Dynamic Properties of Adsorption Layers of κ-Casein Fibrils.

The dynamic surface properties of native κ-casein solutions and aqueous dispersions of its fibrils differ significantly from the corresponding properties of the systems with globular proteins. The dependence of the dynamic surface elasticity of κ-casein solutions on surface pressure has a local maximum, indicating partial displacement of macromolecules from the proximal region of the surface layer to the distal one. This dependence becomes monotonic for fibril dispersions, similar to the results for dispersions of globular protein fibrils, but unlike the latter case, the surface elasticity close to the steady state reaches values that are approximately four times higher than the data for native protein solutions at the same concentrations.

Delayed nucleation in lipid particles.

Metastable states in first-order phase-transitions have been traditionally described by classical nucleation theory (CNT). However, recently an increasing number of systems displaying such a transition have not been successfully modelled by CNT. The delayed crystallization of phospholipids upon super-cooling is an interesting case, since the extended timescales allow access into the dynamics. Herein, we demonstrate the controllable behavior of the long-lived metastable liquid-crystalline phase of dilauroyl-phosphatidylethanolamine (DLPE), arranged in multi-lamellar vesicles, and the ensuing cooperative transition to the crystalline state. Experimentally, we find that the delay in crystallization is a bulk phenomenon, which is tunable and can be manipulated to span two orders of magnitude in time by changing the quenching temperature, solution salinity, or adding a secondary phospholipid. Our results reveal the robust persistence of the metastability, and showcase the apparent deviation from CNT. This distinctive suppression of the transition may be explained by the resistance of the multi-lamellar vesicle to deformations caused by nucleated crystalline domains. Since phospholipids are used as a platform for drug-delivery, a programmable design of cargo hold and release can be of great benefit.

Competing processes of micellization and fibrillization in native and reduced casein proteins.

Kappa-casein (κCN) and beta-casein (ßCN) are disordered proteins present in mammalian milk. In vitro, ßCN self-assembles into core-shell micelles. κCN self assembles into similar micelles, as well as into amyloid-like fibrils. Recent studies indicate that fibrillization can be suppressed by mixing ßCN and κCN, but the mechanism of fibril inhibition has not been identified. Examining the interactions of native and reduced kappa-caseins (N-κCN and R-κCN) with ßCN, we expose a competition between two different self-assembly processes: micellization and fibrillization. Quite surprisingly, however, we find significant qualitative and quantitative differences in the self-assembly between the native and reduced κCN forms. Specifically, thermodynamic analysis reveals exothermic demicellization for ßCN and its mixtures with R-κCN, as opposed to endothermic demicellization of N-κCN and its mixtures with ßCN at the same temperature. Furthermore, with time, R-κCN/ßCN mixtures undergo phase separation into pure ßCN micelles and R-κCN fibrils, while in the N-κCN/ßCN mixtures fibril formation is considerably delayed and mixed micelles persist for longer periods of time. Fibrils formed in N-κCN/ßCN mixtures are shorter and more flexible than those formed in R-κCN/ßCN systems. Interestingly, in the N-κCN/ßCN mixtures, the sugar moieties of N-κCN oligomers seem to organize on the mixed micelles surface in a manner similar to the organization of κCN in milk casein micelles.

Intermolecular chiral assemblies in R(-) and S(+) 2-butanol detected by microcalorimetry measurements.

Supramolecular chiral assemblies of R(-) and S(+) 2-butanol, in their neat form or when dissolved in their nonchiral isomer isobutanol, were evaluated by isothermal titration calorimetry (ITC) ensuing mixing. Dilution of 0.5 M solution of R(-) 2-butanol in isobutanol into the latter liberated heat of several calories per mole, which was approximately double than that obtained in parallel dilutions of S(+) 2-butanol in isobutanol. The ITC dilution profiles indicated an estimate of about 100 isobutanol solvent molecules surrounding each of the 2-butanol enantiomers, presumably arranged in chiral configurations, with different adopted order between the isomers. Mixings of neat R and S 2-butanol were followed by endothermic ITC profiles, indicating that, in racemic 2-butanol, both the supramolecular order and the intermolecular binding energies are lower than in each of the neat chiral isomers. The diversion from symmetrical ITC patterns in these mixings indicated again a subtle difference in molecular organization between the neat enantiomers. It should be noted that the presence of impurities, α-pinene and teterhydrofuran, at a level totaling 0.5%, did not influence the ITC heat flow profiles. The findings of this study demonstrate for the first time that chiral solutes in organic solvents are expected to acquire asymmetric solvent envelopes that may be different between the enantiomers, thus broadening this phenomenon beyond the previously demonstrated cases in aqueous solutions.

Mixed micellization between natural and synthetic block copolymers: ß-casein and Lutrol F-127.

Amphiphilic block copolymers and mixtures of amphiphiles find broad applications in numerous technologies, including pharma, food, cosmetic and detergency. Here we report on the interactions between a biological charged diblock copolymer, ß-casein, and a synthetic uncharged triblock copolymer, Lutrol F-127 (EO(101)PO(56)EO(101)), on their mixed micellization characteristics and the micelles' structure and morphology. Isothermal titration calorimetry (ITC) experiments indicate that mixed micelles form when Lutrol is added to monomeric as well as to assembled ß-casein. The main driving force for the mixed micellization is the hydrophobic interactions. Above ß-casein CMC, strong perturbations caused by penetration of the hydrophobic oxypropylene sections of Lutrol into the protein micellar core lead to disintegration of the micelles and reformation of mixed Lutrol/ß-casein micelles. The negative enthalpy of micelle formation (ΔH) and cooperativity increase with raising ß-casein concentration in solution. ζ-potential measurements show that Lutrol interacts with the protein micelles to form mixed micelles even below its critical micellization temperature (CMT). They further indicate that Lutrol effectively masks the protein charges, probably by forming a coating layer of the ethyleneoxide rich chains. Small-angle X-ray scattering (SAXS) and cryogenic-transmission electron microscopy (cryo-TEM) indicate relatively small changes in the oblate micellar shape, but do show swelling along the small axis of ß-casein micelles in the presence of Lutrol, thereby confirming the formation of mixed micelles.

Impact of Chemical Composition on the Nanostructure and Biological Activity of α-Galactosidase-Loaded Nanovesicles for Fabry Disease Treatment.

Fabry disease is a rare lysosomal storage disorder characterized by a deficiency of α-galactosidase A (GLA), a lysosomal hydrolase. The enzyme replacement therapy administering naked GLA shows several drawbacks including poor biodistribution, limited efficacy, and relatively high immunogenicity in Fabry patients. An attractive strategy to overcome these problems is the use of nanocarriers for encapsulating the enzyme. Nanoliposomes functionalized with RGD peptide have already emerged as a good platform to protect and deliver GLA to endothelial cells. However, low colloidal stability and limited enzyme entrapment efficiency could hinder the further pharmaceutical development and the clinical translation of these nanoformulations. Herein, the incorporation of the cationic miristalkonium chloride (MKC) surfactant to RGD nanovesicles is explored, comparing two different nanosystems-quatsomes and hybrid liposomes. In both systems, the positive surface charge introduced by MKC promotes electrostatic interactions between the enzyme and the nanovesicles, improving the loading capacity and colloidal stability. The presence of high MKC content in quatsomes practically abolishes GLA enzymatic activity, while low concentrations of the surfactant in hybrid liposomes stabilize the enzyme without compromising its activity. Moreover, hybrid liposomes show improved efficacy in cell cultures and a good in vitro/in vivo safety profile, ensuring their future preclinical and clinical development.

In vitro discriminative antipseudomonal properties resulting from acyl substitution of N-terminal sequence of dermaseptin s4 derivatives.

Truncation and acylation were combined to investigate the broad-spectrum bactericidal and hemolytic peptide S4(1-15). Substitution of up to seven residues with dodecanoic acid (C(12)) gradually led to specific antipseudomonal activity: out of 40 bacterial strains tested in vitro, C(12)-S4(8-15) displayed similar minimal inhibitory concentrations (MICs) as S4(1-15) against Pseudomonas aeruginosa sp. (identical MIC(90)) but was practically inactive against most other bacteria or erythrocytes. Surface plasmon resonance and isothermal titration calorimetry experiments revealed the binding properties of S4(1-15) to be consistent with its nonselective activities, while discriminative activities of C(12)-S4(8-15) correlated with high binding affinity to a membrane containing pseudomonal lipopolysaccharides and with lower affinities to membranes containing nonpseudomonal lipopolysaccharides or cholesterol. Various mechanistic studies failed to detect significant differences in secondary structure, bactericidal kinetics, or ability to perturb the cytoplasmic membrane, pointing to a similar mode of action.

Self-assembly of bovine beta-casein below the isoelectric pH.

Beta-casein is an intrinsically unstructured amphiphilic protein that self-assembles into micelles at neutral pH. This paper reports that beta-casein self-organizes into micelles also under acidic conditions. The protein association behavior and micelle characteristics at pH 2.6, well below the p I, are presented. The pH was found to strongly affect the micelle shape and dimensions. Cryogenic transmission electron microscopy (cryo-TEM) experiments revealed disk-like micelles of 20-25 nm in length and approximately 3.5 nm in height in acidic conditions. An aggregation number of 6 was determined by sedimentation equilibrium under these conditions. Isothermal titration calorimetry experiments verified the association below the p I and allowed determination of the micellization enthalpy, the critical micellar concentration, and the micellization relative cooperativity (MR). Small-angle X-ray scattering results at concentrations below the critical micellization concentration (CMC) suggest that the monomeric protein is likely in a premolten globule state at low pH. Calculations of the protein charge at acidic and neutral pH reveal a similar high net charge but considerable differences in the charge distribution along the protein backbone. Overall the results show that beta-casein is amphiphilic at low pH, but the distribution of charge along the protein chain creates packing constraints that affect the micelle organization, leading at concentrations above the CMC to the formation of disk micelles.

The Conformation and Aggregation of Proline-Rich Surfactant-Like Peptides.

The secondary structure of proline-rich surfactant-like peptides is examined for the first time and is found to be influenced by charged end groups in peptides P6K, P6E, and KP6E and an equimolar mixture of P6K and P6E. The peptides exhibit a conformational transition from unordered to polyproline II (PPII) above a critical concentration, detected from circular dichroism (CD) measurements and unexpectedly from fluorescence dye probe measurements. Isothermal titration calorimetry (ITC) measurements provided the Gibbs energies of hydration of P6K and P6E, which correspond essentially to the hydration energies of the terminal charged residues. A detailed analysis of peptide conformation for these peptides was performed using density functional theory calculations, and this was used as a basis for hybrid quantum mechanics/molecular mechanics molecular dynamics (QM/MM MD) simulations. Quantum mechanics simulations in implicit water show both peptides (and their 1:1 mixture) exhibit PPII conformations. However, hybrid QM/MM MD simulations suggest that some deviations from this conformation are present for P6K and P6E in peptide bonds close to the charged residue, whereas in the 1:1 mixture a PPII structure is observed. Finally, aggregation of the peptides was investigated using replica exchange molecular dynamics simulations. These reveal a tendency for the average aggregate size (as measured by the radius of gyration) to increase with increasing temperature, which is especially marked for P6K, although the fraction of the most populated clusters is larger for P6E.

Micellization of bovine beta-casein studied by isothermal titration microcalorimetry and cryogenic transmission electron microscopy.

The association behavior, critical micellization concentration (CMC), and enthalpy of demicellization (DeltaHdemic) of bovine beta-casein were studied, for the first time by isothermal titration calorimetry, in a pH 7.0 phosphate buffer with 0.1 ionic strength and in pure water. In the buffer solutions, the CMC decreased asymptotically from 0.15 to 0.006 mM as the temperature was raised from 16 to 45 degrees C. DeltaHdemic decreased with increasing temperature between 16 and 28 degrees C but increased from 28 to 45 degrees C. Thermodynamic analysis below 30 degrees C is consistent with the Kegeles shell model, which suggests a stepwise association process. At higher temperatures, this model exhibits limitations, and the micellization becomes much more cooperative. The CMC values in water, measured between 17 and 28 degrees C, decreased with increasing temperature and, expectedly, were higher than those found in the buffer solutions. beta-Casein micelles were visualized and characterized, for the first time in their hydrated state, using advanced digital-imaging cryogenic transmission electron microscopy. The images revealed small, oblate micelles, about approximately 13 nm in diameter. The micelles shape and dimensions remained nearly constant in the temperature range of 24-35 degrees C.

From Discs to Ribbons Networks: The Second Critical Micelle Concentration in Nonionic Sterol Solutions.

At the critical micelle concentration (CMC), amphiphiles self-assemble into spherical micelles, typically followed by a transition at the second CMC to cylindrical micelles that are uniform in width but are polydispersed in length and have swollen ends. In this Letter, we report on a new structural path of self-assembly that is based on discoidal (coin-like), rather than spherical, geometry; the nonionic sterol ChEO10 is shown to form monodisperse equilibrium disc assemblies at the first CMC, transitioning at the second CMC into flat ribbons that (like the cylindrical micelles) have uniform width, polydispersed length, and swollen ends. Increase in ChEO10 concentration or the temperature leads to ribbon elongation, branching, and network formation. This self-assembly path reveals that (1) surfactants can form equilibrium nonspherical assemblies at the CMC and (2) aggregate progression around the second CMC is similar for the disc and sphere geometries.

Antioxidant activity of Crataegus aronia aqueous extract used in traditional Arab medicine in Israel.

BACKGROUND: The medicinal use of extracts prepared from plant parts of the genus Crataegus dates back to ancient times. Furthermore, it has been proposed that its antioxidant constituents account for its beneficial therapeutic effects. A decoction of leaves and unripe fruits from Crataegus aronia syn. azarolus (L) (Rosaceae), the indigenous Israeli hawthorn, is used to treat cardiovascular diseases, cancer, diabetes and sexual weakness in Arab traditional medicine. PURPOSE: Because laboratory data on the bioactivity of extracts prepared from the indigenous Israeli hawthorn is lacking, we evaluated the antioxidant and cytotoxic potentials of an extract prepared from leaves and unripe fruits in a variety of cell and cell-free in vitro assays. METHODS: The antioxidant assays measured: (a) its ability to inhibit (i) oxidation of beta-carotene, (ii) 2,2'-azobis(2-amidino-propan) dihydrochloride (AAPH)-induced plasma oxidation and (iii) iron-induced lipid peroxidation in rat liver homogenates; (b) its ability to scavenge the superoxide (O2-) radical; (c) its effects on the enzyme xanthine oxidase (XO) activity; (d) its effect on the redox state of glutathione (GSH) in cultured Hep G2 cells. In addition, we also evaluated the effects of the extract on cell membrane integrity and mitochondrial respiration in cultured Hep G2 cells. RESULTS AND CONCLUSIONS: Water-soluble extracts inhibited (1) oxidation of beta-carotene, (2) AAPH-induced plasma oxidation and (3) Fe(2+)-induced lipid peroxidation in rat liver homogenates. In addition, the extract (4) is an efficient scavenger of the O2- (5) increases intracellular GSH levels and (6) is not cytotoxic. Accordingly, we propose that the therapeutic benefit of Crataegus aronia can be, at least in part, attributed to its effective inhibition of oxidative processes, efficient scavenging of O2- and possible increasing GSH biosynthesis.

Antioxidant activity and cytotoxicity of eight plants used in traditional Arab medicine in Israel.

Ethnopharmacological surveys conducted among herbal practitioners of traditional Arab medicine in Israel and the Palestinian area have revealed a large number of indigenous plant species are used as sources of their herbal therapies. Some of these herbal therapies are used to treat liver disease, jaundice or diabetes, conditions in which oxidative stress is prominent. No laboratory data on the bioactivity of herbal medicines in these settings exist in traditional Arab medicine. We hypothesized that the beneficial effect of these plants might be due to their antioxidant properties. Accordingly, we selected eight plants used to treat these two conditions and assessed their antioxidant potential by measuring their ability to suppress the extent of iron-induced lipid peroxidation in rat liver homogenates and their potential toxicity by evaluating their effects on mitochondrial respiration and cell membrane integrity in cultured PC12 and HepG2 cells. We found that all the extracts can suppress iron-induced lipid peroxidation and are not toxic. Of these extracts, those prepared from Teucrium polium and Pistacia lentiscus were the most effective in suppressing iron-induced lipid peroxidation. Further investigations are now needed to establish the exact mechanism of action and identify the active bio-ingredient(s) of each extract in order to explain their therapeutic efficacy.

Effect of temperature and loading on the structure of ß-casein/ibuprofen assemblies.

ß-Casein is a 24 kDa amphiphilic and unstructured protein that self-assembles into small core-shell micelles at a wide range of concentrations, pH values and temperatures. We recently developed the micelles as nanocarriers for oral delivery of hydrophobic drugs. In this paper we examined the effect of the hydrophobic non-steroidal anti-inflammatory drug (NSAID) ibuprofen on the micellar structure, as a function of temperature and loading. Using cryo-transmission electron microscopy (cryo-TEM) we find two routes of organization ­ mixed micellization and co-assembly (aggregation). The time-dependent events that characterize the second routes has been examined in detail. At 25 °C we find coexistence of small assemblies and larger aggregates of irregular (but defined) structures that contain the drug. Increasing the drug loading increases the relative number of the larger aggregates and their dimensions, leading eventually to the formation of long then branched structures, like in amphiphilic block copolymer solutions. Similar trends were identified for changes in the temperature. Combined, our results suggest that ibuprofen acts as a co-surfactant that possibly is localizes to the interface rather than being encapsulated in the micellar core as other NSAID hydrophobic drugs.

Development and characterization of a novel drug nanocarrier for oral delivery, based on self-assembled ß-casein micelles.

ß-casein is an amphiphilic protein that self-organizes into well-defined core-shell micelles. We developed these micelles as efficient nanocarriers for oral drug delivery. Our model drug is celecoxib, an anti-inflammatory hydrophobic drug utilized for treatment of rheumatoid arthritis and osteoarthritis, now also evaluated as a potent anticancer drug. This system is unique as it enables encapsulation loads >100-fold higher than other ß-casein/drug formulations, and does not require additives as do other formulations that have high loadings. This is combined with the ability to lyophilize the formulation without a cryoprotectant, long-term physical and chemical stability of the resulting powder, and fully reversible reconstitution of the structures by rehydration. The dry dosage form, in which >95% of the drug is encapsulated, meets the daily dose. Cryo-TEM and DLS prove that drug encapsulation results in micelle swelling, and X-ray diffraction shows that the encapsulated drug is amorphous. Altogether, our novel dosage form is highly advantageous for oral administration.

Effect of temperature on self-assembly of bovine beta-casein above and below isoelectric pH. Structural analysis by cryogenic-transmission electron microscopy and small-angle X-ray scattering.

beta-Casein is one of the main proteins in milk, recently classified as an intrinsically unstructured protein. At neutral pH, it is composed of a highly polar N-terminus domain and a hydrophobic C-terminus tail. This amphiphilic block-copolymer-like structure leads to self-organization of the protein monomers into defined micelles. Recently, it has been shown that at room temperature, beta-casein also self-organizes into micelles in an acidic environment, but the effect of temperature on the micelles' formation and properties at the low pH regime were not explored. In the present study, we used two complementary techniques, cryogenic-transmission electron microscopy (cryo-TEM) and small-angle X-ray scattering (SAXS), to characterize at high-resolution the micelles' shape, dimensions, and aggregation numbers and to determine how these properties are affected by temperature between 1 and 40 degrees C. Two different regimes were studied: highly acidic pH where the protein is cationic, and neutral pH, where it is anionic. We found that flat disk-like micelles with low aggregation numbers formed at low temperature in the two pH regimes. Close to neutral pH increase in temperature involves a transition in the micelles' shape and dimensions from flat disks to bulky, almost spheroidal micelles, coupled with a sharp increase in the micelles' aggregation number. In contrast, no effects on the micelles' morphology or aggregation number were detected in the acidic environment within the entire temperature range studied. The self-organization into disk micelles and the lack of effect of temperature in the acidic environment are linked to the unstructured character of the protein and to the charge distribution map. The latter indicates that below the isoelectric pH (pI), beta-casein loses the distinct separation of hydrophobic and hydrophilic domains, thereby suggesting that it may no longer be considered as a classical head-tail block-copolymer amphiphile as in neutral pH.

Aqueous extracts of Teucrium polium possess remarkable antioxidant activity in vitro.

Teucrium polium L. (Lamiaceae) (RDC 1117) is a medicinal plant whose species have been used for over 2000 years in traditional medicine due to its diuretic, diaphoretic, tonic, antipyretic, antispasmodic and cholagogic properties. The therapeutic benefit of medicinal plants is often attributed to their antioxidant properties. We previously reported that an aqueous extract of the leaves and stems of this plant could inhibit iron-induced lipid peroxidation in rat liver homogenate at concentrations that were not toxic to cultured hepatic cells. Others have reported that organic extracts of the aerial components of this plant could inhibit oxidative processes. Against this background, we felt further investigation on the antioxidant action of the extract of T. polium prepared according to traditional Arab medicine was warranted. Accordingly, we assessed (i) its ability to inhibit (a) oxidation of beta-carotene, (b) 2,2'-azobis(2-amidinopropan) dihydrochloride (AAPH)-induced plasma oxidation and (c) iron-induced lipid peroxidation in rat liver homogenates; (ii) to scavenge the superoxide (O2*-) radical and the hydroxyl radical (OH(*)); (iii) its effects on the enzyme xanthine oxidase activity; (iv) its capacity to bind iron; and (v) its effect on cell glutathione (GSH) homeostasis in cultured Hep G2 cells. We found that the extract (i) inhibited (a) oxidation of beta-carotene, (b) AAPH-induced plasma oxidation (c) Fe(2+)-induced lipid peroxidation in rat liver homogenates (IC(50) = 7 +/- 2 mug ml(-1)); (ii) scavenged O2*-(IC(50) = 12 +/- 3 mug ml(-1)) and OH(*) (IC(50) = 66 +/- 20 mug ml(-1)); (iii) binds iron (IC(50) = 79 +/- 17 mug ml(-1)); and (iv) tended to increase intracellular GSH levels resulting in a decrease in the GSSG/GSH ratio. These results demonstrate that the extract prepared from the T. polium possesses antioxidant activity in vitro. Further investigations are needed to verify whether this antioxidant effect occurs in vivo.

Subtle differences in structural transitions between poly-L- and poly-D-amino acids of equal length in water.

Mirror-image asymmetric molecules, i.e., chiral isomers or enantiomers, are classically considered as chemically identical. Recent studies, however, have indicated that parity violation by the nuclear weak force induces a tiny energy difference between chiral isomers. Upon combination with a massive amplification process, expansion of this difference to a detectable macroscopic level may be achieved. Yet, experimental tests of this possibility, where one enantiomer is compared to the other in solution, are hampered by the possible presence of undetectable impurities. In this study we have overcome this problem by comparing structural and dynamic features of synthetic D- and L-polyglutamic acid and polylysine molecules each of 24 identical residues. In these water-soluble polypeptides helix formation is an intramolecular autocatalytic process amplified by each turn, which is actually unaffected by low level of putative impurities in the solvent. The helix and random coil configurations and their transition were determined in this study by circular dichroism (CD) and isothermal titration calorimetry (ITC) in water and deuterium oxide. Distinct differences in structure and transition energies between the enantiomeric polypeptides were detected by both CD and ITC when dissolved in water. Intriguingly, these differences were by and large abolished in deuterium oxide. Our findings suggest that deviation from physical invariance between the D- and L-polyamino acids is induced in part by different hydration in water which is eliminated in deuterium oxide. Based on the recent findings by Tikhonov and Volkov (V. I. Tikhonov and A. A. Volkov, Science 2002, 296, 2363) we suggest that ortho-H(2)O, which constitutes 75% of bulk H(2)O, has a preferential affinity to L-enantiomers. Differential hydration of enantiomers may have played a role in the selection of L-amino acids by early forms of life.

Fertilization-induced changes in growth parameters and antioxidant activity of medicinal plants used in traditional Arab medicine.

In response to increased popularity and greater demand for medicinal plants, a number of conservation groups are recommending that wild medicinal plants be brought into cultivation systems. We collected four medicinal herbs Cichorium pumilum, Eryngium creticum, Pistacia palaestina and Teucrium polium used in traditional Arab medicine for greenhouse cultivation to assess the effects of different fertilization regimes on their growth and antioxidant activity. Wild seedlings were collected and fertilized with either 100% Hoagland solution, 50% Hoagland solution, 20% Hoagland solution or irrigated with tap water. Plant height was measured and the number of green leaves and branches counted weekly. Thereafter, the aboveground parts of plants were harvested for preparing a water-soluble powder extracts of which antioxidant activity was measured by their ability to suppress the oxidation of beta-carotene. Of the fertilization regimes, we found either 20 or 50% Hoagland solution produced the most consistent response of the plant growth parameters. All powders prepared from the four wild growing plants inhibited oxidation of beta-carotene. Increasing the amount of fertilizer caused a significant concentration-dependent increase in antioxidant activity of the cultivated T. polium compared with the wild type. In contrast, increasing the amount of fertilizer caused a significant concentration-dependent reduction in the antioxidant activity of powders prepared from the cultivated E. creticum when compared with wild plants. Our results showed that cultivation success should not rely solely on parameters of growth but should incorporate assessment related to indices of therapeutic potential.