Filip Šupljika

This study investigates the optimization of extraction processes for bioactive compounds and proteins from the mushroom Craterellus cornucopioides by comparing Microwave-Assisted Extraction and Ultrasound-Assisted Extraction. Using Response Surface Methodology, the effects of temperature or amplitude, time, and solvent type were evaluated on total phenols, flavonoids, proteins, glutathione content, and antioxidant capacity measured by DPPH and FRAP assays. Additionally, the antimicrobial potential of the extracts was screened against various pathogens. Results demonstrated that water was the most effective solvent for nearly all parameters across both techniques, providing a unified optimum in the ultrasound system at six minutes and one hundred percent amplitude. However, a notable exception was observed for glutathione recovery in the microwave system, where ethanol proved superior to water. Ultrasound-assisted extraction consistently outperformed microwave extraction in protein yield and overall antioxidant potential, offering a more robust approach regarding process efficiency and bioactive yield. In conclusion, while both green techniques enhance recovery, ultrasound extraction with water establishes itself as the most consistent method for the simultaneous extraction of bioactives.

Mushrooms like Inonotus obliquus and Ganoderma lucidum show significant pharmacological promise. This review analyzes fungi as sources of natural inhibitors against Advanced Glycation End-products (AGEs)—key drivers of diabetes and neurodegeneration. We highlight that extracts from Lignosus rhinocerus and Auricularia auricula exhibit antiglycation potency (IC₅₀ as low as 0.001 mg/mL) superior to aminoguanidine. Inhibitory effects are attributed to bioactive fractions including FYGL proteoglycans, uronic acid-rich polysaccharides, and fungal-specific metabolites like ergothioneine. These compounds act through multi-target mechanisms across the glycation cascade: competitive inhibition of Schiff base formation, trapping reactive dicarbonyls (e.g., methylglyoxal), transition metal chelation, and direct scavenging of reactive oxygen species (ROS). Furthermore, the review addresses the transition from in vitro potency to in vivo efficacy (RAGE pathway modulation), stability during food processing (UV-B irradiation), and critical safety issues regarding heavy metal bioaccumulation. Mushroom-derived inhibitors represent a sustainable therapeutic alternative to synthetic agents, offering broader protection against glycative stress. This synthesis provides a foundation for developing standardized mushroom-based nutraceuticals for managing AGE-related chronic disorders.

Mushrooms have long been valued not only as food but also for their medicinal properties, especially in Eastern European traditional medicine. Species such as Inonotus obliquus, Fomitopsis officinalis, Piptoporus betulinus and Fomes fomentarius have been used to treat gastrointestinal problems, cancers, respiratory ailments and more. Modern research confirms their diverse pharmacological effects, including antitumor, immunomodulatory, antioxidant, antiviral, antibacterial and antidiabetic activities. In addition, mushrooms are widely incorporated into functional foods and nutraceuticals that promote health. Their sustainable cultivation, efficient use of agricultural residues, rapid growth cycles and resilience to environmental stressors make them an environmentally friendly source of food and pharmaceuticals. This review focuses on the potential of fungi to inhibit advanced glycation end products (AGEs)—harmful compounds formed through the non-enzymatic binding of sugars to proteins, lipids or nucleic acids. AGEs are strongly associated with the progression of chronic diseases such as diabetes, cardiovascular disorders, neurodegeneration and aging. Natural AGE inhibitors from mushrooms represent a promising therapeutic alternative to synthetic agents, as they may offer broader mechanisms of action with fewer adverse effects.

Knowledge about the composition (volatile and non-volatile) and functionality of natural extracts from Mediterranean plants serves as a basis for their further application. In this study, five selected plants were used for the extraction of plant metabolites. Leaves and flowers of Critmum maritimum, Rosmarinus officinalis, Olea europea, Phylliera latifolia and Mellisa officinalis were collected, and a total of 12 extracts were prepared. Extractions were performed under microwave-assisted conditions, with two solvent types: water (W) and a hydroalcoholic (ethanolic) solution (HA). Detailed extract analysis was conducted. Phenolics were analyzed by detecting individual bioactive compounds using high-performance liquid chromatography and by calculating total phenolic and total flavonoid content through spectrophotometric analysis. Higher concentrations of total phenolics and total flavonoids were obtained in the hydroalcoholic extracts, with the significantly highest total phenolic and flavonoid values in the rosemary hydroalcoholic extract (3321.21 mg_GAE/L) and sea fennel flower extract (1794.63 mg_QE/L), respectively; and the lowest phenolics in the water extract of olive leaves (204.55 mg_GAE/L) and flavonoids in the water extracts of sea fennel leaves, rosemary, olive and mock privet (around 100 mg_QE/L). Volatile organic compounds (VOC) were detected using HS-SPME/GC–MS (Headspace Solid-Phase Microextraction coupled with Gas Chromatography-Mass Spectrometry), and antioxidant capacity was estimated using DPPH (2,2-diphenyl-1-picrylhydrazyl assay) and FRAP (Ferric Reducing Antioxidant Power) methods. HS-SPME/GC–MS analysis of samples revealed that sea fennel had more versatile profile, with the presence of 66 and 36 VOCs in W and HA sea fennel leaf extracts, 52 and 25 in W and HA sea fennel flower extracts, 57 in rosemary W and 40 in HA, 20 in olive leaf W and 9 in HA, 27 in W mock privet and 11 in HA, and 35 in lemon balm W and 10 in HA extract. The lowest values of chlorophyll a were observed in sea fennel leaves (2.52 mg/L) and rosemary (2.21 mg/L), and chlorophyll b was lowest in sea fennel leaf and flower (2.47 and 2.25 mg/L, respectively), while the highest was determined in olive (6.62 mg/L). Highest values for antioxidant activity, determined via the FRAP method, were obtained in the HA plant extracts (up to 11,216 mg_AAE/L for lemon balm), excluding the sea fennel leaf (2758 mg_AAE/L) and rosemary (2616 mg_AAE/L). Considering the application of these plants for fresh fish preservation, antimicrobial activity of water extracts was assessed against Vibrio fischeri JCM 18803, Vibrio alginolyticus 3050, Aeromonas hydrophila JCM 1027, Moraxella lacunata JCM 20914 and Yersinia ruckeri JCM 15110. No activity was observed against Y. ruckeri and P. aeruginosa, while the sea fennel leaf showed inhibition against V. fisheri (inhibition zone of 24 mm); sea fennel flower was active against M. lacunata (inhibition zone of 14.5 mm) and A. hydrophila (inhibition zone of 20 mm); and rosemary and lemon balm showed inhibition only against V. fisheri (inhibition zone from 18 to 30 mm). This study supports the preparation of natural extracts from Mediterranean plants using green technology, resulting in extracts rich in polyphenolics with strong antioxidant potential, but with no clear significant antimicrobial efficiency at the tested concentrations.

Kombucha is a traditional beverage obtained by the microbial fermentation of tea using a symbiotic culture of bacteria and yeasts. In addition to several documented functional properties, such as anti-inflammatory activity and antioxidant activity, kombucha is often credited with high levels of vitamins, including riboflavin. To our knowledge, the vitamin B2 content in traditionally prepared kombucha has been determined in only two studies, in which the concentration measured by the HPLC technique ranged from 2.2 × 10⁻⁷ to 2.1 × 10⁻⁴ mol dm⁻³. These unexplained differences of three orders of magnitude in the vitamin B2 content prompted us to determine its concentration during the cultivation of kombucha under very similar conditions by spectrofluorimetry. The B2 concentrations during the 10-day fermentation of black tea ranged from 7.6 × 10⁻⁸ to 3.3 × 10⁻⁷ mol dm⁻³.

As a principal pigment in plants, chlorophyll a (Chl a) is widely used to evaluate quality changes and senescence process during storage of leafy vegetables. The determination of Chl a in plant extracts by spectrophotometric methods using various empirical equations is often unreliable. Considering the sensitivity of fluorescence detection, we report here a simple, inexpensive spectrofluorimetric method that can detect and quantify Chl a in plant extracts. The fluorescence standard used for the quantitative determination of Chl a was isolated in our laboratory from the extract of Anthrospira platensis (Spirulina). The method proved to be reliable, fast, and low cost in a study of the influence of the most commonly used domestic storage conditions on Chl a degradation in fresh-cut arugula.

Dipeptidyl peptidase III (DPP III, EC 3.4.14.4) is a monozinc metalloexopeptidase that hydrolyzes dipeptides from the N-terminus of peptides consisting of three or more amino acids. Recently, DPP III has attracted great interest from scientists, and numerous studies have been conducted showing that it is involved in the regulation of various physiological processes. Since it is the only metalloenzyme among the dipeptidyl peptidases, we considered it important to study the process of binding and exchange of physiologically relevant metal dications in DPP III. Using fluorimetry, we measured the K_d values for the binding of Zn²⁺, Cu²⁺, and Co²⁺ to the catalytic site, and using isothermal titration calorimetry (ITC), we measured the K_d values for the binding of these metals to an additional binding site. The structure of the catalytic metal’s binding site is known from previous studies, and in this work, the affinities for this site were calculated for Zn²⁺, Cu²⁺, Co²⁺, and Mn²⁺ using the QM approach. The structures of the additional binding sites for the Zn²⁺ and Cu²⁺ were also identified, and MD simulations showed that two Cu²⁺ ions bound to the catalytic and inhibitory sites exchanged less frequently than the Zn²⁺ ions bound to these sites.

Interactions of an array of nucleic acid structures with a small series of benzothiazole ligands (bis-benzothiazolyl-pyridines—group 1, 2-thienyl/2-benzothienyl-substituted 6-(2-imidazolinyl)benzothiazoles—group 2, and three 2-aryl/heteroaryl-substituted 6-(2-imidazolinyl)benzothiazoles—group 3) were screened by competition dialysis. Due to the involvement of DNA:RNA hybrids and triplex helices in many essential functions in cells, this study’s main aim is to detect benzothiazole-based moieties with selective binding or spectroscopic response to these nucleic structures compared to regular (non-hybrid) DNA and RNA duplexes and single-stranded forms. Complexes of nucleic acids and benzothiazoles, selected by this method, were characterized by UV/Vis, fluorescence and circular dichroism (CD) spectroscopy, isothermal titration calorimetry, and molecular modeling. Two compounds (1 and 6) from groups 1 and 2 demonstrated the highest affinities against 13 nucleic acid structures, while another compound (5) from group 2, despite lower affinities, yielded higher selectivity among studied compounds. Compound 1 significantly inhibited RNase H. Compound 6 could differentiate between B- (binding of 6 dimers inside minor groove) and A-type (intercalation) helices by an induced CD signal, while both 5 and 6 selectively stabilized ATT triplex in regard to AT duplex. Compound 3 induced strong condensation-like changes in CD spectra of AT-rich DNA sequences.

Background: The aim of this study was to evaluate high voltage electrical discharges (HVED) as a green technology, in order to establish the effectiveness of phenolic extraction from olive leaves against conventional extraction (CE). HVED parameters included different green solvents (water, ethanol), treatment times (3 and 9 min), gases (nitrogen, argon), and voltages (15, 20, 25 kV). Methods: Phenolic compounds were characterized by ultra-performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS), while antioxidant potency (total phenolic content and antioxidant capacity) were monitored spectrophotometrically. Data for Near infrared spectroscopy (NIR) spectroscopy, colorimetry, zeta potential, particle size, and conductivity were also reported. Results: The highest yield of phenolic compounds was obtained for the sample treated with argon/9 min/20 kV/50% (3.2 times higher as compared to CE). Obtained results suggested the usage of HVED technology in simultaneous extraction and nanoformulation, and production of stable emulsion systems. Antioxidant capacity (AOC) of obtained extracts showed no significant difference upon the HVED treatment. Conclusions: Ethanol with HVED destroys the linkage between phenolic compounds and components of the plant material to which they are bound. All extracts were compliant with legal requirements regarding content of contaminants, pesticide residues and toxic metals. In conclusion, HVED presents an excellent potential for phenolic compounds extraction for further use in functional food manufacturing.