Linear Furanocoumarins Research Articles

Metabolism of Furanocoumarins by Three Recombinant CYP9A Proteins From the Polyphagous Cotton Bollworm Helicoverpa armigera.

Furanocoumarins are a class of chemical compounds with phototoxic properties. For herbivores, efficient detoxification of such defense compounds is the prerequisite to feed successfully on furanocoumarin-containing plants. The cotton bollworm Helicoverpa armigera is a very important polyphagous pest in agriculture, but how it copes with toxic furanocoumarins in some of its host plants is not well understood. Given that cytochrome P450s are well known for their capacity in xenobiotic metabolism, this study attempted to explore the potential roles of cytochrome P450s in furanocoumarin transformation in this pest. Our data showed that two linear structures (psoralen and xanthotoxin) could be metabolized by three recombinant CYP9A enzymes, but no detectable depletion was observed for the linear one with the 8-dimethylallyloxy substituent on the coumarin moiety (imperatorin) and the angular furanocoumarin (angelicin). Initial epoxidation of the double bond connecting C2' and C3' of the furano ring following by cleavage of the epoxidated furan ring, leading to the formation of more soluble, less reactive and nonphotosensitizing metabolites, was identified as a common mechanism of linear furanocoumarin metabolism using a quadrupole/time-of-flight (Q-TOF) mass spectrometry interfaced with a high performance liquid chromatography (HPLC) system. Our data demonstrated that multiple P450s were involved in the detoxification of linear furanocoumarins in the cotton bollworm. These findings contribute to a better understanding of the biochemical basis of adaptation to plant defense chemicals in this economically important pest.

Heraclenin promotes the osteogenic differentiation of bone marrow stromal cells by activating the RhoA/ROCK pathway.

Osteoporosis is a devastating skeletal disease, the pathogenesis of which is related to abnormal bone metabolism, featured by the imbalance between osteoblastic bone formation and osteoclastic bone resorption. Stem cell-based therapies have been demonstrated to improve osteoporosis treatment. Previously, the linear furanocoumarin heraclenin was reported to enhance osteoblast differentiation and mineralization in mouse mesenchymal stem cells (MSCs), suggesting its potential for osteogenic differentiation and bone regeneration. Our study was designed to confirm the promotive role of heraclenin on osteogenic differentiation of human bone MSCs (BMSCs) and explore the underlying mechanisms. Human BMSCs were treated for 24, 48, and 72h with heraclenin (5, 10, 20, 40, and 80 μM), and cell viability was determined by Cell Counting Kit-8 (CCK-8) assay. To further evaluate the cytotoxicity of heraclenin, cell suspension obtained from BMSCs treated with heraclenin (5, 10, and 20 μM) for 72h was subjected to a MUSE™ cell analyzer for cell viability and count assay. BMSCs were incubated in osteogenic induction medium for 7 days. Then, osteogenic differentiation and mineralization of BMSCs were assessed through alkaline phosphatase (ALP) and Alizarin Red S staining. The expression of osteogenesis markers including ALP, osteocalcin (OCN), osterix (OSX), and runt-related transcription factor 2 (RUNX2) was detected via reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting. The effects of heraclenin on the RhoA/ROCK pathway were estimated through western blotting. Y-27632, the ROCK inhibitor, was used to confirm the role of the RhoA/ROCK pathway in heraclenin-mediated osteogenic differentiation of BMSCs. Heraclenin (5-80 μM) was non-toxic on human BMSCs. Heraclenin treatment (5-20 μM) dose-dependently enhanced ALP activity and calcium deposition. Furthermore, heraclenin promoted ALP, OCN, OSX, and RUNX2 mRNA and protein expression. Mechanically, heraclenin treatment increased RhoA and ROCK1 mRNA expression, stimulated the translocation of ROCK from the cytosolic to the membrane fraction, and elevated the protein levels of phosphorylated cofilin (p-cofilin) and active RhoA. Additionally, treatment with Y-27632 overturned the promotion of heraclenin on ALP activity, calcium deposition, the expression of osteogenesis markers, and the RhoA/ROCK signaling pathway. Heraclenin facilitates the osteogenic differentiation of human BMSCs through the activation of the RhoA/ROCK pathway.

Mechanism-Based Inactivation of CYP2D6 by Phellopterin and Related Drug-Drug Interaction with Metoprolol.

Phellopterin (PLP) is a linear furanocoumarin widely found in citrus fruits and herbal medicines. The study aims to comprehensively investigate the mechanism of inhibition of CYP2D6 enzyme activity by PLP and its alteration of metoprolol pharmacokinetics. PLP was found to irreversibly inhibit CYP2D6 in time-, concentration-, and nicotinamide adenine dinucleotide phosphate-dependent manners. Coincubation with quinidine, which is a competitive inhibitor of CYP2D6, attenuated this time-dependent inhibition. Glutathione (GSH) and catalase/superoxide dismutase failed to reverse the PLP-induced CYP2D6 inactivation. GSH trapping experiments provided strong evidence that PLP metabolic activation produces epoxide or γ-ketoaldehyde intermediates. In addition, pretreatment with PLP resulted in significant increases in Cmax and area under curve of plasma metoprolol in rats.

Angular dihydropyranocoumarins from the flowers of Peucedanum japonicum and their aldo-keto reductase inhibitory activities

Twenty-one angular dihydropyranocoumarins and a linear furanocoumarin, including four previously undescribed compounds (1–4), were isolated from the flowers of Peucedanum japonicum (Umbelliferae). The structures of 1–4, along with their absolute stereochemistry, were determined to be (3′S,4′S)-3′-O-propanoyl-4′-O-(3‴-methyl-2‴-butenoyl)khellactone (1), (3′S,4′S)-3′-O-propanoyl-4′-O-(2‴-methyl-2‴Z-butenoyl)khellactone (2), (3′S,4′S)-3′-O-propanoyl-4′-O-(2‴-methylbutanoyl)khellactone (3), and (3′S,4′S)-3′-O-(2″-methylpropanoyl)-4′-O-(3‴-methyl-2‴-butenoyl)khellactone (4) using one- and two-dimensional nuclear magnetic resonance, high-resolution electrospray ionization mass spectroscopy, and electronic circular dichroism spectroscopy. In addition, the absolute configuration of the three angular dihydropyranocoumarins (5–7) was determined for the first time in this study. Among the previously reported compounds isolated in this study, 8 and 9 were isolated for the first time from the genus Peucedanum, whereas 10 and 11 were previously unreported and had not been isolated from P. japonicum to date. Furthermore, all isolated compounds were evaluated for their aldo–keto reductase 1C1 inhibitory activities on A549 human non-small-cell lung cancer cells. Compounds 10 and 12 exhibited substantial AKR1C1 inhibitory activities with IC50 values of 35.8 ± 0.9 and 44.2 ± 1.5 μM, respectively.

The genome of the toxic invasive species Heracleum sosnowskyi carries an increased number of genes despite absence of recent whole-genome duplications.

Heracleum sosnowskyi, belonging to a group of giant hogweeds, is a plant with large effects on ecosystems and human health. It is an invasive species that contributes to the deterioration of grassland ecosystems. The ability of H. sosnowskyi to produce linear furanocoumarins (FCs), photosensitizing compounds, makes it very dangerous. At the same time, linear FCs are compounds with high pharmaceutical value used in skin disease therapies. Despite this high importance, it has not been the focus of genetic and genomic studies. Here, we report a chromosome-scale assembly of Sosnowsky's hogweed genome. Genomic analysis revealed an unusually high number of genes (55106) in the hogweed genome, in contrast to the 25-35 thousand found in most plants. However, we did not find any traces of recent whole-genome duplications not shared with its confamiliar, Daucus carota (carrot), which has approximately thirty thousand genes. The analysis of the genomic proximity of duplicated genes indicates on tandem duplications as a main reason for this increase. We performed a genome-wide search of the genes of the FC biosynthesis pathway and surveyed their expression in aboveground plant parts. Using a combination of expression data and phylogenetic analysis, we found candidate genes for psoralen synthase and experimentally showed the activity of one of them using a heterologous yeast expression system. These findings expand our knowledge on the evolution of gene space in plants and lay a foundation for further analysis of hogweed as an invasive plant and as a source of FCs.

Ruta chalepensis L. In Vitro Cultures as a Source of Bioactive Furanocoumarins and Furoquinoline Alkaloids

Recently, due to the decreasing areas of cultivation and climate change, the use of biotechnological methods to obtain biomass, which is a source of valuable bioactive metabolites, is becoming more and more interesting. In this study, Ruta chalepensis in vitro cultures were investigated in RITA® temporary immersion bioreactors. Biomass growth and the production of secondary metabolites in 4- and 5-week growth cycles on three variants of the Linsmaier and Skoog (LS) medium (naphthyl-1-acetic acid/6-benzylaminopurine (NAA/BAP): 0.5/1.0, 0.1/0.1, and 1.0/1.0 mg/L) were analyzed. Using high-performance liquid chromatography of methanolic extracts of biomass, the presence of linear furanocoumarins (bergapten, isoimperatorin, isopimpinellin, psoralen, and xanthotoxin) and furoquinoline alkaloids (γ-fagarine, 7-isopentenyloxy-γ-fagarine, and skimmianine) was confirmed. The highest content of linear furanocoumarins (1170 mg/100 g DW (dry weight)) was observed in the LS medium variant containing 0.5/1.0 mg/L NAA/BAP (4-week growth cycle). The highest content of furoquinoline alkaloids (449 mg/100 g DW) was observed in the LS medium variant containing 0.1/0.1 mg/L NAA/BAP (5-week growth cycle). Hence, R. chalepensis bioreactor cultures may be used as a biotechnological source of linear furanocoumarins (xanthotoxin and bergapten) and furoquinoline alkaloids (skimmianine and γ-fagarine).

The Influence of Exogenous Phenylalanine on the Accumulation of Secondary Metabolites in Agitated Shoot Cultures of Ruta graveolens L.

This study aimed to examine the influence of the addition of a precursor (phenylalanine) on the accumulation of secondary metabolites in agitated shoot cultures of Ruta graveolens. Cultures were grown on Linsmaier and Skoog (LS) medium, with plant growth regulators (0.1 mg/L α-naphthaleneacetic acid-NAA-and 0.1 mg/L 6-benzylaminopurine-BAP). Phenylalanine was added to the cultures at a concentration of 1.25 g/L after 4 and 5 weeks of growth cycles. Biomass was collected after 2, 4, and 7 days of precursor addition. Both control and experimental cultures had the same secondary metabolites accumulated. Using the HPLC method, linear furanocoumarins (bergapten, isoimperatorin, isopimpinellin, psoralen, and xanthotoxin), furoquinoline alkaloids (γ-fagarine, 7-isopentenyloxy-γ-fagarine, and skimmianine), and catechin were detected and quantified in the methanolic extracts. In turn, phenolic acids, such as gallic acid, protocatechuic acid, p-hydroxybenzoic acid, syringic acid, p-coumaric acid, and ferulic acid were detected in hydrolysates. The production of phenolic acids and catechin (1.5-fold) was significantly increased by the addition of precursor, while there was no significant effect on the production of coumarins and alkaloids. The highest total content of phenolic acids (109 mg/100 g DW) was obtained on the second day of phenylalanine addition (the fourth week of growth cycles). The dominant phenolic compounds were p-coumaric acid (maximum content 64.3 mg/100 g DW) and ferulic acid (maximum content 35.6 mg/100 g DW). In the case of catechins, the highest total content (66 mg/100 g DW) was obtained on the third day of precursor addition (the fourth week of growth cycles). This study is the first to document the effect of feeding the culture medium with phenylalanine on the accumulation of bioactive metabolites in in vitro cultures of R. graveolens.

Degree of Dietary Specialization on Furanocoumarin-Containing Hostplants in a Newly Invasive Web Building Caterpillar.

The genus Depressaria (Lepidoptera: Depressariidae) mostly comprises specialist herbivores with varying capacity for detoxification of defensive phytochemistry. Depressaria depressana, a Eurasian moth recently introduced into North America, is a family-level specialist of the Apiaceae, whose hosts include more than a dozen species in multiple tribes; Depressaria radiella is a super-specialist of Eurasian origin that feeds exclusively on species in the genera Pastinaca and Heracleum throughout its native and introduced range. In eastern North America, it feeds upon Pastinaca sativa, an invasive European species, and Heracleum maximum, a native species. We determined whether differences in furanocoumarin metabolism exist between D. depressana and two isolated populations of D. radiella, feeding exclusively on either P. sativa or H. maximum. We also compared gravimetric estimates of feeding efficiency to assess D. depressana larval performance on different diets. Both populations of D. radiella metabolized furanocoumarins at a greater rate than D. depressana. Although there was no difference in rates of metabolism of linear furanocoumarins in the two populations of D. radiella, individuals collected from H. maximum metabolized angular furanocoumarins more rapidly. The gravimetric assessments of feeding efficiencies revealed that D. depressana exhibited highest efficiencies consuming Daucus carota; moreover, this species survived to pupation consuming fruits of Zizia aurea, an apiaceous species native to North America. Our preliminary phylogenetic analysis, building on an earlier morphological analysis, incorporates mitochondrial cytochrome oxidase subunit 1 data from the BOLD database and revealed that the presence or absence of furanocoumarins is not a strong predictor of species-level evolution in Depressaria.

Two O-Methyltransferases Mediate Multiple Methylation Steps in the Biosynthesis of Coumarins in Cnidium monnieri.

Coumarins with methoxy groups such as osthole (1), xanthotoxin (2), bergapten (3), and isopimpinellin (4) are typical bioactive ingredients of many medicinal plants. The methylation steps remain widely unknown. Herein, we report the discovery of two methyltransferases in the biosynthesis of O-methyl coumarins in Cnidium monnieri by transcriptome mining, heterologous expression, and in vitro enzymatic assays. The results reveal that (i) CmOMT1 catalyzes the methylation of osthenol (8) as the final step in the biosynthesis of 1, (ii) CmOMT2 shows the highest efficiency and preference for methylating xanthotoxol (11) to form 2, and (iii) CmOMT1 and CmOMT2 also efficiently transform bergaptol (10) and 8-hydroxybergapten (13) into 3 or 4, suggesting the CmOMTs mediate multistep methylations in the biosynthesis of linear furanocoumarins in C. monnieri.

Phytochemical Characterization, and Antioxidant and Antimicrobial Properties of Agitated Cultures of Three Rue Species: Ruta chalepensis, Ruta corsica, and Ruta graveolens.

The in vitro cultures of the following three species of the genus Ruta were investigated: R. chalepensis, R. corsica, and R. graveolens. The dynamics of biomass growth and accumulation of secondary metabolites in the 3-, 4-, 5-, 6-, and 7-week growth cycle were analysed. The antioxidant capacity of the methanol extracts obtained from the biomass of the in vitro cultures was also assessed by different in vitro assays: 1,1-diphenyl-2-picrylhydrazil (DPPH), reducing power, and Fe2+ chelating activity assays. Moreover, a preliminary screening of the antimicrobial potential of the extracts was performed. The extracts were phytochemically characterized by high-performance liquid chromatography (HPLC), which highlighted the presence of linear furanocoumarins (bergapten, isoimperatorin, isopimpinellin, psoralen, and xanthotoxin) and furoquinoline alkaloids (γ-fagarine, 7-isopentenyloxy-γ-fagarine, and skimmianine). The dominant group of compounds in all the cultures was coumarins (maximum content 1031.5 mg/100 g DW (dry weight), R. chalepensis, 5-week growth cycle). The results of the antioxidant tests showed that the extracts of the three species had varied antioxidant capacity: in particular, the R. chalepensis extract exhibited the best radical scavenging activity (IC50 = 1.665 ± 0.009 mg/mL), while the R. graveolens extract displayed the highest chelating property (IC50 = 0.671 ± 0.013 mg/mL). Finally, all the extracts showed good activity against Staphylococcus aureus with MIC values of 250 μg/mL for the R. corsica extract and 500 μg/mL for both R. graveolens and R. chalepensis extracts.

Coumarins and lignans from the roots of Zanthoxylum nitidum

The phytochemical investigation of the Chinese medicine Zanthoxylum nitidum led to the isolation of a new coumarin glycoside (1), seven known coumarins (2–8) and two known lignan glycosides (9–10). Their structures were elucidated based on extensive spectroscopic methods (HRESIMS, 1D and 2D NMR experiments) and by comparison with literature data. The new compound (1) was a linear furanocoumarin glycoside. Among these known compounds, Compound 10 was isolated from Z. nitidum for the first time, while compounds 2, 4, 5, and 9 were firstly recovered from the Rutaceae family. The lignan glycosides (9–10), which possessed a 4-hydroxy-3,5-dimethoxycinnamic ester at the glycoside portion, were rare in plants. The chemotaxonomic significance of these isolated compounds has also been discussed.

Exploring Cheminformatic Toolsets for Predicting the Dermal Toxicity of Furanocoumarins

Linear furanocoumarins are skin sensitizers and anticancer agents whose appeal in skincare therapeutics is widely exploited. Owing to the need to predict the biological activities of medicines, this work aimed the investigate the predicted dermal toxicity of linear furanocoumarins through chemoinformatic approaches . Therefore, eight major linear furanocoumarins of interest in medicine were selected, and their pharmacophores / toxicophores were modelled and inputted in several databases and cheminformatic toolsets previously described in the literature. Moreover, Principal Components Analysis was performed to allow multivariable comparisons. Results showcased that the first two PCs accounted for 95.48% of all variance in the model, and molecular weight and polar surface showcased a positive correlation to Log P and Log K p , which may be involved in skin penetration. Moreover, the pharmacophore modelling evidenced superimposition between linear furanocoumarins, ethidium bromide and acridine orange, thereby suggesting that these compounds share similar biological effects, supported by their acknowledged DNA intercalating activities. Therefore, this work showcased the application of various cheminformatic tools to screen the dermal toxicity of chemicals.

Oxypeucedanin: Chemotaxonomy, Isolation, and Bioactivities

The present review comprehensively gathered phytochemical, bioactivity, and pharmacokinetic reports on a linear furanocoumarin, namely oxypeucedanin. Oxypeucedanin (OP), which structurally contains an epoxide ring, has been majorly isolated from ethyl acetate-soluble partitions of several genera, particularly Angelica, Ferulago, and Prangos of the Apiaceae family; and Citrus, belonging to the Rutaceae family. The methanolic extract of Angelica dahurica roots has been analytically characterized as the richest natural OP source. This naturally occurring secondary metabolite has been described to possess potent antiproliferative, cytotoxic, anti-influenza, and antiallergic activities, as assessed in preclinical studies. In order to explore potential drug candidates, oxypeucedanin, its derivatives, and semi-synthetically optimized analogues can be considered for the complementary assessments of biological assays.

Determination of benchmark doses for linear furanocoumarin consumption associated with inhibition of cytochrome P450 1A2 isoenzyme activity in healthy human adults.

Millions of individuals globally consume traditional herbal medicines (THMs), which contain abundant amounts of linear furanocoumarins. Linear furanocoumarins (i.e., 8-methoxypsoralen, 5-methoxypsoralen, and isopimpinellin) are inhibitors of cytochrome P450 (CYP) isoenzymes including 1A2, a major enzyme involved in drug metabolism and carcinogen bioactivation. Despite the high consumption of furanocoumarin-containing THMs, no studies have measured the furanocoumarin consumption level that triggers an inhibition to CYP1A2 activity in humans. The first objective was to verify if the potencies of the three furanocoumarins are additive towards the inhibition of CYP1A2 activity in vitro using concentration-addition and whole-mixture chemical-mixture-assessment models. A second objective was to determine the benchmark dose (BMD) with the mixtures of furanocoumarin oral doses, expressed as 8-MOP equivalents, and to assess the in vivo CYP1A2 activity, expressed as inhibition percentages. The in vitro results indicated that the three furanocoumarin inhibitory potencies were additive in the THM extracts, validating the use of the concentration-addition model in total furanocoumarin dose-equivalent calculations. Using the USEPA BMD software, the BMD was 18.9 μg 8-MOP equivalent/kg body weight. This information is crucial for furanocoumarin-related health-assessment studies and the regulation of THMs. Further studies should be performed for the remaining major metabolic enzymes to complete the safety profile of furanocoumarin-containing THMs and to provide accurate warning labelling.

Crude plant extract versus single compounds for vitiligo treatment: Ex vivo intestinal permeability assessment on Brosimum gaudichaudii Trécul

Roots´ bark extract of Brosimum gaudichaudii Trécul (EBGT) is traditionally used for photochemotherapy of vitiligo due to the presence of furanocoumarins psoralen (PSO) and 5-methoxypsoralen (5-MOP) as major compounds. Though plant extracts may provide additional highly permeable psoralens-like substances which may act synergically on vitiligo’s therapy. Thus, the aim of this work was to develop an LC–MS/MS method for screening new highly permeable furanocoumarins from B. gaudichaudii and to compare biomarkers permeability and solubility provided as single compounds or as crude extract, according to BCS. An optimized LC–MS/MS method showed twelve permeable and bioactive compounds, among which 9 furanocoumarins, 2 pyranocoumarins and 1 dihydrocinnamic acid derivative were detected in EBGT samples. Solubility of PSO and 5-MOP was found to be, respectively, six- and eleven-fold higher in crude extract than as pure compounds. Permeability (Papp) of PSO and 5-MOP in EBGT were higher than metoprolol, the low/high BCS permeability class boundary reference compound. Hence, both biomarkers were considered as highly permeable (BCS2) compounds. Their permeability were concentration-dependent displaying values from 30.26 ± 5.13–8.21 ± 2.16 × 10−6 cm/s and 10.72 ± 1.73–6.07 ± 1.27 × 10−6 cm/s, respectively, over a wide range (2.3–200.0 mg mL-1). Thus, a carrier-mediated absorption process is suggested as the main mechanism. Accordingly, all additional permeated coumarins, identified by LC–MS/MS, showed to be at comparable amount of biomarkers in the permeated samples inferring similar high permeability rate. Moreover, biomarkers and other highly absorbable and bioactive linear furanocoumarins from EBGT may be used for vitiligo´s photochemotherapy. Taken together, these findings bring additional evidences for using crude plant extract when aiming synergistic effects of bioactive compounds on melanogenic therapies.

Ducrosia spp., Rare Plants with Promising Phytochemical and Pharmacological Characteristics: An Updated Review.

The rare genus Ducrosia (Apiaceae family) consists of six species, which are mainly native to Asia, specifically to Iran and Iraq. The aerial parts of D. anethifolia, as the most common species, have been traditionally consumed to relieve headache, backache and colic pain, and have also been used as an anxiolytic, an antidepressant, and for treating insomnia. The antispasmodic and carminative effects of D. assadii, and the analgesic activity of D. flabellifolia, along with the insecticidal activities and use as a remedy of skin infections of D. ismaelis, have been previously documented. Among the 49 non-volatile secondary metabolites identified from D. anethifolia and D. ismaelis, 17 linear furanocoumarins and 8 flavonoids have been characterized. The essential oil compositions of four species, including D. anethifolia, D. assadii, D. flabellifolia and D. ismaelis, have been analyzed, whereby aldehyde hydrocarbons, including decanal (10.1–74.0%) and dodecanal (7.2–33.41%), and α-pinene (4.0–70.3%), were identified as the main aroma constituents. From the species of the genus, the bioactivities of D. anethifolia, as well as D. ismaelis, D. assadii and D. flabellifolia, have been previously investigated. Except one clinical trial, all the pharmacological data are derived from preclinical tests, predominantly focusing on antimicrobial, antioxidant, antiproliferative and cytotoxic activities in vitro, and neuroprotective, antidiabetic and analgesic effects in vivo. Considering the vast ethnobotanical uses of the plants in Iranian folk medicine, the phytochemical and pharmacological analysis of un-investigated species might be promising. Furthermore, due to extensive consumption of the Ducrosia genus, more scientific data are needed to support the safety and efficacy of these plants.

Vascular Protective Effects of Xanthotoxin and Its Action Mechanism in Rat Aorta and Human Vascular Endothelial Cells

Objective: Xanthotoxin (XAT) is a linear furanocoumarin mainly extracted from the plants Ammi majus L. XAT has been reported the apoptosis of tumor cells, anti-convulsant, neuroprotective effect, antioxidative activity, and vasorelaxant effects. This study aimed to investigate the vascular protective effects and underlying molecular mechanisms of XAT. Methods: XAT’s activity was studied in rat thoracic aortas, isolated with aortic rings, and human umbilical vein endothelial cells (HUVECs). Results: XAT induced endothelium-dependent vasodilation in a concentration-dependent manner in the isolated rat thoracic aortas. Removal of endothelium or pretreatment of aortic rings with L-NAME, 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one, and wortmannin significantly inhibited XAT-induced relaxation. In addition, treatment with thapsigargin, 2-aminoethyl diphenylborinate, Gd³⁺, and 4-aminopyridine markedly attenuated the XAT-induced vasorelaxation. XAT increased nitric oxide production and Akt- endothelial NOS (eNOS) phosphorylation in HUVECs. Moreover, XAT attenuated the expression of TNF-α-induced cell adhesion molecules such as intercellular adhesion molecule, vascular cell adhesion molecule-1, and E-selectin. However, this effect was attenuated by the eNOS inhibitors L-NAME and asymmetric dimethylarginine. Conclusions: This study suggests that XAT induces vasorelaxation through the Akt-eNOS-cGMP pathway by activating the K_V channel and inhibiting the L-type Ca²⁺ channel. Furthermore, XAT exerts an inhibitory effect on vascular inflammation, which is correlated with the observed vascular protective effects.

Constructing Microbial Hosts for the Production of Benzoheterocyclic Derivatives.

Natural products containing benzoheterocyclic skeletons are widely found in plants and exhibit various pharmacological activities. To address the current limited availability of these compounds, we herein demonstrate the production of benzopyran, furanocoumarins, and pyranocoumarins in Streptomyces xiamenensis by employing prenyltransferases and two substrate-promiscuous enzymes, XimD and XimE. To avoid the degradation in S.xiamenensis, furanocoumarins and pyranocoumarins were also successfully produced in Escherichia coli. The production of linear furanocoumarins (marmesin) and angular pyranocoumarins (decursinol) reached 3.6 and 3.7 mg/L in shake flasks, respectively. To the best of our knowledge, this is the first report of the microbial production of the plant metabolites furanocoumarins and pyranocoumarins. Our study complements the missing link in the biosynthesis of pyranocoumarins by leveraging the catalytic promiscuity of microbial enzymes.

Imperatorin as a Promising Chemotherapeutic Agent Against Human Larynx Cancer and Rhabdomyosarcoma Cells.

Naturally occurring coumarins are bioactive compounds widely used in Asian traditional medicine. They have been shown to inhibit proliferation, induce apoptosis, and/or enhance the cytotoxicity of currently used drugs against a variety of cancer cell types. The aim of our study was to examine the antiproliferative activity of different linear furanocoumarins on human rhabdomyosarcoma, lung, and larynx cancer cell lines, and dissolve their cellular mechanism of action. The coumarins were isolated from fruits of Angelica archangelica L. or Pastinaca sativa L., and separated using high-performance counter-current chromatography (HPCCC). The identity and purity of isolated compounds were confirmed by HPLC-DAD and NMR analyses. Cell viability and toxicity assessments were performed by means of methylthiazolyldiphenyl-tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays, respectively. Induction of apoptosis and cell cycle progression were measured using flow cytometry analysis. qPCR method was applied to detect changes in gene expression. Linear furanocoumarins in a dose-dependent manner inhibited proliferation of cancer cells with diverse activity regarding compounds and cancer cell type specificity. Imperatorin (IMP) exhibited the most potent growth inhibitory effects against human rhabdomyosarcoma and larynx cancer cell lines owing to inhibition of the cell cycle progression connected with specific changes in gene expression, including CDKN1A. As there are no specific chemotherapy treatments dedicated to laryngeal squamous cell carcinoma and rhabdomyosarcoma, and IMP seems to be non-toxic for normal cells, our results could open a new direction in the search for effective anti-cancer agents.

Substrate-specificity of cytochrome P450-mediated detoxification as an evolutionary strategy for specialization on furanocoumarin-containing hostplants: CYP6AE89 in parsnip webworms.

The parsnip webworm, Depressaria pastinacella, is restricted to two hostplant genera containing six structurally diverse furanocoumarins. Of these, imperatorin is detoxified by a specialized cytochrome P450, CYP6AB3. A previous whole-larva transcriptome analysis confirmed the presence of nine transcripts that belong to the CYP6AE subfamily. Here, by examining midgut-specific gene expression patterns we determined that CYP6AE89 transcripts were highly expressed and furanocoumarin-inducible. Computer docking and energy-minimization of a CYP6AE89 model with all six furanocoumarins showed that 5-methoxylated bergapten and 8-methoxylated xanthotoxin had the smallest distances from the heme to the proton-donor residue in the catalytic I-helix, and that the 5,8-dimethoxylated isopimpinellin and bergapten had the smallest energy-minimized distance from the heme oxygen to the furan ring double bond. To evaluate this prediction, we expressed the CYP6AE89 protein in an Escherichia coli system, and used it to detect high catalytic activity against the two mono-methoxylated linear furanocoumarins - bergapten and xanthotoxin - and weak activity against isopimpinellin. Thus, CYP6AE89, like CYP6AB3, is probably specialized for detoxifying only a subset of hostplant furanocoumarins. A maximum-likelihood tree built with six representative lepidopterans with manually annotated cytochrome P450s shows that CYP6AE89 may have evolved much faster than the other CYP6AE proteins, possibly indicative of host selection pressure.