New indole derivatives from endophytic fungus Colletotruchum sp. HK-08 originated from leaves of Nerium indicum.

Objective: To study secondary metabolites from endophytic fungus Colletotruchum sp. HK-08 originated from the leaves of Nerium indicum. Methods: The compounds were isolated by various column chromatographic techniques, and their structures were elucidated by spectroscopic techniques [high resolution electrospray ionization mass spectroscopy (HRESIMS), one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance spectroscopy (NMR)], as well as comparison with literature data. The Ellman method was used to determine the acetylcholinesterase (AChE) inhibitory activity. Results: Four indole derivatives were identified from Colletotruchum sp. HK-08, including 6'-hydroxymonaspiloindole (1), 2-(2-oxoindolin-3-yl)ethyl 2-(4-hydroxyphenyl) acetate (2), 2-(2-oxoindolin-3-yl)ethyl 2-(2-hydroxyphenyl)acetate (3), and monaspiloindole (4). Compound 4 presented weak AChE inhibitory activity with IC50 value of (69.30 ± 6.27) µmol/L [tacrine as the positive control, with IC50 value of (0.61 ± 0.07) µmol/L]. Conclusion: Compounds 1-3 were new compounds, and compound 4 had weak AChE inhibitory activity.

Supramolecular assembly of dendronized diacetylenes into thermoresponsive chiral fibers and their covalent fixation through topochemical polymerization.

By combination of dendritic topological structures with photopolymerizable diacetylene, here we report on supramolecular chiral assembly of the dendronized diacetylenes in water. These dendronized diacetylenes are constituted with three-fold dendritic oligoethylene glycols (OEGs), bridged with a dipeptide from phenylalanine and glycine. These dendronized amphiphiles exhibit intensive propensity to aggregate in water and form helical fibers, which show characteristic thermoresponsive behavior with phase transition temperatures dominated by hydrophilicity of the dendritic OEGs. Topochemical polymerization of these supramolecular fibers through UV irradiation transfers them into the covalent helical dendronized polydiacetylenes. Chirality of these dendronized polydiacetylenes can be mediated through the thermally-induced phase transitions, but is also intriguingly dependent on vortex via stirring. Through stirring the solutions, chiralities of the dendronized polydiacetylenes are inverted, which can be reversibly recovered after keeping still the solution. Hydrogels are formed from these dendronized diacetylenes through concentration-enhanced interactions between the supramolecular fibers. Their mechanical properties can be greatly increased through thermally-enhanced interactions between the fibers with storage moduli increased from 20 Pa to a few hundred Pa. In addition, through photo-polymerization, the supramolecular fibers are transferred into covalent dendronized polydiacetylenes, and the corresponding hydrogels show much improved mechanical properties with storage moduli about 10 kPa.

Bi-2-(2-phenethyl)chromone derivatives from agarwood of Aquilaria filaria in the Philippines.

Twelve undescribed 2-(2-phenethyl)chromone dimers (1-12) were isolated from EtOAc extract of agarwood originating from Aquilaria filaria in the Philippines, guided by a UHPLC-MS analysis. Their structures were elucidated by 1D NMR, 2D NMR, and HR-ESI-MS spectra. The absolute configuration of 2-(2-phenylethyl)chromone dimers was determined by single-crystal X-ray diffraction analysis and comparison of the experimental and calculated ECD spectra. Compounds 1, 2, 5 and 9-12 exhibited potent to moderate anti-inflammatory activity with IC50 values in the range of 22.43 ± 0.86 to 53.88 ± 4.06 µM.

Molecular evolution and characterization of type III polyketide synthase gene family in Aquilaria sinensis.

2-(2-Phenylethyl) chromone (PEC) and its derivatives are markers of agarwood formation and are also related to agarwood quality. However, the biosynthetic and regulatory mechanisms of PECs still remain mysterious. Several studies suggested that type III polyketide synthases (PKSs) contribute to PEC biosynthesis in Aquilaria sinensis. Furthermore, systematic studies on the evolution of PKSs in A. sinensis have rarely been reported. Herein, we comprehensively analyzed PKS genes from 12 plant genomes and characterized the AsPKSs in detail. A unique branch contained only AsPKS members was identified through evolutionary analysis, including AsPKS01 that was previously indicated to participate in PEC biosynthesis. AsPKS07 and AsPKS08, two tandem-duplicated genes of AsPKS01 and lacking orthologous genes in evolutionary models, were selected for their transient expression in the leaves of Nicotiana benthamiana. Subsequently, PECs were detected in the extracts of N. benthamiana leaves, suggesting that AsPKS07 and AsPKS08 promote PEC biosynthesis. The interaction between the promoters of AsPKS07, AsPKS08 and five basic leucine zippers (bZIPs) from the S subfamily indicated that their transcripts could be regulated by these transcription factors (TFs) and might further contribute to PECs biosynthesis in A. sinensis. Our findings provide valuable insights into the molecular evolution of the PKS gene family in A. sinensis and serve as a foundation for advancing PEC production through the bioengineering of gene clusters. Ultimately, this contribution is expected to shed light on the mechanism underlying agarwood formation.

Six unprecedented 2-(2-phenethyl)chromone dimers from agarwood of Aquilaria filaria.

Six new dimeric 2-(2-phenylethyl)chromones (1-6) were successfully isolated from the ethanol extract of agarwood of Aquilaria filaria from Philippines under HPLC-MS guidance. Compounds 1-6 are all dimers formed by linking 5,6,7,8-tetrahydro-2-(2-phenylethyl)chromone and flindersia 2-(2-phenylethyl)chromone via a single ether bond, and the linkage site (C5-O-C8'') of compound 2 is extremely rare. A variety of spectroscopic methods were used to ascertain their structures, including extensive 1D and 2D NMR spectroscopic analysis, HRESIMS, and comparison with literature. The in vitro tyrosinase inhibitory and anti-inflammatory activities of each isolate were assessed. Among these compounds, compound 2 had a tyrosinase inhibition effect with an IC50 value of 27.71 ± 2.60 µM, and compound 4 exhibited moderate inhibition of nitric oxide production in lipopolysaccharide-stimulated RAW264.7 cells with an IC50 value of 35.40 ± 1.04 µM.

Aquilaria sinensis: An Upstart Resource for Cucurbitacin Production Offers Insights into the Origin of Plant Bitter (Bi) Gene Clusters.

Cucurbitacins, oxygenated tetracyclic triterpenoids that are found mainly in the Cucurbitaceae family, play essential roles as defensive compounds, serving as allomones against herbivores and pathogens and as signals for insect-parasite recognition. These compounds also exhibit various pharmacological effects. The biosynthesis of cucurbitacins is largely regulated by the bitter (Bi) gene, encoding an oxidosqualene cyclase, which catalyzes the conversion of 2,3-oxidosqualene into cucurbitadienol, a common precursor for cucurbitacin synthesis. Previous studies focused on uncovering the Bi gene clusters in Cucurbitaceae, but their presence in other cucurbitacin-producing plants remained unexplored. Here, the evolutionary history of Bi genes and their clusters were investigated in twenty-one plant genomes spanning three families based on chemotaxonomy. Nineteen Bi genes were identified in fourteen Cucurbitaceae, four Begoniaceae, and one Aquilaria species. Phylogenetic analysis suggested that the genome of Aquilaria sinensis contained the earliest Bi gene clusters in this dataset. Moreover, the genomic analysis revealed a conserved microsynteny of pivotal genes for cucurbitacin biosynthesis in Cucurbitaceae, while interspersed Bi gene clusters were observed in Begoniaceae, indicating rearrangements during plant Bi gene cluster formation. The bitter gene in A. sinensis was found to promote cucurbitadienol biosynthesis in the leaves of Nicotiana benthamiana. This comprehensive exploration of plant Bi genes and their clusters provides valuable insights into the genetic and evolutionary underpinnings of cucurbitacin biosynthesis. These findings offer prospects for a deeper understanding of cucurbitacin production and potential genetic resources for their enhancement in various plants.

Sesquiterpenoids and bibenzyl derivative from Dendrobium hercoglossum.

Three new sesquiterpenoids, dendrohercoglin A - C (1-3), and one new bibenzyl derivative, dendronbiline D (4), together with nine known sesquiterpenoids (5-13) were isolated from Dendrobium hercoglossum. The structures of the new compounds were elucidated by extensive spectroscopic analysis as well as NMR and ECD calculations. All the compounds were evaluated for their neuroprotective and anti-inflammatory activities. Compounds 2 and 3 increased the H2O2-damaged SH-SY5Y cell viabilities from 43.3% to 58.6% and 68.4%, respectively. Compound 4 exhibited pronounced anti-inflammatory activity with IC50 value of 9.5 ± 0.45 µM which was superior to the reference compound quercetin (IC50: 15.7 ± 0.89 µM).

Identification of volatile compounds and their bioactivities from unpolar fraction of Alpinia oxyphylla Miq. and mining key genes of nootkatone biosynthesis.

In this study, analysis of the chemical constituents and bioactivities of the unpolar fractions [petroleum ether (PE) and chloroform (C)] of fruits and leaves of Alpinia oxyphylla Miq. were carried out, as well as the bioactivities of the main compounds nootkatone and valencene. From PE and C fractions of the fruits, and PE fraction of the leaves, 95.80%, 59.30%, and 82.11% of the chemical constituents respectively were identified by GC-MS. Among these identified compounds, nootkatone was the main compound in all of three fractions, while valencene was the second main compound in the PE fractions of the fruits and leaves. The bioactivities results showed that all of the fractions and the major compound nootkatone showed tyrosinase inhibitory, as well as inhibitory effect on NO production in LPS-stimulated RAW264.7 cells. While valencene only presented inhibitory activity on NO production in RAW264.7 cells. The critical genes involved in nootkatone biosynthesis in A. oxyphylla were identified from the public transcriptome datasets, and protein sequences were preliminarily analyzed. Our studies develop the usage of the unpolar fractions of A. oxyphylla, especially its leaves as the waste during its production, and meanwhile provide the gene resources for nootkatone biosynthesis.

Two new anti-inflammatory trachylobane diterpenoids from Euphorbia atoto.

Two new rare trachylobane euphoratones A-B (1-2), together with five known diterpenoids (compounds 3-7), were isolated from the aerial parts of Euphorbia atoto. Their structures were unambiguously elucidated through HRESIMS, 1D and 2D NMR spectral analysis. Compounds 1, 3, 4 and 7 showed weak anti-inflammatory activities (IC50 77.49 ± 6.34, 41.61 ± 14.49, 16.00 ± 1.71 and 33.41 ± 4.52 µM, respectively), compared to the positive control quercetin (IC50 15.23 ± 0.65 µM).

Comprehensive Analysis of NAC Transcription Factors Reveals Their Evolution in Malvales and Functional Characterization of AsNAC019 and AsNAC098 in Aquilaria sinensis.

NAC is a class of plant-specific transcription factors that are widely involved in the growth, development and (a)biotic stress response of plants. However, their molecular evolution has not been extensively studied in Malvales, especially in Aquilaria sinensis, a commercial and horticultural crop that produces an aromatic resin named agarwood. In this study, 1502 members of the NAC gene family were identified from the genomes of nine species from Malvales and three model plants. The macroevolutionary analysis revealed that whole genome duplication (WGD) and dispersed duplication (DSD) have shaped the current architectural structure of NAC gene families in Malvales plants. Then, 111 NAC genes were systemically characterized in A. sinensis. The phylogenetic analysis suggests that NAC genes in A. sinensis can be classified into 16 known clusters and four new subfamilies, with each subfamily presenting similar gene structures and conserved motifs. RNA-seq analysis showed that AsNACs presents a broad transcriptional response to the agarwood inducer. The expression patterns of 15 AsNACs in A. sinensis after injury treatment indicated that AsNAC019 and AsNAC098 were positively correlated with the expression patterns of four polyketide synthase (PKS) genes. Additionally, AsNAC019 and AsNAC098 were also found to bind with the AsPKS07 promoter and activate its transcription. This comprehensive analysis provides valuable insights into the molecular evolution of the NAC gene family in Malvales plants and highlights the potential mechanisms of AsNACs for regulating secondary metabolite biosynthesis in A. sinensis, especially for the biosynthesis of 2-(2-phenyl) chromones in agarwood.

Supramolecular assembly of dendronized spiropyrans in aqueous solutions into nanospheres with photo- and thermo-responsive chiralities.

Tailoring the amphiphilicity of a molecule through external stimuli can alter the balance between self-association and repulsion, resulting in different propensities for its assembly. Here we report on the supramolecular assembly of a series of dendronized spiropyrans (DSPs) in water. These DSPs carry 3-fold dendritic oligoethylene glycols (OEGs) with either methoxyl or ethoxyl terminals for different hydrophilicities, and contain an Ala-Gly dipeptide to provide the chirality. These dendronized amphiphiles form supramolecular nanospheres in aqueous solutions with remarkable induced chirality to a level of 1.0 × 106 deg cm2 dmol-1. They can be tuned reversibly through photoisomerization of the spiropyran moieties from the hydrophobic SP form into the hydrophilic MC form, and can even become chirally silent through thermally mediated collapse of the dendritic OEGs. Photoisomerization of the spiropyran moieties in these DSPs is accompanied by simultaneous changes of UV absorption, fluorescence emission, supramolecular chirality and aqueous solution colors. These supramolecular nanospheres exhibit characteristic thermoresponsive behavior due to thermal collapse of the dendritic OEGs with their cloud point temperatures (Tcps) being dependent on the overall hydrophilicity of the molecules and also the aggregate morphologies resulting from how dendritic OEGs are wrapped around the aggregates. Both photo-irradiation-mediated isomerization of the spiropyran moieties and thermally mediated dehydration and collapse of the dendritic OEGs influence the amphiphilicity of these DSPs and their solvation by water, leading to varied driving forces for their assembly. NMR, circular dichroism (CD) and fluorescence spectroscopy techniques, as well as DLS and AFM techniques are combined to follow the supramolecular assembly and illustrate the aggregation mechanism. All experimental results demonstrate that the reversible chirality of the aggregates originates from the balance between dendritic OEGs and spiropyran moieties against water solvation.

Evaluating the Antioxidant Properties of the Leaves and Stems of Alpinia oxyphylla In Vitro and Its Growth-Promoting, Muscle Composition Change, and Antioxidative Stress Function on Juvenile Litopenaeus vannamei.

Alpinia oxyphylla is a homology of medicine and food. This study aims to investigate the dominant chemical composition and explore the antioxidant properties of the ethanol extract of the leaves and stems of A. oxyphylla (AOE) on juvenile shrimp, Litopenaeus vannamei. An in vitro test showed that AOE and its dominant chemical composition procyanidin B-2 (1) and epicatechin (2) presented DPPH and ABTS radical scavenging activities. A shrimp feeding supplement experiment revealed that shrimp growth parameters and muscle composition were improved significantly when fed with a 200 mg/Kg AOE additive. Meanwhile, the activities of antioxidant enzymes (CAT, GSH-Px, SOD, and T-AOC) in serum and the liver and the expression of related genes (LvMn-SOD, LvCAT, LvproPo, and LvGSH-Px) were enhanced with various degrees in different AOE additive groups while the content of MDA was significantly decreased. Moreover, the antioxidative effect of AOE additive groups on shrimp was also observed in an acute ammonia nitrogen stress test.

Systematic evolution of bZIP transcription factors in Malvales and functional exploration of AsbZIP14 and AsbZIP41 in Aquilaria sinensis.

Introduction: Agarwood, the dark-brown resin produced by Aquilaria trees, has been widely used as incense, spice, perfume or traditional medicine and 2-(2-phenethyl) chromones (PECs) are the key markers responsible for agarwood formation. But the biosynthesis and regulatory mechanism of PECs were still not illuminated. The transcription factor of basic leucine zipper (bZIP) presented the pivotal regulatory roles in various secondary metabolites biosynthesis in plants, which might also contribute to regulate PECs biosynthesis. However, molecular evolution and function of bZIP are rarely reported in Malvales plants, especially in Aquilaria trees. Methods and results: Here, 1,150 bZIPs were comprehensively identified from twelve Malvales and model species genomes and the evolutionary process were subsequently analyzed. Duplication types and collinearity indicated that bZIP is an ancient or conserved TF family and recent whole genome duplication drove its evolution. Interesting is that fewer bZIPs in A. sinensis than that species also experienced two genome duplication events in Malvales. 62 AsbZIPs were divided into 13 subfamilies and gene structures, conservative domains, motifs, cis-elements, and nearby genes of AsbZIPs were further characterized. Seven AsbZIPs in subfamily D were significantly regulated by ethylene and agarwood inducer. As the typical representation of subfamily D, AsbZIP14 and AsbZIP41 were localized in nuclear and potentially regulated PECs biosynthesis by activating or suppressing type III polyketide synthases (PKSs) genes expression via interaction with the AsPKS promoters. Discussion: Our results provide a basis for molecular evolution of bZIP gene family in Malvales and facilitate the understanding the potential functions of AsbZIP in regulating 2-(2-phenethyl) chromone biosynthesis and agarwood formation.

Anti-inflammatory and α-glucosidase inhibitory constituents from Dendrobium nobile Lindl.

Four new compounds ((±)-1-3), including one pair of enantiomers ((±)-1), along with 11 known bibenzyls (4-14) were isolated from Dendrobium nobile. The structures of the new compounds were elucidated by spectroscopic methods including 1D and 2D NMR as well as HRESIMS. The configurations of (±)-1 were established via the electronic circular dichroism (ECD) calculations. Compounds (+)-1 and 13 displayed pronounced α-glucosidase inhibitory activities with IC50 values of 16.7 ± 2.3 and 13.4 ± 0.2 µM, respectively, which were comparable to that of genistein (IC50, 8.54 ± 0.69 µM). Kinetic studies revealed that (+)-1 and 13 were non-competitive inhibitors against α-glucosidase and molecular docking simulations illuminated their interactions with α-glucosidase. All the isolates were also evaluated for their anti-inflammatory activities. Compounds 4, 5, and 11 exhibited superior inhibition activity with IC50 values ranging from 9.2 to 13.8 µM to that of quercetin (IC50, 16.3 ± 1.1 µM).

Systematic investigation of the R2R3-MYB gene family in Aquilaria sinensis reveals a transcriptional repressor AsMYB054 involved in 2-(2-phenylethyl)chromone biosynthesis.

Trees in the genus Aquilaria produce agarwood, a valuable resin used in medicine, perfumes, and incense. 2-(2-Phenethyl)chromones (PECs) are characteristic components of agarwood; however, molecular mechanisms underlying PEC biosynthesis and regulation remain largely unknown. The R2R3-MYB transcription factors play important regulatory roles in the biosynthesis of various secondary metabolites. In this study, 101 R2R3-MYB genes in Aquilaria sinensis were systematically identified and analyzed at the genome-wide level. Transcriptomic analysis revealed that 19 R2R3-MYB genes were significantly regulated by an agarwood inducer, and showed significant correlations with PEC accumulation. Expression and evolutionary analyses revealed that AsMYB054, a subgroup 4 R2R3-MYB, was negatively correlated with PEC accumulation. AsMYB054 was located in the nucleus and functioned as a transcriptional repressor. Moreover, AsMYB054 could bind to the promoters of the PEC biosynthesis related genes AsPKS02 and AsPKS09, and inhibit their transcriptional activity. These findings suggested that AsMYB054 functions as a negative regulator of PEC biosynthesis via the inhibition of AsPKS02 and AsPKS09 in A. sinensis. Our results provide a comprehensive understanding of the R2R3-MYB subfamily in A. sinensis and lay a foundation for further functional analyses of R2R3-MYB genes in PEC biosynthesis.

Cytotoxic steroids from the stems of Strophanthus divaricatus.

Phytochemical investigation on the stems of Strophanthus divaricatus led to the isolation of four undescribed cardiac glycosides and one undescribed C21 pregnane, together with eleven known steroids. Their structures were elucidated by a comprehensive analysis of HRESIMS, 1D and 2D NMR spectra. The absolute configuration of 16 was determined by comparison of the experimental and computed ECD spectra. Compounds 1-13 and 15 displayed potent to significant cytotoxicity against human cancer cell lines K562, SGC-7901, A549 and HeLa with IC50 values of 0.02-16.08, 0.04-23.13, 0.06-22.31 and 0.06-15.13 µM, respectively.

New Isocoumarins from the Marine Fungus Phaeosphaeriopsis sp. WP-26.

Five new isocoumarins, phaeosphaerins A-E (1-5), were isolated from the fermentation broth of the marine fungus Phaeosphaeriopsis sp. WP-26, along with one known isocoumarin, 6,8-dihydroxy-7-methoxy-3-methylisocoumarin (6), and two known pimarane-type diterpenes, diaportheins A (7) and B (8). Their structures were elucidated via NMR experiments, X-ray diffraction analysis, and comparison of the experimental and computed ECD curves. Compounds 1-7 displayed weak neuroprotective effects against H2O2-induced damage in SH-SY5Y cells. Moreover, compound 8 showed cytotoxicity against BEL-7402, SGC-7901, K562, A549, and HL-60 cell lines.

Integrated analysis of Dendrobium nobile extract Dendrobin A against pancreatic ductal adenocarcinoma based on network pharmacology, bioinformatics, and validation experiments.

Background: Dendrobium nobile (D. nobile), a traditional Chinese medicine, has received attention as an anti-tumor drug, but its mechanism is still unclear. In this study, we applied network pharmacology, bioinformatics, and in vitro experiments to explore the effect and mechanism of Dendrobin A, the active ingredient of D. nobile, against pancreatic ductal adenocarcinoma (PDAC). Methods: The databases of SwissTargetPrediction and PharmMapper were used to obtain the potential targets of Dendrobin A, and the differentially expressed genes (DEGs) between PDAC and normal pancreatic tissues were obtained from The Cancer Genome Atlas and Genotype-Tissue Expression databases. The protein-protein interaction (PPI) network for Dendrobin A anti-PDAC targets was constructed based on the STRING database. Molecular docking was used to assess Dendrobin A anti-PDAC targets. PLAU, one of the key targets of Dendrobin A anti-PDAC, was immunohistochemically stained in clinical tissue arrays. Finally, in vitro experiments were used to validate the effects of Dendrobin A on PLAU expression and the proliferation, apoptosis, cell cycle, migration, and invasion of PDAC cells. Results: A total of 90 genes for Dendrobin A anti-PDAC were screened, and a PPI network for Dendrobin A anti-PDAC targets was constructed. Notably, a scale-free module with 19 genes in the PPI indicated that the PPI is highly credible. Among these 19 genes, PLAU was positively correlated with the cachexia status while negatively correlated with the overall survival of PDAC patients. Through molecular docking, Dendrobin A was found to bind to PLAU, and the Dendrobin A treatment led to an attenuated PLAU expression in PDAC cells. Based on clinical tissue arrays, PLAU protein was highly expressed in PDAC cells compared to normal controls, and PLAU protein levels were associated with the differentiation and lymph node metastatic status of PDAC. In vitro experiments further showed that Dendrobin A treatment significantly inhibited the proliferation, migration, and invasion, inducing apoptosis and arresting the cell cycle of PDAC cells at the G2/M phase. Conclusion: Dendrobin A, a representative active ingredient of D. nobile, can effectively fight against PDAC by targeting PLAU. Our results provide the foundation for future PDAC treatment based on D. nobile.

Seven new 2-(2-phenethyl)chromone derivatives from agarwood of Aquilaria walla.

Two rare flavonoid-2-(2-phenylethyl)chromones and five new dimeric 2-(2-phenylethyl)chromones were isolated from ethanol extract of agarwood of Aquilaria walla by LC-MS-guided fractionation procedure. Their structures were established based on extensive spectroscopic methods including HRESIMS, 1D and 2D NMR, as well as by comparison with the literature. Compound 1 showed cytotoxic activity against five human cancer cell lines with IC50 values ranging from 13.40 to 28.96 µM with cisplatin as the positive control.

Dimeric 2-(2-phenethyl)chromones from agarwood of Aquilaria filaria.

Four new 2-(2-phenethyl)chromone dimers (1-4) were isolated from EtOAc extract of agarwood originating from Aquilaria filaria from Philippines. Their structures were elucidated by spectroscopic analysis (1D and 2D NMR, and HRESIMS) and comparison of the experimental and computed ECD curves. Compounds 1-4 exhibited inhibition of nitric oxide production in lipopolysaccharide-stimulated RAW264.7 cells with IC50 values in the range from 33.94 to 57.53 µM.