A Comparative Study of the Chemical Properties and Antibacterial Activity of Four Different Ozonated Oils for Veterinary Purposes.

Infectious skin diseases are quite common in veterinary medicine. These diseases can be caused by both bacteria and pathogenic fungi. Antimicrobial drugs are usually used for treatment. An alternative to these drugs could be ozonated oils with antibacterial and antifungal properties. Four different ozonated oils (linseed, hemp seed, sunflower, and olive) were tested in order to develop an optimal pharmaceutical form for the treatment of skin infections in animals. Chemical parameters such as acid and acidity value, iodine and peroxide value, viscosity, and infrared spectres were analysed. The ozonation of oils resulted in changes in their chemical composition. The antimicrobial activity of the tested oils was evaluated by determining the minimum inhibitory concentrations and zones of inhibition in agar. After ozonation, the acid content increased in all the tested oils. The highest acidity was found in linseed oil (13.00 ± 0.11 mg KOH/g; 6.1%). Hemp oil, whose acidity was also significant (second only to linseed oil), was the least acidified by ozonation (11.45 ± 0.09 mg KOH/g; 5.75%). After ozonation, the iodine value in oils was significantly reduced (45-93%), and the highest amounts of iodine value remained in linseed (47.50 ± 11.94 g Iodine/100 g oil) and hemp (44.77 ± 1.41 Iodine/100 g oil) oils. The highest number of peroxides after the ozonation of oils was found in sunflower oil (382 ± 9.8 meqO2/kg). It was found that ozonated hemp and linseed oils do not solidify and remain in liquid form when the temperature drops. The results showed a tendency for the reference strains of S. aureus, E. faecalis, and E. coli to have broader zones of inhibition (p < 0.001) than clinical strains. Overall, ozonated linseed oil had the highest antibacterial activity, and ozonated olive oil had the lowest, as determined by both methods. It was found that ozonated linseed oil was the most effective on bacteria, while the most sensitive were S. aureus ATCC 25923, MRSA, and S. pseudointermedius (MIC 13.5 mg/mL, 4.6 mg/mL, and 13.5 mg/mL, respectively, and sterile zones 20.67 ± 0.98 mm, 20.25 ± 0.45 mm, and 18.25 ± 0.45 mm, respectively). The aim and new aspect of this work is the characterisation of selected ozonated vegetable oils, especially hemp oil, according to chemical and antibacterial parameters, in order to select suitable candidates for preclinical and clinical animal studies in the treatment of bacterial or fungal skin infections in terms of safety and efficacy.

Application of Poloxamer for In Situ Eye Drop Modeling by Enrichment with Propolis and Balsam Poplar Buds Phenolic Compounds.

In situ poloxamer-based gels are increasingly being explored as ocular drug delivery carriers to extend the release of active substances, thereby enhancing bioavailability. The objective of this study was to develop thermally stable in situ gels incorporating balsam poplar bud extract, propolis extract, and p-coumaric acid solution and to evaluate the physicochemical parameters of these gelified eye drops. This research assessed the compatibility of poloxamer-based eye drops with active components, their physicochemical properties, stability post-sterilization and during storage, and the release profiles of the active compounds. Fifteen eye drop formulations were prepared and categorized into three groups based on active components. One of the active components was propolis extract. As an alternative to propolis, eye drops containing the plant precursor, balsam poplar bud extract, were developed. The third group's active component was p-coumaric acid, a dominant phenolic acid in propolis and balsam poplar bud extracts. The study reported phenolic contents of 76.63 CAE mg/g for propolis and 83.25 CAE mg/g for balsam poplar bud aqueous extracts, with balsam poplar bud extracts showing higher SPF values (14.0) compared to propolis (12.7), while p-coumaric acid solution exhibited the highest SPF values (25.5). All eye drops were transparent, with pH values meeting the requirements for ocular drops. Formulations containing 8-10% poloxamer 407 met the criteria for in situ gels. All formulations remained stable for 90 days. Conclusion: The study results indicate that the formulated gels possess suitable physicochemical properties, are resistant to applied autoclaving conditions, and exhibit an extended release of active compounds with an increase in poloxamer content.

Edible Gels with Cranberry Extract: Evaluation of Anthocyanin Release Kinetics.

The bioactive compounds found in cranberry fruit are natural antioxidants, and their consumption reduces the risk of diabetes, cardiovascular disease, cancers, and urinary tract infections. Oral gels with cranberry fruit extract are a promising product that can ensure accurate dosage and release of the active compounds and are suitable for people with dysphagia. The aim of this study was to determine the effect of polymeric materials on the dissolution kinetics of cranberry fruit anthocyanins from gel formulations. Gel formulations were prepared using freeze-dried cranberry fruit extract with different gelling excipients: chitosan (G1-G3), sodium carboxymethylcellulose (G4-G6), and sodium carboxymethylcellulose combined with carbomers (G7-G9). The dissolution test showed that the release of anthocyanins from gel formulations G1-G6 and G9 was most intense within the first 10 min, with little change in the anthocyanin content of the acceptor medium afterwards. For the formulations based on carboxymethyl cellulose and carbomers G7 and G8, the amount of anthocyanins released into the acceptor medium gradually increased, which prolonged the release time of the active compounds. The test for the release of anthocyanins from the semi-solid systems through a hydrophilic membrane revealed that within the first hour, the total amount of anthocyanins released from the modeled gel formulations (G1-G9) was within the range of 6.02%-13.50%. The 1% chitosan (G1) gel formulation released the fastest and highest amount of anthocyanins (70% within 6 h). The other formulations showed a slower release of anthocyanins, and after 6 h, the amount of anthocyanins released from formulations G2-G9 was <57%.

Balsam Poplar Buds Extracts-Loaded Gels and Emulgels: Development, Biopharmaceutical Evaluation, and Biological Activity In Vitro.

Balsam poplar buds have been used for wound healing and treating irritated skin in traditional medicine. Balsam poplar buds extracts exhibit anti-inflammatory, antioxidant, and antimicrobial effects. In recent years, scientific research has begun to validate some of these traditional uses, leading to an increased interest in balsam poplar buds as a potential source of natural remedies in modern medicine. The study aims to simulate semi-solid pharmaceutical forms with balsam poplar buds extract and evaluate their quality through biopharmaceutical research. The active compounds identified in Lithuanian poplar buds were p-coumaric acid, cinnamic acid, caffeic acid, galangin, pinocembrin, pinobanksin, and salicin. In gels, pH values ranged from 5.85 ± 0.05 to 5.95 ± 0.07. The determined pH values of emulgels ranged from 5.13 ± 0.05 to 5.66 ± 0.15. After 6 h, the release of active compounds from gels and emulgels ranged from 47.40 ± 2.41% to 71.17 ± 3.54. p-coumaric acid dominates in the balsam poplar buds extracts. The pH values of the prepared sem-solid pharmaceutical forms are suitable for use on the skin. The viscosity of the formulations depends on the amount of gelling agent. All formulations showed antioxidant activity. It is relevant to conduct a more extensive study on the influence of the chosen carrier on the release of active compounds from semi-solid formulations with an extract of balsam poplar buds.

Optimization of Delivery and Bioavailability of Encapsulated Caffeic Acid.

Caffeic acid is a widely distributed phenolic acid. It is described in the scientific literature that caffeic acid has poor solubility. The aim of this study was to improve the solubility of caffeic acid for better dissolution kinetics when administered orally. During the study, oral capsules of different compositions were modeled. The results of the disintegration test revealed that the excipients affected the disintegration time of the capsules. The excipient hypromellose prolonged the disintegration time and dissolution time of caffeic acid. The dissolution kinetics of caffeic acid from capsules depend on the chosen excipients. P407 was more effective compared to other excipients and positively affected the dissolution kinetics of caffeic acid compared to other excipients. When the capsule contained 25 mg of ß-cyclodextrin, 85% of the caffeic acid was released after 60 min. When the capsule contained 25-50 mg poloxamer 407, more than 85.0% of the caffeic acid was released from capsules after 30 min. The research results showed that in order to improve the dissolution kinetics of caffeic acid, one of the important steps is to improve its solubility.

Formulation and Biopharmaceutical Evaluation of Capsules Containing Freeze-Dried Cranberry Fruit Powder.

Cranberry fruits are an important source of anthocyanins and anthocyanidins. The aim of the present study was to investigate the effect of excipients on the solubility of cranberry anthocyanins and their dissolution kinetics as well as on the disintegration time of the capsules. Selected excipients (sodium carboxymethyl cellulose, beta-cyclodextrin and chitosan) were found to affect the solubility and release kinetics of anthocyanins in freeze-dried cranberry powder. Capsule formulations N1-N9 had a disintegration time of less than 10 min, and capsule formulation N10 containing 0.200 g of freeze-dried cranberry powder, 0.100 g of Prosolv (combination of microcrystalline cellulose and colloidal silicon dioxide), and 0.100 g of chitosan had a capsule disintegration time of over 30 min. The total amount of anthocyanins released into the acceptor medium ranged from 1.26 ± 0.06 mg to 1.56 ± 0.03 mg. Capsule dissolution test data showed that the time to release into the acceptor medium was statistically significantly longer for the chitosan-containing capsule formulations compared to the control capsules (p < 0.05). Freeze-dried cranberry fruit powder is a potential source of anthocyanin-rich dietary supplements, and the choice of excipient chitosan could be a suitable solution in capsule formulations providing greater anthocyanin stability and modified release in the gastrointestinal tract.

Correction: Stanciauskaite et al. Balsam Poplar Buds: Extraction of Potential Phenolic Compounds with Polyethylene Glycol Aqueous Solution, Thermal Sterilization of Extracts and Challenges to Their Application in Topical Ocular Formulations. Antioxidants 2022, 11, 1771.

In the original publication [...].

Comparison of the Formulation, Stability and Biological Effects of Hydrophilic Extracts from Black Elder Flowers (Sambucus nigra L.).

Elderflower preparations have long been used to treat colds and flu, but their use is undeservedly reduced, and only dried flower teas, less often ethanolic extracts, can be purchased in pharmacies. In the case of homemade teas, the medicinal plant material is extracted with hot water for a relatively short time, thus only a small part of the active substances is extracted. The industrially produced ethanolic extract is rich in active substances, but its use is limited since ethanol in many countries is undesirable and unsuitable for children and geriatric patients. Therefore, the aim of this work was to produce extracts from elder flowers using water as extractant and a mixture of water + polyethylene glycol (PEG) 20%, to compare their chemical composition and stability, and to study the ability to neutralize reactive oxygen species (ROS) and to sustain the viability of C6 glial cells under oxidative stress conditions. The ethanolic extract was used as a standard. Thus, the extract with PEG contained more than two times higher amount of total phenolics (PC) than the aqueous one, and the stability at 6-8 °C was comparable to the stability of ethanolic extract. All three extracts showed an antioxidant effect in a concentration-dependent manner in vitro. However, only the PEG containing extract (at 20-40 µg/mL PC) was the most effective in reducing the intracellular level of ROS and sustaining the viability of glial cells. The results suggest that the co-solvent PEG increases the yield of phenolics in the extract, prolongs the stability, and enhances positive biological effects.

Evaluation of Chemical Composition, Sun Protection Factor and Antioxidant Activity of Lithuanian Propolis and Its Plant Precursors.

The growing interest in polyphenols of natural origin and their plant sources encourages the study of their chemical composition and biological activity. Propolis is widely used as a source of phenolic compounds. The aim of this study is to evaluate and compare the chemical composition, antioxidant activity and sun protection factor (SPF) of the ethanolic extracts of the poplar buds, birch buds and pine buds of propolis plant precursors collected in Lithuania. The IC50 concentration of the extracts was evaluated using DPPH and ABTS methods. Extracts of poplar buds, birch buds and propolis showed a lower IC50 concentration by ABTS and DPPH methods compared with pine buds extracts. Poplar buds and propolis extracts showed the highest SPF value, while birch and pine buds extracts showed a lower SPF value. High-performance liquid chromatography (HPLC) analysis results showed that phenolic acids, such as p-coumaric acid and cinnamic acid, and flavonoids, such as pinobanksin and pinocembrin, were identified in all the tested extracts. Salicin has been identified only in poplar buds extracts. The results of antioxidant activity showed that propolis poplar and birch buds are a promising source of biologically active polyphenols.

Balsam Poplar Buds: Extraction of Potential Phenolic Compounds with Polyethylene Glycol Aqueous Solution, Thermal Sterilization of Extracts and Challenges to Their Application in Topical Ocular Formulations.

Phenolic compounds of natural origin have been valued for their beneficial effects on health since ancient times. During our study, we performed the extraction of phenolic compounds from balsam poplar buds using different concentrations of aqueous polyethylene glycol 400 solvents (10-30% PEG400). The aqueous 30% PEG400 extract showed the best phenolic yield. The stability of the extract during autoclave sterilization was evaluated. The extract remained stable under heat sterilization. Ophthalmic formulations are formed using different concentrations (8-15%) of poloxamer 407 (P407) together with hydroxypropyl methylcellulose (0.3%), sodium carboxymethyl cellulose (0.3%) or hyaluronic acid (0.1%). Physicochemical parameters of the formulations remained significantly unchanged after sterilization. Formulations based on 12% P407 exhibited properties characteristic of in situ gels, the gelation point of the formulations was close to the temperature of the cornea. After evaluating the amount of released compounds, it was found that, as the concentration of polymers increases, the amount of released compounds decreases. Formulations based on 15% P407 released the least biologically active compounds. Sterilized formulations remained stable for 30 days.

Dermal Penetration Studies of Potential Phenolic Compounds Ex Vivo and Their Antioxidant Activity In Vitro.

Phenolic compounds with miscellaneous biological activities are an interesting component in dermatology and cosmetology practices. The aim of our study was to determine the phenolic compounds released from emulsion, emulgel, gel, ointment, and oleogel formulations penetration into human skin layers, both the epidermis and dermis, and estimate their antioxidant activity. The ex vivo penetration study was performed using Bronaugh type flow-through diffusion cells. Penetration studies revealed that, within 24 h, the chlorogenic acid released from the oleogel penetrated into skin layers to a depth of 2.0 ± 0.1 µg/mL in the epidermis and 1.5 ± 0.07 µg/mL in the dermis. The oleogel-released complex of phenolic compounds penetrating into epidermis showed the strongest DPPH free radical scavenging activity (281.8 ± 14.1 µM TE/L). The study estimated a strong positive correlation (r = 0.729) between the amount of quercetin penetrated into epidermis and the antioxidant activity detected in the epidermis extract. Plant based phenolic compounds demonstrated antioxidant activity and showed great permeability properties through the skin.

Formulation of Gels and Emulgels with Malus domestica Borkh: Apple Extracts and Their Biopharmaceutical Evaluation In Vitro.

Phenolic compounds that estimate apple extracts with multifaceted biological effects are potentially valuable for protection against skin disorders. The purpose of our research was to formulate gels and emulgels containing a complex of phenolic compounds of apple extracts and to perform a biopharmaceutical evaluation of semi-solid pharmaceutical forms, determining their antioxidant activity in vitro. HPLC analyses of phenolic compounds were performed. The total amount of phenolic compounds found in the sample of apples from the 'Paprastasis antaninis' cultivar was 1455.5 ± 72.8 µg/g. The release of phenolics from gels and emulgels was assessed by Franz-type diffusion cells. The in vitro release test revealed that phenolic compounds were released from the gel (G1-G6) formulations (70.6-73.8%) compared to the amounts (77.2-83.9%) released from the emulgel (E1-E6) formulations. The largest amount (83.9%) of phenolic compounds was released from the E5 formulation, while the smallest amounts (70.6%) were released from the formulations G3 and G5. The antioxidant activity evaluated by the DPPH and FRAP methods observed in all gel (G1-G6) and emulgel (E1-E6) formulations after 6 h were the strongest, compared to the activities observed in the formulations after 2 or 4 h. Gels and emulgels, which are rich in apple extracts, have strong antioxidant properties and may be promising choices for the development of new, innovative pharmaceutical forms or cosmetics.

Comparison of Ethanolic and Aqueous Populus balsamifera L. Bud Extracts by Different Extraction Methods: Chemical Composition, Antioxidant and Antibacterial Activities.

The balsam poplar (Populus balsamifera L.) buds that grow in Lithuania are a polyphenol-rich plant material with a chemical composition close to that of propolis. In order to potentially adapt the extracts of this plant's raw material for therapeutic purposes, it is important to carry out detailed studies on the chemical composition and biological activity of balsam poplar buds. An important step is to evaluate the yield of polyphenols by different extraction methods and using different solvents. According to our research, extracts of balsam poplar buds collected in Lithuania are dominated by p-coumaric (496.9-13,291.2 µg/g), cinnamic acid (32.9-11,788.5 µg/g), pinobanksin (34.9-1775.5 µg/g) and salicin (215.3-1190.7 µg/g). The antioxidant activity of poplar buds was evaluated by the ABTS (2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), DPPH (2,2-diphenyl-1-picrylhydrazyl) and FRAP (ferric-reducing antioxidant power) methods, all extracts showed antioxidant activity and the obtained results correlated with the obtained amounts of total phenolic compounds in the extracts (ABTS r = 0.974; DPPH r = 0.986; FRAP r = 0.955, p < 0.01). Studies of antimicrobial activity have shown that ethanolic extracts have an antimicrobal activity effect against Staphylococcus aureus, Enterococcus faecalis and Escherichia coli. The extracts showed a better antimicrobal activity against gram-positive bacteria.

Ophthalmic In Situ Gels with Balsam Poplar Buds Extract: Formulation, Rheological Characterization, and Quality Evaluation.

Balsam poplar buds are a raw material with a high content of polyphenols. Various polyphenols are known for their anti-inflammatory and antioxidant properties. In this study, an aqueous extract of balsam poplar buds was prepared in order to use environmentally friendly and non-aggressive solvents. The aqueous extract was lyophilized, and a 1% aqueous solution of lyophilized balsam poplar buds extract (L1) was prepared. L1 solution was used as a source of polyphenols for the production of ophthalmic in situ gels, so as to develop a product featuring antioxidant properties. Poloxamer 407 (P407) and hydroxypropyl methylcellulose (HPMC) were selected as gelling agents for the in situ gels. In order to select the formulations with the best conditions of use, formulations of different polymer concentrations (P407-10%, 12%, 15%; HPMC-0.5%, 0.75%) were prepared, choosing the same amount of the active polyphenol source L1. The physicochemical properties, rheological parameters, stability, and irritant effect on the rabbit corneal cell line (SIRC) were evaluated. Formulations in which P407 and HMPC concentrations were 10/0.75% and 12%/0.75% reached a gelation point close to the ocular surface temperature; the gels remained stable for 30 days and did not cause an irritant effect on the SIRC cell line.

Formulation of Ocular In Situ Gels with Lithuanian Royal Jelly and Their Biopharmaceutical Evaluation In Vitro.

Royal jelly is a natural substance produced by worker bees that possesses a variety of biological activities, including antioxidant, anti-inflammatory, antibacterial, and protective. Although fresh royal jelly is kept at low temperatures, to increase its stability, it needs to be incorporated into pharmaceutical formulations, such as in situ gels. The aim of this study was to formulate in situ ocular gels containing Lithuanian royal jelly for topical corneal use in order to increase the retention time of the formulation on the ocular surface and bioavailability. Gels were evaluated for physicochemical characteristics (pH, rheological properties, refractive index) and in vitro drug release measuring the amount of 10-hydroxy-2-decenoic acid (10-HDA). An ocular irritation test and cell viability tests were performed using the SIRC (Statens Seruminstitut Rabbit Cornea) cell culture line. Results indicated that all the in situ gels were within an acceptable pH and refractive index range close to corneal properties. Rheology studies have shown that the gelation temperature varies between 25 and 32 °C, depending on the amount of poloxamers. The release studies have shown that the release of 10-HDA from in situ gels is more sustained than royal jelly suspension. All gel formulations were non-irritant according to the short-time exposure test (STE) using the SIRC cell culture line, and long-term cell viability studies indicated that the formulations used in small concentrations did not induce cell death. Prepared in situ gels containing royal jelly have potential for ocular drug delivery, and they may improve the bioavailability, stability of royal jelly, and formation of non-irritant ocular formulations.

Extracts of Poplar Buds (Populus balsamifera L., Populus nigra L.) and Lithuanian Propolis: Comparison of Their Composition and Biological Activities.

Balsam poplar and black poplar (Populus balsamifera L. and Populus nigra L.) buds that grow in Lithuania are the primary source of propolis, therefore it is proper to evaluate and compare the composition of these raw plant materials and propolis quantitatively and qualitatively. Propolis and balsamic poplar bud extract are dominated by p-coumaric acid and black poplar-caffeic acid. Antioxidant activity was evaluated by DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), FRAP (ferric-reducing antioxidant power) and CUPRAC (cupric reducing antioxidant capacity) methods and all extracts showed antioxidant activity, and obtained results correlated with the obtained amounts of phenolic compounds and flavonoids in the extracts. Studies of antimicrobial activity have shown that all extracts have a growth inhibitory effect against Staphylococcus aureus and Candida albicans, but the extract of balsam poplar buds showed the most significant effect of such kind. Considering the results of the research, it can be stated that balsam poplar buds cultured in Lithuania are the primary raw material of propolis, which is rich in phenolic compounds with antioxidant properties and is a promising raw material for pharmaceutical purposes.

Formulation of Liposomes Containing Royal Jelly and Their Quality Assessment.

Royal jelly, a gelatinuous consistency bee product produced and secreted by the hypopharyngeal and mandibular glands of worker honeybees, is beneficial in the treatment of dermatological conditions, likely through its content of the fatty acid 10-hydroxy-2-decenoic acid (10-HDA). However, 10-HAD poorly penetrates into skin. Thus, in this work, we produced royal jelly incorporated liposomes with the aim of increasing skin penetration of 10-HDA. Lipid nanocarriers were prepared by the thin lipid-film hydration method. Size and polydispersity index of the nanocarrier particles, and their stability over 30 days were measured. The effects of royal jelly and 10-HDA liposomal formulations on the viability of immortalized human keratinocyte cells were tested with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The skin penetration of 10-HDA from liposomal formulations and royal jelly solution was studied in vitro with Franz type vertical diffusion cells using porcine skin as limiting membrane. As result, small liposomes were achieved, and the efficacy of the obtained nanoformulations was examined by means of in vitro cell assays with a HaCaT immortalized human keratinocyte cell culture line. Finally, the skin penetration experiments showed that liposomal incorporation greatly increased 10-HDA penetration into skin layers.

Biopharmaceutical Evaluation of Capsules with Lyophilized Apple Powder.

Apples are an important source of biologically active compounds. Consequently, we decided to model hard gelatin capsules with lyophilized apple powder by using different excipients and to evaluate the release kinetics of phenolic compounds. The apple slices of "Ligol" cultivar were immediately frozen in a freezer (at -35°C) with air circulation and were lyophilized with a sublimator at the pressure of 0.01 mbar (condenser temperature, -85°C). Lyophilized apple powder was used as an active substance filled into hard gelatin capsules. We conducted capsule disintegration and dissolution tests to evaluate the quality of apple lyophilizate-containing capsules of different encapsulating content. Individual phenolic compounds can be arranged in the following descending order according to the amount released from the capsules of different compositions: chlorogenic acid > rutin > avicularin > hyperoside > phloridzin > quercitrin > (-)-epicatechin > isoquercitrin. Chlorogenic acid was the compound that was released in the highest amounts from capsules of different encapsulating content: its released amounts ranged from 68.4 to 640.3 µg/mL. According to the obtained data, when hypromellose content ranged from 29% to 41% of the capsule mass, the capsules disintegrated within less than 30 min, and such amounts of hypromellose did not prolong the release of phenolic compounds. Based on the results of the dissolution test, the capsules can be classified as fast-dissolving preparations, as more than 85% of the active substances were released within 30 min.

Propolis - quality analysis and use in topical formulations.

The aim of this study was to produce propolis extracts, assess their quality and effect on skin cells and determine the penetration of active ingredients from designed semi-solid topical formulations. The use of higher-concentration ethanol and a larger amount of raw material allows extracting a larger quantity of active ingredients from raw propolis. Ultrasound extraction is an effective method for the production of aqueous extracts of propolis. The results show that depending on concentration, propolis extracts reduce the viability of keratinocytes. The phenolic compounds under observation penetrated the epidermis and dermis from designed formulations. The base of semi-solid formulation influences the efficacy of propolis preparations. The overall quantity of phenolic compounds that penetrated the skin was around 2 % from the ointment and 1.5 % from the cream.

The Role of Mitochondria in Brain Cell Protection from Ischaemia by Differently Prepared Propolis Extracts.

Mitochondria are both the primary targets and mediators of ischaemic damage in brain cells. Insufficient oxygen causes reactive oxygen species that damage the mitochondria, leading to the loss of functionality and viability of highly energy-demanding neurons. We have recently found that aqueous (AqEP), polyethylene glycol-aqueous (Pg-AqEP) and ethanolic propolis extracts (EEP) can modulate mitochondria and ROS production in C6 cells of astrocytic origin. The aim of this study was to investigate the effect of the extracts on viability, mitochondrial efficiency and superoxide generation, and inflammatory cytokine release in primary rat cerebellar neuronal-glial cell cultures affected by ischaemia (mimicked by hypoxia +/- deoxyglucose). AqEP and Pg-AqEP (15-60 µg/mL of phenolic compounds, or PC) significantly increased neuronal viability in ischaemia-treated cultures, and this was accompanied by a reduction in mitochondrial superoxide levels. Less extended protection against ischaemia-induced superoxide production and death was exhibited by 2 to 4 µg/mL of PC EEP. Both Pg-AqEP and Ag-EP (but not EEP) significantly protected the cultures from hypoxia-induced elevation of TNF-α, IL-1ß and IL-6. Only Pg-AqEP (but not AqEP or EEP) prevented hypoxia-induced loss of the mitochondrial basal and ATP-coupled respiration rate, and significantly increased the mitochondrial respiratory capacity. Summarising, the study revealed that hydrophilic propolis extracts might protect brain cells against ischaemic injury by decreasing the level of mitochondrial superoxide and preventing inflammatory cytokines, and, in the case of Pg-AqEP, by protecting mitochondrial function.