Plant Coumarins Research Articles

Multi-omics analysis reveals genetic architecture and local adaptation of coumarins metabolites in Populus

BackgroundAccumulation of coumarins plays key roles in response to immune and abiotic stress in plants, but the genetic adaptation basis of controlling coumarins in perennial woody plants remain unclear.ResultsWe detected 792 SNPs within 334 genes that were significantly associated with the phenotypic variations of 15 single-metabolic traits and multiple comprehensive index, such as principal components (PCs) of coumarins metabolites. Expression quantitative trait locus mapping uncovered that 337 eQTLs associated with the expression levels of 132 associated genes. Selective sweep revealed 55 candidate genes have potential selective signature among three geographical populations, highlighting that the coumarins biosynthesis have been encountered forceful local adaptation. Furthermore, we constructed a genetic network of seven candidate genes that coordinately regulate coumarins biosynthesis, revealing the multiple regulatory patterns affecting coumarins accumulation in Populus tomentosa. Validation of candidate gene variations in a drought-tolerated population and DUF538 heterologous transformation experiments verified the function of candidate genes and their roles in adapting to the different geographical conditions in poplar.ConclusionsOur study uncovered the genetic regulation of the coumarins metabolic biosynthesis of Populus, and offered potential clues for drought-tolerance evaluation and regional improvement in woody plants.

Deep eutectic solution elution assisted ligand affinity assay: A useful tool for the active coumarins screening from Fructus cnidii

BackgroundMany of the ligand affinity analyses are presented in water environment, and the hydrophilic solution such as methanol is used for dissociating the bound compounds. The obtained dissociation solution needs to be concentrated for improving the sensitivity of the assay. However, it is not good for the analysis of hydrophobic and volatile compounds such as coumarins. The solubility of them in water environment is low. And they may be lost in the sample preparation process. In this work, a deep eutectic solvent elution assisted ligand affinity assay for the screening of active coumarins in plants was proposed. ResultsFor the first time, deep eutectic solvent was used as a dissociation solution for dissociating the compounds that bind with acetylcholinesterase immobilized magnetic beads. Analytes could be enriched during the phase separation process of deep eutectic solvent. The concentration of the analytes in sample solution was therefore increased. It is good for the detection of coumarins. The LOD and LOQ of the proposed assay were 0.50 (μ g/mL) and 1.50 (μ g/mL), respectively. The relative recovery of the assay was 99.4 ± 7.2 %. The intra-day and inter-day precision were 7.25 % and 6.80 %. Using the assay as a tool, the active coumarins in Fructus cnidii were analyzed. And two coumarins with acetylcholinesterase inhibition activity such as imperatorin and osthole were found. SignificanceCoumarins have good solubility in the selected deep eutectic solvent, and could be enriched into hydrophobic phase during the phase separation process. The extra concentration process is not needed. Compared with traditional methods, the proposed method has the benefits of simplified operation process, high sensitivity, and is friendly to the volatile and hydrophobic compounds.

Combined metabolome and transcriptome reveal HmF6’H1 regulating simple coumarin accumulation against powdery mildew infection in Heracleum moellendorffii Hance

BackgroundPowdery mildew, caused by Eeysiphe heraclei, seriously threatens Heracleum moellendorffii Hance. Plant secondary metabolites are essential to many activities and are necessary for defense against biotic stress. In order to clarify the functions of these metabolites in response to the pathogen, our work concentrated on the variations in the accumulation of secondary metabolites in H. moellendorffii during E. heraclei infection.ResultsFollowing E. heraclei infection, a significant upregulation of coumarin metabolites—particularly simple coumarins and associated genes was detected by RNA-seq and UPLC-MS/MS association analysis. Identifying HmF6’H1, a Feruloyl CoA 6’-hydroxylase pivotal in the biosynthesis of the coumarin basic skeleton through ortho-hydroxylation, was a significant outcome. The cytoplasmic HmF6’H1 protein was shown to be able to catalyze the ortho-hydroxylation of p-coumaroyl-CoA and caffeoyl-CoA, resulting in the formation of umbelliferone and esculetin, respectively. Over-expression of the HmF6’H1 gene resulted in increased levels of simple coumarins, inhibiting the biosynthesis of furanocoumarins and pyranocoumarins by suppressing PT gene expression, enhancing H. moellendorffii resistance to powdery mildew.ConclusionsThese results established HmF6’H1 as a resistance gene aiding H. moellendorffii in combatting E. heraclei infection, offering additional evidence of feruloyl-CoA 6’-hydroxylase role in catalyzing various types of simple coumarins. Therefore, this work contributes to our understanding of the function of simple coumarins in plants’ defense against powdery mildew infection.

Anabolic metabolism of autotoxic substance coumarins in plants

Background Autotoxicity is an intraspecific manifestation of allelopathy in plant species. The specialized metabolites and their derivatives that cause intraspecific allelopathic inhibition in the plant are known as autotoxic substances. Consequently, autotoxic substances production seriously affects the renewal and stability of ecological communities. Methods This article systematically summarizes the types of autotoxic substances present in different plants. They mainly include phenolic compounds, terpenoids, and nitrogenous organic compounds. Phenolic coumarins are the main autotoxic substances in many plants. Therefore, we also discuss differences in coumarin types and content among plant varieties, developmental stages, and tissue parts, as well as their mechanisms of autotoxicity. In addition, we review the metabolic pathways involved in coumarin biosynthesis, the key enzymes, genes, and transcription factors, as well as factors affecting coumarin biosynthesis. Results Coumarin biosynthesis involves three stages: (1) the formation of the coumarin nucleus; (2) acylation, hydroxylation, and cyclization; (3) structural modification. The key enzymes involved in the coumarin nuclear formation stage include PAL, C4H, 4CL, HCT, CAOMT, COSY, F6’H, and CCoAOMT1, and the key genes involved include BGA, CYP450 and MDR, among others. Ortho-hydroxylation is a key step in coumarin biosynthesis and PS, COSY and S8H are the key enzymes involved in this process. Finally, UGTs are responsible for the glycosylation modification of coumarins, and the MaUGT gene may therefore be involved in coumarin biosynthesis. Conclusion It is important to elucidate the autotoxicity and anabolic mechanisms of coumarins to create new germplasms that produce fewer autotoxic substances.

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.

Biosynthesis of the Plant Coumarin Osthole by Engineered Saccharomyces cerevisiae.

Osthole is a coumarin compound found in the traditional Chinese medicine Cnidium monnieri. Extensive studies have shown that osthole exhibits many medicinal properties, and recently, researchers have found that it possesses potent airway-relaxation activity by inhibiting phosphodiesterase 4D activity, making it a potential novel bronchodilator that does not target β2-adrenoceptors for asthma treatment. Here, we report the complete biosynthesis of osthole in engineered yeast. We created an umbelliferone (UMB)-producing strain by reconstituting the complete UMB pathway in yeast. We found that coumarin synthase (COSY) is essential for the conversion of 2',4'-dihydroxycinnamoyl-CoA into UMB in yeast; this conversion has been treated as a spontaneous step in previously reported UMB-producing microbials. By introducing downstream prenyltransferase and methyltransferase genes and addressing problems such as protein expression and cofactor supply to fulfill the downstream steps, complete biosynthesis of osthole was achieved. Finally, through metabolic engineering, to ensure precursor supply, and the debugging of rate-limited steps, the osthole titer reached 108.10 mg/L in shake flasks and 255.1 mg/L in fed-batch fermentation. Our study is the first to produce osthole using engineered microbes, providing a blueprint for the supply of plant-derived osthole via microbial fermentation, which will remove the barriers of resource limitations for osthole-based drug development.

Emergence of a proton exchange-based isomerization and lactonization mechanism in the plant coumarin synthase COSY

Plants contain rapidly evolving specialized enzymes that support the biosynthesis of functionally diverse natural products. In coumarin biosynthesis, a BAHD acyltransferase-family enzyme COSY was recently discovered to accelerate coumarin formation as the only known BAHD enzyme to catalyze an intramolecular acyl transfer reaction. Here we investigate the structural and mechanistic basis for COSY’s coumarin synthase activity. Our structural analyses reveal an unconventional active-site configuration adapted to COSY’s specialized activity. Through mutagenesis studies and deuterium exchange experiments, we identify a unique proton exchange mechanism at the α-carbon of the o-hydroxylated trans-hydroxycinnamoyl-CoA substrates during the catalytic cycle of COSY. Quantum mechanical cluster modeling and molecular dynamics further support this key mechanism for lowering the activation energy of the rate-limiting trans-to-cis isomerization step in coumarin production. This study unveils an unconventional catalytic mechanism mediated by a BAHD-family enzyme, and sheds light on COSY’s evolutionary origin and its recruitment to coumarin biosynthesis in eudicots.

Plant Coumarins with Anti-HIV Activity: Isolation and Mechanisms of Action.

This review summarizes and systematizes the literature on the anti-HIV activity of plant coumarins with emphasis on isolation and the mechanism of their antiviral action. This review summarizes the information on the anti-HIV properties of simple coumarins as well as annulated furano- and pyranocoumarins and shows that coumarins of plant origin can act by several mechanisms: inhibition of HIV reverse transcriptase and integrase, inhibition of cellular factors that regulate HIV-1 replication, and transmission of viral particles from infected macrophages to healthy ones. It is important to note that some pyranocoumarins are able to act through several mechanisms or bind to several sites, which ensures the resistance of these compounds to HIV mutations. Here we review the last two decades of research on the anti-HIV activity of naturally occurring coumarins.

SHSY5Y NÖROBLASTOMA HÜCRELERİNDE SKOPOLETİN NÖROTOKSİSİTESİNE BAĞLI EMPEDANS DEĞİŞİKLİKLERİNİN GERÇEK ZAMANLI ANALİZİ

Plant coumarins are important components of the human diet and a number of them are considered to possess chemopreventive and therapeutic properties against cancer. Scopoletin, a natural coumarin component has been shown to inhibit the growth of many cancer cells. In this study, the cytotoxic activity of scopoletin was studied against human neuroblastoma cells SHSY5Y. The activity of scopoletin was evaluated by Sulphorhodamine B (SRB) assay and xCELLigence system. It was demonstrated that scopoletin reduced human SHSY5Y neuroblastoma cell viability. Scopoletin inhibited the growth of the cell line and the IC50 values were 91.82 and 79.19 μM for 48 h and 72 h, respectively. The findings from this study demonstrated that the growth inhibitory and cytotoxic effects of scopoletin on SHSY5Y cells may prove essential in the development of novel treatment regimens for neuroblastoma cancer.

REAL-TIME ANALYSIS OF IMPEDANCE ALTERATIONS BY THE NEUROTOXICITY OF SCOPOLETIN ON SH-SY5Y NEUROBLASTOMA CELLS

Plant coumarins are important components of the human diet and a number of them are considered to possess chemo-preventive and therapeutic properties against cancer. Scopoletin, a natural coumarin component has been shown to inhibit the growth of many cancer cells. In this study, the cytotoxic activity of scopoletin was studied against human neuroblastoma cells SH-SY5Y. The activity of scopoletin was evaluated by Sulphorhodamine B (SRB) assay and xCELLigence system. It was demonstrated that scopoletin reduced human SH-SY5Y neuroblastoma cell viability. Scopoletin inhibited the growth of the cell line and the IC50 values were 91.82 and 79.19 μM for 48-h and 72-h, respectively. The findings from this study demonstrated that the growth inhibitory and cytotoxic effects of scopoletin on SH-SY5Y cells may prove essential in the development of novel treatment regimens for neuroblastoma cancer.

Preliminary Screening of Phytoconstituents and Anti-Bacterial Examination of Annona muricata and Cucumbita maxima Leaves

Dried leaves of Annona muricata and Cucumbita maxima were extracted using chloroform, methanol and hexane solvents. The extracts were analysed for phytoconstituents using standard procedures. A preliminary test was also carried out on the anti-bacterial properties of the different solvent extracts against S. aureus, V. cholera and E. coli. Phytochemical analysis of Annona muricata and Cucumbita maxima showed the presence of alkaloids, fats and oil, tannins, saponins, flavonoids, terpenoids, steroids and proteins. However, glycosides were not detected in any of the plant extracts and coumarins were not detected in A. muricata and flavonoids were not detected Cucumbita maxima. The growth of S. aureus was inhibited in chloroform and hexane extracts of A muricata and hexane and methanol extract of C. maxima. The growth of V. cholera was inhibited to varying degrees by the extracts from all the solvents, while E. coli was only inhibited in hexane extracts of both plants. The results observed revealed that both plants can serve useful purposes in traditional medicine and can be harnessed for scientific design in drug synthesis and production. Keywords: Phytochemicals, screening, solvent extracts, Annona muricata and Cucumbita maxima, anti-bacterial

Innovation and Application of the Type III Secretion System Inhibitors in Plant Pathogenic Bacteria.

Many Gram-negative pathogenic bacteria rely on a functional type III secretion system (T3SS), which injects multiple effector proteins into eukaryotic host cells, for their pathogenicity. Genetic studies conducted in different host-microbe pathosystems often revealed a sophisticated regulatory mechanism of their T3SSs, suggesting that the expression of T3SS is tightly controlled and constantly monitored by bacteria in response to the ever-changing host environment. Therefore, it is critical to understand the regulation of T3SS in pathogenic bacteria for successful disease management. This review focuses on a model plant pathogen, Dickeya dadantii, and summarizes the current knowledge of its T3SS regulation. We highlight the roles of several T3SS regulators that were recently discovered, including the transcriptional regulators: FlhDC, RpoS, and SlyA; the post-transcriptional regulators: PNPase, Hfq with its dependent sRNA ArcZ, and the RsmA/B system; and the bacterial second messenger cyclic-di-GMP (c-di-GMP). Homologs of these regulatory components have also been characterized in almost all major bacterial plant pathogens like Erwinia amylovora, Pseudomonas syringae, Pectobacterium spp., Xanthomonas spp., and Ralstonia spp. The second half of this review shifts focus to an in-depth discussion of the innovation and development of T3SS inhibitors, small molecules that inhibit T3SSs, in the field of plant pathology. This includes T3SS inhibitors that are derived from plant phenolic compounds, plant coumarins, and salicylidene acylhydrazides. We also discuss their modes of action in bacteria and application for controlling plant diseases.

Osthole, a Natural Plant Derivative Inhibits MRGPRX2 Induced Mast Cell Responses.

Mast cells are tissue-resident innate immune cells known for their prominent role in mediating allergic reactions. MAS-related G-protein coupled receptor-X2 (MRGPRX2) is a promiscuous G-protein coupled receptor (GPCR) expressed on mast cells that is activated by several ligands that share cationic and amphipathic properties. Interestingly, MRGPRX2 ligands include certain FDA-approved drugs, antimicrobial peptides, and neuropeptides. Consequently, this receptor has been implicated in causing mast cell-dependent pseudo-allergic reactions to these drugs and chronic inflammation associated with asthma, urticaria and rosacea in humans. In the current study we examined the role of osthole, a natural plant coumarin, in regulating mast cell responses when activated by the MRGPRX2 ligands, including compound 48/80, the neuropeptide substance P, and the cathelicidin LL-37. We demonstrate that osthole attenuates both the early (Ca2+ mobilization and degranulation) and delayed events (chemokine/cytokine production) of mast cell activation via MRGPRX2 in vitro. Osthole also inhibits MrgprB2- (mouse ortholog of human MRGPRX2) dependent inflammation in in vivo mouse models of pseudo-allergy. Molecular docking analysis suggests that osthole does not compete with the MRGPRX2 ligands for interaction with the receptor, but rather regulates MRGPRX2 activation via allosteric modifications. Furthermore, flow cytometry and confocal microscopy experiments reveal that osthole reduces both surface and intracellular expression levels of MRGPRX2 in mast cells. Collectively, our data demonstrate that osthole inhibits MRGPRX2/MrgprB2-induced mast cell responses and provides a rationale for the use of this natural compound as a safer alternative treatment for pseudo-allergic reactions in humans.

A Study of Plant Coumarins. 18. Conjugates of Coumarins with Lupane Triterpenoids and 1,2,3-Triazoles: Synthesis and Anti-Inflammatory Activity

In the reaction of coumarin peuruthenicin with α,ω-dibromoalkanes, the corresponding 7-(ω-bromoalkyloxy)coumarins have been synthesized. The interaction of the resulting compounds with sodium azide has yielded 7-(ω-azidoalkyl)-substituted peuruthenicin derivatives, which exhibited high activity in the Cu(I)-catalyzed Huisgen cycloaddition reaction with the propargyl ester of betulonic acid. As a result of the reaction, 28-O-(chromenoalkyl-triazolylmethyl)lupenones have been synthesized. The reaction of betulonic acid propargyl ester with 2-azidooreoselones in the presence of aqueous copper sulfate and sodium ascorbate has led to the corresponding (furochromen-triazolyl)-20(29)-lupen-3-ones. The newly synthesized hybrid compound triterpenoid furocoumarin containing a triazole linker exhibits anti-inflammatory activity in a model of histamine inflammation.

Design and Synthesis of 3‐(N‐Substituted)aminocoumarins as Anticancer Agents from 3‐Bromopeuruthenicin

AbstractA convenient protocol for the rapid and efficient synthesis of 3‐bromopeuruthenicin 2 from plant coumarin peuruthenicin 1 is described. Coumarin 2 have been successfully reacted with N‐methylpiperazine, or 5‐aminoisoquinoline under reflux in chloroform with the formation of 3‐(N‐substituted)aminoumbelliferons. The easiness of formation of the mentioned compounds can be explained by occurring of the tautomerization processes in 3‐bromo‐7‐hydroxycoumarin 2 in the reaction conditions. For obtaining high yields of 3‐(arylamino)coumarins in the reaction of 3‐bromocoumarin 2 with substituted anilines the palladium‐catalyzed C–N coupling reaction was studied. The reaction proceeded cleanly in the presence of the Pd(OAc)2‐Xantphos catalytic system with the formation of the corresponding coupling products. The Suzuki cross‐coupling reaction of 3‐(3‐bromophenylamino)coumarin 8 f with aryl‐ and (hetaryl)boronic acids 11,13 and 14 using PdCl2(dppf) as the catalyst provided the formation of 3‐(N‐(aryl‐hetaryl))aminocoumarins 12, 15 and 16 in good yields. The cytotoxicity of new umbelliferone derivatives was evaluated against human cancer cells using the conventional MTT assays. The data revealed that compounds 12, 15 and 16 possessed most promising cytotoxic potential; aminocoumarins 12 and 16 shown selectivity toward the breast cancer cells MCF‐7. The cytotoxicity of 3‐(N‐substituted)aminocoumarins 12 and 16 on this cell lines was comparable to those of standard drug Doxorubicin.

Evolutionary Developments in Plant Specialized Metabolism, Exemplified by Two Transferase Families.

Plant specialized metabolism emerged from the land colonization by ancient plants, becoming diversified along with plant evolution. To date, more than 1 million metabolites have been predicted to exist in the plant kingdom, and their metabolic processes have been revealed on the molecular level. Previous studies have reported that rates of evolution are greater for genes involved in plant specialized metabolism than in primary metabolism. This perspective introduces topics on the enigmatic molecular evolution of some plant specialized metabolic processes. Two transferase families, BAHD acyltransferases and aromatic prenyltransferases, which are involved in the biosynthesis of paclitaxel and meroterpenes, respectively, have shown apparent expansion. The latter family has been shown to beinvolved in the biosynthesis of a variety of aromatic substances, including prenylated coumarins in citrus plants and shikonin in Lithospermum erythrorhizon. These genes have evolved in the development of each special subfamily within the plant lineage. The broadness of substrate specificity and the exon-intron structure of their genes may provide hints to explain the evolutionary process underlying chemodiversity in plants.

Temperature Dependent Green Synthesis of 3-Carboxycoumarins and 3,4-unsubstituted Coumarins.

Because of the low abundance of 3,4-unsubstituted coumarins in plants combined with the complex purification process required, synthetic routes towards 3,4-unsubstituted coumarins are especially valuable. In the present work, we explore the possibilities of a solvent-free Green Knoevenagel condensation on various 2-hydroxybenzaldehyde derivatives and malonic acid without the use of toxic organocatalysts like pyridine and piperidine but only use ammonium bicarbonate as the catalyst. To investigate the scope of the Green Knoevenagel condensation for the synthesis of 3,4-unsubstituted coumarins, various 2-hydroxybenzaldehyde derivatives were screened as starting material in the optimized two-step procedure developed for 2-hydroxybenzaldehyde. This study shows that the intramolecular esterification and the decarboxylation are in competition, but show different temperature optima. In order to suppress premature decarboxylation and maximize the yield of coumarin, a two-step procedure was adopted. The reaction mixture containing ammonium bicarbonate is initially kept at 90ºC for 1 hour. After completion of the cyclization, the temperature of the reaction mixture is increased to 140ºC for 2 hours. Following this protocol, coumarin could be isolated with a yield of 95%. A two-step procedure for the solvent-free synthesis of several 3,4-unsubstituted coumarins was developed using ammonium bicarbonate, resulting in high yields of the desired products. Moreover, this procedure has a low E-factor and is, therefore an environmental friendly reaction in line with the principles of Green Chemistry. It was shown that by initially capping the temperature at 90ºC, premature decarboxylation can be suppressed. After full conversion to the intermediate 3-carboxycoumarin, the temperature can be increased to 140ºC finalizing the reaction. Ammonium bicarbonate was shown to catalyze both the Green Knoevenagel condensation and the decarboxylation step.

41.柑橘およびマメ科植物におけるクマリン化合物生合成酵素の探索(口頭発表)

41.柑橘およびマメ科植物におけるクマリン化合物生合成酵素の探索(口頭発表)

A study of plant coumarins 16*. Synthesis and transformations of 7-alkynylcoumarins

7-Alkynyl-substituted coumarins were synthesized by a Sonogashira reaction of 6-substituted 7-(trifluoromethylsulfonyloxy)coumarins with terminal acetylenes. The reaction of 7-ethynyl-substituted coumarins with azidobenzoic acids in the presence of copper(II) sulfate and sodium ascorbate was used to synthesize the respective 7-[(1-carboxyphenyl)-1H-1,2,3-triazol-4-yl]coumarins.

Rapid Access to Oxazine Fused Furocoumarins and in vivo and in silico Studies of theirs Biological Activity.

The synthesis of 1,2-oxazine-fused linear furocoumarins was performed involving the transition metal catalysis reaction of plant coumarin oreoselone derivatives. The Pd-catalyzed desulfonative cross-coupling reactions of 2-(tosyl)oreoselone with terminal alkynes and the successive treatment of the obtained 2-(arylethynyl)furocoumarins with an excess of hydroxylamine gave the expected (Z,E)-3-(hydroxyimino)-2-(arylethynyl)furocoumarins with an (Z:E) ratio of about 1:0.5. The gold(III)-catalyzed cycloisomerization of furocoumarin β,γ-acetylenic (Z)-oximes led to a new group of heterocyclic compounds - chromeno[6',7':4,5]furo[3,2-c][1,2]oxazine. The (E)-isomer in this condition was transformed into (E)-3-(hydroxyimino)-2-(propan-2-ylidene) furocoumarin. Pharmacological screening of the synthesized 1,2-oxazine-fused linear furocoumarins for anti-inflammatory and analgesic activity in vivo revealed that this compounds possessed high activity which was depend on the substitution in the aromatic ring of the oxazine unit. The results of experimental studies were found to be in accordance with that of the in silico docking results. The moderate toxicity of compounds (LD50 value was more than 2000 mg/kg) encouraged the further design of therapeutically relevant analogues based on this novel type of fused linear furocoumarins.