Lipophilized epigallocatechin gallate (EGCG) derivatives as novel antioxidants

Epigallocatechin gallate (EGCG) is the major polyphenol in green tea and known to render many health benefits associated with tea consumption. EGCG was modified structurally to improve its lipophilicity, expand its application in lipophilic media, and enhance its cellular absorption in vivo. Esterification of the water-soluble EGCG with selected long-chain saturated and unsaturated fatty acids was carried out, followed by a purification process. Ester derivatives of EGCG with stearic acid (SA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) were prepared, and their enhanced lipophilicity was confirmed by octanol-water partition coefficient. The chemical structures of the EGCG derivatives, determined by HPLC-MS and 1H and 13C NMR, were EGCG-3',5',3',5'-O-tetraesters of SA, EPA, and DHA. The lipophilized EGCG derivatives exhibited greater antioxidant activity in scavenging the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical than EGCG itself. The results suggest that EGCG derivatives may be used as potential lipophilic antioxidants in the food, cosmetic, and medicinal industries.     

Synthesis, structure analyses, and characterization of novel epigallocatechin gallate (EGCG) glycosides using the glucansucrase from Leuconostoc mesenteroides B-1299CB

In this study, three epigallocatechin gallate glycosides were synthesized by the acceptor reaction of a glucansucrase produced by Leuconostoc mesenteroides B-1299CB with epigallocatechin gallate (EGCG) and sucrose. Each of these glycosides was then purified, and the structures were assigned as follows: epigallocatechin gallate 7-O-alpha-D-glucopyranoside (EGCG-G1); epigallocatechin gallate 4'-O-alpha-D-glucopyranoside (EGCG-G1'); and epigallocatechin gallate 7,4'-O-alpha-D-glucopyranoside (EGCG-G2). One of these compounds (EGCG-G1) was a novel compound. The EGCG glycosides exhibited similar or slower antioxidant effects, depending on their structures (EGCG > or = EGCG-G1 > EGCG-G1' > EGCG-G2), and also manifested a higher degree of browning resistance than was previously noted in EGCG. Also, EGCG-G1, EGCG-G1', and EGCG-G2 were 49, 55, and 114 times as water soluble, respectively, as EGCG.     

Thermal degradation kinetics of bixin in an aqueous model system

The kinetics of the thermal degradation of the natural cis carotenoid bixin in a water/ethanol (8:2) solution was studied as a function of temperature (70-125 degrees C), using high-performance liquid chromatography. The curves for the decay of bixin and formation of products (e.g., di-cis and all-trans isomers and a C17 degradation compound) did not adjust well to a first-order rate law, but very good fits were obtained using a biexponential model. This mathematical modeling gave the rate constant values for the formation of the primary products from bixin, and the energy barrier for each step was obtained. The di-cis isomers were formed immediately (15 kcal/mol) together with the decay of bixin, followed by a slow consumption, indicating their role as reaction intermediates. In fact, the di-cis isomers could easily revert to bixin (Ea approximately 3 kcal/mol) or yield the primary C17 degradation product, with an energy barrier of 6.5 kcal/mol. In turn, 24 kcal/mol was necessary for the Bix --> all-trans step, explaining its slower formation.     

Effect of reduced air pressure on soil thermal conductivity over a wide range of water content and temperature

To clarify the role of air molecules in coupled heat and mass transfer in soil, we measured the thermal conductivity of three kinds of soil (Ando soil, Red Yellow soil, and Toyoura sand) under reduced air pressure over a wide range of water content and temperature (10–75°C). The thermal conductivity increased sharply under reduced air pressure above a critical water content of the soil, becoming several times larger than that under normal pressure (101 kPa). The maximum thermal conductivity for each soil was obtained below 75°C and was similar to the thermal conductivity of some metals such as Mn, Hg and stainless steel. When the soil was drier than its critical water content, the thermal conductivity did not increase under reduced air pressure. The hydraulic diffusivity at the critical water content for each soil was of the order of 10^?8 m² s^?1. This suggests that the latent heat transfer is enhanced by the circulation of the condensed water. However, very little is known about the effect of circulating water on the latent heat transfer under reduced air pressure. To make this clear, the thermal conductivity would need to be measured in the steady state under reduced air pressure.     

Temperature dependence of thermal conductivity of soil over a wide range of temperature (5–75°C)

The coupled heat and mass transfer in soil can be analysed by examining the temperature dependence of thermal conductivity. We have measured the thermal conductivity of two kinds of soil (Ando soil and Red Yellow soil) as a function of both temperature (5–75°C) and water content by the twin heat probe method. From our results we concluded that the thermal conductivity resulting from the latent heat transfer can be separated from the apparent thermal conductivity by subtracting the thermal conductivity at a temperature near 0°C from that at a higher temperature. The relation between the phenomenological enhancement factor (β) and the volumetric air‐filled porosity was divided into two parts: β increases linearly as the volumetric air‐filled porosity increases from zero (that is, water saturation), to the point at which soil water potential corresponds to ?320 J kg^?1; from that point to oven‐dry condition, β decreased logistically with the volumetric air‐filled porosity. From these results, we could generalize the behaviour of β.     

Isomerization and degradation kinetics of hop (Humulus lupulus) acids in a model wort-boiling system

The rate of isomerization of alpha acids to iso-alpha acids (the compounds contributing bitter taste to beer) was determined across a range of temperatures (90-130 degrees C) to characterize the rate at which iso-alpha acids are formed during kettle boiling. Multiple 12 mL stainless steel vessels were utilized to heat samples (alpha acids in a pH 5.2 buffered aqueous solution) at given temperatures, for varying lengths of time. Concentrations of alpha acids and iso-alpha acids were quantified by high-pressure liquid chromatography (HPLC). The isomerization reaction was found to be first order, with reaction rate varying as a function of temperature. Rate constants were experimentally determined to be k1 = (7.9 x 10(11)) e(-11858/T) for the isomerization reaction of alpha acids to iso-alpha acids, and k2 = (4.1 x 10(12)) e(-12994/T) for the subsequent loss of iso-alpha acids to uncharacterized degradation products. Activation energy was experimentally determined to be 98.6 kJ per mole for isomerization, and 108.0 kJ per mole for degradation. Losses of iso-alpha acids to degradation products were pronounced for cases in which boiling was continued beyond two half-lives of alpha-acid concentration.     

Phase transitions of pea starch over a wide range of water content

The phase transitions of pea starch over a wide range of water content were investigated by differential scanning calorimetry (DSC). Swelling of starch granules increased progressively with increasing water content. The main endotherm G broadened progressively with increasing water content up to 94.5 wt % (water:starch ratio 15:1), above which it became too broad to define. The corresponding peak and conclusion temperatures and enthalpy change increased with increasing water content. Scanning electron microscopy (SEM) showed that, at a water:starch ratio of 2:1 (water content of 70.7 wt %), starch granules only swelled partially with discernible granular contours still clearly evident. The results of swelling power tests and SEM images revealed that the endotherm G obtained at a water:starch ratio of 2:1 represented only partial swelling of starch granules. The transition from a narrow to broad endotherm G was interpreted to reflect the thermal transition behavior progressing from limited swelling to maximum swelling and then partial dissolution and leaching of starch polymers from the granules.     

Metabolic fate of (-)-[4-(3)H]epigallocatechin gallate in rats after oral administration

After oral administration of [4-(3)H]EGCg to rats, the radioactivity in blood, major tissues, urine, and feces was measured over time. The radioactivity in blood and most tissues remained low for 4 h postdose, began to increase after 8 h, peaked at 24 h, and then decreased. Major urinary excretion of radioactivity occurred in the 8-24 h period, and the cumulative radioactivity excreted by 72 h was 32.1% of the dose. The radioactivity in the feces was 35.2% of the dose within 72 h postdose. In the case of rats pretreated with antibiotics (antibiotic-pretreated rats), the radioactivity levels of the blood and urine were definitely lower than those in rats not pretreated with antibiotics (normal rats). The radioactivity recovered in the antibiotic-pretreated rat urine was estimated to be only (1)/(100) of that in the normal rat urine. These results clearly demonstrated that the radioactivity detected in the blood and urine of normal rats mostly originated from degradation products of EGCg produced by intestinal bacteria. Furthermore, a main metabolite in the normal rats was purified and identified as 5-(5'-hydroxyphenyl)-gamma-valerolactone 3'-O-beta-glucuronide (M-2). In feces of the normal rats, EGC (40.8% of the fecal radioactivity) and 5-(3',5'-dihydroxyphenyl)-gamma-valerolactone (M-1, 16.8%) were detected. These results suggested that M-1 was absorbed in the body after degradation of EGCg by intestinal bacteria, yielding M-1 with EGC as an intermediate. Furthermore, M-2 was thought to be formed from M-1 in the intestinal mucosa and/or liver, then to enter the systemic circulation, and finally to be excreted in the urine. Taking into account all of the above findings, a possible metabolic route of EGCg orally administered to rats is proposed.     

Aggregation of a proline-rich protein induced by epigallocatechin gallate and condensed tannins: effect of protein glycosylation

Astringency is one of the most important organoleptic qualities of numerous beverages, including red wines. It is generally thought to originate from interactions between tannins and salivary proline-rich proteins (PRPs). In this work interactions between a glycosylated PRP, called II-1, and flavan-3-ols were studied in aqueous solutions and at a colloidal level, by dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS). The flavan-3-ols were a monomer, epigallocatechin gallate (EGCG), and polymerized flavan-3-ol fractions extracted from grape seeds. In aqueous solutions containing EGCG and protein II-1, protein aggregation took place when protein concentration and the EGCG/protein ratio exceeded a threshold. The aggregates had a small size, comparable with the dimensions of protein monomers, and formed stable dispersions (no phase separation). Most proteins remained free in solution. This behavior is in sharp contrast with the phase separation observed for nonglycoslated PRP in the same conditions. Moreover, this slight aggregation of II-I in the presence of EGCG was disrupted by the addition of 12% ethanol. Increasing the flavan-3-ol molecular weight strongly enhanced II-I/tannin aggregation: the threshold was at a lower protein concentration (0.2 mg/mL) and a lower tannin/protein ratio. Still, in most cases, and in contrast with that observed with a nonglycosylated PRP, the aggregates remained of discrete size and stable. Only at low ethanol content (2%) did the addition of tannin polymers finally lead to phase separation, which occurred when the molar ratio of tannins to proteins exceeded 12. This systematic effect of ethanol confirmed the strong effect of cosolvents on protein/tannin interactions.     

A comparison of measured and theoretical soil acidity diffusion coefficients over a wide range of pH

The soil acidity diffusion coefficients have been measured at two P_co2 levels with values in the range 2–70 × 10^?9 cm² s^?1. The coefficient passed through an ill-defined minimum in the pH range 5–6.5. Theoretical values, calculated on the basis that the only significant acid-base carriers in the soil were the H₃O⁺ -H₂O and H₂CO₃-HCO^?₃ pairs, agreed well with the experimental values over the whole pH range at P_co2= 0.005 atm. Agreement was not so good however at a CO₂ pressure of 0.0003 atm., especially in the neutralalkaline pH range. This was thought to be due to difficulties in maintaining this level of CO₂ throughout the soil samples.     

Synthesis of (-)-[4-3H]epigallocatechin gallate and its metabolic fate in rats after intravenous administration

Because a great deal of attention has been focused on the metabolism of (-)-epigallocatechin gallate (EGCg), quantitative analysis of this compound is required. For this purpose we developed a method of chemical synthesis of [4-(3)H]EGCg. Synthesized [4-(3)H]EGCg showed 99.5% radiochemical purity and a specific activity of 13 Ci/mmol. To clarify the excretion route of EGCg, the radioactivity levels of bile and urine were quantified after intravenous administration of [4-(3)H]EGCg to bile-duct-cannulated rats. Results showed that the radioactivity of the bile sample excreted within 48 h accounted for 77.0% of the dose, whereas only 2.0% of the dose was recovered in the urine. The excretion ratio of bile to urine was calculated to be about 97:3. These results clearly showed that bile was the major excretion route of EGCg. Time-course analysis of the radioactivity in blood was also performed to estimate the pharmacokinetic parameters following intravenous administration of [4-(3)H]EGCg. In addition, EGCg metabolites excreted in the bile within 4 h after the intravenous dose of [4-(3)H]EGCg were analyzed by HPLC. The results showed that 4',4"-di-O-methyl-EGCg was present in the conjugated form and made up about 14.7% of the administered radioactivity.     

Effects of concentration,incubation temperature,and repeated applications on degradation kinetics of dicyandiamide (DCD) in model experiments with a silt loam soil

Summary The kinetics of dicyandiamide (DCD) decomposition were studied (at 80% water-holding capacity) in pretreated and non-pretreated soils, using model experiments. DCD was added in different concentrations (6.7, 16.7, and 33.3 g DCD-N g^–1 dry soil) and incubated at various temperatures (10°, 20°, and 30°C). Additionally, DCD decomposition was examined in sterile soil (with or without Fe₂O₃) after inoculation with a DCD-enrichment culture. In the sterile variant, (30°C)the applied dicyandiamide concentration remained constant, even after 36 days. In the sterilized and reinoculated variant, DCD disappeared within 7 days. Addition of Fe₂O₃ powder to the sterilized soil had no effect on DCD degradation. In the pretreated soils, DCD mineralization started immediately at all temperatures and concentrations without a lag phase. A temperature increase of 10°C doubled the mineralization rate. The mineralization rates were independent of the initial concentrations. In the non-pretreated soils (except at 30°C with 16.7 and 33.3 g DCD-N g^–1 dry soil) DCD decreased only after a short (30°C) or a long (10°C) lag phase. These results suggest that an inducible metabolic degradation occurred, following zeroorder kinetics.     

Interaction mechanism and kinetics of ferrous sulfide and manganese oxides in aqueous system

Journal of Soils and Sediments - The oxidation of ferrous sulfide (FeS) causes soil acidification and the release of toxic heavy metal ions. Manganese oxides usually participate in the oxidation of...     

Influence of temperature on mineralization kinetics with a nitrification inhibitor (mixture of dicyandiamide and ammonium thiosulphate)

Summary The influence of temperature on the action of a dicyandiamide nitrification inhibitor was studied during a laboratory incubation after the addition of ammonium sulphate labelled with ¹⁵N. In the control treatment, nitrification was only slightly affected by temperature and was rapid; on the 42nd day, two-thirds of the ¹⁵N was incorporated into the nitrate fraction while no further tracer was found in ammoniacal form. With the addition of dicyandiamide, the process was slowed down considerably when the temperature was maintained at 10°C, and only about 10% of the ¹⁵N was nitrified in 6 months. After 1 month of incubation at 10°C, a temperature increase to 15°C for 4 weeks modified the nitrification kinetics only slightly. However, as soon as the temperature reached 20°C, the beginning of dicyandiamide decomposition and an increase in the quantity of NO ₃ ^- -N was observed. The inhibition was measured by the nitrification index, which was greater than 80% as long as the temperature did not exceed 15°C, and decreased to 10% after 6 months; this value was reached only after 1 year in soil maintained at 10°C. The half-life of the NH ₄ ⁺ was decreased by raising the temperature. In the experimental conditions described, nitrification was inhibited by the dicyandiamide for at least 6 months provided the temperature did not exceed 15°C.     

Effects of ozone and oxygen on the degradation of carotenoids in an aqueous model system

The effects of ozone and oxygen on the degradation of carotenoids in an aqueous model system were studied. All-trans beta-carotene, 9-cis beta-carotene, beta-cryptoxanthin, and lycopene were adsorbed onto a C(18) solid phase and exposed to a continuous flow of water saturated with oxygen or ozone at 30 degrees C. Carotenoids were analyzed using HPLC with a C(30) column and a photodiode array detector. Approximately 90% of all-trans beta-carotene, 9-cis beta-carotene, and beta-cryptoxanthin were lost after exposure to ozone for 7 h. A similar loss of lycopene occurred in only 1 h. When exposed to oxygen, all carotenoids, except beta-cryptoxanthin, degraded at lower rates. The degradation of all the carotenoids followed zero-order reaction kinetics with the following relative rates: lycopene > beta-cryptoxanthin > all-trans beta-carotene > 9-cis beta-carotene. The major degradation products of beta-carotene were tentatively identified on the basis of their elution on the HPLC column, UV-Vis spectra, and electrospray LC-MS. Predominant isomers of beta-carotene were 13-cis, 9-cis, and a di-cis isomer. Products resulting from cleavage of the molecule were beta-apo-13-carotenone and beta-apo-14'-carotenal, whereas epoxidation yielded beta-carotene 5,8-epoxide and beta-carotene 5, 8-endoperoxide.     

Heat-induced, metal-catalyzed oxidative degradation of quercetin and rutin (Quercetin 3-O-rhamnosylglucoside) in aqueous model systems

The oxidative degradation of quercetin and rutin in phosphate buffer solutions, pH 8.0, at 97 degrees C, was studied by means of UV-vis spectroscopy and reversed-phase high-performance liquid chromatography (HPLC). The effect of the transition metal ions Fe(2+) and Cu(2+) on degradation rate and browning development was also assessed. It was shown that both flavonols are very labile to thermally induced degradation under oxidative conditions. Fe(2+) and Cu(2+) caused an increase in the degradation rate, as well as an increase in browning (A(420)). Significant differences were observed in the degradation mechanisms, as implied by HPLC analyses. It is postulated that metal ions promote flavonol oxidation through reactive oxygen species formation, whereas increases in browning could be ascribed to oxidation and metal-polyphenol interactions.     

Erratum to: Interaction mechanism and kinetics of ferrous sulfide and manganese oxides in aqueous system

Journal of Soils and Sediments -     

Identification of biliary metabolites of (-)-epigallocatechin gallate in rats

After oral administration of (-)-epigallocatechin gallate (EGCg) to rats, its biliary metabolites were examined. Although a large part of the biliary metabolites was found to exist in conjugated forms, it was difficult to separate the conjugated forms. Thus the free form of biliary metabolites was prepared by beta-glucuronidase/sulfatase treatment and was purified by HPLC. Six compounds purified were subjected to FABeta-MS and NMR analyses. The six metabolites thus obtained were shown to be EGCg, 3'-O-methyl-EGCg, 4'-O-methyl-EGCg, 3' '-O-methyl-EGCg, 4' '-O-methyl-EGCg, and 4',4' '-di-O-methyl-EGCg, respectively. The six EGCg metabolites and their conjugates excreted during a 4-h period were estimated to be roughly 0.1% and 3.3% of the administered EGCg, respectively. In addition, 4' '-O-methyl-EGCg and 4',4' '-di-O-methyl-EGCg were estimated to exist only in the sulfate form, but the other four metabolites existed in both glucuronide (and/or sulfoglucuronide) and sulfate forms.     

The temperature dependence of dicyandiamide (DCD) degradation in soils: A data synthesis

Dicyandiamide (DCD, C₂H₄N₄) is a nitrification inhibitor that has been studied for more than 80 years. However, there are few papers that have examined the use of DCD on dairy farms where cattle graze pasture and where urine is the primary form of nitrogen (N) deposited onto soils. After DCD was applied (10 kg DCD ha^?1) with bovine urine (700–1200 kg N ha^?1) to five soils throughout New Zealand, the reduction in direct nitrous oxide (N₂O) emissions was significant and remarkably consistent (71 ± 8%, average ± standard error). The application of DCD to these soils occurred in autumn and winter; daily average soil temperature (T) was reported but these data were not further analysed. Perusal of the literature suggested no consensus on the temperature dependence of DCD degradation in soils. Based on published data from controlled-environment studies of soils sampled in four countries, we quantified the relation between T and the time for DCD concentration in soils to decline to half its application value (t_½) as t_½ (T) = 168e^?0.084T with parameter standard errors of ±16 d and ±0.011 d^?1, respectively (n = 16). For example, at 5 °C a 1 °C increase in T reduced t_½ from 110 to 101 d whereas at 25 °C the reduction was 20–19 d. Analysing T data from the New Zealand trials using our t_½ (T) function, over 43–89 d when direct N₂O emissions from treated plots became indistinguishable from the controls, the estimated percentage of applied DCD remaining in the soil averaged 43 ± 10%. These calculations suggested the apparently remaining DCD was ineffective with respect to direct N₂O emissions. In the absence of measurements, explanations for this interpretation included vertical displacement of the DCD and sorption onto organic matter in soils. The consistent DCD efficacy from these trials corresponded with T generally <10 °C, so it is suggested as an application criteria for the reduction of direct N₂O emissions from pastoral soils subjected to urine excretion by grazing cattle.     

Reaction of lizard populations to a catastrophic wildfire in a central Arizona mountain range

In April 1996, the Lone Fire denuded over 90% of the vegetation in 130 km² around the Four Peaks area of the Mazatzal Mountains in central Arizona. To understand the reaction of a relatively immobile guild of species to a wildfire, we pit-trapped lizards from 1996 to 1999 in both burned and unburned interior chaparral and Madrean evergreen forest. In 26,214 trap nights, we found relative abundance was up to 10 times greater in burned than unburned vegetation. Species richness and diversity values were also greater in burned sites. Our data indicate a rapid settlement of burned areas primarily by individuals that survived the fire. Increased capture rate, diversity and richness values in 1998 and 1999 indicate that many species of lizards may even prefer early successional stages in chaparral and Madrean evergreen forests. Resident species of Teiidae and Sceloporus undulatus were more adapted to disturbed habitats than other resident Phrynosomatidae or Crotophytidae.