Barbara Pilawa

This article reviews the application of the methods of electron paramagnetic resonance (EPR) spectroscopy and ultraviolet–visible (UV-Vis) spectrophotometry in the examination of plant antioxidants. It proposes their use in obtaining antioxidant raw materials from plants particularly those living in harsh environments exposed to extreme external factors. It presents the principles of the operation of the EPR spectrometer and UV-Vis spectrophotometer, as well as the role of spectroscopic parameters in analysing free radicals and antioxidants for medical purposes. In this article, an X-band (9.3 GHz) EPR spectroscopy is described as well as the role of DPPH (2,2-diphenyl-1-picrylhydrazyl) free radicals in the analysis of plant antioxidant properties. Antioxidants quench the EPR and UV-Vis spectra of DPPH. EPR and UV-Vis spectra decrease with increasing time of interaction of DPPH free radicals with the antioxidant plant extracts until the minimum is reached. After that the magnitude of the spectra remains constant. Extracts with stronger antioxidant properties characterise the lower minimum values of the EPR spectra amplitude and UV-Vis absorbance. The described spectroscopic methods are helpful in finding plant extracts with strong antioxidant properties for medical purposes. This work includes examples of quenching of the EPR and UV-Vis spectra of DPPH free radicals caused by their interactions with plant extracts, including examples of extracts obtained from the single raw plant materials and their combinations. We demonstrate the influence of habitat conditions on the antioxidant properties of the plant extracts. The importance of developing research into plant products obtained from disturbed environments was emphasised.

Background: Folic acid (FA), also known as vitamin B9, functions as a co-factor in many cellular processes. Ultraviolet radiation (UV) has been shown to cause the formation of free radicals, and chronic exposure of the skin to UV radiation has been demonstrated to result in many adverse effects. Skin protection against harmful environmental factors is one of the aims of cosmetic products. One such substance is folic acid. However, aqueous FA solutions decompose after exposure to UV radiation, and the decomposition products can exhibit variable pro/anti-oxidative roles depending on the cell type and its environment. Objectives: The objective of the present study was to demonstrate the effectiveness of folic acid as a UV-protective agent in vitro cell culture model. Methods: The experimental model comprised an in vitro culture of normal human fibroblasts derived from adult skin (NHDF-Ad). Paramagnetic electron resonance (EPR) was used to assess the interaction of folic acid with free radicals after exposure to UV radiation. RT-qPCR was utilized to evaluate the impact of ultraviolet (UV) radiation on the expression of selected cell cycle regulatory genes (CCND1, P53, BAX, and BCL-2) in vitro cultured fibroblasts that were protected by folic acid. Results: EPR studies revealed the antioxidant properties of folic acid. Free radical forms of folic acid are induced during UV irradiation. The strong effect of UV irradiation on interactions of folic acid with free radicals was observed. The interaction was found to be weaker for the irradiated samples. Molecular studies have demonstrated a decline in the BAX/BCL-2 ratio in cells that have been treated with folic acid and exposed to UV radiation in comparison to the BAX/BCL-2 ratio observed in cells that have been exposed exclusively to UV radiation and not treated with folic acid. Conclusions: Whilst molecular and EPR studies both confirm the effectiveness of folic acid as a UV-protective ingredient in cosmetics and pharmaceutical products, further research in this area is required.

This comprehensive review paper describes how stressful environmental conditions affect the amounts and types of secondary metabolites synthetized by plants, with particular emphasis on plants that spontaneously grow on post-mining sites. Secondary metabolites are compounds that are not directly necessary for the performance of basic life functions by plants but play an important role in the protection against adverse biotic and abiotic factors. Stress conditions stimulate the synthesis of secondary metabolites. The challenging post-mining sites are spontaneously colonized by many plant species, including medical plants. This observation inspired us to conduct the present review study. Apart from the abiotic conditions, the synthesis of secondary metabolites is also influenced by symbionts such as mycorrhizal fungi. A common effect of abiotic stressors is oxidative damage caused by reactive oxygen species (ROS). Metabolites such as antioxidants maintain the level of ROS at a level safe for the organism. This article presents the current state of knowledge about the impact of habitat conditions on the synthesis of secondary metabolites, which could impact the plant species growing spontaneously in post-mining areas. It considers the possibility of using such post-mining, mineral habitats to enhance these physiological mechanisms for synthesizing secondary metabolites.

Background/objectives: Interactions of infusions of Ginkgo biloba, ginseng, Yerba Mate, and green tea, with free radicals, were examined. The aim of these studies was to determine quenching of free radicals by the extracts from the selected plant raw materials that are useful in the treatment of Alzheimer’s disease. Methods: The interactions were tested by an X-band (9.3 GHz) EPR spectroscopy and UV-Vis spectrophotometry. The model DPPH free radicals were used. The magnitude and changes with time of EPR and UV-Vis spectra of DPPH by the tested extracts were measured. Results: EPR and UV-Vis lines of DPPH free radicals decrease with increasing time of interactions of the extracts with DPPH, and after reaching the minimum value, it does not change with time. Ginseng infusion quenched free radicals the least. Ginkgo biloba extract quenches free radicals a little stronger than ginseng extract. Taking into account the tested extracts, Ginkgo biloba and ginseng extracts interact with free radicals less effectively compared to extracts of Yerba mate and green tea. Ginkgo biloba and ginseng extracts quench free radicals weaker than Yerba Mate and green tea extracts. Conclusions: Yerba Mate extract definitely had the strongest antioxidant properties. This extract quenches free radicals most effectively, what can be useful in the case of Alzheimer’s disease. Given its strong antioxidant properties, green tea extract can also be particularly recommended in the case of Alzheimer’s disease.

Tigecycline is an antibiotic belonging to the glycylcycline group of tetracyclines. Similar to other tetracycline derivatives, tigecycline is used in dermatology because of its bacteriostatic effect. Despite an overall favorable safety profile, tetracyclines are associated with a spectrum of cutaneous adverse effects, notably pigmentary disorders and phototoxic reactions. These dermatologic manifestations are presumed to result from tigecycline’s affinity for melanin biopolymer and its subsequent accumulation within the pigment-containing tissues. This study aimed to assess the impact of tigecycline on human normal skin cell homeostasis varied by melanin content. The study was conducted on HEMn-LP melanocytes and human dermal fibroblasts. The aim was achieved by determining the cell number, cell cycle, mitochondrial potential, and redox homeostasis and determining in silico the possibility of binding tigecycline to melanin biopolymers. In this study, it was shown that the cells more sensitive to tigecycline were HEMn-LP melanocytes. The obtained results showed that tigecycline decreased cell number in a dose-dependent manner. In addition, tigecycline was shown to reduce mitochondrial potential, increase the level of oxidized thiols, and increase ROS content in melanocytes, contributing to oxidative stress. In silico studies have shown that the binding of tigecycline to melanin may play a role in the induction of the toxic effects of tigecycline on the skin.

Free radicals in G-361 human melanoma malignum control cells and the cells cultured with simvastatin were examined by EPR spectroscopy. The proliferation of the cells was determined. The aim of this work was to examine the influence of simvastatin used at different concentrations in the G-361 cell culture on its free radicals. The concentrations of simvastatin—0.1 μM, 1 μM, 3 μM, and 5 μM—were tested. EPR spectra of free radicals were measured by an X-band (9.3 GHz) spectrometer. Amplitudes, integral intensities, linewidths, and g factors were determined. Melanin biopolymers are the main source of o-semiquinone free radicals in G-361 human melanoma malignum cells, for which the EPR lines show characteristic g values of 2.0046–2.0059, but also, free radicals occurring in other cellular structures may contribute to these signals. The amount of free radicals decreases after interactions of simvastatin with the G-361 cells, and this effect depends on the concentration of simvastatin. The highest amounts of free radicals exist in G-361 cells cultured with simvastatin at concentrations of 3 μM and 5 μM. The relatively lower amounts of free radicals occur in G-361 cells cultured with simvastatin at concentrations of 0.1 μM and 1 μM. The fast spin–lattice relaxation processes exist in the control G-361 cells and in the cells cultured with simvastatin, regardless of simvastatin concentration.

The pharmaceutical product is a powdered tablets containing bisoprolol fumarate that is used in the treatment of circulatory system diseases. They were examined by X-band (9.3 GHz) electron paramagnetic resonance spectroscopy. The aim of this work was to determine the influence of the physical conditions of storage on the properties and content of free radicals in this pharmaceutical product. The product was subjected to a temperature of 50 °C, UVA radiation, and UVA radiation and then a temperature of 50 °C. The amplitude, integral intensity, linewidth of EPR lines, and g factor, were analyzed. Free radicals were formed in all tested samples; thus, the product containing bisoprolol fumarate should not be stored at a temperature of 50 °C, and it should be protected from UVA radiation, which is in line with the manufacturer’s requirements. The content of free radicals in the examined product was highest after treatment at a temperature of 50 °C. The lowest free radical content characterized the product after the interaction of both UVA radiation and a temperature of 50 °C. EPR lines were not microwave saturated below a power of 70 mW, which indicates fast spin-lattice relaxation processes in the product. It has been demonstrated that free radical formation in the product containing bisoprolol fumarate depends on the type of physical factor.

Electron paramagnetic resonance (EPR) examination of the effect of storage conditions on properties and contents of free radicals formed in the pharmaceutical preparation containing spironolactone, taking into account physical factors such as UVA radiation and the higher temperature, was conducted. The changes in amplitude (A), integral intensity (I) and EPR linewidth (ΔB_pp) of the EPR spectra and free radical contents were determined. It was obtained that free radicals are formed during storage of this preparation under three different treatments: 50 °C, under UVA radiation, and under UVA radiation and a temperature of 50 °C. During storage, the preparation containing spironolactone should be protected from temperatures 50 °C and from UVA radiation. A higher content of free radicals is formed in the tested preparation exposed to both UVA and a temperature of 50 °C, than in the preparation exposed only to UVA or only to a temperature of 50 °C. Similar integral intensities (I) were obtained for the tested preparation exposed to a temperature of 50 °C or exposed to UVA radiation. Fast spin-lattice relaxation processes occur in all tested pharmaceutical preparation samples containing spironolactone. The usefulness of EPR spectral analysis in the study of free radicals formed in pharmaceutical preparations has been confirmed.

New active ingredients, including those of plant origin, which could protect the skin against various harmful factors, such as UV radiation and free radicals responsible for skin ageing, are still being sought. The present study was focused on the antioxidant activity of Hippophaë rhamnoides L. and Vaccinium oxycoccos L. fruit glycolic extracts. Investigations were also carried out to evaluate the effect of UVA radiation and the storage of the sea buckthorn and European cranberry extracts at an elevated temperature of 50 °C on their interactions with free radicals. The kinetics of the interactions of the extracts with DPPH were assessed using electron paramagnetic resonance (EPR) spectroscopy. The sea buckthorn and European cranberry extracts quench the EPR signal of DPPH free radicals, which indicates their antioxidant potential. The EPR method further showed that a mixture of sea buckthorn and cranberry extracts in a volume ratio of 2:1 was more potent in quenching free radicals compared to a mixture of these extracts in a ratio of 1:2. Our findings demonstrate that long-term UVA radiation exposure reduces the ability of sea buckthorn and cranberry extracts to interact with free radicals. Moreover, storage at elevated temperatures does not affect the interaction of sea buckthorn extract with free radicals, while it alters the ability of cranberry extract to interact with free radicals. This study has demonstrated that an important factor in maintaining the ability to scavenge radicals is the storage of raw materials under appropriate conditions. H. rhamnoides and V. oxycoccos extracts can be used as valuable raw materials with antioxidant properties in the pharmaceutical and cosmetic industries.

Cladosporium cladosporioides are the pigmented soil fungi containing melanin. The aim of this work was to determine the influence of amphotericin B on free radicals in the natural melanin isolated from pigmented fungi Cladosporium cladosporioides and to compare it with the effect in synthetic DOPA-melanin. Electron paramagnetic resonance (EPR) spectra were measured at X-band (9.3 GHz) with microwave power in the range of 2.2–70 mW. Amplitudes, integral intensities, linewidths of the EPR spectra, and g factors, were analyzed. The concentrations of free radicals in the tested melanin samples were determined. Microwave saturation of EPR lines indicates the presence of pheomelanin in addition to eumelanin in Cladosporium cladosporioides. o-Semiquinone free radicals in concentrations ~10²⁰ [spin/g] exist in the tested melanin samples and in their complexes with amphotericin B. Changes in concentrations of free radicals in the examined synthetic and natural melanin point out their participation in the formation of amphotericin B binding to melanin. A different influence of amphotericin B on free radical concentration in Cladosporium cladosporioides melanin and in DOPA-melanin may be caused by the occurrence of pheomelanin in addition to eumelanin in Cladosporium cladosporioides. The advanced spectral analysis in the wide range of microwave powers made it possible to compare changes in the free radical systems of different melanin polymers. This study is important for knowledge about the role of free radicals in the interactions of melanin with drugs.