Transcriptome responses of Arabidopsis to necrotrophic fungus Alternaria brassicae reveal pathways and candidate genes associated with resistance.

Alternaria leaf blight (ALB), caused by a necrotrophic fungus Alternaria brassicae is a serious disease of oleiferous Brassicas resulting in significant yield losses worldwide. No robust resistance against A. brassicae has been identified in the Brassicas. Natural accessions of Arabidopsis show a spectrum of responses to A. brassicae ranging from high susceptibility to complete resistance. To understand the molecular mechanisms of resistance/ susceptibility, we analysed the comparative changes in the transcriptome profile of Arabidopsis accessions with contrasting responses- at different time points post-infection. Differential gene expression, GO enrichment, pathway enrichment, and weighted gene co-expression network analysis (WGCNA) revealed reprogramming of phenylpropanoid biosynthetic pathway involving lignin, hydroxycinnamic acids, scopoletin, anthocyanin genes to be highly associated with resistance against A. brassicae. T-DNA insertion mutants deficient in the biosynthesis of coumarin scopoletin exhibited enhanced susceptibility to A. brassicae. The supplementation of scopoletin to medium or exogenous application resulted in a significant reduction in the A. brassicae growth. Our study provides new insights into the transcriptome dynamics in A. brassicae-challenged Arabidopsis and demonstrates the involvement of coumarins in plant immunity against the Brassica pathogen A. brassicae.

Genes to specialized metabolites: accumulation of scopoletin, umbelliferone and their glycosides in natural populations of Arabidopsis thaliana.

BACKGROUND: Scopoletin and umbelliferone belong to coumarins, which are plant specialized metabolites with potent and wide biological activities, the accumulation of which is induced by various environmental stresses. Coumarins have been detected in various plant species, including medicinal plants and the model organism Arabidopsis thaliana. In recent years, key role of coumarins in maintaining iron (Fe) homeostasis in plants has been demonstrated, as well as their significant impact on the rhizosphere microbiome through exudates secreted into the soil environment. Several mechanisms underlying these processes require clarification. Previously, we demonstrated that Arabidopsis is an excellent model for studying genetic variation and molecular basis of coumarin accumulation in plants. RESULTS: Here, through targeted metabolic profiling and gene expression analysis, the gene-metabolite network of scopoletin and umbelliferone accumulation was examined in more detail in selected Arabidopsis accessions (Col-0, Est-1, Tsu-1) undergoing different culture conditions and characterized by variation in coumarin content. The highest accumulation of coumarins was detected in roots grown in vitro liquid culture. The expression of 10 phenylpropanoid genes (4CL1, 4CL2, 4CL3, CCoAOMT1, C3'H, HCT, F6'H1, F6'H2,CCR1 and CCR2) was assessed by qPCR in three genetic backgrounds, cultured in vitro and in soil, and in two types of tissues (leaves and roots). We not only detected the expected variability in gene expression and coumarin accumulation among Arabidopsis accessions, but also found interesting polymorphisms in the coding sequences of the selected genes through in silico analysis and resequencing. CONCLUSIONS: To the best of our knowledge, this is the first study comparing accumulation of simple coumarins and expression of phenylpropanoid-related genes in Arabidopsis accessions grown in soil and in liquid cultures. The large variations we detected in the content of coumarins and gene expression are genetically determined, but also tissue and culture dependent. It is particularly important considering that growing plants in liquid media is a widely used technology that provides a large amount of root tissue suitable for metabolomics. Research on differential accumulation of coumarins and related gene expression will be useful in future studies aimed at better understanding the physiological role of coumarins in roots and the surrounding environments.

Synergistic induction of phytoalexins in Nicotiana attenuata by jasmonate and ethylene signaling mediated by NaWRKY70.

Production of the phytoalexins scopoletin and scopolin is regulated by jasmonate (JA) and ethylene signaling in Nicotiana species in response to Alternaria alternata, the necrotrophic fungal pathogen that causes brown spot disease. However, how these two signaling pathways are coordinated to control this process remains unclear. In this study, we found that the levels of these two phytoalexins and transcripts of their key enzyme gene, feruloyl-CoA 6'-hydroxylase 1 (NaF6'H1), were synergistically induced in Nicotiana attenuata by co-treatment with methyl jasmonate (MeJA) and ethephon. By combination of RNA sequencing and virus-induced gene silencing, we identified a WRKY transcription factor, NaWRKY70, which had a similar expression pattern to NaF6'H1 and was responsible for A. alternata-induced NaF6'H1 expression. Further evidence from stable transformed plants with RNA interference, knock out and overexpression of NaWRKY70 demonstrated that it is a key player in the synergistic induction of phytoalexins and plant resistance to A. alternata. Electrophoretic mobility shift, chromatin immunoprecipitation-quantitative PCR, and dual-luciferase assays revealed that NaWRKY70 can bind directly to the NaF6'H1 promoter and activate its expression. Furthermore, the key regulator of the ethylene pathway, NaEIN3-like1, can directly bind to the NaWRKY70 promoter and activate its expression. Meanwhile, NaMYC2s, important JA pathway transcription factors, also indirectly regulate the expression of NaWRKY70 and NaF6'H1 to control scopoletin and scopolin production. Our data reveal that these phytoalexins are synergistically induced by JA and ethylene signaling during A. alternata infection, which is largely mediated by NaWRKY70, thus providing new insights into the defense responses against A. alternata in Nicotiana species.

Scopoletin alleviates high glucose-induced toxicity in human renal proximal tubular cells via inhibition of oxidative damage, epithelial-mesenchymal transition, and fibrogenesis.

BACKGROUND: Recent years of evidence suggest the crucial role of renal tubular cells in developing diabetic kidney disease. Scopoletin (SCOP) is a plant-based coumarin with numerous biological activities. This study aimed to determine the effect of SCOP on renal tubular cells in developing diabetic kidney disease and to elucidate mechanisms. METHODS AND RESULTS: In this study, SCOP was evaluated in vitro using renal proximal tubular (HK-2) cells under hyperglycemic conditions to understand its mechanism of action. In HK-2 cells, SCOP alleviated the high glucose-generated reactive oxygen species (ROS), restored the levels of reduced glutathione, and decreased lipid peroxidation. High glucose-induced alteration in the mitochondrial membrane potential was markedly restored in the SCOP-treated cells. Moreover, SCOP significantly reduced the high glucose-induced apoptotic cell population in the Annexin V-FITC flow cytometry study. Furthermore, high glucose markedly elevated the mRNA expression of fibrotic and extracellular matrix (ECM) components, namely, transforming growth factor (TGF)-ß, alfa-smooth muscle actin (α-SMA), collagen I, and collagen III, in HK-2 cells compared to the untreated cells. SCOP treatment reduced these mRNA expressions compared to the high glucose-treated cells. Collagen I and TGF-ß protein levels were also significantly reduced in the SCOP-treated cells. Further findings in HK-2 cells revealed that SCOP interfered with the epithelial-mesenchymal transition (EMT) in the high glucose-treated HK-2 cells by normalizing E-cadherin and downregulating the vimentin and α-SMA proteins. CONCLUSIONS: In conclusion, SCOP modulates the high glucose-generated renal tubular cell oxidative damage and accumulation of ECM components and may be a promising molecule against diabetic nephropathy.

Identification of natural small molecule modulators of MurB from Salmonella enterica serovar Typhi Ty2 strain using computational and biophysical approaches.

The increase of antibiotic-resistant bacterial pathogens has created challenges in treatment and warranted the design of antibiotics against comparatively less exploited targets. The peptidoglycan (PG) biosynthesis delineates unique pathways for the design and development of a novel class of drugs. Mur ligases are an essential component of bacterial cell wall synthesis that play a pivotal role in PG biosynthesis to maintain internal osmotic pressure and cell shape. Inhibition of these enzymes can interrupt bacterial replication and hence, form attractive targets for drug discovery. In the present work, we focused on the PG biosynthesis pathway enzyme, UDP-N-acetylpyruvylglucosamine reductase, from Salmonella enterica serovar Typhi (stMurB). Biophysical characterization of purified StMurB was performed to gauge the molecular interactions and estimate thermodynamic stability for determination of attributes for possible therapeutic intervention. The thermal melting profile of MurB was monitored by circular dichroism and validated through differential scanning calorimetry experiment. Frequently used chemical denaturants, GdmCl and urea, were employed to study the chemical-induced denaturation of stMurB. In the search for natural compound-based inhibitors, against this important drug target, an in silico virtual screening based investigation was conducted with modeled stMurB structure. The three top hits (quercetin, berberine, and scopoletin) returned were validated for complex stability through molecular dynamics simulation. Further, fluorescence binding studies were undertaken for the selected natural compounds with stMurB alone and with NADPH bound form. The compounds scopoletin and berberine, displayed lesser binding to stMurB whereas quercetin exhibited stronger binding affinity than NADPH. This study suggests that quercetin can be evolved as an inhibitor of stMurB enzyme.

Engineered coumarin accumulation reduces mycotoxin-induced oxidative stress and disease susceptibility.

Coumarins can fight pathogens and are thus promising for crop protection. Their biosynthesis, however, has not yet been engineered in crops. We tailored the constitutive accumulation of coumarins in transgenic Nicotiana benthamiana, Glycine max and Arabidopsis thaliana plants, as well as in Nicotiana tabacum BY-2 suspension cells. We did so by overexpressing A. thaliana feruloyl-CoA 6-hydroxylase 1 (AtF6'H1), encoding the key enzyme of scopoletin biosynthesis. Besides scopoletin and its glucoside scopolin, esculin at low level was the only other coumarin detected in transgenic cells. Mechanical damage of scopolin-accumulating tissue led to a swift release of scopoletin, presumably from the scopolin pool. High scopolin levels in A. thaliana roots coincided with reduced susceptibility to the root-parasitic nematode Heterodera schachtii. In addition, transgenic soybean plants were more tolerant to the soil-borne pathogenic fungus Fusarium virguliforme. Because mycotoxin-induced accumulation of reactive oxygen species and cell death were reduced in the AtF6'H1-overexpressors, the weaker sensitivity to F. virguliforme may be caused by attenuated oxidative damage of coumarin-hyperaccumulating cells. Together, engineered coumarin accumulation is promising for enhanced disease resilience of crops.

Identification of a Novel Coumarins Biosynthetic Pathway in the Endophytic Fungus Fusarium oxysporum GU-7 with Antioxidant Activity.

Coumarins are generally considered to be produced by natural plants. Fungi have been reported to produce coumarins, but their biosynthetic pathways are still unknown. In this study, Fusarium oxysporum GU-7 and GU-60 were isolated from Glycyrrhiza uralensis, and their antioxidant activities were determined to be significantly different. Abundant dipeptide, phenolic acids, and the plant-derived coumarins fraxetin and scopoletin were identified in GU-7 by untargeted metabolomics, and these compounds may account for its stronger antioxidant activity compared to GU-60. Combined with metabolome and RNA sequencing analysis, we identified 24 potentially key genes involved in coumarin biosynthesis and 6 intermediate metabolites. Interestingly, the best hit of S8H, a key gene involved in hydroxylation at the C-8 position of scopoletin to yield fraxetin, belongs to a plant species. Additionally, nondestructive infection of G. uralensis seeds with GU-7 significantly improved the antioxidant activity of seedlings compared to the control group. This antioxidant activity may depend on the biological characteristics of endophytes themselves, as we observed a positive correlation between the antioxidant activity of endophytic fungi and that of their nondestructively infected seedlings. IMPORTANCE Plant-produced coumarins have been shown to play an important role in assembly of the plant microbiomes and iron acquisition. Coumarins can also be produced by some microorganisms. However, studies on coumarin biosynthesis in microorganisms are still lacking. We report for the first time that fraxetin and scopoletin were simultaneously produced by F. oxysporum GU-7 with strong free radical scavenging abilities. Subsequently, we identified intermediate metabolites and key genes in the biosynthesis of these two coumarins. This is the first report on the coumarin biosynthesis pathway in nonplant species, providing new strategies and perspectives for coumarin production and expanding research on new ways for plants to obtain iron.

Estimation of scopoletin from a polyherbal formulation composition using HPLC coupled with fluorescence detector through the concept of Design of Experiment.

High-performance liquid chromatography (HPLC) coupled with a fluorescence detector was used to analyse bioactive phytoconstituent scopoletin from a polyherbal composition derived from the extract prepared from roots of Argyreia nervosa, roots of Withania somnifera, and fruits of Tribulus terrestris. This analytical method was developed as a quality control tool for standardization of the composition to be formulated to enhance spermatogenesis. Chromatographic separation was achieved using Luna® (250 mm × 4.6 mm, 100 Å, 5 µm) C18 column as a stationary phase, and water (0.01 M glacial acetic acid):methanol: acetonitrile (60:20:20, %v/v/v) as the mobile phase; passed through the column at a set flow rate of 1.0 ml min-1 . The elute in the flow cell was excited at 345 nm and the chromatogram was recorded at 444 nm as the emission wavelength. As a part of the analytical Quality by Design approach, systemic studies were conducted to identify potential risks affecting the critical attributes (area, resolution, retention time) of the analytical method, and mitigating the potential risks after optimizing the chromatographic parameters with the help of the Design of Experiment approach. The developed analytical method was subjected to the validation studies, which showed a linear relationship (r2 = 0.9982) between the concentration and the area corresponding to scopoletin peak in the concentration range 10-130 ng ml-1 . The method was found selective, sensitive, and precise. The recovery of the scopoletin was found in a range 99.53-102.13%; confirming the accuracy of the analytical method. The amount of scopoletin was estimated to be 0.146%w/w from the polyherbal composition.

Scopoletin Reactivates Latent HIV-1 by Inducing NF-κB Expression without Global T Cell Activation.

Reversing HIV-1 latency promotes the killing of infected cells and is essential for cure strategies. However, current latency-reversing agents (LRAs) are not entirely effective and safe in activating latent viruses in patients. In this study, we investigated whether Scopoletin (6-Methoxy-7-hydroxycoumarin), an important coumarin phytoalexin found in plants with multiple pharmacological activities, can reactivate HIV-1 latency and elucidated its underlying mechanism. Using the Jurkat T cell model of HIV-1 latency, we found that Scopoletin can reactivate latent HIV-1 replication with a similar potency to Prostratin and did so in a dose- and time-dependent manner. Moreover, we provide evidence indicating that Scopoletin-induced HIV-1 reactivation involves the nuclear factor kappa B (NF-κB) signaling pathway. Importantly, Scopoletin did not have a stimulatory effect on T lymphocyte receptors or HIV-1 receptors. In conclusion, our study suggests that Scopoletin has the potential to reactivate latent HIV-1 without causing global T-cell activation, making it a promising treatment option for anti-HIV-1 latency strategies.

Design and Synthesis of Scopoletin Sulfonate Derivatives as Potential Insecticidal Agents.

(1) Background: Scopoletin and scoparone, two naturally occurring coumarins, have garnered considerable attention and have been introduced to the market in China due to their high insecticidal efficacy and low toxicity. To investigate the structure-activity relationship of these coumarins, a series of scopoletin derivatives with aryl sulfate at C7 and different substitutes at C3 were designed and synthesized, and their insecticidal activity was studied. (2) Methods: A total of 28 new scopoletin derivatives were designed and synthesized. Most target compounds exhibited moderate insecticidal activity against the phytophagous mite Tetranychus cinnabarinus and the brine shrimp Artemia salina. (3) Results: Among these compounds, compounds 5a and 5j possessed the best insecticidal activities against T. cinnabarinus, with LC50 values of 57.0 and 20.0 µg/mL, respectively, whereas that of the control drug was 15.0 µg/mL. Compound 4j exhibited selective insecticidal activities against A. salina, with an LC50 value of 9.36 µg/mL, whereas its LC50 value against T. cinnabarinus was 93.0 µg/mL. The enzymatic inhibitory activity on acetylcholinesterase (AChE) showed a consistent tendency with the insecticidal activity. Further molecular docking analyses predicted the binding conformations of these compounds, which showed a good correlation between the insecticidal activity and the binding scores. (4) Conclusions: In general, a decreased electron cloud density of the Δ3,4 olefinic bond is beneficial for improving the insecticidal activity against both T. cinnabarinus and A. salina. In addition, naphthyl or benzene groups with a sulfate ester at the C7 position could further improve the insecticidal activity against A. salina. AChE was implied to be a site of action for potential insecticidal activity. The results provide insight into the rational design of a new generation of effective coumarin insecticides.

Scopoletin and umbelliferone protect hepatocytes against palmitate- and bile acid-induced cell death by reducing endoplasmic reticulum stress and oxidative stress.

BACKGROUND: The number of patients with non-alcoholic fatty liver disease (NAFLD) is rapidly increasing due to the growing epidemic of obesity. Non-alcoholic steatohepatitis (NASH), the inflammatory stage of NAFLD, is characterized by lipid accumulation in hepatocytes, chronic inflammation and hepatocyte cell death. Scopoletin and umbelliferone are coumarin-like molecules and have antioxidant, anti-cancer and anti-inflammatory effects. Cytoprotective effects of these compounds have not been described in hepatocytes and the mechanisms of the beneficial effects of scopoletin and umbelliferone are unknown. AIM: To investigate whether scopoletin and/or umbelliferone protect hepatocytes against palmitate-induced cell death. For comparison, we also tested the cytoprotective effect of scopoletin and umbelliferone against bile acid-induced cell death. METHODS: Primary rat hepatocytes were exposed to palmitate (1 mmol/L) or the hydrophobic bile acid glycochenodeoxycholic acid (GCDCA; 50 µmol/L). Apoptosis was assessed by caspase-3 activity assay, necrosis by Sytox green assay, mRNA levels by qPCR, protein levels by Western blot and production of reactive oxygen species (ROS) by fluorescence assay. RESULTS: Both scopoletin and umbelliferone protected against palmitate and GCDCA-induced cell death. Both palmitate and GCDCA induced the expression of ER stress markers. Scopoletin and umbelliferone decreased palmitate- and GCDCA-induced expression of ER stress markers, phosphorylation of the cell death signaling intermediate JNK as well as ROS production. CONCLUSION: Scopoletin and umbelliferone protect against palmitate and bile acid-induced cell death of hepatocytes by inhibition of ER stress and ROS generation and decreasing phosphorylation of JNK. Scopoletin and umbelliferone may hold promise as a therapeutic modality for the treatment of NAFLD.

An overview of the pharmacological activities of scopoletin against different chronic diseases.

Chronic diseases are considered a major public health concern worldwide, and most of these diseases like cancer, cardiovascular, metabolic, and neurological disorders occur due to atypical regulation of multiple signaling pathways. It has also been observed that most of the currently approved therapies for these diseases fail to show prolonged efficacy due to their mono-targeted nature and are associated with the development of chemoresistance, thus restricting their utility. The plant-derived compounds, on the other hand, show multi-targeted nature, and thus these phytochemicals have gained wide attention as they offer negligible side effects. The present review aims to recapitulate the potential effects of one such phytochemical, Scopoletin, which was found to have a diverse range of pharmacological activities such as anti-cancer, anti-diabetic, anti-inflammatory, cardioprotective, hepatoprotective, etc. Scopoletin modulated multiple molecular signatures in cancer, including AMPK, EGFR, MAPK/ ERK, NF-κB, PI3K/Akt/ mTOR, and STAT3; regulated the levels of critical markers of metabolic diseases such as ALT, AST, TG, and TC; inflammatory diseases such as ILs and TNFs; neurological diseases such as AChE, etc. thus relieving the symptoms and severity associated with these diseases. Further, this compound has a non-toxic nature and possesses an excellent pharmacokinetic property, which warrants further investigation in clinical settings for developing it as a potential drug.

Advances in biosynthesis of scopoletin.

Scopoletin is a typical example of coumarins, which can be produced in plants. Scopoletin acts as a precursor for pharmaceutical and health care products, and also possesses promising biological properties, including antibacterial, anti-tubercular, anti-hypertensive, anti-inflammatory, anti-diabetic, and anti-hyperuricemic activity. Despite the potential benefits, the production of scopoletin using traditional extraction processes from plants is unsatisfactory. In recent years, synthetic biology has developed rapidly and enabled the effective construction of microbial cell factories for production of high value-added chemicals. Herein, this review summarizes the progress of scopoletin biosynthesis in artificial microbial cell factories. The two main pathways of scopoletin biosynthesis are summarized firstly. Then, synthetic microbial cell factories are reviewed as an attractive improvement strategy for biosynthesis. Emerging techniques in synthetic biology and metabolic engineering are introduced as innovative tools for the efficient synthesis of scopoletin. This review showcases the potential of biosynthesis of scopoletin in artificial microbial cell factories.

Scopoletin inhibits PDGF-BB-induced proliferation and migration of airway smooth muscle cells by regulating NF-κB signaling pathway.

BACKGROUND: Asthma is a common chronic inflammatory disease of the airway, and airway remodeling and the proliferation mechanism of airway smooth muscle cells (ASMCs) is of great significance to combat this disease. OBJECTIVE: To assess possible effects of scopoletin on asthma and the potential signaling pathway. MATERIALS AND METHODS: ASMCs were treated PDGF-BB and scopoletin and subjected to cell viability detection by CCK-8 assay. Cell migration of ASMCs was determined by a wound closure assay and transwell assay. The protein level of MMP2, MMP9, calponin and α-SMA were measured using western blot. The levels of NF-κB signaling pathway were detected by Western blotting. RESULTS: Scopoletin inhibited proliferation of PDGF-BB - induced ASMCs. Also it suppressed the migration and invasion of PDGF-BB - induced ASMCs. We further showed that Scopoletin regulated phenotypic transition of ASMCs. Mechanically, Scopoletin inhibited proliferation and invasion of ASMCs by regulating NF-κB signaling pathway. CONCLUSIONS: We therefore thought Scopoletin could serve as a promising drug for the treatment of asthma.

Knockdown of p-Coumaroyl Shikimate/Quinate 3'-Hydroxylase Delays the Occurrence of Post-Harvest Physiological Deterioration in Cassava Storage Roots.

Cassava storage roots are an important source of food, feed, and material for starch-based industries in many countries. After harvest, rapid post-harvest physiological deterioration (PPD) reduces their palatability and marketability. During the PPD process, vascular streaking occurs through over-accumulation of coumarins, the biosynthesis of which involves the key enzyme p-coumaroyl shikimate/quinate 3'-hydroxylase (C3'H). Repression of MeC3'H expression by RNA interference in transgenic cassava plants caused a significant delay in PPD by decreasing scopoletin and scopolin accumulation in field-harvested storage roots. This study demonstrates that MeC3'H is the key enzyme participating in coumarin biosynthesis during PPD and shows that MeC3'H is a useful target gene for editing to prolong the shelf life of cassava storage roots.

Soil modification with organic amendments and organo-clays: Effects on sorption, degradation, and bioactivity of the allelochemical scopoletin.

We assessed the effect of three organic amendments and two organo-clays on sorption, persistence, and phytotoxicity of scopoletin, an allelochemical compound with potential as bioherbicide, in a Mediterranean alkaline soil. The aim was to elucidate whether the phytotoxicity of scopoletin could be expressed better in amended than unamended soil. The three organic amendments were fresh solid olive-mill waste (OMW), composted solid olive-mill waste (OMWc), and biochar (BC) prepared from OMWc. The two organo-clays were a commercial organo-montmorillonite (Cloi10) and lab-synthesized oleate-modified hydrotalcite (HT-OLE). The amendments enhanced sorption of scopoletin by the soil consistently with their individual affinities for the allelochemical: Cloi10 ≫ OMW > BC > OMWc > HT-OLE. The soil persistence of scopoletin increased significantly because of the addition of Cloi10, OMW, and BC. This increase was attributed to a combination of sorption, which protected the allelochemical from rapid biodegradation, and microbial activity changes. Although the inhibitory effect produced by the amendments themselves obscured the phytotoxicity of scopoletin to Lactuca sativa L. in soil treated with OMW and Cloi10, applying scopoletin to BC-amended soil led to a marked reduction in root length and aerial biomass of the emerged seedlings even though BC alone did not negatively affect these parameters. This inhibitory effect of scopoletin in BC-amended soil was in contrast to the negligible effect exerted by the allelochemical when applied to unamended soil. The results show that soils treated with suitable amendments, such as BC, might provide a scenario in which the herbicidal properties of 7-hydroxycoumarins could be better expressed.

Comparative Analysis of the Phenolic Profile of Lycium barbarum L. Fruits from Different Regions in China.

Lycium barbarum L. (LB) fruits have high nutritive values and therapeutic effects. The aim of this study was to comprehensively evaluate the differences in phenolic composition of LB fruits from different geographical regions. Different methods of characterization and statistical analysis of data showed that different geographic sources of China could be significantly separated from each other. The highest total phenolic compound (TPC) content was observed in LB fruits from Ningxia (LBN), followed by those from Gansu (LBG) and Qinghai (LBQ). The Fourier transform infrared (FTIR) spectra of LB fruits revealed that LBQ had a peak at 2972 cm-1 whereas there was no similar peak in LBG and LBQ. A new HPLC method was established for the simultaneous determination of 8 phenolic compounds by quantitative analysis of multiple components by a single marker (QAMS), including 4 phenolic acids (chlorogenic acid, caffeic acid, 4-hydroxycinnamic acid, and ferulic acid), 1 coumarin (scopoletin), and 3 flavonoids (kaempferol-3-O-rutinoside, rutin, and narcissoside). It was showed that rutin was the most dominant phenolic compound in LBQ, although the average content of 4 phenolic acids was also high in LBQ, and scopoletin was the richest in LBG. UHPLC-Q-TOF-MS was used to qualitatively analyze the phenolics, which showed LBN was abundant in phenolic acids, LBQ was rich in flavonoids, and coumarins were the most plentiful in LBG. In conclusion, this study can provide references for the quality control and evaluation of phenolics in LB fruits and their by-products.

Challenges in the Heterologous Production of Furanocoumarins in Escherichia coli.

Coumarins and furanocoumarins are plant secondary metabolites with known biological activities. As they are present in low amounts in plants, their heterologous production emerged as a more sustainable and efficient approach to plant extraction. Although coumarins biosynthesis has been positively established, furanocoumarin biosynthesis has been far more challenging. This study aims to evaluate if Escherichia coli could be a suitable host for furanocoumarin biosynthesis. The biosynthetic pathway for coumarins biosynthesis in E. coli was effectively constructed, leading to the production of umbelliferone, esculetin and scopoletin (128.7, 17.6, and 15.7 µM, respectively, from tyrosine). However, it was not possible to complete the pathway with the enzymes that ultimately lead to furanocoumarins production. Prenyltransferase, psoralen synthase, and marmesin synthase did not show any activity when expressed in E. coli. Several strategies were tested to improve the enzymes solubility and activity with no success, including removing potential N-terminal transit peptides and expression of cytochrome P450 reductases, chaperones and/or enzymes to increase dimethylallylpyrophosphate availability. Considering the results herein obtained, E. coli does not seem to be an appropriate host to express these enzymes. However, new alternative microbial enzymes may be a suitable option for reconstituting the furanocoumarins pathway in E. coli. Nevertheless, until further microbial enzymes are identified, Saccharomyces cerevisiae may be considered a preferred host as it has already been proven to successfully express some of these plant enzymes.

Lysozyme inhibits postharvest physiological deterioration of cassava.

After harvest, cassava (Manihot esculenta Crantz) storage roots undergo rapid postharvest physiological deterioration, producing blue-brown discoloration in the vasculature due to the production of polyphenolics (mainly quinones and coumarins) by enzymes such as polyphenol oxidase (PPO). Here, we report the application of hen egg-white lysozyme (HEWL), a natural PPO inhibitor, in transgenic cassava to repress the symptoms of postharvest physiological deterioration. The HEWL-expressing transgenic plants had lower levels of the two main cassava coumarins tested, scopoletin and scopolin, compared with wild type. HEWL-expressing cassava also showed increased tolerance of oxidative stress. Overall, the lysozyme-PPO system proved to be functional in plants for repressing PPO-mediated commercial product browning.

[Differences in intestinal absorption characteristics of Cynanchum auriculatum extract based on in situ intestinal circulation perfusion model in two states].

The present study aimed to investigate the intestinal absorption characteristics of six components(syringic acid, scopoletin, baishouwu benzophenone, caudatin, qingyangshengenin, and deacylmetaplexigenin) in Cynanchum auriculatum extract. In situ intestinal circulation perfusion model was employed to investigate the differences in intestinal absorption characteristics of C. auriculatum extract under the influence of different intestinal segments, different drug concentrations, and bile in the normal and functional dyspepsia(FD) states. The results showed that the absorption of baishouwu benzophenone decreased with the increase in the concentration of C. auriculatum extract(P<0.01), while the absorption of syringic acid and other components increased in a dose-independent manner, suggesting that baishouwu benzophenone might follow active absorption, while other components might not be on a single absorption pattern. The main absorption sites of each component in the normal state were different from those in the FD state. The cumulative absorption conversion rates in the FD state were generally lower than those in the normal state, and bile inhibited the absorption of other components except for scopoletin in both states(P<0.05). As revealed, the small intestine showed selectivity to the absorption of drugs, and the pathological state(such as FD) and bile both affected the absorption of the main components, which provides a theoretical basis for the development of new drugs and further development of C. auriculatum.