Biosynthesis of Scopoletin in Sweet Potato Confers Resistance against Fusarium oxysporum.

Fusarium wilt is a severe fungal disease caused by Fusarium oxysporum in sweet potato. We conducted transcriptome analysis to explore the resistance mechanism of sweet potato against F. oxysporum. Our findings highlighted the role of scopoletin, a hydroxycoumarin, in enhancing resistance. In vitro experiments confirmed that scopoletin and umbelliferone had inhibitory effects on the F. oxysporum growth. We identified hydroxycoumarin synthase genes IbF6'H2 and IbCOSY that are responsible for scopoletin production in sweet potatoes. The co-overexpression of IbF6'H2 and IbCOSY in tobacco plants produced the highest scopoletin levels and disease resistance. This study provides insights into the molecular basis of sweet potato defense against Fusarium wilt and identifies valuable genes for breeding wilt-resistant cultivars.

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.

Functional analysis of a down-regulated transcription factor-SoxNeuroA gene involved in the acaricidal mechanism of scopoletin against spider mites.

BACKGROUND: Insight into the mode of action of plant-derived acaricides will help in the development of sustainable control strategies for mite pests. Scopoletin, a promising plant-derived bioactive compound, displays prominent acaricidal activity against Tetranychus cinnabarinus. The transcription factor SoxNeuroA plays a vital role in maintaining calcium ion (Ca2+ ) homeostasis. Down-regulation of SoxNeuroA gene expression occurs in scopoletin-exposed mites, but the functional role of this gene remains unknown. RESULTS: A SoxNeuroA gene from T. cinnabarinus (TcSoxNeuroA) was first cloned and identified. Reverse transcription polymerase chain reaction (RT-PCR), quantitative real-time polymerase chain reaction (qPCR), and Western blotting assays all confirmed that the gene expression and protein levels of TcSoxNeuroA were significantly reduced under scopoletin exposure. Furthermore, RNA interference silencing of the weakly expressed SoxNeuroA gene significantly enhanced the susceptibility of mites to scopoletin, suggesting that the acaricidal mechanism of scopoletin was mediated by the weakly expressed SoxNeuroA gene. Additionally, yeast one-hybrid (Y1H) and dual-luciferase reporter assays revealed that TcSoxNeuroA was a repressor of Orai1 Ca2+ channel gene transcription, and the key binding sequence was ATCAAAG (positions -361 to -368 of the Orai1 promoter). Importantly, site-directed mutagenesis and microscale thermophoresis assays further indicated that ASP185, ARG189, and LYS217, which were key predicted hydrogen-bonding sites in the molecular docking model, may be the vital binding sites for scopoletin in TcSoxNeuroA. CONCLUSION: These results demonstrate that the acaricidal mechanism of scopoletin involves inhibition of the transcription factor SoxNeuroA, thus inducing the activation of the Orai1 Ca2+ channel, eventually leading to Ca2+ overload and lethality. Elucidation of the transcription factor-targeted mechanism for this potent plant-derived acaricide has vital implications for the design of next-generation green acaricides with novel targets. © 2023 Society of Chemical Industry.

Scopoletin a potential phytochemical therapy for antitubercular treatment drug induced liver injury (ATT-DILI) model in Wistar rats.

OBJECTIVES: The hepatoprotective properties of scopoletin have been explored in carbon tetrachloride (CCl4) induced liver injury but not in drug-induced liver injury (DILI) scenarios. Only N-acetyl-cysteine (NAC) has proven efficacy in DILI treatment. Accordingly, we conducted a study to assess the hepatoprotective action of scopoletin in the anti-tubercular treatment (ATT)-DILI model in Wistar rats, if any. METHODS: A total of 36 rats were evaluated, with six in each group. A 36-day ATT at 100 mg/kg dose for isoniazid, 300 mg/kg for rifampicin and 700 mg/kg for pyrazinamide were fed to induce hepatotoxicity in rats. Group I and II-VI received normal saline and ATT, respectively. Oral scopoletin (1,5 and 10 mg/kg) and NAC 150 mg/kg were administered in groups III, IV, V and VI, respectively, once daily for the last 15 days of the experiment. LFT monitoring was performed at baseline, days 21, 28, and 36. Rats were sacrificed for the histopathology examination. RESULTS: Aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP) and bilirubin levels were significantly increased in group II (receiving ATT) compared to normal control on day 28 and day 36 (p<0.05). All three doses of scopoletin and NAC groups led to the resolution of AST, ALT, ALP, and bilirubin changes induced by ATT medications effect beginning by day 28 and persisting on day 36 (p<0.01). An insignificant effect was observed on albumin and total protein levels. The effect was confirmed with antioxidants and histopathology analysis. CONCLUSIONS: The study confirms the hepatoprotective efficacy of scopoletin in a more robust commonly encountered liver injury etiology.

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.

Scopoletin protects INS-1 pancreatic ß cells from glucotoxicity by reducing oxidative stress and apoptosis.

This study investigated whether scopoletin could protect INS-1 pancreatic ß cells from apoptosis and oxidative stress caused by high glucose. Cells were pretreated with glucose (5.5 or 30 mM) and then treated with 0, 5, 10, 25, or 50 µM Scopoletin. Cell viability and insulin secretion were measured in addition to ROS, TBARS, NO and antioxidant enzymes. Western blot analysis and flow cytometric assessment of apoptosis were also carried out. High glucose of 30 mM caused glucotoxicity and cell death in INS-1 pancreatic ß cells. However, 5, 10, 25 or 50 µM scopoletin increased the level of cell viability as concentrations increased. The levels of ROS, TBARS, and NO increased by high glucose were significantly decreased after scopoletin treatment. Scopoletin also raised antioxidant enzyme activities up against oxidative stress produced by high glucose. These effects influenced the apoptosis pathway, raising levels of anti-apoptotic protein, Bcl-2, and reducing levels of pro-apoptotic proteins, including JNK, Bax, cytochrome C, and caspase 9. Annexin V/propidium staining indicated that scopoletin significantly lowered high glucose-produced apoptosis. These results indicate that scopoletin can protect INS-1 pancreatic ß cells from glucotoxicity caused by high glucose and have potential as a pharmaceutical material to protect the pancreatic ß cells.

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.

Multi-omics reveal key enzymes involved in the formation of phenylpropanoid glucosides in Artemisia annua.

Although mainly known for producing artemisinin, Artemisia annua is enriched in phenylpropanoid glucosides (PGs) with significant bioactivities. However, the biosynthesis of A. annua PGs is insufficiently investigated. Different A. annua ecotypes from distinct growing environments accumulate varying amounts of metabolites, including artemisinin and PGs such as scopolin. UDP-glucose:phenylpropanoid glucosyltransferases (UGTs) transfers glucose from UDP-glucose in PG biosynthesis. Here, we found that the low-artemisinin ecotype GS produces a higher amount of scopolin, compared to the high-artemisinin ecotype HN. By combining transcriptome and proteome analyses, we selected 28 candidate AaUGTs from 177 annotated AaUGTs. Using AlphaFold structural prediction and molecular docking, we determined the binding affinities of 16 AaUGTs. Seven of the AaUGTs enzymatically glycosylated phenylpropanoids. AaUGT25 converted scopoletin to scopolin and esculetin to esculin. The lack of accumulation of esculin in the leaf and the high catalytic efficiency of AaUGT25 on esculetin suggest that esculetin is methylated to scopoletin, the precursor of scopolin. We also discovered that AaOMT1, a previously uncharacterized O-methyltransferase, converts esculetin to scopoletin, suggesting an alternative route for producing scopoletin, which contributes to the high-level accumulation of scopolin in A. annua leaves. AaUGT1 and AaUGT25 responded to induction of stress-related phytohormones, implying the involvement of PGs in stress responses.

Comparative Pharmacokinetics of Scoparone and its Metabolite Scopoletin in Normal and ANIT-induced Intrahepatic Cholestatic Rats.

BACKGROUND: Scoparone, the principal natural active ingredient of Artemisia capillaries (Yin Chen), can effectively treat cholestatic diseases, but the pharmacokinetic properties of scoparone are rarely studied in intrahepatic cholestatic rats. OBJECTIVE: A sensitive and rapid LC-MS/MS method was established to detect scoparone and its metabolite of scopoletin in rat plasma and then compare their plasma pharmacokinetic differences between the normal and ANITinduced cholestasis rats. METHODS: Positive ionization was used to separate scoparone and scopoletin using acetonitrile and 0.1 % formic acid water as the mobile phase on a Hypersil ODS-BP column. RESULTS: The calibration curves presented good linearity (R=0.9983 and 0.9989) in the concentration range of 10- 10000 ng/mL and 0.5-500 ng/mL for scoparone and scopoletin, respectively. The precision of ≤ 9.4% and the accuracy ranged from -6.4% to 6.8% were recorded over three validation runs, and the recovery was higher than 83.9%. Under different storage conditions, scoparone and scopoletin were stable. Therefore, we studied the pharmacokinetic properties of scoparone and scopoletin in rats after a single oral administration with the above method. According to the results, the pharmacokinetic parameters of AUC, t1/2, and Cmax values of scoparone in the ANIT group were increased by 106%, 75%, and 44%, respectively, while these values of scopoletin were increased by 142%, 62%, and 65%. CONCLUSION: The findings indicated that the pharmacokinetic properties of scoparone and scopoletin were significantly different between the normal and ANIT-induced cholestasis rats, which suggested that the clinical application dosage of scoparone should be adjusted according to the liver function of patients.

Screening quality markers (Q-markers) of Xiaoer Chaige Tuire Oral Liquid by in vitro sequential metabolism and in vivo biopharmaceutical analysis.

BACKGROUND: Xiaoer Chaige Tuire Oral Liquid (XCT) is a preparation composed of 7 traditional Chinese medicines including Bupleuri Radix, Puerariae Lobatae Radix, Scutellariae Radix, Gypsum Fibrosum, Artemisiae Annuae Herba, Paeoniae Radix Alba and Glycyrrhizae Radix Et Rhizoma Praeparata Cum Melle in proportion. According to traditional Chinese medicine theory, it has the function of dispelling wind evil and relieving exterior syndrome, clearing summer heat and dampness, and reducing internal heat. So, it is indicated for pediatric upper respiratory tract infection caused by exogenous wind-heat. Modern pharmacological studies have indicated that XCT has a variety of activities such as anti-inflammation and antivirus. PURPOSE: To screen potential quality markers (Q-markers) of XCT by tracking in vivo bioactive compounds concomitantly using in vitro sequential metabolism and in vivo biopharmaceutical analysis. METHODS: In vitro metabolic models including artificial gastric juice, intestinal juice, intestinal microbiota, Caco-2 cell monolayer and liver S9 were employed to simulate metabolism of main compounds of XCT in the body. High performance liquid chromatography with diode-array detection (HPLC-DAD) was used to quantitatively determine main components of XCT preparation and its sequential metabolism samples. Ultra performance liquid chromatography with QExactive Orbitrap tandem mass spectrometry (UPLC-QExactive-HF-x-Orbitrap-MS) was used to qualitatively determine in vivo components of XCT preparation in rat plasma and metabolites obtained with liver S9 fraction of rats. RESULTS: Twenty-five compounds were identified from the preparation of XCT. Sequential in vitro metabolism studies indicated that most of these compounds except baicalin and baicalein were stable in artificial gastric juice, albiflorin, glycyrrhizic acid, gallic acid and baicalein were unstable in artificial intestinal juice, daidzin, liquiritin and genistin were hydrolyzed into their aglycones daidzein, liquiritigenin and genistein by intestinal microbiota, and 7 compounds thereout including benzoic acid, puerarin, 3'-methoxypuerarin, paeoniflorin, scopoletin, daidzein and liquiritigenin were shown to be well absorbed with Caco-2 cell monolayer model. These 7 compounds were demonstrated to be metabolized via hydroxylation and glycosylation by liver S9 system. Ten components of XCT preparation including puerarin, baicalin, wogonoside, benzoic acid, daidzein, baicalein, wogonin, oroxylin A, isoscopoletin and isoliquiritigenin were identified from rat plasma by in vivo biopharmaceutical analysis. Most of the compounds screened with both in vitro and in vivo metabolic studies were shown to be active against inflammation and influenza virus. CONCLUSIONS: A screening strategy for potential quality markers (Q-markers) of XCT preparation based on tracking in vivo bioactive compounds using the combination of in vitro sequential metabolism and in vivo biopharmaceutical analysis was established. With this strategy, a total of 12 compounds including puerarin, daidzein, benzoic acid, baicalin, baicalein, wogonoside, wogonin, oroxylin A, 3'-methoxypuerarin, paeoniflorin, scopoletin and liquiritigenin were screened to be potential Q-markers of XCT, which provides a material basis for quality control and development of XCT.

Metabolite fingerprinting of cassava (Manihot esculenta Crantz) landraces assessed for post-harvest physiological deterioration (PPD).

Cassava landraces are impacted by post-harvest physiological deterioration (PPD). 34 primary/secondary metabolites (carotenes, flavonols, indols, phenolic, hydroxycinnamic, and organic acids) were analysed using HPLC/GC-MS in 72 landraces harvested 8 months after planting (MAP) to clarify whether these compounds may play a role in PPD tolerance. Cluster analysis differentiated a first group with high organic acids contents, citric acid being dominant, a second group with landraces high in tryptophan, a third group including landraces with high phenolic and hydroxycinnamic acids content, and a fourth group characterised by 8 carotenoids. PPD tolerant and susceptible landraces were present in each group. To determine if PPD is related to age of harvest, 174 landraces were harvested at 6, 8, 10 and 12 MAP. Scopoletin, sucrose and glucose were analysed. PPD was positively correlated with DMC and negatively correlated with scopoletin at all ages of harvest. Scopoletin is a useful biomarker to characterize landraces.

Dose-Response Evaluation of Scopoletin, a Phytochemical, in a High-Fructose High-Fat Diet-Induced Dyslipidemia Model in Wistar Rats.

The putative hypolipidemic properties of scopoletin have not been fully confirmed due to a lack of validation in an irreversible chronic hyperlipidemia animal model. The druggability also needs to be studied in terms of bioavailability in the vascular compartment. Accordingly, we conducted a study to assess the hypolipidemic and pharmacokinetic behavior of scopoletin in the high-fructose high-fat diet (HFHFD)-induced dyslipidemia model in Wistar rats. A total of 42 rats were studied, with 6 in each of the 7 groups. A 60-day HFHFD opted for induction of dyslipidemia. Group I and groups II-VII received normal rat chow diet and HFHFD, respectively. Oral scopoletin (1, 5, 10 mg/kg) and atorvastatin 5 mg/kg were administered in groups III-VI, respectively, once daily for the next 15 days. A separate group, group VII, was used for the pharmacokinetic assessment comparing the scopoletin 10 mg/kg intraperitoneally (IP) in group VII versus the oral (group V). Pharmacokinetic blood sampling was performed on the 10th day of continuous once-daily therapy. Rats were sacrificed for the histological examination. All three scopoletin dosages significantly decreased the total cholesterol, low-density lipoproteins, and triglycerides (P < .05 for all), but not in a dose-dependent manner. Atherogenic Index of plasma, Castelli's risk indices, and histopathological findings confirmed the protective effect of scopoletin. The IP administration showed a 23.18% higher exposure than the oral route (P < .001 for area under the curve and P < .05 for concentration-maximum). This study confirms the hypolipidemic efficacy of scopoletin in a more robust irreversible model of dyslipidemia. Scopoletin's gut absorption in the disease state may also boost the initial phase exploratory clinical trial.

Scopoletin Improves Glucose Homeostasis in the High-Fructose High-Fat Diet-Induced Diabetes Model in Wistar Rats.

Antihyperglycemic action of scopoletin needs to be validated before considering it for clinical trials. The present study explored antihyperglycemic action of scopoletin in high-fructose high-fat diet (HFHFD)-induced diabetes in rats. The animal study was performed using 48 rats, 6 in each group. HFHFD was administered for model induction for 74 days. Rats in Group I (normal control [NC]) and group II (experimental control [EC]) received normal saline and HFHFD, respectively, throughout the study. Groups III, IV, V, and VI received oral scopoletin (1 mg/kg [low dose, LD], 5 mg/kg [medium dose, MD], 10 mg/kg [high dose, HD]), and metformin (250 mg/kg; positive control [PC] for efficacy), respectively, once daily from day 60 to 74, in addition to HFHFD. Group VII (10 mg/kg oral scopoletin safety group) and VIII (0.1 mg/kg oral warfarin; PC for safety) were separately used for bleeding time-clotting time (BTCT) assessment on days 60, 68, and 74. Groups I, VII, and VIII rats were studied for safety assessment. Later, animals were sacrificed for histological examination. Scopoletin-treated groups showed a significant decline in glucose levels, especially in the MD (5.18 ± 0.12) and HD group (5.271 ± 0.11) in comparison to the EC (6.37 ± 0.05) on day 74 (P < .05). Two weeks after scopoletin treatment, ß-cell function significantly improved (53.073 ± 4.67) in the MD group versus 29.323 ± 8.505 in the NC group (P < .05). A statistically significant difference was observed when the MD group (53.07 ± 4.67) was compared to the metformin-treated group (24.80 ± 3.24; P < .05). The safety assessment in the form of BTCT findings did not observe a difference among groups I, VII, and VIII (P > .05). The study showed that scopoletin dose-independently reversed insulin resistance. Consequently, scopoletin can be a potential candidate for antidiabetic drug development.

[Expression of ß-glucosidase An-bgl3 from Aspergillus niger for conversion of scopolin].

Scopoletin is a coumarin compound with various biological activities including detumescence and analgesic, insecticidal, antibacterial and acaricidal effects. However, interference with scopolin and other components often leads to difficulties in purification of scopoletin with low extraction rates from plant resource. In this paper, heterologous expression of the gene encoding ß-glucosidase An-bgl3 derived from Aspergillus niger were carried out. The expression product was purified and characterized with further structure-activity relationship between it and ß-glucosidase analyzed. Subsequently, its ability for transforming scopolin from plant extract was studied. The results showed that the specific activity of the purified ß-glucosidase An-bgl3 was 15.22 IU/mg, the apparent molecular weight was about 120 kDa. The optimum reaction temperature and pH were 55 ℃ and 4.0, respectively. Moreover, 10 mmol/L metal ions Fe2+ and Mn2+ increased the enzyme activity by 1.74-fold and 1.20-fold, respectively. A 10 mmol/L solution containing Tween-20, Tween-80 and Triton X-100 all inhibited the enzyme activity by 30%. The enzyme showed affinity towards scopolin and tolerated 10% methanol and 10% ethanol solution, respectively. The enzyme specifically hydrolyzed scopolin into scopoletin from the extract of Erycibe obtusifolia Benth with a 47.8% increase of scopoletin. This demonstrated that the ß-glucosidase An-bgl3 from A. niger shows specificity on scopolin with good activities, thus providing an alternative method for increasing the extraction efficiency of scopoletin from plant material.

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.

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.

Estimation of Scopoletin from Roots of Argyreia nervosa (Burm. fill.) Bojer Using a Validated HPLC-Fluorescence Method Optimized Using the Design of Experiment Approach.

BACKGROUND: Argyreia nervosa (Burm. Fil.) Bojer., a woody climber, is indicated in Ayurveda to treat debilities of the male reproductive system, diseases of the nervous system, and chronic ulcers. OBJECTIVE: A sensitive analytical method was developed to estimate bioactive scopoletin from methanolic extract prepared from the medicinally active dried roots of Argyreia nervosa (Burm. fil.) Bojer using HPLC equipped with a fluorescence detector. METHODS: Chromatographic separation was achieved using a LunaTM (C18, 250 × 4.6 mm, id: 5 µm) column using an isocratic mobile phase comprising phosphate buffer (pH 3.5)-acetonitrile (80 + 20, by volume) at a flow rate of 1.0 mL/min. The excitation wavelength was 345 nm, and the emission wavelength was 444 nm. The chromatographic parameters were optimized using the design of the experiment approach after determining the combined effects of selected independent variables on area, retention time, and tailing factor (TF) for the peak corresponding to scopoletin, and the experimental design was validated by navigating through the design space. RESULTS: The developed method was found linear in the range 10-140 ng/mL. The results of the studies confirmed the accuracy, precision, and robustness of the developed analytical method. The plant material was found to contain 0.0125 ± 0.0001% w/w scopoletin on a dried weight basis when estimated using the developed method. CONCLUSION: The method was developed using the HPLC-fluoresence detection by adopting the design of experiment approach and simple sample preparation for the estimation of scopoletin from roots of A. nervosa. HIGHLIGHT: This extremely sensitive analytical method with one-step sample preparation has the potential to be adapted for routine QC procedures.

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.

Subfunctionalization of a monolignol to a phytoalexin glucosyltransferase is accompanied by substrate inhibition.

Uridine diphosphate-dependent glycosyltransferases (UGTs) mediate the glycosylation of plant metabolites, thereby altering their physicochemical properties and bioactivities. Plants possess numerous UGT genes, with the encoded enzymes often glycosylating multiple substrates and some exhibiting substrate inhibition kinetics, but the biological function and molecular basis of these phenomena are not fully understood. The promiscuous monolignol/phytoalexin glycosyltransferase NbUGT72AY1 exhibits substrate inhibition (Ki) at 4 µM scopoletin, whereas the highly homologous monolignol StUGT72AY2 is inhibited at 190 µM. We therefore used hydrogen/deuterium exchange mass spectrometry and structure-based mutational analyses of both proteins and introduced NbUGT72AY1 residues into StUGT72AY2 and vice versa to study promiscuity and substrate inhibition of UGTs. A single F87I and chimeric mutant of NbUGT72AY1 showed significantly reduced scopoletin substrate inhibition, whereas its monolignol glycosylation activity was almost unaffected. Reverse mutations in StUGT72AY2 resulted in increased scopoletin glycosylation, leading to enhanced promiscuity, which was accompanied by substrate inhibition. Studies of 3D structures identified open and closed UGT conformers, allowing visualization of the dynamics of conformational changes that occur during catalysis. Previously postulated substrate access tunnels likely serve as drainage channels. The results suggest a two-site model in which the second substrate molecule binds near the catalytic site and blocks product release. Mutational studies showed that minor changes in amino acid sequence can enhance the promiscuity of the enzyme and add new capabilities such as substrate inhibition without affecting existing functions. The proposed subfunctionalization mechanism of expanded promiscuity may play a role in enzyme evolution and highlights the importance of promiscuous enzymes in providing new functions.