Systematical screening of the metabolic profile of marine fungus Aspergillus sp. EGF 15-0-3 using OSMAC-GNPS molecular networking cascade followed by target isolation of the environmental-induced products resulted the identification of eight unprecedented indole diketopiperazine-based hybrids (1-8) featuring three distinct chemical backbones. The structures of the obtained compounds were established by combination of extensive spectroscopic analyses, X-ray crystallography, and ECD calculations. All these environmental-induced metabolites were demonstrated to be unusual tyrosyl-DNA phosphodiesterase 2 inhibitors. Compound 3, as the most outstanding example, showed selective synergistic antitumor effect when combined with the chemotherapeutic drugs.

Hyperactivation of DNA repairing pathway is highly associated with the chemosensitivity and chemoresistance of cancer cells. In this manuscript, guided by cascaded one strain many compounds-global natural products social molecular networking (OSMAC-GNPS) strategy, a pair of epimeric environmental-induced metabolites were isolated from Aspergillus sp. EGF 15-0-3. Structurally, sterpiperazines A (1) and B (2) represent the first steroid-based indole alkaloids with unprecedented backbones. Biologically, compound 1 could be identified as a novel tyrosyl-DNA phosphodiesterase 1 (Tdp1) inhibitor with a unique mechanism distinct from the reported modulators, and was able to significantly enhance the sensitivity of NCI-H460 cells to the clinic chemotherapeutic drug through inhibiting the DNA repairment and enhanced the DNA damage of cancer cells.

2-Phenylethylchromones (PECs) and 2-styrylchromones (SCs) are the primary components responsible for the delightful fragrance and bioactivity of agarwood, a highly valuable aromatic resinous heartwood. PECs are derived from a common precursor with a diarylpentanoid skeleton (C6-C5-C6). However, the biosynthesis of SCs remains unclear. In this study, based on the successful conversion of the PEC skeleton, rather than a dehydrogenated diarylpentanoid, into SCs by Aquilaria sinensis suspension cells, we demonstrated that double bond formation of the styryl group in SCs occurs after the creation of the PEC skeleton, not before this step from a dehydrogenated diarylpentanoid precursor. Through transcriptomic data mining, transient expression in Nicotiana benthamiana and A. sinensis suspension cells, we identified a new 2-oxoglutarate-dependent oxygenase (As2OG1) that plays a crucial role in the conversion of PECs into SCs. Further protein structure prediction and mutagenesis studies, combined with probing of the catalytic potential of As2OG1 using chemically synthesized hydroxylated intermediates, suggested that As2OG1 possibly uses diradical or carbocation intermediates, rather than hydroxylated intermediates, to install double bonds in SCs. The results not only provide insights into the molecular mechanism of agarwood formation but also facilitate the overproduction of pharmaceutically important SCs using metabolic engineering approaches.

Two new benzaldehyde derivatives, aspergitones A (1) and B (2), along with seven known analogues 3-9, were isolated from the soft coral-associated epiphytic fungus Aspergillus sp. EGF 15-0-3. Their structures were elucidated using 1D and 2D nuclear magnetic resonance (NMR) spectroscopy and high-resolution electrospray ionization mass spectrometry (HRESIMS), whereas the absolute configuration of 1 was determined through the experimental and computational ECD analysis. On the basis of the molecular docking and activity assays against the antitumor targets tyrosyl-DNA phosphodiesterases 1 and 2 (TDP1 and TDP2), compound 7 was found to be a potential dual inhibitor against TDP1 and TDP2, whereas 5 showed the strongest inhibition against TDP1.

Rational: Aconiti Lateralis Radix Praeparata (AC) is a traditional Chinese medicine with a long history of use. However, the current research on the material basis of AC and its processed products is still not comprehensive, especially the changes in lipo-diterpenoid alkaloids (LDAs) that can be hydrolyzed into diester-diterpenoid alkaloids in AC before and after processing. This study aimed to provide material basis guidance for the clinical use of AC and its processed products by comprehensively analyzing the changes in substances between AC and its processed products. Methods: An ultra-high-performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF-MS/MS) approach was optimized to chemical profiling. The MS data were processed using molecular networking combined with the in-house library database to fast characterize the compounds. Multivariate statistical methods were adopted to determine the dissimilarities of components in AC and its processed products. Results: A total of 310 compounds were tentatively identified from AC, including 109 potential new alkaloids, of which 98 were potential novel LPAs. A metabolomics approach was applied to find the characteristic marker components. As a result, 52 potential chemical markers were selected to distinguish the AC samples of different extraction methods and 42 potential chemical markers for differentiating between AC and its processed products were selected. Conclusion: The results indicate that UHPLC/Q-TOF-MS/MS and Global Natural Products Social Molecular Networking coupled with multivariate analysis strategies was a powerful tool to rapidly identify and screen the chemical markers of alkaloids between the AC samples and its processed products. These results also indicate that the toxicity of water extracts of AC and its processed products were decreased. This research not only guides the clinical safe use of AC and its processed products, but also extends the application of the molecular networking strategy in traditional herbal medicine.

Penicilloneines A (1) and B (2) are the first reported quinolone-citrinin hybrids. They were isolated from the starfish-derived fungus Penicillium sp. GGF16-1-2, and their structures were elucidated using spectroscopic, chemical, computational, and single-crystal X-ray diffraction methods. Penicilloneines A (1) and B (2) share a common 4-hydroxy-1-methyl-2(1H)-quinolone unit; however, they differ in terms of citrinin moieties, and these two units are linked via a methylene bridge. Penicilloneines A (1) and B (2) exhibited antifungal activities against Colletotrichum gloeosporioides, with lethal concentration 50 values of 0.02 and 1.51 μg/mL, respectively. A mechanistic study revealed that 1 could inhibit cell growth and promote cell vacuolization and consequent disruption of the fungal cell walls via upregulating nutrient-related hydrolase genes, including putative hydrolase, acetylcholinesterase, glycosyl hydrolase, leucine aminopeptidase, lipase, and beta-galactosidase, and downregulating their synthase genes 3-carboxymuconate cyclase, pyruvate decarboxylase, phosphoketolase, and oxalate decarboxylase.

2-(2-Phenylethyl)chromones (PECs) are the primary constituents responsible for the promising pharmacological activities and unique fragrance of agarwood. However, the O-methyltransferases (OMTs) involved in the formation of diverse methylated PECs have not been reported. In this study, we identified one Mg2+-dependent caffeoyl-CoA-OMT subfamily enzyme (AsOMT1) and three caffeic acid-OMT subfamily enzymes (AsOMT2-4) from NaCl-treated Aquilaria sinensis calli. AsOMT1 not only converts caffeoyl-CoA to feruloyl-CoA but also performs nonregioselective methylation at either the 6-OH or 7-OH position of 6,7-dihydroxy-PEC. On the other hand, AsOMT2-4 preferentially utilizes PECs as substrates to produce structurally diverse methylated PECs. Additionally, AsOMT2-4 also accepts nonPEC-type substrates such as caffeic acid and apigenin to generate methylated products. Protein structure prediction and site-directed mutagenesis revealed that residues of L313 and I318 in AsOMT3, as well as S292 and F313 in AsOMT4 determine the distinct regioselectivity of these two OMTs toward apigenin. These findings provide important biochemical evidence of the remarkable structural diversity of PECs in agarwood.

Global Natural Products Social (GNPS) molecular networking platform was applied to discovery the undescribed compounds from the common marine fungi Aspergillus versicolor CGF9-1-2, ultimately resulting in isolation of four new polyketides, decumbenone E (1), decumbenone F (2), 2'-epi-8-O-methylnidurufin (6), (-)-phomoindene A (7), one new nucleoside, 3-methyl-9-(2-methylbutene)-xanthine (8), and five known analogues. Their structures were elucidated based on 1D/2D NMR spectroscopic and HRESIMS data analyses, meanwhile, the absolute configurations of new compounds were established based on the X-ray crystallographic experiments, as well as the electronic circular dichroism (ECD) analysis. All compounds were predicted pharmaceutical chemistry with ten commonly disease-related proteins by molecular docking. In addition, all compounds against TDP1 were performed in vitro, which was consistent with the docking result, and compound 6 shown a weak inhibitory activity.

Two new disubstituted maleimides, aspergteroids G–H (1–2), and two trisubstituted butenolides aspergteroids I–J (3–4), along with four known analogs, were isolated and structurally identified from the fermentation extract of soft-coral-associated symbiotic and epiphytic fungus Aspergillus terreus EGF7-0-1. The structures of the new compounds were established mainly via spectroscopic data analyses, and their absolute configurations were determined via X-ray diffraction analysis and comparison of the calculated and experimental electronic circular dichroism. Myocardial protection assays showed that compounds 1, 2, 5, and 6 possess protective effects against tert-butyl hydroperoxide (TBHP)-induced H9c2 (rat myocardial cells) apoptosis at low concentrations. Based on the analyses of the protein–protein interaction (PPI) network and Western blotting, compound 1 may inhibit the apoptosis and inflammatory response of cardiomyocytes after TBHP induction and improve the antioxidant capacity of cardiomyocytes. We speculate that the anti-inflammatory response of compound 1 is suppressed by the glycogen synthase kinase-3 beta (GSK-3β), downregulated by the NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome activation, and suppressed by the expression of cysteinyl aspartate specific proteinase-3 (caspase-3) and B-cell lymphoma-2 associated X protein (Bax).

Xanthocillin is a unique natural product with an isonitrile group and shows remarkable antibacterial activity. In this study, the genome of an endophytic fungus Penicillium chrysogenum MT-40 isolated from Huperzia serrata was sequenced, and the gene clusters with the potential to synthesize xanthocillin analogues were mined by local BLAST and various bioinformatics analysis tools. As a result, a biosynthetic gene cluster (named for) responsible for the biosynthesis of xanthocillin analogues was identified by further heterologous expression of the key genes in Aspergillus oryzae NSAR1. Specifically, the ForB catalyzes the synthesis of 2-formamido-3-(4-hydroxyphenyl) acrylic acid, and the ForG catalyzes the dimerization of 2-formamido-3-(4-hydroxyphenyl) acrylic acid to produce the xanthocillin analogue N, N'-(1, 4-bis (4-hydroxyphenyl) buta-1, 3-diene-2, 3-diyl) diformamide. The results reported here provide a reference for further discovery of xanthocillin analogues from fungi.

Two new indole diketopiperazine alkaloids (IDAs), (+)19-epi-sclerotiamide (1) and (-)19-epi-sclerotiamide (2), along with 13 known analogs (3-15), were isolated from a soft coral-associated epiphytic fungus Aspergillus versicolor CGF 9-1-2. The structures of two new compounds were established based on the combination of HR-ESI-MS, 1D and 2D NMR spectroscopy, optical rotation measurements and quantum chemical 13 C-NMR, the absolute configurations were determined by experimental and electronic circular dichroism (ECD) calculations. The results of molecular docking showed that all the compounds had a good binding with TDP1, TDP2, TOP1, TOP2, Ache, NLRP3, EGFR, EGFR L858R, EGFR T790M and EGFR T790/L858. Biological evaluation of compounds 3, 6, 8, 11 showed that 3 exerted a strong inhibitory effect on TDP2 with a rate of 81.72 %.

Objective: To study the chemical constituents of the roots of Angelica dahurica, a well-known Chinese her-bal medicine named Baizhi in Chinese.Methods: Compounds were separated by various chromatographies, and the structures of new com-pounds were elucidated based on the analysis of their spectroscopic and spectrometric data (1D, 2D NMR, HRESI MS, IR, and UV). The absolute configurations of new compounds were determined by the cal-culated electronic circular dichroism and chemical derivatization. The inhibitory activities of all isolates against nitric oxide (NO) production were evaluated using lipopolysaccharide-activated RAW 264.7 macrophage cells.Results: Seven new 3,4-dihydro-furanocoumarin derivatives (1a/1b, 2a/2b, 3a/3b, 4) together with a known furanocoumarin (5) were isolated from the roots of A. dahurica. The new compounds included three pairs of enantiomers, (4S, 2"R)-angelicadin A (1a)/(4R, 2"S)-angelicadin A (1b), (4S, 2"S)-angelicadin A (2a)/(4R, 2"R)-angelicadin A (2b), and (4S, 2"S)-secoangelicadin A (3a)/(4R, 2"R)-secoangelicadin A (3b), together with (4R, 2"R)-secoangelicadin A methyl ester (4). The known xantho-toxol (5) inhibited the NO production with the half-maximal inhibitory concentration (IC50) value of (32.8 +/- 0.8) lmol/L, but all the new compounds showed no inhibitory activities at the concentration of 100 lmol/L.Conclusion: This is the first report of the discovery of 3,4-dihydro-furanocoumarins from A. dahurica. The results are not only meaningful for the understanding of the chemical constituents of A. dahurica, but also enrich the reservoir of natural products.(c) 2023 Tianjin Press of Chinese Herbal Medicines. Published by ELSEVIER B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Dihydroflavonol 4-reductase（DFR） plays an essential role in the biosynthesis of anthocyanin and regulation of plant flower color. Based on the transcriptome data of Cistanche tubulosa（Schenk） Wight, a fulllength cDNA sequence of CtDFR gene was cloned by reverse transcription-polymerase chain reaction（RT-PCR）.CtDFR contains an open reading frame（ORF） of 1 263 bp which encodes 420 amino acids with a predicted molecular weight of 47.5 kDa. The sequence analysis showed that CtDFR contains a nicotinamide adenine dinucleotide phosphate（NADPH） binding domain and a specific substrate binding domain. The expression analysis indicated that CtDFR was highly expressed in red and purple flowers, and the relative expression levels were 4.04and 19.37 times higher than those of white flowers, respectively. The recombinant CtDFR protein was expressed in E.coli BL21（DE3） using vector pET-28a-CtDFR and was purified. In vitro enzyme activity analysis, CtDFR could reduce three types of dihydroflavonols including dihydrokaempferol, dihydroquercetin, and dihydromyricetin to leucopelargonidin, leucocyanidin and leucodelphinidin. Subcellular localization analysis showed that CtDFR was mainly localized in the cytoplasm. These results demonstrate that CtDFR plays an important role in regulation of flower color in C. tubulosa and make a valuable contribution for the further investigation on the regulation mechanism of C. tubulosa（Schenk） Wight flower color.

Objective To explore the mass spectrometric cleavage patterns of the alkaloids in Sophora flavescens and their variation in different S. flavescens samples, including aseptic seedlings（roots, leaves, and stems） and callus, by LC-MS analysis, so as to provide the basis for the identification of the key enzymes involved in the biosynthetic pathway of S. flavescens alkaloids using comparative transcriptome sequencing approach. Methods UPLC-Q-Exactive-Orbitrap MS was used to comprehensively identify the alkaloids in the roots of 5-year-old S. flavescens. UPLC-QQQ-MS/MS was used to quantify the alkaloids in different tissues（roots, stems, leaves） of S. flavescens aseptic seedling and callus. Results A total of 47 alkaloids components were identified from the roots of 5-year-old S. flavescens, and 17 of them were quantified by UPLC-QQQ-MS/MS. The results revealed that the alkaloids in different S. flavescens samples were significantly different, and the content of alkaloids in tissues of S. flavescens aseptic seedling was relatively high. Conclusion In this study, a rapid qualitative and quantitative method for the alkaloids in S. flavescens was developed, resulting in the identification of 47 alkaloids from S. flavescens roots, and 17 alkaloids in different S. flavescens tissue samples were quantified, which are helpful to the subsequent mining and identification of key enzymes involved in the biosynthesis of alkaloids in S. flavescens by using comparative transcriptome sequencing technology.

Qualitative and quantitative analysis of 2-(2-phenylethyl) chromones in sodium chloride(NaCl)-treated suspension cells of Aquilaria sinensis was conducted by UPLC-Q-Exactive-MS and UPLC-QQQ-MS/MS. Both analyses were performed on a Waters T3 column(2.1 mm×50 mm, 1.8 μm) with 0.1% formic acid aqueous solution(A)-acetonitrile(B) as mobile phases at gradient elution. MS data were collected by electrospray ionization in positive ion mode. Forty-seven phenylethylchromones was identified from NaCl-treated suspension cell samples of A. sinensis using UPLC-Q-Exactive-MS, including 22 flindersia-type 2-(2-phenylethyl) chromones and their glycosides, 10 5,6,7,8-tetrahydro-2-(2-phenylethyl) chromones and 15 mono-epoxy or diepoxy-5,6,7,8-tetrahydro-2-(2-phenylethyl) chromones. Additionally, 25 phenylethylchromones were quantitated by UPLC-QQQ-MS/MS. Overall, the rapid and efficient qualitative and quantitative analysis of phenylethylchromones in NaCl-treated suspension cells of A. sinensis by two LC-MS techniques, provides an important reference for the yield of phenylethylchromones in Aquilariae Lignum Resinatum using in vitro culture and other biotechnologies.

细胞色素P450还原酶(cytochrome P450 reductase,CPR)是细胞色素P450酶电子传递链的组成部分,在生物体内起着重要的电子传递作用.本研究从白木香(Aquilaria sinensis)愈伤组织细胞中克隆得到1个CPR基因(Ascpr1),Ascpr1含有一个2 124 bp的开放阅读框,编码707个氨基酸残基,其蛋白分子质量为78.82 kD.系统进化分析显示,AsCPR1为Ⅱ型CPR蛋白,与来源于可可树(Theobroma cacao)的CPR蛋白亲缘关系较近.跨膜预测结果显示该蛋白的第52～71位氨基酸为跨膜区,在大肠杆菌Transetta(DE3)中成功表达了N-端缺失67个氨基酸残基的融合蛋白,经亲和色谱纯化后获得对细胞色素C具有还原能力的融合蛋白AsCPR1.本研究结果为进一步研究P450酶参与的白木香防御物质合成及CPR在白木香防御反应中的作用奠定基础.

Prenyltransferases (PTs) play important roles in the biosynthesis and structural diversification of natural products. In the present study, two new PTs were characterized from a medicinal plant Murraya exotica. MePT1 unprecedentedly catalyses the formation of two C-geranylated products 8/6-C-geranylumbelliferone together with a trace product 7-O-geranylumbelliferone from umbelliferone. MePT2 regio-specifically catalyses the formation of C-3 dimethylallylated products from quinolone alkaloids. This is the first report that a plant PT catalyses the simultaneous formation of C- and O-prenylated products, and a plant PT specifically utilizes quinolone alkaloids as prenyl acceptors. The results not only provide important insight into the functional diversity of plant PTs and the biosynthesis of the prenylated coumarins, quinolone and carbazole alkaloids in Murraya plants, but also pave the way for the overproduction of the prenylated coumarins and alkaloids using metabolic engineering approaches.

The biosynthetic gene cluster of atlantinone B (10) was discovered in Penicillium chrysogenum MT-40. A multifunctional cytochrome P450 (AtlD) encoded by the cluster is responsible for the formation of the unique lactone-bridged ring and the 16β-hydroxyl of atlantinone B, and a new terpene cyclase (AtlC) can unprecedentedly accept the demethylated substrate epoxyfarnesyl-DMOA (4a) to generate three bicyclic meroterpenoids (5a-5c). This study paves the way for combinatorial synthesis of structurally diverse meroterpenoids for drug discovery.

Chlorogenic acid (5-CQA), neochlorogenic acid (3-CQA), and cryptochlorogenic acid (4-CQA), usually simultaneously exist in many traditional Chinese medicines (TCMs). However, insufficient attentions have been paid to the comparative metabolism study on these three isomeric constituents with similar effects on anti-inflammation until now. In this study, a novel strategy was established to perform comparative analysis of their metabolic fates in rats and elucidate the pharmacological mechanism of anti-inflammation. Firstly, diagnostic product ions (DPIs) deduced from the representative reference standards were adopted to rapidly screen and characterize the metabolites in rat plasma, urine and faeces using UHPLC-Q-TOF MS. Subsequently, Network pharmacology was utilized to elucidate their anti-inflammatory mechanism. Consequently, a total of 73 metabolites were detected and characterized, including 50, 47 and 43 metabolites for 5-CQA, 4-CQA and 3-CQA, orderly. Moreover, the network pharmacology study indicated that these three isomeric constituents and their major metabolites with similar in vivo metabolic pathways exerted anti-inflammatory effects through co-owned 20 biological processes, which involved 10 major signal pathways and 159 potential targets. Our study shed light on the similarities and differences of the metabolic profiling and anti-inflammatory activity among these three isomeric constituents and set an example for the further researches on the active mechanism of isomeric constituents existing in TCMs based on comparative metabolism study.

目的 利用UPLC-LTQ-Orbitrap-MS技术对4种秦艽药材的化学成分进行定性分析.方法 采用Waters Acquity UPLC HSS T3色谱柱(2.1 mm×50 mm,1.7 μm),在电喷雾正负离子模式下采集数据.将母离子列表(PIL)-MS2和高分辨质谱结合,用于秦艽药材中化学成分的鉴别.结果 结合质谱数据和文献数据共鉴定、推断出66个成分,其中包括环烯醚萜类36个、木脂素类5个、黄酮类5个、三萜类12个、其他类8个.其中19个成分为首次报道.结论 4种秦艽成分大致相同,利用UPLC-LTQ-Orbitrap-MS技术结合母离子列表的方法可以快速筛选《中国药典》规定的4种秦艽中的化学成分,为秦艽化学成分的提取分离及其药效物质基础研究提供参考.