Optimization of conditions for the enzymatic hydrolysis of phytoestrogen conjugates in urine and plasma

Optimal pH, temperature, and concentration of enzyme conditions for the rate of hydrolysis of five isoflavone conjugates (daidzein, O-desmethylangolensin, equol, genistein, and glycitein) and two lignans (enterodiol and enterolactone) from two biological matrices (urine and plasma) were studied using beta-glucuronidase from Helix pomatia. In addition, the use of mixtures of beta-glucuronidase and sulfatase enzymes from different sources was investigated to find enzyme preparations that contained lower amounts of naturally present phytoestrogens. Quantification of aglycones spiked with (13)C(3)-labeled internal standards was carried out by LC-MS/MS. In urine, all of the phytoestrogen conjugates hydrolyzed within 2h under standard hydrolysis conditions (24mul H. pomatia, pH 5, 37 degrees C). Hydrolysis rates were improved at 45 degrees C and by doubling the enzyme concentration and may be used to further reduce hydrolysis times down to 100min. In plasma, a 16-h hydrolysis was required to ensure complete hydrolysis of all conjugates. As with urine, the use of increased temperature or increased enzyme concentration reduced hydrolysis times for most analytes. However, the rate of hydrolysis in plasma was significantly slower than that in urine for all analytes except enterodiol, for which the reverse was true. Neither increased temperature nor increased enzyme concentration increased the rate of hydrolysis of enterolactone. Hydrolysis at pH 6 proved to be detrimental to hydrolysis of phytoestrogen conjugates, especially those in plasma. Other enzyme preparations from different sources, such as beta-glucuronidase from Escherichia coli, were found to contain lower amounts of contaminating phytoestrogens and showed increased enzyme activity for isoflavones, but lower activity for lignans, when used with other sulfatase enzymes. In addition, this involved complicating the analytical procedure through using mixtures of enzymes. Therefore, the use of beta-glucuronidase from H. pomatia combined with an enzyme "blank" to correct for phytoestrogen contamination was shown to be a suitable method for hydrolysis of phytoestrogens.     

Phenylpropanoids and lignans from Prunus tomentosa seeds as efficient β-amyloid (Aβ) aggregation inhibitors

Alzheimer’s disease (AD) is characterized by the progressive accumulation of extracellular β-amyloid (Aβ) aggregates. Recently, lignans and phenylpropanoids are attracting increasing attention to discovery useful agents of inhibition on Aβ aggregation. In the present study, to develop potential agents for slowing the progression of AD, Prunus tomentosa seeds were selected as a raw material for bioactive compounds, which led to the separation of two pairs of new enantiomeric lignans and phenylpropanoids using chiral HPLC. The planar structures of these compounds were elucidated by spectroscopic data analyses. And their absolute configurations were determined by comparing of experimental and calculated electronic circular dichroism (ECD). The biosynthesis pathway was also discussed. Additionally, the inhibitory activity on Aβ aggregation of all optical pure compounds was tested by thioflavin T (ThT) assay. The isolates (1a, 1b, 2a and 2b) showed more potent inhibitory activity than positive control curcumin with inhibitory rate of 73.89 ± 3.41% 78.69 ± 1.50%, 63.25 ± 2.68%, and 67.13 ± 0.90% at 20 μM, respectively. More importantly, the inhibition profiles were explained by molecular dynamics and docking simulation studies.     

Two highly oxygenated eudesmanes and 10 lignans from Achillea holosericea

Two new highly oxygenated eudesmanes and 10 known lignans were isolated from the aerial parts of Achillea holosericea. Their structures were elucidated by extensive application of one- and two-dimensional 1H and 13C NMR spectroscopy.     

Structure-radical scavenging activity relationships of phenolic compounds from traditional Chinese medicinal plants

Traditional Chinese medicinal plants associated with anticancer contain a wide variety of natural phenolic compounds with various structural features and possessing widely differing antioxidant activity. The structure-radical scavenging activity relationships of a large number of representative phenolic compounds (e.g., flavanols, flavonols, chalcones, flavones, flavanones, isoflavones, tannins, stilbenes, curcuminoids, phenolic acids, coumarins, lignans, and quinones) identified in the traditional Chinese medicinal plants were evaluated using the improved ABTS*+ and DPPH methods. Different categories of tested phenolics showed significant mean differences in radical scavenging activity. Tannins demonstrated the strongest activity, while most quinones, isoflavones, and lignans tested showed the weakest activity. This study confirmed that the number and position of hydroxyl groups and the related glycosylation and other substitutions largely determined radical scavenging activity of the tested phenolic compounds. The differences in radical scavenging activity were attributed to structural differences in hydroxylation, glycosylation and methoxylation. The ortho-dihydroxy groups were the most important structural feature of high activity for all tested phenolic compounds. Other structural features played a modified role in enhancing or reducing the activity. Within each class of phenolic compounds, the structure-activity relationship was elucidated and discussed. This study reveals the structure-activity relationships of a large series of representative natural phenolic compounds more systematically and fully than previous work. Structure-radical scavenging activity relationships of some natural phenolics identified in the medicinal plants were evaluated for the first time.     

RNAi-mediated pinoresinol lariciresinol reductase gene silencing in flax (Linum usitatissimum L.) seed coat: Consequences on lignans and neolignans accumulation

RNAi technology was applied to down regulate LuPLR1 gene expression in flax (Linum usitatissimum L.) seeds. This gene encodes a pinoresinol lariciresinol reductase responsible for the synthesis of (+)-secoisolariciresinol diglucoside (SDG), the major lignan accumulated in the seed coat. If flax lignans biological properties and health benefits are well documented their roles in planta remain unclear. This loss of function strategy was developed to better understand the implication of the PLR1 enzyme in the lignan biosynthetic pathway and to provide new insights on the functions of these compounds. RNAi plants generated exhibited LuPLR1 gene silencing as demonstrated by quantitative RT-PCR experiments and the failed to accumulate SDG. The accumulation of pinoresinol the substrate of the PLR1 enzyme under its diglucosylated form (PDG) was increased in transgenic seeds but did not compensate the overall loss of SDG. The monolignol flux was also deviated through the synthesis of 8-5′ linked neolignans dehydrodiconiferyl alcohol glucoside (DCG) and dihydro-dehydrodiconiferyl alcohol glucoside (DDCG) which were observed for the first time in flax seeds.     

Dibenzylbutane and aryltetralone lignans from seeds of Virola sebifera

Two lignans rel-(8R, 8'R)-3,4:3',4'-bis-(methylenedioxy)-7.7'-dioxo-lignan and (7'R,8'S,8S)-2'-hydroxy-3,4:4',5'-bis-(methylenedioxy)-7-oxo-2,7'-cyclolignan were isolated from seeds of Virola sebifera. The cyclolignan showed two atropisomers as determined by 1H NMR spectroscopy at low temperature.