Effect of isoflavone administration on age-related hepatocyte changes in old ovariectomized femal Wistar rats

Aging seems to be due to the accumulation of oxidative damage in cells and molecules. On the other hand, menopause and ovariectomy induce deleterious effects on different organs and systems that have been shown to be counteracted by estrogens and in a not so evident form also with phytoestrogens. The present study has investigated whether the administration of a commercial soy extract that contains approximately 10% isoflavones was able to modify some parameters related to oxidative stress and inflammation in hepatocytes isolated from old ovariectomized female Wistar rats. Eighteen 22-month-old animals that had been previously ovariectomized at 12 months of age were divided into four groups: ovariectomized control rats, estradiol-treated ovariectomized females and ovariectomized rats treated with isoflavones. Six intact female rats of 2 months of age were used as reference group. Hepatocytes were isolated and cultured, and carbon monoxide (CO) and nitric oxide (NO) release, as well as adenosyl triphosphate (ATP), cyclic guanosyl monophosphate (cGMP), phosphatidylcholine (PC) and lipid peroxide (LPO) content of cells were evaluated. Uterus was also removed and weighed. Hepatocytes isolated from old ovariectomized rats showed a decrease in ATP content as compared to young animals. Age also induced an increase in LPO cell content. NO, CO and cGMP were augmented with age, and PC synthesis showed a dramatic reduction. Treatment with either estradiol or isoflavones were able to improve all the mentioned parameters altered in hepatocytes isolated from old ovariectomized rats, and the magnitude of the improvement was similar for both treatments. Ovariectomy induced a significant reduction in uterine weight, which was significantly counteracted by estradiol treatment but not by isoflavone administration. In conclusion, the administration of a soy extract containing isoflavones seems to prevent oxidative changes in hepatocytes isolated from old ovariectomized female rats, without modifying uterus weight.     

Sustainable production of genistin from glycerol by constructing and optimizing Escherichia coli

Genistin is one of the bioactive isoflavone glucosides found in legumes, which have great nutraceutical and pharmaceutical significance. The market available isoflavones are currently produced by direct plant extraction. However, its low abundance in plant and structural complexity hinders access to this phytopharmaceutical via plant extraction or chemical synthesis. Here, the E. coli cell factory for sustainable production of genistin from glycerol was constructed. First, we rebuilt the precursor genistein biosynthesis pathway in E. coli, and its titer was then increased by 668% by identifying rate-limiting steps and applying an artificial protein scaffold system. Then de novo production of genistin from glycerol was achieved by functional screening and introduction of glycosyl-transferases, UDP-glucose pathway and specific genistin efflux pumps, and 48.1 mg/L of genistin was obtained. A further engineered E. coli strain equipped with an improved malonyl-CoA pathway, alternative glycerol-utilization pathways, acetyl-CoA carboxylase (ACC), and CRISPR interference (CRISPRi) mediated regulation produced up to 137.8 mg/L of genistin in shake flask cultures. Finally, 202.7 mg/L genistin was achieved through fed-batch fermentation in a 3-L bioreactor. This study represents the de novo genistin production from glycerol for the first time and will lay the foundation for low-cost microbial production of glucoside isoflavones. In addition, the multiphase workflow may provide a reference for engineering the biosynthetic pathways in other microbial hosts as well, for green manufacturing of complex natural products.     

植物异黄酮合酶研究进展

植物异黄酮是一类具有增强植物抗病、诱导根瘤形成以及预防激素相关肿瘤发生、缓解女性更年期综合症的活性次生代谢产物,其合成关键酶是异黄酮合酶(IFS)。介绍了IFS的催化机理、基因克隆与表达调控的研究进展,讨论了开展IFS代谢工程研究对提高植物抗病虫害能力和改善农作物营养保健功效的意义。     

Improvement of isoflavone aglycones production using β-glucosidase secretory produced in recombinant Aspergillus oryzae

β-Glucosidase (BGL1) from Aspergillus oryzae was efficiently produced in recombinant A. oryzae using sodM promoter-mediated expression system. The yield of BGL1 was 960 mg/l in liquid culture, which is 20-fold higher than the yield of BGL1 produced using the yeast Saccharomyces cerevisiae. Recombinant BGL1 converted isoflavone glycosides into isoflavone aglycones more efficiently than β-glucosidase from almond. In addition, BGL1 produced isoflavone aglycones even in the presence of the insoluble form of isoflavone glycosides.     

Naturally occurring polyphenolic inhibitors of amyloid beta aggregation

Alzheimer’s disease is the most common neurodegenerative disease and is one of the main causes of death in developed countries. Consumption of foods rich in polyphenolics is strongly correlated with reduced incidence of Alzheimer’s disease. Our study has investigated the biological activity of previously untested polyphenolic compounds in preventing amyloid β aggregation. The anti-aggregatory potential of these compounds was assessed using the Thioflavin-T assay, transmission electron microscopy, dynamic light scattering and size exclusion chromatography. Two structurally related compounds, luteolin and transilitin were identified as potent inhibitors of Aβ fibril formation. Computational docking studies with an X-ray derived oligomeric structure offer a rationale for the inhibitory activity observed and may facilitate development of improved inhibitors of Aβ aggregation and toxicity.     

Synthesis,structure–activity relationships and biological evaluation of barbigerone analogues as anti-proliferative and anti-angiogenesis agents

A series of barbigerone analogues (7a–7w, 13a–13x) were designed, synthesized and biologically evaluated for their anti-proliferative and anti-angiogenic activities. Among these compounds, compound 13a exhibited the most potent inhibitory effect on the proliferation of HUVECs, HepG2, A375, U251, B16, and HCT116 cells (IC₅₀ = 3.80, 0.28, 1.58, 3.50, 1.09 and 0.68 μM, respectively). Compound 13a inhibited the angiogenesis in zebrafish embryo assay in a concentration-dependent manner. Furthermore, 13a also effectively inhibited the migration and capillary like tube formation of human umbilical vein endothelial cell in vitro. These results support the further investigation of this class of compounds as potential anti-proliferative and anti-angiogenesis agents.     

Construction of a high-density genetic map based on large-scale markers developed by specific length amplified fragment sequencing (SLAF-seq) and its application to QTL analysis for isoflavone content in Glycine max

Background

Quantitative trait locus (QTL) mapping is an efficient approach to discover the genetic architecture underlying complex quantitative traits. However, the low density of molecular markers in genetic maps has limited the efficiency and accuracy of QTL mapping. In this study, specific length amplified fragment sequencing (SLAF-seq), a new high-throughput strategy for large-scale SNP discovery and genotyping based on next generation sequencing (NGS), was employed to construct a high-density soybean genetic map using recombinant inbred lines (RILs, Luheidou2 × Nanhuizao, F_5:8). With this map, the consistent QTLs for isoflavone content across various environments were identified.

Results

In total, 23 Gb of data containing 87,604,858 pair-end reads were obtained. The average coverage for each SLAF marker was 11.20-fold for the female parent, 12.51-fold for the male parent, and an average of 3.98-fold for individual RILs. Among the 116,216 high-quality SLAFs obtained, 9,948 were polymorphic. The final map consisted of 5,785 SLAFs on 20 linkage groups (LGs) and spanned 2,255.18 cM in genome size with an average distance of 0.43 cM between adjacent markers. Comparative genomic analysis revealed a relatively high collinearity of 20 LGs with the soybean reference genome. Based on this map, 41 QTLs were identified that contributed to the isoflavone content. The high efficiency and accuracy of this map were evidenced by the discovery of genes encoding isoflavone biosynthetic enzymes within these loci. Moreover, 11 of these 41 QTLs (including six novel loci) were associated with isoflavone content across multiple environments. One of them, qIF20-2, contributed to a majority of isoflavone components across various environments and explained a high amount of phenotypic variance (8.7% - 35.3%). This represents a novel major QTL underlying isoflavone content across various environments in soybean.

Conclusions

Herein, we reported a high-density genetic map for soybean. This map exhibited high resolution and accuracy. It will facilitate the identification of genes and QTLs underlying essential agronomic traits in soybean. The novel major QTL for isoflavone content is useful not only for further study on the genetic basis of isoflavone accumulation, but also for marker-assisted selection (MAS) in soybean breeding in the future.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1086) contains supplementary material, which is available to authorized users.     

Structure-guided design and synthesis of isoflavone analogs of GW4064 with potent lipid accumulation inhibitory activities

Our group has previously reported a series of isoflavone derivatives with antidyslipidemic activity. With this background, a series of isoflavone analogs of GW4064 were designed, synthesized and evaluated the lipid-lowering activity of analogs. As a result, most of compounds significantly reduced the lipid accumulation in 3T3-L1 adipocytes and four of them (10a, 11, 15c and 15d) showed stronger inhibitory than GW4064. The most potent compound 15d exhibited promising agonistic activity for FXR in a cell-based luciferase reporter assay. Meanwhile, 15d up-regulated FXR, SHP and BSEP gene expression and down-regulated the mRNA expression of lipogenesis gene SREBP-1c. Besides, an improved safety profile of 15d was also observed in a HepG2 cytotoxicity assay compared with GW4064. The obtained biological results were further confirmed by a molecular docking study showing that 15d fitted well in the binding pocket of FXR and interacted with some key residues simultaneously.     

Anti-inflammatory isoflavones and isoflavanones from the roots of Pongamia pinnata (L.) Pierre

A phytochemical study on the roots of Pongamia pinnata afforded five new isoflavone and isoflavanone derivatives (1–5), including two previously undescribed phenylisoflavones possessing an 1,2-oxetane ring, along with 21 known compounds (6–26) among which compound 18 is the first time to be isolated from nature. The structures of the isolated compounds were determined on the basis of 1D, 2D NMR, and HRESIMS. The absolute configurations of the compounds were assigned via analysis of the specific rotations and circular dichroism (CD) spectra, as well as by comparison of the calculated and experimental electronic circular dichroism (ECD) data. All the isolated compounds were evaluated for their inhibitory effects on NO production in LPS-stimulated BV-2 microglial cells. Twelve compounds exhibited different levels of inhibitory effects against NO production, and compound 1 showed the best activity with an IC₅₀ value at 9.0?μM.