass ratio (0.15-1.20) on water and polyphenols transport properties was investigated. Swelling ratio of dry particles (68-230 µm) in water ranged from 280% to 530%, depending on the formulation. The decrease in swelling was observed with increased GA/Ch mass ratio (i.e. crosslinking degree) at the same chitosan concentration, or with increased chitosan concentration at the same GA/Ch mass ratio. The increase in GA/Ch mass ratio was also manifested by increased particle compactness i.e. decreased size and reduced surface roughness. The sorption capacity for polyphenols seems to be a complex interplay of swelling behaviour and interactions chitosan-glutaraldehyde-polyphenols identified by Fourier transmission infrared analysis. An increase in crystallinity of chitosan was observed upon crosslinking with glutaraldehyde and encapsulation of polyphenols, as observed by X-ray diffraction analysis. The results obtained from release kinetics of selected polyphenolic compounds (caffeic acid, rosmarinic acid, total flavonoids, and total phenol content) showed that polyphenols were released at a lower amount (2-4 times) in water, but more rapidly (45-120 min) in comparison with the release in gastric followed by intestinal simulated fluid (SGF-SIF) (120-240 min). The experimental results of the time-dependent swelling in water and polyphenols release in both, water and SGF-SIF, were analyzed with several mathematical models. The results depicted Fickian diffusion as the water transport mechanism. In the case of polyphenols, only empirical Weibull model could be suggested for describing release kinetics.

Chitosan microbeads for encapsulation of thyme (Thymus serpyllum L.) polyphenols.

In this work chitosan microbeads were prepared by emulsion technique and loaded with thyme polyphenols by diffusion from an external aqueous solution of Thymus serpyllum L. The effects of concentrations of chitosan (1.5-3% (w/v)) and GA (glutaraldehyde) (0.1-0.4% (v/v)), as a crosslinking agent on the main properties of microbeads were assessed. The obtained microgel beads from ∼ 220 to ∼ 790 µm in diameter were exposed to controlled drying process at air (at 37 °C) after which they contracted to irregular shapes (∼ 70-230 µm). The loading of dried microbeads with polyphenols was achieved by swelling in the acidic medium. The swelling rate of microbeads decreased with the increase in GA concentration. Upon this rehydration, thyme polyphenols were effectively encapsulated (active load of 66-114 mg GAE g(beads)(-1)) and the microbeads recovered a spherical shape. Both, the increase in the amount of the crosslinking agent and the presence of polyphenols, contributed to a more pronounced surface roughness of microbeads. The release of encapsulated polyphenols in simulated gastrointestinal fluids was prolonged to 3h.

Encapsulation of thyme (Thymus serpyllum L.) aqueous extract in calcium alginate beads.

BACKGROUND: Encapsulation of Thymus serpyllum L. aqueous extract within calcium alginate beads was studied in order to produce dosage formulations containing polyphenolic compounds. Electrostatic extrusion was applied for encapsulation of thyme aqueous extract in alginate gel beads. In addition to hydrogel beads, heat-dried and freeze-dried forms of beads were examined. METHODS: Encapsulation systems were examined and compared in order to choose the optimal one with respect to entrapment efficiency, preservation of antioxidant activity and thermal behaviour under heating conditions simulating the usual food processing. RESULTS: The beads obtained with approximately 2 mg g⁻¹ of gallic acid equivalents encapsulated in 0.015 g mL⁻¹ of alginate were spheres of a uniform size of about 730 µm. Encapsulation efficiency varied in the range 50-80% depending on the encapsulation method. Besides, the analysis reveals that the encapsulation process and the material used did not degrade the bioactive compounds, as the total antioxidant content remained unchanged. This was verified by Fourier transform infrared analysis, which proved the absence of chemical interactions between extracted compounds and alginate. Addition of a filler substance, such as sucrose and inulin, in the dried product reduced its collapse and roundness distortion during drying process. CONCLUSION: This study demonstrates the potential of using hydrogel material for encapsulation of plant poplyphenols to improve their functionality and stability in food products.