Synthetic Studies toward 5,6,7,3',4'-Monomethoxytetrahydroxyflavones: Synthesis of Pedalitin.

During the synthetic studies toward 5,6,7,3',4'-monomethoxytetrahydroxyflavones, a concise pedalitin synthesis procedure was achieved. As previously reported, 6-hydroxy-2,3,4-trimethoxyacetophenone was prepared by Friedel-Crafts acylation of 1,4-dihydroxy-2,6-dimethoxybenzene with boron trifluoride diethyl etherate in acetic acid. When aldol condensation of 6-hydroxy-2,3,4-trimethoxyacetophenone 2b with vanillin was performed in basic conditions, it produced 2'-hydroxychalcone 3b, and, surprisingly, along with 3-hydroxyflavone 4 in a considerable amount. We propose that this oxidative cyclization is presumably due to the contribution of a quinone methide, likely to be subjected to aerobic oxidation. The chalcone was then subjected to oxidative cyclization with iodine in dimethyl sulfoxide to afford flavone 5 in good yield. To our delight, serial demethylation of the three methoxy groups at the 5-, 6-, and 3'-positions of 5 proceeded smoothly to produce pedalitin 1, under hydrogen bromide solution (30% in acetic acid). The crystal structures of 3-hydroxyflavone 4 and pedalitin tetraacetate 6 were unambiguously determined by X-ray crystallography.

Integrative analysis of metabolite and transcriptome reveals biosynthetic pathway and candidate genes for eupatilin and jaceosidin biosynthesis in Artemisia argyi.

Artemisia argyi (A. argyi) is a medicinal plant belonging to the Asteraceae family and Artemisia genus. Flavonoids abundant in A. argyi are associated with anti-inflammatory, anticancer, and antioxidative effects. Eupatilin and jaceosidin are representative polymethoxy flavonoids with medicinal properties significant enough to warrant the development of drugs using their components. However, the biosynthetic pathways and related genes of these compounds have not been fully explored in A. argyi. This study comprehensively analyzed the transcriptome data and flavonoids contents from four different tissues of A. argyi (young leaves, old leaves, trichomes collected from stems, and stems without trichomes) for the first time. We obtained 41,398 unigenes through the de-novo assembly of transcriptome data and mined promising candidate genes involved in the biosynthesis of eupatilin and jaceosidin using differentially expressed genes, hierarchical clustering, phylogenetic tree, and weighted gene co-expression analysis. Our analysis led to the identification of a total of 7,265 DEGs, among which 153 genes were annotated as flavonoid-related genes. In particular, we were able to identify eight putative flavone-6-hydroxylase (F6H) genes, which were responsible for providing a methyl group acceptor into flavone basic skeleton. Furthermore, five O-methyltransferases (OMTs) gene were identified, which were required for the site-specific O-methylation during the biosynthesis of eupatilin and jaceosidin. Although further validation would be necessary, our findings pave the way for the modification and mass-production of pharmacologically important polymethoxy flavonoids through genetic engineering and synthetic biological approaches.

Golden Gate Cloning-Compatible DNA Replicon/2A-Mediated Polycistronic Vectors for Plants.

The expression of multiple proteins and high-throughput vector assembly system are highly relevant in the field of plant genetic engineering and synthetic biology. Deployment of the self-cleaving 2A peptide that mediates polycistronic gene expression has been an effective strategy for multigene expression, as it minimizes issues in coordinated transgene regulation and trait staking in plants. However, efficient vector assembly systems optimized for 2A peptide-mediated polycistronic expression are currently unavailable. Furthermore, it is unclear whether protein expression levels are influenced by the transgene position in the polycistronic expression cassette. In this article, we present Golden Gate cloning-compatible modular systems allowing rapid and flexible construction of polycistronic expression vectors applicable for plants. The genetic modules comprised 2A peptides (T2A and P2A)-linked tricistron expression cassette and its acceptor backbones, named pGO-DV1 and pGO-DV2. While both acceptor backbones were binary T-DNA vectors, pGO-DV2 was specially designed to function as a DNA replicon enhancing gene expression levels. Using the Golden Gate cloning, a set of six tricistronic vectors was constructed, whereby three transgenes encoding fluorescent proteins (mCherry, eYFP, and eGFP) were combinatorially placed along the expression cassette in each of the binary vectors. Transient expression of the construct in tobacco leaves revealed that the expression levels of three fluorescent proteins were comparable each other regardless of the gene positions in the tricistronic expression cassette. pGO-DV2-based constructs were able to increase protein expression level by up to 71%, as compared to pGO-DV1-based constructs.

Gymnaster Koraiensis Extract Alleviated Metabolic Syndrome Symptoms and Stimulated UCP1-Independent Energy Consumption via AMPK Activation in White Adipose Tissue.

SCOPE: Metabolic syndrome and obesity are rising worldwide concerns that are accompanied by adverse health consequences. Here, it is hypothesized that the ethanol extract from Gymnaster koraiensis (GK), an edible Korean plant known for its anti-cancer and hepatoprotective properties, could attenuate metabolic syndrome-related symptoms in high-fat dietary-induced obese (DIO) mice. METHODS AND RESULTS: Administration of 100 mg kg-1 GK extract to DIO mice effectively reduces body and white adipose tissue (WAT) weight. It also reduces cardiovascular disease risk and improves insulin resistance by lowering the fasting blood glucose levels and mitigating oxidative stress and inflammation. Moreover, supplementation with GK causes elevated energy expenditure in WAT by increasing the mitochondrial oxidative capacity and lipid catabolism through upregulated adenosine monophosphate-activated protein kinase (AMPK) signaling. Orlistat is used as a positive control drug due to its widespread use in previous studies. It is found that GK extract causes weight loss, similar to Orlistat, and it additionally shows unique functions, such as upregulation of energy consumption in WAT. CONCLUSION: GK extract treatment prominently reduces obesity and its associated metabolic complications, such as hyperlipidemia, hyperglycemia, and insulin resistance. Hence, It can be used as a promising multi-target functional food that can improve metabolic syndrome-related symptoms.

Rapid securing of reference substances from Peucedanum japonicum Thunberg by recycling preparative high-performance liquid chromatography.

Among the most critical needs of natural product chemistry is a complete library of pure reference substances. Some khellactone-type isomers of pharmacological importance are either still lacking reference substances or references are only available in limited amounts. To address this need, a recycling high-performance liquid chromatography (R-HPLC) strategy was adopted to improve the isomer separation efficiency from Peucedanum japonicum. Under the optimal isolation conditions, we obtained isomerically pure substances, particularly khellactone coumarins with different substituent groups. Isolated compounds attained purities greater than 98% as determined by ultra-performance liquid chromatography-charged aerosol detector (UPLC-CAD) and photodiode array (PDA). The structures of these compounds were identified according to their mass patterns and 2D NMR spectra. The proposed methods of single-column recycling obtained the same amount of product as conventional systems while being simple, increasing efficiency and reducing cost.

Isotrifoliol inhibits pro-inflammatory mediators by suppression of TLR/NF-κB and TLR/MAPK signaling in LPS-induced RAW264.7 cells.

Soybeans, produced by Glycine max (L.) Merr., contain high levels of isoflavones, such as genistein and daidzein. However, soy leaves contain more diverse and abundant flavonol glycosides and coumestans, as compared to the soybean. This study investigated the anti-inflammatory effects of the major coumestans present in soy leaf (coumestrol, isotrifoliol, and phaseol) in lipopolysaccharide (LPS)-induced RAW264.7 cells. Coumestans significantly reduced LPS-induced nitric oxide (NO), prostaglandin E2 (PGE2), and reactive oxygen species (ROS) production; isotrifoliol had the most potent anti-inflammatory activity. Isotrifoliol reduced LPS-mediated induction of mRNA expression of inducible nitric-oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin (IL)-1ß, IL-6, tumor necrosis factor alpha (TNFα), and chemokines, such as chemokine (C-C motif) ligand (CCL) 2, CCL3, and CCL4. Isotrifoliol prevented NF-κB p65 subunit activation by reducing the phosphorylation and degradation of the inhibitor of NF-κB. And isotrifoliol significantly suppressed phosphorylation of the extracellular signal-regulated protein kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK). Furthermore, isotrifoliol suppressed LPS-induced Toll-like Receptor (TLR) signaling pathway, including mRNA expression of TNF receptor associated factor 6, transforming growth factor beta-activated kinase 1 (TAK1), TAK1 binding protein 2 (TAB2), and TAB3. These results demonstrate that isotrifoliol exerts an anti-inflammatory effect by suppressing the expression of inflammatory mediators via inhibition of TLR/NF-κB and TLR/MAPK signaling in LPS-induced RAW264.7 macrophages. Therefore, isotrifoliol can be used as an anti-inflammatory agent, and coumestan-rich soy leaf extracts may provide a useful dietary supplement.