(−)-Epigallocatechin gallate suppresses the cytotoxicity induced by trichothecene mycotoxins in mouse cultural macrophages

Trichothecene mycotoxins are toxic secondary metabolites produced by a number of fungi including Fusarium species, which adversely affect lymphocytes. Deoxynivalenol (DON) and HT-2 toxin (HT-2) belong to the trichothecene group of mycotoxins and the occurrence of cereals and foodstuffs with these compounds are serious health problems. The aim of this study was to examine the effect of (−)-epigallocatechin gallate (EGCG), one of the main components in green tea catechins, on DON- or HT-2-induced cytotoxicity in mouse macrophages. EGCG had protective effects against the trichothecene-induced cytotoxicities of both mycotoxins. Additionally, EGCG suppressed the DON-induced activation of caspase-3/7, which is an indicator of apoptosis. These results indicate that EGCG might be useful in protection against DON- or HT-2-induced cell death, suggesting that EGCG could contribute to reducing the toxicities of trichothecenes.     

Structure–activity studies of (−)-epigallocatechin gallate derivatives as HCV entry inhibitors

Preventing viral entry into cells is a recognized approach for HIV therapy and has attracted attention for use against the hepatitis C virus (HCV). Recent reports described the activity of (−)-epigallocatechin gallate (EGCG) as an inhibitor of HCV entry with modest potency. EGCG is a polyphenolic natural product with a wide range of biological activity and unfavorable pharmaceutical properties. In an attempt to identify more drug-like EGCG derivatives with improved efficacy as HCV entry inhibitors, we initiated structure–activity investigations using semi-synthetic and synthetic EGCG analogs. The data show that there are multiple regions in the EGCG structure that contribute to activity. The gallate ester portion of the molecule appears to be of particular importance as a 3,4-difluoro analog of EGCG enhanced potency. This derivative and other active compounds were shown not to be cytotoxic in Huh-7 cell culture. These data suggest that more potent, non-cytotoxic EGCG analogs can be prepared in an attempt to identify more drug-like candidates to treat HCV infection by this mechanism.     

Increasing Doxycycline Hyclate Photostability by Complexation with β-Cyclodextrin

Doxycycline hyclate (DOX) is a highly photosensitive drug, a feature that limits the stability of the corresponding dosage forms. The main objectives of this work were the preparation and characterization of an inclusion complex of DOX with β-cyclodextrin (βCD) and to investigate if this approach could improve the photostability of the drug. Guest-host interactions were investigated using nuclear magnetic resonance, which were afterwards combined with molecular modeling methods to study the complex formation and its three-dimensional structure was proposed. A freeze-drying method was applied to obtain the complex in the solid state, which was further confirmed by thermal and spectroscopic techniques. To evaluate the complexation effect on DOX integrity, the photostability of the inclusion complex was studied, with a significant decrease in the photodegradation of DOX being found in aqueous solution upon complexation. Finally, the photoprotection produced by the complexation was evaluated by means of an antimicrobial assay. Overall, the presented results suggest that the formulation of DOX complexed with βCD constitutes an interesting approach for the preparation of pharmaceutical dosage forms of DOX with enhanced stability properties.KEY WORDS: β-cyclodextrin, doxycycline hyclate, microbiological assay, molecular modeling, photostability     

Template-assisted lateral growth of amyloid-β42 fibrils studied by differential labeling with gold nanoparticles

Amyloid-β protein (Aβ) aggregation into amyloid fibrils is central to the origin and development of Alzheimer's disease (AD), yet this highly complex process is poorly understood at the molecular level. Extensive studies have shown that Aβ fibril growth occurs through fibril elongation, whereby soluble molecules add to the fibril ends. Nevertheless, fibril morphology strongly depends on aggregation conditions. For example, at high ionic strength, Aβ fibrils laterally associate into bundles. To further study the mechanisms leading to fibril growth, we developed a single-fibril growth assay based on differential labeling of two Aβ42 variants with gold nanoparticles. We used this assay to study Aβ42 fibril growth under different conditions and observed that bundle formation is preceded by lateral interaction of soluble Aβ42 molecules with pre-existing fibrils. Based on this data, we propose template-assisted lateral fibril growth as an additional mechanism to elongation for Aβ42 fibril growth.     

Promotion of Neuronal Plasticity by (−)-Epigallocatechin-3-Gallate

The consumption of (−)-epigallocatechin-3-gallate (EGCG), the major polyphenolic compound found in green tea, has been associated with various neurological benefits including cognitive improvement. The physiological basis for this effect is unknown. In this study, we used synaptic transmission between the CA3 and CA1 regions (Schaffer collateral) of the mouse hippocampus to examine the effects of EGCG on neuronal plasticity. We found that the level of high frequency stimulation-evoked long-term potentiation (LTP) was significantly enhanced when hippocampal slices were pre-incubated with 10 μM EGCG for 1 h prior to the experiment. EGCG incubation also enabled hippocampal slices prepared from Ts65Dn mice, a Down syndrome mouse model deficient in LTP, to express LTP to a level comparable to the normal controls. EGCG treatment did not alter the degree of pair-pulse inhibition; therefore, the enhancement effect of EGCG is unlikely to involve the attenuation of this inhibitory mechanism.     

Mathematical calculations of iron complex stoichiometry by direct UV–Vis spectrophotometry

The effects of iron-chelating agents on miscellaneous pathologies are currently largely tested. Due to various indications, different properties for chelators are required. A stoichiometry of the complex in relation to pH is one of the crucial factors. Moreover, the published data on the stoichiometry, especially concerning flavonoids, are equivocal.In this study, a new complementary approach was employed for the determination of stoichiometry in 10 iron-chelating agents, including clinically used drugs, by UV–Vis spectrophotometry at relevant pH conditions and compared with the standard Job’s method.This study showed that the simple approach based on absorbance at the wavelength of complex absorption maximum was sufficient when the difference between absorption maximum of substance and complex was high. However, in majority of substances this difference was much lower (9–73 nm). The novel complementary approach was able to determine the stoichiometry in all tested cases. The major benefit of this method compared to the standard Job’s approach seems to be its capability to reveal a reaction stoichiometry in chelators with moderate affinity to iron.In conclusion, using this complementary method may explain several previous contradictory data and lead to a better understanding of the underlying mechanisms of chelator’s action.     

Structural basis of protein–protein interaction studied by NMR

This paper describes efforts of the structural genomics project in the nuclear magnetic resonance (NMR) laboratory at the University of Science and Technology of China. This structural genomics project is biological-functional driven. Targets are mainly selected from two systems: proteins related with regulation of gene expression in humans and other eukaryotes, and proteins existing in the cell junction in humans. The majority of proteins selected from these two systems are related with human health and diseases, and some are potential drug targets. Twenty-five protein structures from Homo sapiens and other eukaryotes have been determined during last 5 years in this laboratory. Nuclear magnetic resonance (NMR) spectroscopy is highly suited to investigate molecular interactions at a close physiological condition and is particularly suited for the study of low-affinity, transient complexes. It can provide information on protein surface interaction, their complex structure, and their dynamic properties during protein recognition. Several examples are given in this paper.     

Conformational transition of an α-helix studied by molecular dynamics

Molecular dynamics simulations were performed on a 20-residue polyalanine helix and a spontaneous transition from a kinked to a straight conformation was observed. The kinetics of the transition was analyzed within the framework of the Kramers model for chemical reactions and within a random walk model. The Kramers model which is based on diffusion along a one-dimensional reaction pathway and the crossing of an energy barrier was found to be inadequate. Instead, a random walk model based on diffusion in the high-dimensional phase space of the system was found to be compatible with the data. The high dimensionality of the phase space permits the system to circumvent high energy barriers and diffuse rapidly at about constant energy, but decelerates the reaction since in the labyrinth of pathways the transition state is reached rarely.     

Microbial Hydroxylation of (−)-α-Santonin by Aspergillus niger

The microbiological transformation of a sesquiterpene lactone, (−)-α-santonin was carried out by using Aspergillus niger MIL 5024 and MIL 5025. Strain MIL 5024 brings about the transformation of (−)-α-santonin (400 mg) to 11-hydroxy-(−)-α-santonin (P1) (50.4 mg), 3,6,9-trihydroxy-9,10-seco-selina-1,3,5(10)-trien-12-oic acid-12,6-lactone (P2) (22.4 mg), and 3,6-dihydroxy-9,10-seco-selina-1,3,5(10)-trien-9,12-dioic acid-12,6-lactone (P3) (27.1 mg), which were isolated and characterized by UV, IR, mass and ¹H-NMR spectroanalyses. All of these products are described for the first time. Results similar to those with MIL 5024 were also obtained from the transformation of (−)-α-santonin by the other isolate, strain MIL 5025.     

Complexation of Tocotrienol with γ-Cyclodextrin Enhances Intestinal Absorption of Tocotrienol in Rats

To determine the bioavailability of tocotrienol complex with γ-cyclodextrin, the effects of tocotrienol/γ-cyclodextrin complex on tocotrienol concentration in rat plasma and tissues were studied. Rats were administered by oral gavage an emulsion containing tocotrienol, tocotrienol with γ-cyclodextrin, or tocotrienol/γ-cyclodextrin complex. At 3 h after administration, the plasma γ-tocotrienol concentration of the rats administered tocotrienol/γ-cyclodextrin complex was higher than that of the rats administered tocotrienol and γ-cyclodextrin. In order to determine the effect of complexation on tocotrienol absorption, rats were injected with Triton WR1339, which prevents the catabolism of triacylglycerol-rich lipoprotein by lipoprotein lipase, and then administered by oral gavage an emulsion containing tocotrienol, tocotrienol with γ-cyclodextrin, or tocotrienol/γ-cyclodextrin complex. The plasma γ-tocotrienol concentration of the Triton-treated rats administered tocotrienol/γ-cyclodextrin complex was higher than that of the other Triton-treated rats. These results suggest that complexation of tocotrienol with γ-cyclodextrin elevates plasma and tissue tocotrienol concentrations by enhancing intestinal absorption.     

(−)-Epigallocatechin gallate inhibits growth and activation of the VEGF/VEGFR axis in human colorectal cancer cells

(?)-Epigallocatechin gallate (EGCG), the major constituent of green tea, inhibits the growth of colorectal cancer cells by inhibiting the activation of various types of receptor tyrosine kinases (RTKs). The RTK vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) axis induces tumor angiogenesis in colorectal cancer. This study examined the effects of EGCG on the activity of the VEGF/VEGFR axis and the expression of hypoxia-inducible factor (HIF)-1α, which promotes angiogenesis by elevating VEGF levels, in human colorectal cancer cells. Total and phosphorylated (i.e., activated) form (p-VEGFR-2) of VEGFR-2 proteins were overexpressed in a series of human colorectal cancer cell lines. Within 3 h, EGCG caused a decrease in the expression of HIF-1α protein and VEGF, HIF-1α, insulin-like growth factor (IGF)-1, IGF-2, epidermal growth factor (EGF), and heregulin mRNAs in SW837 colorectal cancer cells, which express a constitutively activated VEGF/VEGFR axis. A decrease was also observed in the expression of VEGFR-2, p-VEGFR-2, p-IGF-1 receptor, p-ERK, and p-Akt proteins within 6 h after EGCG treatment. Drinking EGCG significantly inhibited the growth of SW837 xenografts in nude mice, and this was associated with the inhibition of the expression and activation of VEGFR-2. The consumption of EGCG also inhibited activation of ERK and Akt, both of which are downstream signaling molecules of the VEGF/VEGFR axis, and reduced the expression of VEGF mRNA in xenografts. These findings suggest that EGCG may exert, at least in part, growth-inhibitory effects on colorectal cancer cells by inhibiting the activation of the VEGF/VEGFR axis through suppressing the expression of HIF-1α and several major growth factors. EGCG may therefore be useful in the chemoprevention and/or treatment of colorectal cancer.     

Improvement of Aripiprazole Solubility by Complexation with (2-Hydroxy)propyl-β-cyclodextrin Using Spray Drying Technique

Due to the fact that the number of new poorly soluble active pharmaceutical ingredients is increasing, it is important to investigate the possibilities of improvement of their solubility in order to obtain a final pharmaceutical formulation with enhanced bioavailability. One of the strategies to increase drug solubility is the inclusion of the APIs in cyclodextrins. The aim of this study was to investigate the possibility of aripiprazole solubility improvement by inclusion in (2-hydroxy)propyl-β-cyclodextrin (HPBCD) and simultaneous manipulation of pH of the medium and addition of polyvinylpyrrolidone. Aripiprazole-HPBCD complexes were prepared by spray drying aqueous drug-HPBCD solutions, and their properties were compared with those prepared by solvent-drop co-grinding and physical mixing. The obtained powders were characterized by thermoanalytical methods (TGA and DSC), FTIR spectroscopy, their dissolution properties were assessed, while the binding of aripiprazole into the cavity of HPBCD was studied by molecular docking simulations. The solubilization capacity was found to be dependent on pH as well as the buffer solution's ionic composition. The presence of PVP in the formulation could affect the solubilization capacity significantly, but further experimentation is required before its effect is fully understood. On the basis of solubility studies, the drug/HPBCD stoichiometry was found to be 1:3. The spray-dried products were free of crystalline aripiprazole, they possessed higher solubility and dissolution rate, and were stable enough over a prolonged period of storage. Spray drying of cyclodextrin solutions proved to be an appropriate and efficient technique for the preparation of highly soluble inclusion compounds of aripiprazole and HPBCD.     

Interactions of (−)-epigallocatechin-3-gallate with model lipid membranes

(?)-Epigallocatechin-3-gallate (EGCG) is a flavonoid known for its good antioxidant potential and health benefits. It is one of the most intriguing flavonoids, especially because of its specific interactions with model lipid membranes. It was noticed that EGCG might form EGCG rich domains/rafts at certain compositions of lipid membranes. In this article, we investigate whether EGCG forms EGCG rich domains when incorporated in 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) liposomes. Our results show that EGCG decreases lipid ordering parameter in ordered membranes and increases it in the case of disordered ones. Also, incorporation of EGCG does not affect the zeta-potential and shape of the liposomes, but it can induce aggregation of liposomes. Our study also demonstrates that liposomes with incorporated EGCG are highly protected against UV-light induced oxidation.     

Antimycobacterial activity generated by the amide coupling of (−)-fenchone derived aminoalcohol with cinnamic acids and analogues

Aminoethyl substituted 2-endo-fenchol prepared from (−)-fenchone was used as scaffold for the synthesis of series of 31 amide structures by N-acylation applying cinnamic acids and analogues. The evaluation of their in vitro activity against Mycobacterium tuberculosis H₃₇Rv showed for some of them promising activity—up to 0.2 μg/ml, combined with relatively low cytotoxicity of the selected active compounds.     

Conversion of (−)-Carvone and (+)-Carvone by a Strain of Aspergillus niger

The conversion of (?)-carvone and (+)-carvone by a strain of Aspergillus niger was studied as one of the series of biochemical reduction of terpenes.

(?)-Carvone was found to be reduced essentially to (+)-neodihydrocarveol, although (+)-dihydrocarvone and (+)-isodihydrocarvone were also formed in small amounts, whereas (+)-carvone was converted to (?)-isodihydrocarvone, (?)-isodihydrocarveol, (?)-neoisodihydrocarveol, (?)-dihydrocarvone, (?)-neodihydrocarveol, and (+)-dihydrocarveol, of which the former three were the major products.

The metabolic pathways for (?)-carvone and (+)-carvone by the strain of Aspergillus niger are discussed and the results on microbial and chemical reductions of carvone and dihydrocarvone are summarized.