The Effect of the New Lupeol Derivatives on Human Skin Cells as Potential Agents in the Treatment of Wound Healing.

Skin barrier damage can be the result of various external factors including heat, radiation, chemicals and many others. Any interruption of the skin barrier integrity causes the exposure of the organism to harmful environmental factors. Therefore, there is an urgent need to develop novel therapeutics characterized by high bioavailability and effectiveness in skin damage recovery. Birch bark is known as a clinically proven, traditional medicinal remedy to accelerate wound healing. Lupeol, one of the main birch bark ingredients, shows a wide range of biological activity beneficial to the skin. The purpose of the research was to determine the influence of new lupeol derivatives on keratinocyte and fibroblast migration and proliferation, as well as to investigate various mechanisms of their antioxidant activity. The chemical modification of lupeol structure was intended to obtain more effective therapeutics characterized by higher bioavailability, permeability and safety of use. The novel triterpenes presented in this study were evaluated as the potential active ingredients preventing skin tissue degradation. Lupeol esters influence skin cells' motility and proliferation. Importantly, they are able to reduce reactive oxygen species and act indirectly by protecting the skin protein structure from being oxidized by free radicals.

Incorporation of bioflavonoids from Bidens tripartite into micelles of non-ionic surfactants - experimental and theoretical studies.

Classical extraction methods used for an isolation of active substances from the plant material are expensive, complicated and often environmentally unfriendly. The ultrasonic assistance micelle-mediated extraction method (UAMME) seems to be an interesting alternative. The aim of this work was to compare an efficiency of water solutions of three non-ionic surfactants: C9-11 Pareth-5, PPG-6 Steareth-7 and PPG-4 Laureth-5 in UAMME of Bidens tripartita. Subsequently, the obtained extracts were separated into two immiscible phases, a polyphenols rich surfactants phase and an aqueous phase by its heating above surfactants cloud points (CPC) or by salting out with NaCl. Along with decreasing the Hydrophilic/Lipophilic Balance (HLB) factor value of surfactants, i.e. increase of the hydrophobicity, a significant decreasing of the flavonoid content was observed. While polyphenols content and antioxidant activity increased. The good surface properties of all surfactants correspond to the high content of phenolic compounds in extracts and both concentration methods resulted in even a 50-fold increase of polyphenols content. Dynamic light scattering measurements (DLS) provided that solubilization of polyphenols, i.e. their incorporation into surfactants' micelles, occurred with significant enlarging of particle size. Based on the molecular dynamic simulations, the mechanism of polyphenols incorporation into micelles was discussed.

New lupeol esters as active substances in the treatment of skin damage.

The purpose of the research was to obtain new derivatives of natural triterpene lupeol and to evaluate their potential as active substances in the treatment of skin damage. Four new lupeol esters (propionate, succinate, isonicotinate and acetylsalicylate) and lupeol acetate were obtained using an eco-friendly synthesis method. In the esterification process, the commonly used hazardous reagents in this type of synthesis were replaced by safe ones. This unconventional, eco-friendly, method is particularly important because the compounds obtained are potentially active substances in skin care formulations. Even trace amounts of hazardous reagents can have a toxic effect on damaged or irritated tissues. The molecular structure of the esters were confirmed by 1H NMR, 13C NMR and IR spectroscopy methods. Their crystal structures were determined using XRD method. To complete the analysis of their characteristics, physicochemical properties (melting point, lipophilicity, water solubility) and biological activity of the lupeol derivatives were studied. Results of an irritant potential test, carried out on Reconstructed Human Epidermis (RHE), confirmed that the synthesized lupeol derivatives are not cytotoxic and they stimulate a process of human cell proliferation. The safety of use for tested compounds was determined in a cell viability test (cytotoxicity detection kit based on the measurement of lactate dehydrogenase activity) for keratinocytes and fibroblasts. The results obtained showed that the modification of lupeol structure improve its bioavailability and activity. All of the esters penetrate the stratum corneum and the upper layers of the dermis better than the maternal lupeol. Lupeol isonicotinate, acetate and propionate were the most effective compounds in a stimulation of the human skin cell proliferation process. This combination resulted in an increase in the concentration of cells of more than 30% in comparison to control samples. The results indicate that the chemical modification of lupeol allows to obtain promising active substances for treatment of skin damage, including thermal, chemical and radiation burns.

NLCs as a potential carrier system for transdermal delivery of forskolin.

Nanostructured lipid carriers (NLC) composed of the substances generally recognized as safe (GRAS) were obtained by using a hot high-pressure homogenization technique (HPH). The influence of the number of homogenization cycles and concentration of a decyl glucoside surfactant on the NLC properties were studied. The system's stability was assessed by macroscopic observation, light backscattering and zeta potential measurements. NLC particle size was measured using dynamic light scattering (DLS). The kinetically stable formulations were loaded with forskolin and selected for in vitro drug permeation study using the Franz cell method. Concentration of forskolin in the receptor solution (i.e. ethanol/PBS mixture) was analyzed with high performance liquid chromatography (HPLC) with UV detection. The obtained results have shown that NLC formulations could be used as effective carriers for forskolin permeation through the skin.

The Release of Perillyl Alcohol from the Different Kind of Vehicles.

BACKGROUND: Skin cancers are the most common malignancy in humans, and the number of cases has increased dramatically in the past few decades. Therefore, it is very important to carry out studies concerning new and safer anticancer natural agents (e.g. perillyl alcohol) and modern drug delivery systems, such as nanoformulations, which increase their bioavailability. OBJECTIVES: The aim of this work was to obtain different kinds of topical vehicles formulation and compare their efficiency in the release of perillyl alcohol. The release kinetics was determined by using certain selected mathematical models. METHOD: Formulations of a hydrogel, O/W nanoemulsion, O/W macroemulsion and nanostructured lipid carrier were developed as carriers for perillyl alcohol - one of the promising anticancer natural agents. The release study of the active agents was carried out using the Spectra/Por Standard Regenerated Cellulose membrane, at temperature T=320C. The concentration of active agents in the receptor solution was analyzed by high performance liquid chromatography. The release kinetics was determined by using selected mathematical models. RESULTS: The results of our release studies have shown that the highest and comparable amount of perillyl alcohol was released from hydrogel (35.72 ± 0.21%), NLC (35.54 ± 1.48%) and nanoemulsion (34.87 ± 4.49%). The release was found to follow Fickian diffusion in the case of hydrogel and macroemulsion, while an anomalous mechanism was observed in the case of nanoformulations. Nevertheless, the obtained nanoformulations, as well as a conventional hydrogel, may be considered potential vehicles in topical delivery of perillyl alcohol.

A micelle mediated extraction as a new method of obtaining the infusion of Bidens tripartita.

In this paper, the extracts of three-part beggarticks (Bidens tripartita) were prepared using ultrasonic assisted micelle-mediated extraction method (UAMME). Different kind of surfactants and WPC (whey protein concentrate) were applied. In the B. tripartita extracts twenty polyphenols were identified by UPLC-DAD-MS. Both aqueous and UAMME extracts mainly contained chlorogenic acid, caftaric acid and its derivatives as well as luteolin 7-O-glucoside. The luteolin was extracted with Rokanol B2 and Triton X-100. Furthermore, antioxidative properties of the extracts were analyzed with two methods: reactions with di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium (DPPH) reagent and Follin's method. The DPPH radical scavenging by micellar extracts was in general comparable with the antioxidant activity of conventional extracts. The most active was sample with Tego Care CG90, reducing about 73% of the radical. Obtained results confirmed that the UAMME might be an alternative method, to the liquid-liquid or solid-liquid extraction, of obtaining specified extracts rich in active compounds. Selecting a suitable surfactant may thus provide the expected composition of the extract.

Study of O/W micro- and nano-emulsions based on propylene glycol diester as a vehicle for geranic acid.

Nano- and microemulsions containing as the oil phase caprylic/capric propylene glycol diesters (Crodamol PC) were investigated as potential vehicle for controlled release of geranic acid. The influence of emulsifiers and co-surfactants on stability of the emulsions was investigated. Different kind of polysorbates (ethoxylated esters of sorbitan and fatty acids) were applied as the emulsifiers. The short-chain alcohols (ethanol, 1-propanol, 1-butanol) were used as co-surfactants. The emulsions were prepared at ambient temperature (25°C), by the phase inversion composition method (PIC). The stable O/W high dispersed emulsion systems based on Crodamol PC, of mean droplets size less than 200 nm, were prepared. Microemulsions stabilized by the mixture of Polisorbat 80 and 1-butanol were characterized by the largest degree of dispersion (137 nm) and the lowest PDI value (0.094), at surfactant/co-surfactant: oil weight ratio 90:10. The stable nano-emulsion (mean droplet size of 33 nm) was obtained for surfactant: oil (S:O) weight ratio 90:10, without co-surfactant addition. This nano-emulsion was chosen to release studies. The obtained results showed that the prepared stable nano-emulsion can be used as a carrier for controlled release of geranic acid. The active substance release from the nano-emulsion and the oil solution, after 24 hours was 22%.

Production of triterpenoids with cell and tissue cultures.

Triterpenes are group of biologically active compounds which can be found in higher plants. Their main source are plants bark, leaves, twigs, fruits, resins or oils. The biological activity of triterpens is very diversified and many studies have already confirmed the following therapeutic effects: anti-inflammatory, antimicrobial, antiviral, antifungial, immunomodulatory, and hepatoprotective. Synthesis of triterpenes derivates can be performed by chemical or enzymatic reactions, however biotransformation is more specific and eliminates the side products and the molecule alterations. These processes use isolated enzymes or microorganisms. Cell culture in vitro eliminates problems like extract variability as well as instability of the compounds being obtained during the extraction process. What is more, it ensures high reproducibility and optimal regio- and enantioselectivity. The most widely used technique is a classical screening of a series of microbial strains. Studies on triterpene biotransformation give a lot of information about new biologically active compounds and let predict the metabolism of biological compounds. This review presents most important advancements in the metabolic engineering of microorganisms for the production of triterpenoids. Moreover, the review highlights general strategies to obtain rich biochemical diversity of plants by employing the biocatalysts produced by microorganisms or tissue cultures.

Influence of process parameters on properties of Nanostructured Lipid Carriers (NLC) formulation.

Nanostructured lipid carriers (NLC) are stable colloidal formulations with notable advantages for drug delivery systems. Thanks to their physicochemical stability, biocompatibility, biodegradability and controlled drug release, they have received increasing attention for the last several years. The aim of the study was to prepare and characterize nanostructured lipid carriers (NLC). Both, the effect of the process parameters and the effect of the preemulsion composition on the NLC properties were investigated. In the work, different type of surfactants (i.e. decyl glucoside, Poloxamer188, Tween 80, sodium cholate) and their combinations were used to stabilize NLC dispersions. Moreover, several kinds of solid lipids (modified beeswax, gliceryl behenate, cetyl palmitate and berry wax) and liquid lipids (caprilic/capric triglyceride and decyl oleate) were applied. An ultrasonication method using a probe type sonicator was used to obtain NLC, and the time and energy of the process were modified throuhout. The physicochemical properties of the formulations, such as particle size, size distribution, polidispersity index were studied using the dynamic light scattering (DLS) method. The electrophoretic mobility of obtained particles was also measured, using the Zetasizer Nano ZS Malvern Instrument based on the Laser Doppler Velocimetry (LDV) technique. Knowing the value of electrophoretic mobility of particles for given conditions, the zeta potential was determined. The obtained results showed that the process parameters and the composition of the preemulsion had significant impact on the nanoparticles structure. The optimal formulations size ranged between 60 and 80 nm, and the value of their zeta potential was up to -30mV. The stability of these systems was further confirmed by macroscopic observation.

Studies on the formation of O/W nano-emulsions, by low-energy emulsification method, suitable for cosmeceutical applications.

The formation of oil/water (O/W) nano-emulsions suitable for cosmeceutical application was studied. Nano-emulsions were prepared by using phase inversion composition (PIC) method, one of the low-energy emulsification methods. The process consist of stepwise water addition to oil/surfactant mixture, at T = 25°C. Caprylic/capric triglycerides (GTCC), propylene glycol dicaprylate/dicaprate (PC) and oleic acid (OA) were applied as an oil phase. Polysorbate 80 was used as the surfactant. Kinetic stability of the nano-emulsions was analyzed by measuring droplet size as a function of time for different oil/surfactant ratio. The particles size distribution was analyzed by means DLS measurement technique (Dynamic Light Scattering), using Zetasizer Nano ZS (Malvern Instruments, UK). One of triterpenoic acid, practically non-water soluble substance was selected as an active and incorporated into the stable formulation. The obtained results proved that the nanoemulsion NE-T80-GTCC-20:80 based on caprylic/capric triglycerides with the oil/surfactant ratio O/S = 2 0:80 and the droplet size r = 25 nm was the most stable one and additionally showed the highest solubilisation capacity for the triterpene.

Micelle-mediated extraction of elderberry blossom by whey protein and naturally derived surfactants.

Classical methods of the extraction of active ingredients from the plant material are expensive, complicated and often environmentally unfriendly. The micelle-mediated extraction method (MME) seems to be a good alternative. In this work, extractions of elderberry blossoms (Flos Sambuci) were performed using MME methods. Several popular surfactants and whey protein concentrate (WPC) was applied in the process. The obtained results were compared with those obtained in extraction by means of water. Antioxidant properties of the extracts were analyzed by using two different methods: reaction with di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium (DPPH) reagent and Follin's method. Furthermore, the flavonoid content in the extracts was determined. The results confirmed that the MME method with using whey protein might be an alternative method for obtaining, rich in natural antioxidants, plant extracts.

In situ UV-Vis DRS evidence of Cr2+ species oxidation by CO2.

In situ UV-Vis DRS investigations have revealed that the Cr(2+) species, which dominate on the surface of a CO-reduced Cr/SBA-1 catalyst, can be oxidized by CO(2) at a wide range of temperatures. Such an oxidation is a multistep process proceeding probably via carboxylate intermediates.