Discovery of plant chemical defence mediated by a two-component system involving ß-glucosidase in Panax species.

Plants usually produce defence metabolites in non-active forms to minimize the risk of harm to themselves and spatiotemporally activate these defence metabolites upon pathogen attack. This so-called two-component system plays a decisive role in the chemical defence of various plants. Here, we discovered that Panax notoginseng, a valuable medicinal plant, has evolved a two-component chemical defence system composed of a chloroplast-localized ß-glucosidase, denominated PnGH1, and its substrates 20(S)-protopanaxadiol ginsenosides. The ß-glucosidase and its substrates are spatially separated in cells under physiological conditions, and ginsenoside hydrolysis is therefore activated only upon chloroplast disruption, which is caused by the induced exoenzymes of pathogenic fungi upon exposure to plant leaves. This activation of PnGH1-mediated hydrolysis results in the production of a series of less-polar ginsenosides by selective hydrolysis of an outer glucose at the C-3 site, with a broader spectrum and more potent antifungal activity in vitro and in vivo than the precursor molecules. Furthermore, such ß-glucosidase-mediated hydrolysis upon fungal infection was also found in the congeneric species P. quinquefolium and P. ginseng. Our findings reveal a two-component chemical defence system in Panax species and offer insights for developing botanical pesticides for disease management in Panax species.

Identification of a diarylpentanoid-producing polyketide synthase in the biosynthesis of 2-(2-phenylethyl)chromones in agarwood.

Agarwood has been valued as an exquisite, high-grade fragrant wood since ancient times. Due to the scarcity of high-quality agarwood, it is quite expensive, and the number of original plants has been drastically reduced due to overharvesting, including illegal logging. Despite this, a reliable method of agarwood cultivation has yet to be developed. Thus, identifying the biosynthetic pathways of the fragrant components in agarwood might help developers to optimize the culture conditions and create artificial agarwood, by monitoring the expression of the biosynthetic enzymes or their genes. This review presents the characteristics of our recently identified key enzyme, 2-(2-phenylethyl)chromone precursor synthase (PECPS), which generates the common precursor of 2-(2-phenylethyl)chromones (PECs), the main fragrances in agarwood, as well as our reasoning to reach these conclusions. We also discuss the biosynthetic pathway of PECs, unveiled following the identification of PECPS.

Seven new 3,4-dihydro-furanocoumarin derivatives from Angelica dahurica.

Objective: To study the chemical constituents of the roots of Angelica dahurica, a well-known Chinese herbal medicine named Baizhi in Chinese. Methods: Compounds were separated by various chromatographies, and the structures of new compounds 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 calculated 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 xanthotoxol (5) inhibited the NO production with the half-maximal inhibitory concentration (IC50) value of (32.8 ± 0.8) µmol/L, but all the new compounds showed no inhibitory activities at the concentration of 100 µmol/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.

[LC-MS analysis of 2-(2-phenylethyl) chromones in sodium chloride-treated suspension cells of Aquilaria sinensis].

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.

[Research progress on application of multi-enzyme-catalyzed cascade reactions in enzymatic synthesis of natural products].

As a biocatalyst, enzyme has the advantages of high catalytic efficiency, strong reaction selectivity, specific target products, mild reaction conditions, and environmental friendliness, and serves as an important tool for the synthesis of complex organic molecules. With the continuous development of gene sequencing technology, molecular biology, genetic manipulation, and other technologies, the diversity of enzymes increases steadily and the reactions that can be catalyzed are also gradually diversified. In the process of enzyme-catalyzed synthesis, the majority of common enzymatic reactions can be achieved by single enzyme catalysis, while many complex reactions often require the participation of two or more enzymes. Therefore, the combination of multiple enzymes together to construct the multi-enzyme cascade reactions has become a research hotspot in the field of biochemistry. Nowadays, the biosynthetic pathways of more natural products with complex structures have been clarified, and secondary metabolic enzymes with novel catalytic activities have been identified, discovered, and combined in enzymatic synthesis of natural/unnatural molecules with diverse structures. This study summarized a series of examples of multi-enzyme-catalyzed cascades and highlighted the application of cascade catalysis methods in the synthesis of carbohydrates, nucleosides, flavonoids, terpenes, alkaloids, and chiral molecules. Furthermore, the existing problems and solutions of multi-enzyme-catalyzed cascade method were discussed, and the future development direction was prospected.

Biosynthesis Investigations of Terpenoid, Alkaloid, and Flavonoid Antimicrobial Agents Derived from Medicinal Plants.

The overuse of antibiotics in the past decades has led to the emergence of a large number of drug-resistant microorganisms. In recent years, the infection rate caused by multidrug-resistant microorganisms has been increasing, which has become one of the most challenging problems in modern medicine. Plant-derived secondary metabolites and their derivatives have been identified to display significant antimicrobial abilities with good tolerance and less adverse side effects, potentially having different action mechanisms with antibiotics of microbial origin. Thus, these phyto-antimicrobials have a good prospect in the treatment of multidrug-resistant microorganisms. Terpenoids, alkaloids, and flavonoids made up the predominant part of the currently reported phytochemicals with antimicrobial activities. Synthetic biology research around these compounds is one of the hotspot fields in recent years, which not only has illuminated the biosynthesis pathways of these phyto-antimicrobials but has also offered new methods for their production. In this review, we discuss the biosynthesis investigations of terpenoid, alkaloid, and flavonoid antimicrobial agents-using artemisinin and oleanolic acid (terpenoids), berberine and colchicine (alkaloids), and baicalin (flavonoids) as examples-around their antimicrobial action mechanisms, biosynthesis pathway elucidation, key enzyme identification, and heterologous production, in order to provide useful hints for plant-derived antimicrobial agent discovery and development.

Characterization of a coumarin C-/O-prenyltransferase and a quinolone C-prenyltransferase from Murraya exotica.

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.

Two new diterpenoids from Penicillium chrysogenum MT-12, an endophytic fungus isolated from Huperzia serrata.

Two new diterpenoids, penicichrysogene A (1) and penicichrysogene B (2), were isolated from the solid substrate fermentation cultures of Penicillium chrysogenum MT-12, an endophytic fungus isolated from the medicinal plant of Huperzia serrata. Their structures were elucidated on the basis of extensive spectroscopic and spectrometric data (1D and 2D NMR, UV, IR, and HRESIMS). The absolute configurations of 1 and 2 were assigned on the basis of experimental and calculated electronic circular dichroism spectra. Compound 1 exhibited inhibitory activity on ATP release of thrombin-activated platelets with IC50 = 42.7 ± 3.5 µM.

Molecular cloning and biochemical characterization of a new coumarin glycosyltransferase CtUGT1 from Cistanche tubulosa.

UDP-glycosyltransferases (UGTs) are an important and functionally diverse family of enzymes involved in secondary metabolite biosynthesis. Coumarin is one of the most common skeletons of natural products with candidate pharmacological activities. However, to date, many reported GTs from plants mainly recognized flavonoids as sugar acceptors. Only limited GTs could catalyze the glycosylation of coumarins. In this study, a new UGT was cloned from Cistanche tubulosa, a valuable traditional tonic Chinese herb, which is abundant with diverse glycosides such as phenylethanoid glycosides, lignan glycosides, and iridoid glycosides. Sequence alignment and phylogenetic analysis showed that CtUGT1 is phylogenetically distant from most of the reported flavonoid UGTs and adjacent to phenylpropanoid UGTs. Extensive in vitro enzyme assays found that although CtUGT1 was not involved in the biosynthesis of bioactive glycosides in C. tubulosa, it could catalyze the glucosylation of coumarins umbelliferone 1, esculetine 2, and hymecromone 3 in considerable yield. The glycosylated products were identified by comparison with the reference standards or NMR spectroscopy, and the results indicated that CtUGT1 can regiospecifically catalyze the glucosylation of hydroxyl coumarins at the C7-OH position. The key residues that determined CtUGT1's activity were further discussed based on homology modeling and molecular docking analyses. Combined with site-directed mutagenesis results, it was found that H19 played an irreplaceable role as the crucial catalysis basis. CtUGT1 could be used in the enzymatic preparation of bioactive coumarin glycosides.

[Isolation and identification of endophytic fungi from Huperzia serrata and their metabolites' inhibitory activities against acetylcholinesterase and anti-inflammatory activities].

A total of 27 endophytic fungal strains were isolated from Huperzia serrata,which were richly distributed in the stems and leaves while less distributed in roots. The 27 strains were identified by Internal Transcribed Spacer( ITS) r DNA molecular method and one of the strains belongs to Basidiomycota phylum,and other 26 stains belong to 26 species,9 general,6 families,5 orders,3 classes of Ascomycota Phylum. The dominant strains were Colletotrichum genus,belonging to Glomerellaceae family,Glomerellales order,Sordariomycetes class,Ascomycota Phylum,with the percentage of 48. 15%. The inhibitory activities of the crude extracts of 27 endophytic fungal strains against acetylcholinesterase( ACh E) and nitric oxide( NO) production were evaluated by Ellman's method and Griess method,respectively. Crude extracts of four fungi exhibited inhibitory activities against ACh E with an IC50 value of 42. 5-62. 4 mg·L~(-1),and some fungi's crude extracts were found to inhibit nitric oxide( NO) production in lipopolysaccharide( LPS)-activated RAW264. 7 macrophage cells with an IC50 value of 2. 2-51. 3 mg·L~(-1),which indicated that these fungi had potential anti-inflammatory activities.The chemical composition of the Et OAc extract of endophytic fungus HS21 was also analyzed by LCMS-IT-TOF. Seventeen compounds including six polyketides,four diphenyl ether derivatives and seven meroterpenoids were putatively identified.

Polyketides from Alternaria alternata MT-47, an endophytic fungus isolated from Huperzia serrata.

Four new polyketides, alternatains A-D (1-4), along with 17 known compounds (5-21) were obtained from the solid substrate fermentation cultures of Alternaria alternata MT-47, an endophytic fungus isolated from the medicinal plant of Huperzia serrata. Their structures were elucidated by extensive spectroscopic and spectrometric techniques (1D and 2D NMR, IR, and HRESIMS) and calculated electronic circular dichroism (ECD) method. Compounds 4, 6, 15, and 21 exhibited inhibitory activities on ATP release of thrombin-activated platelets with IC50 values in the range of 18.2-68.8 µM.

Lignan Glycosides from Urena lobata.

Four new lignan glycosides; urenalignosides A-D (1-4), along with 12 known ones (5-16) were isolated from Urena lobata. Their structures were determined on the basis of extensive spectroscopic and spectrometric data (1D and 2D NMR; IR; CD; and HRESIMS). Compounds 2-4; 6; 7; 10; and 11 showed inhibition of nitric oxide production in lipopolysaccharide-induced RAW 264.7 macrophage cells with IC50 values in the range of 25.5-98.4 µM (positive control; quercetin; IC50 = 7.2 ± 0.2 µM).

Lycopodium alkaloids from Huperzia serrata.

Five new Lycopodium alkaloids, huperzine Y1 (1), huperzine Y2 (2), huperzine Y3 (3), (+)-huperzine Z (4a) and (-)-huperzine Z (4b) as well as ten known alkaloids (5-14) were isolated from Huperzia serrata. The structures of the new compounds were established using extensive spectroscopic (1D and 2D NMR, IR, and HRESIMS) and calculated electronic circular dichroism (ECD) methods. Compounds 4a and 4b were a pair of enantiomers successfully separated by chiral HPLC resolution. Compounds 2 and 3 indicated inhibitory activities against acetylcholinesterase with IC50 value of 57.1 ±â€¯1.6 and 32.7 ±â€¯1.0 µΜ, respectively. However, no compound showed inhibitory effect on butyrocholinesterase at the concentration of 100 µΜ.

Pyrrole 2-carbaldehyde derived alkaloids from the roots of Angelica dahurica.

Six new pyrrole 2-carbaldehyde derived alkaloids, dahurines A-F (1-6), along with five known ones (7-11) and butyl 2-pyrrolidone-5-carboxylate (12) were isolated from the roots of Angelica dahurica. Their structures were determined by extensive spectroscopic and spectrometric data (1D and 2D NMR, IR, and HRESIMS) and calculated electronic circular dichroism (ECD) methods. Although compounds 7 and 8 have been chemically synthesized, they were obtained from natural materials for the first time. Compounds 2, 3, 4, 10, and 11 exhibited acetylcholinesterase inhibitory activity with IC50 values in the range of 47.5-52.5 µM. Pyrrole 2-carbaldehyde derived alkaloids from the roots of Angelica dahurica.

LC-MS-guided isolation of anti-inflammatory 2-(2-phenylethyl)chromone dimers from Chinese agarwood (Aquilaria sinensis).

Fifteen previously undescribed 2-(2-phenylethyl)chromone dimers, along with two known analogues were isolated from Chinese agarwood (Aquilaria sinensis) by a LC-MS-guided fractionation procedure. Their structures were elucidated on the basis of spectroscopic and spectrometric data (1D and 2D NMR, IR, and HRESIMS). The isolated compounds exhibited significant inhibition of nitric oxide production in lipopolysaccharide-stimulated RAW264.7 cells with IC50 values in the range 0.6-37.1 µM.

Radix Salviae miltiorrhizae improves bone microstructure and strength through Wnt/ß-catenin and osteoprotegerin/receptor activator for nuclear factor-κB ligand/cathepsin K signaling in ovariectomized rats.

Although radix Salviae miltiorrhizae (RSM) is reported to exhibit the antiosteoporotic effect in preclinical study, the underlying mechanism is unclear. To this end, ovariectomized (OVX) rats were employed with administration of RSM (5 g/kg) for 14 weeks. The disturbed serum levels of alkaline phosphatase (ALP), osteoprotegerin (OPG), tartrate-resistant acid phosphatase, and receptor activator of nuclear factor-κB ligand (RANKL) in OVX rats were improved by RSM treatment. Furthermore, supplement of RSM to OVX rats resulted in an increase in femoral bone mineral density and bone strength as well as an improvement in bone microstructures. Moreover, the decreased expression of phosphor (p)-LRP6, insulin-like growth factor-1(IGF-1), ALP, and OPG, as well as increased expression of RANKL and cathepsin K in the tibias and femurs of OVX rats were shifted by RSM treatment. Additionally, RSM reversed the decreased ratio of p-glycogen synthase kinase 3ß (GSK3ß) to GSK3ß and increased ratio of p-ß-catenin to ß-catenin in OVX rats. Altogether, it is suggestive that RSM improves bone quantity and quality by favoring Wnt/ß-catenin and OPG/RANKL/cathepsin K signaling pathways in OVX rats thereby suggesting the potential of this herb to be a novel source of antiosteoporosis drugs.

Megastigmane glycosides from Urena lobata.

Five new megastigmane glycosides, urenalobasides A-E (1-5), together with 11 known ones (6-16) were isolated from Urena lobata. Their structures were determined by extensive spectroscopic and spectrometric data (1D and 2D NMR, IR, and HRESIMS) and calculated electronic circular dichroism method. Compounds 1 and 2 are two unusual megastigmanes structurally containing a 6/5 fused ring system. Compound 3 exhibits inhibition of nitric oxide production in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells with IC50 value of 53.7 ±â€¯1.0 µM (positive control, dexamethasone, IC50 = 16.6 ±â€¯0.8 µM).

[Cloning and expression analysis of transcription factor AsMYB1 and AsMYB2 from Aquilaria sinensis].

The MYB gene family comprises one of the richest groups of transcription factors in plants. The full length of two MYB genes were isolated through heterologous screening of Aquilaria sinensis calli transcriptome data, and the reverse transcription PCR was performed to obstain the corrected MYB clones, named AsMYB1, AsMYB2. The MYB transmembrane domain and phylogenetic analysis were predicted by different software to analyze the bioinformatics of MYB proteins. The transcript level of AsMYB1, AsMYB2 was performed by real-time quantitative RT-PCR in different tissues and in responds to abiotic stresses including salt, cold, metal and drought stress, and hormone treatments including abscisic acid (ABA), salicylic acid (SA), gibberellins (GA3) and methyl jasmonate (MeJA) treatment. The AsMYB1 cDNA sequence had an ORF of 1 063 nucleotides, encoding a protein of 353 amino acids. The largest AsMYB2 ORF was 1 081 nucleotides, and its predicted translation products consisted of 359 amino acids. Two MYB genes had a tissues-specific pattern in A. sinensis. Moreover, the expression level of AsMYB1 and AsMYB2 was regulated by different abiotic stresses and hormone treatments, suggesting the transcription factors AsMYB1 and AsMYB2 play an important role in plant defense and hormone signal transduction in A. sinensis.

Nitric oxide inhibitory polyketides from Penicillium chrysogenum MT-12, an endophytic fungus isolated from Huperzia serrata.

Twelve new polyketides, penicichrysogenins A-L (1-10, 11a, and 11b) along with five known compounds (12a, 12b, and 13-15) were isolated from the solid substrate fermentation cultures of a Huperzia serrata endophytic fungus Penicillium chrysogenum MT-12. The structures of the new compounds were established using extensive spectroscopic (1D and 2D NMR, IR, and HRESIMS) and calculated electronic circular dichroism (ECD) methods. Compounds 11a/11b and 12a/12b were two pairs of enantiomers successfully separated by chiral HPLC resolution. Compounds 4, 5, 8, 9, 11a/11b, and 12a/12b exhibited inhibition of nitric oxide production in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells with IC50 values in the range of 17.5-98.4µM.

3,5-Dimethylorsellinic Acid Derived Meroterpenoids from Penicillium chrysogenum MT-12, an Endophytic Fungus Isolated from Huperzia serrata.

Eight new chrysogenolides (A-H (1-8)) and seven known (9-15) 3,5-dimethylorsellinic acid derived meroterpenoids were isolated from the solid substrate fermentation cultures of a Huperzia serrata endophytic fungus, Penicillium chrysogenum MT-12. The structures of the new compounds were elucidated by interpretation of spectroscopic and spectrometric data (1D and 2D NMR, IR, and HRESIMS). The absolute configurations of 1-4 were determined by single-crystal X-ray crystallographic analysis, and those of 5-8 were assigned on the basis of experimental and calculated electronic circular dichroism spectra. Compounds 3, 4, 6, 11, and 12 showed inhibition of nitric oxide production in lipopolysaccharide-activated RAW 264.7 macrophage cells with IC50 values in the range of 4.3-78.2 µM (positive control, indomethacin, IC50 = 33.6 ± 1.4 µM).