Kristina Ramanauskienė

Release of Propolis Phenolic Acids from Semisolid Formulations and Their Penetration into the Human Skin In Vitro

Antioxidant and free radical scavenging effects are attributed to phenolic compounds present in propolis, and when delivered to the skin surface and following penetration into epidermis and dermis, they can contribute to skin protection from damaging action of free radicals that are formed under UV and premature skin aging. This study was designed to determine the penetration of phenolic acids and vanillin into the human skin in vitro from experimentally designed vehicles. Results of the study demonstrated the ability of propolis phenolic acids (vanillic, coumaric, caffeic, and ferulic acids) and vanillin to penetrate into skin epidermis and dermis. The rate of penetration and distribution is affected both by physicochemical characteristics of active substances and physical structure and chemical composition of semisolid vehicle. Vanillin and vanillic acid demonstrated relatively high penetration through epidermis into dermis where these compounds were concentrated, coumaric and ferulic acids were uniformly distributed between epidermis and dermis, and caffeic acid slowly penetrated into epidermis and was not determined in dermis. Further studies are deemed relevant for the development of semisolid topically applied systems designed for efficient delivery of propolis antioxidants into the skin.

Flavonoids of willow herb (Chamerion angustifolium (L.) Holub) and their radical scavenging activity during vegetation

PURPOSE Willow herb has been traditionally used in folk medicine and currently it is a potential raw material for production of phytopharmaceuticals. The aim of this work was to determine the highest amount of flavonoids and the highest radical scavenging activity of willow herb, which was collected in different vegetation phases (intensive growing, bud, massive blooming, ripening of fruits (seeds) and the end of vegetation) and in different parts of the plant (blooms, leaves and stems). MATERIAL/METHODS Raw material was collected at Kaunas Botanical garden of Vytautas Magnus University. Willow herb was extracted using methanol/water mixture (75/25 v/v, %). Methanolic extracts were purified using solid-phase extraction. For determination of the radical scavenging activity of compounds the HPLC system with the on-line post-column DPPH (1,1-diphenyl-2-picrylhydrazyl) radical reaction detection was used. RESULTS Five flavonoids were identified and their quantitative distribution and radical scavenging activity were evaluated. The highest total amount of flavonoids and radical scavenging activity were determined in willow herb collected during the massive blooming phase (11.12 ± 0.34 mg/g and 8.71 ± 0.29 mg/g, respectively). CONCLUSIONS The highest amount of flavonoids and radical scavenging activity was determined for raw material collected during the massive blooming phase. Evaluation of different parts of the plant during the massive blooming phase revealed that the highest amount of flavonoids and radical scavenging activity are characteristic for blooms of the plant.

Total Phenolic Content and Antimicrobial Activity of Different Lithuanian Propolis Solutions

The manufacture of ethanol-free propolis solutions offers a broader application. A few trials with Lithuanian propolis have been conducted. The aims of the study are to manufacture propolis water and water-free solutions and evaluate the quality and antimicrobial activity of these solutions. The studied solutions containing 2.5%, 5%, and 10% propolis are prepared. As solvents, purified water, 70% v/v ethanol, 96.3% v/v ethanol, propylene glycol, and their systems were used. Determination of total levels of phenolic compounds (FAE mg/g) is based on colour oxidation-reduction reaction using Folin-Ciocalteu reagent under alkaline conditions and performed at 765 nm wavelength using UV spectrophotometer. The highest content of phenolic compounds was determined in solutions containing 10% propolis extracts, and the lowest amounts in 2.5% propolis extracts. The water extracted the lowest amount of phenolic compounds from crude propolis, ethanol extracted the highest amount, and propylene glycol ranked the middle position. It is determined that technological parameters (stirring, temperature) contribute to content of phenolic compounds. During microbiological study, MICs were determined. The studies showed that water extracted propolis solutions and solvents mixture did not inhibit the growth of the studied microorganisms, and propolis solutions in propylene glycol were found to have antimicrobial activity.

Design and Formulation of Optimized Microemulsions for Dermal Delivery of Resveratrol

The objective of this study was to formulate optimal formulations of microemulsions (MEs) and evaluate their feasibility for delivery of resveratrol into human skin ex vivo. Oil-in-water MEs were formulated using surfactant (S) PEG-8 caprylic/capric glycerides and cosurfactant (CoS) polyglyceryl-6-isostearate. Ethyl oleate was used as an oily phase. MEs were formulated using 5 : 1, 6 : 1, and 7 : 1 surfactant and cosurfactant (S : CoS) weight ratios. Pseudoternary phase diagrams were constructed and optimal compositions of MEs were obtained using Design Expert software. Mean droplet size for optimized ME formulations was determined to be 68.54 ± 1.18 nm, 66.08 ± 0.16 nm, and 66.66 ± 0.56 nm for systems with S : CoS weight ratios 5 : 1, 6 : 1, and 7 : 1, respectively. Resveratrol loading resulted in mean droplet size increase. The distribution of droplet size between fractions changed during storage of formulated MEs. Results demonstrated the increase of number of droplets and relative surface area when S : CoS weight ratios were 6 : 1 and 7 : 1 and the decrease when S : CoS weight ratio was 5 : 1. The highest penetration of resveratrol into the skin ex vivo was determined from ME with S : CoS weight ratio 5 : 1. It was demonstrated that all MEs were similar in their ability to deliver resveratrol into the skin ex vivo.

Assessment of Lemon Balm (Melissa officinalis L.) Hydrogels: Quality and Bioactivity in Skin Cells

The aim of the study was to design gels with lemon balm extract, assess their quality, and investigate the effect of rosmarinic acid on skin cells in normal conditions and under oxidative stress. Methods. The quantities of rosmarinic acid (RA) released from gels were evaluated by applying the HPLC technique. HaCaT cell viability was assessed by using the MTT method. ROS generation was measured using DCFH-DA dye. The results showed that the gelling material affected the release of RA content from gels. Lower and slower RA content release was determined in carbomer-based gels. After 6 hours of biopharmaceutical research in vitro, at least 4% of RA was released from the gel. The results of the biological studies on HaCaT cells demonstrated that, in the oxidative stress conditions, RA reduced intracellular ROS amounts to 28%; 0.25–0.5 mg/mL of RA increased cell viability by 10–24% and protected cells from the damage caused by H2O2. Conclusions. According to research results, it is appropriate to use a carbomer as the main gelling material, and its concentration should not exceed 1.0%. RA, depending on the concentration, reduces the amount of intracellular ROS and enhances cell viability in human keratinocytes in oxidative stress conditions.

Testing of resveratrol microemulsion photostability and protective effect against UV induced oxidative stress

Abstract Resveratrol is well known for its antioxidant activity and susceptibility to ultraviolet radiation. Development of formulations providing improved stability and relevant drug delivery of resveratrol is still a challenging task. The aim of this study was to determine protective characteristics of formulated microemulsions by evaluating photoisomerization of resveratrol and to investigate the effects of resveratrol on human keratinocyte cells under oxidative stress caused by ultraviolet radiation. Incorporation of resveratrol into microemulsions resulted in increased photostability of active compounds and the results demonstrated that photodegradation of resveratrol was significantly delayed. Results of biopharmaceutical evaluation in vitro demonstrated that up to 60 % of resveratrol was released from microemulsions within 6 hours under a constant release rate profile. In vivo biological testing confirmed the ability of resveratrol to protect cells from oxidative stress and to increase cell viability. It was concluded that microemulsions might be considered in the development of UV light sensitive compounds.

Formulation of Propolis Phenolic Acids Containing Microemulsions and Their Biopharmaceutical Characterization

Microemulsions (MEs) were formulated using PEG-8 caprylic/capric glycerides and ethanolic propolis extracts. Characterization of MEs was performed by determining mean droplet size, polydispersity index, stability under varying external factors, and formulation effect on delivery of phenolic compounds into the skin ex vivo. Essential oils were included into the formulations of MEs and their influence on physical characteristics of the nanostructured systems as well as penetration into epidermis and dermis were evaluated. The droplet size, their distribution, and stability of the formulated MEs were not affected. Presence of essential oils in the formulation increased penetration of phenolic compounds in general, but only the amount of ferulic acid increased significantly. Mean droplet size increased with increase of oily phase amount, suggesting that phenolic compounds and components of essential oils were not modifying the formation of the interphase film composition and/or structure. Phenolic compounds were predominantly located in the lipid phase of the MEs thus minimizing their availability at the surface of the skin.

Release of rosmarinic acid from semisolid formulations and its penetration through human skin ex vivo

Abstract The aim of this study was to evaluate the release of rosmarinic acid (RA) from the experimental topical formulations with the Melissa officinalis L. extract and to evaluate its penetration through undamaged human skin ex vivo. The results of the in vitro release study showed that higher amounts of RA were released from the emulsion vehicle when lemon balm extract was added in its dry form. An inverse correlation was detected between the released amount of RA and the consistency index of the formulation. Different penetration of RA into the skin may be influenced by the characteristics of the vehicle as well as by the form of the extract. The results of penetration assessment showed that the intensity of RA penetration was influenced by its lipophilic properties: RA was accumulating in the epidermis, while the dermis served as a barrier, impeding its deeper penetration.

Analysis of antiproliferative effect of Chamerion angustifolium water extract and its fractions on several breast cancer cell lines

PURPOSE To evaluate the antiproliferative effect of the aerial part of Chamerion angustifolium (L.) Holub. (Onagraceae) extract and its fractions in vitro. This is the first study on the anti-proliferative effect of C. angustifolium on 3 distinct breast cancer cell lines. MATERIAL/METHODS Breast cancer cell lines MCF7, MDA-MB-468 and MDA-MB-231 were exposed to different concentrations of the water extract of C. angustifolium, where DPPH radical scavenging activity was 0.018-0.443mg/ml, expressed in rutin equivalents. Cell growth was analyzed after 24, 48 and 72h of incubation. Solid-phase extraction was applied for the fractionation of C. angustifolium water extract and MDA-MB-468 cell line growth was tested using different fractions. RESULTS The concentrations corresponding to radical scavenging activity of 0.117 and 0.266mg/ml caused MCF7 cells growth inhibition, while in the samples exposed to the highest concentration (0.355 and 0.443mg/ml) no proliferation was register, suggesting cell death. MDA-MB-468 cell analysis showed similar responses. MDA-MB-231 demonstrated cell growth inhibition following the exposure to all analyzed high extract doses (0.117-0.443mg/ml). MDA-MB-468 cells were selected to evaluate the effect of fractions. In the samples exposed to the fraction containing the highest amount (91%) of oenothein B, at the concentration of 0.117mg/ml a pronounced cell growth inhibition while at higher concentrations (0.266 and 0.443mg/ml) no cell proliferation was observed. CONCLUSIONS The consumption of C. angustifolium herb can be advantageous, alongside with conventional breast cancer treatment.

Application of HPLC-ELSD for the Quantification of 5-Aminolevulinic Acid after Penetration into Human Skin Ex Vivo

5-Aminolevulinic acid (5-ALA) has been used for treatment of different skin diseases (e.g., skin cancer, actinic keratosis (AK), psoriasis, and acne). The quality of the treatment is directly associated to the amount of 5-ALA that penetrates into skin. 5-ALA was extracted from skin samples after performing the experiment with Bronaugh cells for 4 hours. Two methods were developed by applying different column systems and mobile phase compositions: YMC-Pack Hydrosphere C18 column (250 × 4 mm, 5 µm) in series with Hichrom Hypersil H5ODS (150 × 4 mm, 5 µm), mobile phase consisted of 0.1% TFA in water with 2% of ACN (method A); ACE 3AQ column (150 × 4 mm, 3 µm) eluted with 0.05% TFA in water (method B). Exploratory fluorimetric analysis requiring pre-column or post-column derivatization did not warrant the expectations of simple and effective analysis; therefore, ELS detection for 5-ALA was considered. Parallel chromatography was applied for double-validation of the methods in order to correctly evaluate the possibility of ELSD application for 5-ALA detection and the quantification amount in skin extracts. The retention time and adequate lowest limit of quantification (LLOQ) of 5-ALA were determined in both methods: 5.5 min and 20 µg/ml (method A); 6.2 min and 8.4 µg/ml (method B). Both HPLC-ELSD methods proved to be simple, accurate, precise, reliable, and showed reproducible values in the concentration range of 20–200 µg/ml in human skin extracts.