Heterodimers of Aromadendrane Sesquiterpenoid with Benzoquinone from the Chinese Liverwort Mylia nuda.

Mylnudones A-G (1-7), unprecedented 1,10-seco-aromadendrane-benzoquinone-type heterodimers, and a highly rearranged aromadendrane-type sesquiterpenoid (8), along with four known analogs (9-12), were isolated from the liverwort Mylia nuda. Compounds 1-6 and 7, bearing tricyclo[6.2.1.02,7] undecane and tricyclo[5.3.1.02,6] undecane backbones, likely formed via a Diels-Alder reaction and radical cyclization, respectively. Their structures were determined by spectroscopic analysis, computational calculation, and single-crystal X-ray diffraction analysis. Dimeric compounds displayed cytoprotective effects against glutamic acid-induced neurological deficits.

Efficient Production of Flavonoid Glucuronides in Escherichia coli Using Flavonoid O-Glucuronosyltransferases Characterized from Marchantia polymorpha.

As a model liverwort, Marchantia polymorpha contains various flavone glucuronides with cardiovascular-promoting effects and anti-inflammatory properties. However, the related glucuronosyltransferases have not yet been reported. In this study, two bifunctional UDP-glucuronic acid/UDP-glucose:flavonoid glucuronosyltransferases/glucosyltransferases, MpUGT742A1 and MpUGT736B1, were identified from M. polymorpha. Extensive enzymatic assays found that MpUGT742A1 and MpUGT736B1 exhibited efficient glucuronidation activity for flavones, flavonols, and flavanones and showed promiscuous regioselectivity at positions 3, 6, 7, 3', and 4'. These enzymes catalyzed the production of a variety of flavonoid glucuronides with medicinal value, including apigenin-7-O-glucuronide and scutellarein-7-O-glucuronide. With the use of MpUGT736B1, apigenin-4'-O-glucuronide and apigenin-7,4'-di-O-glucuronide were prepared by scaled-up enzymatic catalysis and structurally identified by NMR spectroscopy. MpUGT742A1 also displayed glucosyltransferase activity on the 7-OH position of the flavanones using UDP-glucose as the sugar donor. Furthermore, we constructed four recombinant strains by combining the pathway for increasing the UDP-glucuronic acid supply with the two novel UGTs MpUGT742A1 and MpUGT736B1. When apigenin was used as a substrate, the extracellular apigenin-4'-O-glucuronide and apigenin-7,4'-di-O-glucuronide production obtained from the Escherichia coli strain BB2 reached 598 and 81 mg/L, respectively. Our study provides new candidate genes and strategies for the biosynthesis of flavonoid glucuronides.

Interaction of PKR with STCS1: an indispensable step in the biosynthesis of lunularic acid in Marchantia polymorpha.

Unlike bibenzyls derived from the vascular plants, lunularic acid (LA), a key precursor for macrocyclic bisbibenzyl synthesis in nonvascular liverworts, exhibits the absence of one hydroxy group within the A ring. It was hypothesized that both polyketide reductase (PKR) and stilbenecarboxylate synthase 1 (STCS1) were involved in the LA biosynthesis, but the underlined mechanisms have not been clarified. This study used bioinformatics analysis with molecular, biochemical and physiological approaches to characterize STCS1s and PKRs involved in the biosynthesis of LA. The results indicated that MpSTCS1s from Marchantia polymorpha catalyzed both C2→C7 aldol-type and C6→C1 Claisen-type cyclization using dihydro-p-coumaroyl-coenzyme A (CoA) and malonyl-CoA as substrates to yield a C6-C2-C6 skeleton of dihydro-resveratrol following decarboxylation and the C6-C3-C6 type of phloretin in vitro. The protein-protein interaction of PKRs with STCS1 (PPI-PS) was revealed and proved essential for LA accumulation when transiently co-expressed in Nicotiana benthamiana. Moreover, replacement of the active domain of STCS1 with an 18-amino-acid fragment from the chalcone synthase led to the PPI-PS greatly decreasing and diminishing the formation of LA. The replacement also increased the chalcone formation in STCS1s. Our results highlight a previously unrecognized PPI in planta that is indispensable for the formation of LA.

Identification of a flavonoid C-glycosyltransferase from fern species Stenoloma chusanum and the application in synthesizing flavonoid C-glycosides in Escherichia coli.

BACKGROUND: Flavonoid C-glycosides have many beneficial effects and are widely used in food and medicine. However, plants contain a limited number of flavonoid C-glycosides, and it is challenging to create these substances chemically. RESULTS: To screen more robust C-glycosyltransferases (CGTs) for the biosynthesis of flavonoid C-glycosides, one CGT enzyme from Stenoloma chusanum (ScCGT1) was characterized. Biochemical analyses revealed that ScCGT1 showed the C-glycosylation activity for phloretin, 2-hydroxynaringenin, and 2-hydroxyeriodictyol. Structure modeling and mutagenesis experiments indicated that the glycosylation of ScCGT1 may be initiated by the synergistic action of conserved residue His26 and Asp14. The P164T mutation increased C-glycosylation activity by forming a hydrogen bond with the sugar donor. Furthermore, when using phloretin as a substrate, the extracellular nothofagin production obtained from the Escherichia coli strain ScCGT1-P164T reached 38 mg/L, which was 2.3-fold higher than that of the wild-type strain. Finally, it is proved that the coupling catalysis of CjFNS I/F2H and ScCGT1-P164T could convert naringenin into vitexin and isovitexin. CONCLUSION: This is the first time that C-glycosyltransferase has been characterized from fern species and provides a candidate gene and strategy for the efficient production of bioactive C-glycosides using enzyme catalysis and metabolic engineering.

Unprecedented 4,9-Seco-Oplopanane and Seven Drimane Sesquiterpenoids from the Chinese Liverwort Lejeunea flava (Sw.) Nees.

An unprecedented 4,9-seco-oplopanane (1), two undescribed drimane epimers (2 and 3), and five known drimane sesquiterpenoids (4-8) were isolated from the Chinese liverwort Lejeunea flava (Sw.) Nees. The structures of the new sesquiterpenoids were determined using nuclear magnetic resonance spectroscopy, electronic circular dichroism calculations, and single-crystal X-ray diffraction measurements. The inhibitory capacity of the new compounds against nitric oxide production in lipopolysaccharide-induced RAW 264.7 murine macrophages, along with the cytotoxicity of the new compounds against A549 and HepG-2 human cancer cell lines, were discussed.

Cytotoxic Activities of 9,10-seco-Cycloartane-Type Triterpenoids from the Chinese Liverwort Lepidozia reptans.

Ten new triterpenoids, including nine 9,10-seco-cycloartanes (1-9) and one 9,19-cyclolanostane (10), as well as one sesquiterpenoid (11) and four known compounds (12-15), were extracted and purified from the whole plant of the Chinese liverwort Lepidozia reptans. Multiple techniques (NMR, HRESIMS, IR, and X-ray crystallographic analysis) were applied to determine the structures of the isolated compounds. Bioassay determinations showed that compound 7, which contains an α,ß-unsaturated carbonyl moiety in its structure, inhibited the growth of a panel of cancer cell lines with IC50 values ranging from 4.2 ± 0.2 to 5.7 ± 0.5 µM. Further investigation revealed that compound 7 induces PC-3 cell death via mitochondrial-related apoptosis.

Diverse Prenylated Bibenzyl Enantiomers from the Chinese Liverwort Radula apiculata and Their Cytotoxic Activities.

An EtOH extract of the Chinese liverwort Radula apiculata showed cytotoxic activity against the A549 lung cancer cell line. Bioassay-guided fractionation led to the isolation of 19 prenylated bibenzyls, including eight previously unknown dimeric prenylated bibenzyls [radulapins A-H (1-8)], four new prenylated bibenzyls (9-12), and seven known compounds (13-19). Compounds 1-11 were analyzed as racemates by chiral-phase separation. Their structures were determined by detailed analysis of their spectroscopic data and by single-crystal X-ray diffraction, chiral resolutions, and electronic circular dichroism measurements. Using an MTT assay, these dimers (1-8) showed significant cytotoxic activity against a panel of human cancer cell lines. Further investigation revealed that compound 4 induces PC-3 cell death via mitochondrial-derived apoptosis.

Enantioselective Total Syntheses of Manginoids†A and†C and Guignardones†A and†C.

An enantioselective synthetic approach for preparing manginoids and guignardones, two types of biogenetically related meroterpenoids, is reported. This bioinspired and divergent synthesis employs an oxidative 1,3-dicarbonyl radical-initiated cyclization and cyclodehydration of the common precursor to forge the central ring of the manginoids and guignardones, respectively, at a late stage. Key synthetic steps include silica-gel-promoted semipinacol rearrangement to form the 6-oxabicyclo[3.2.1]octane skeleton and the Suzuki-Miyaura reaction of vinyl bromide to achieve fragment coupling. This synthesis protocol enables the asymmetric syntheses of four fungal meroterpenoids from commercially available materials.

Terpenoids from the Liverwort Plagiochila fruticosa and Their Antivirulence Activity against Candida albicans.

Fourteen new terpenoids plagicosins A-N (1-14), including seven sesquiterpenoids (1-7) consisting of six ent-bicyclogermacrenes and one ent-2,3-seco-aromadendrane, as well as seven diterpenoids (8-14) comprising five fusicoccanes, a eunicellane, and a rare gersemiane, were isolated from the Chinese liverwort Plagiochila fruticosa Mitt. The structures of these terpenoids were determined on the basis of comprehensive analysis of MS and NMR spectroscopic data coupled with electronic circular dichroism (ECD) and coupling constant calculations. Plagicosin F (6) displayed potent antivirulence activity through inhibiting the hyphal morphogenesis, adhesion, and biofilm formation of Candida albicans. The genes related to hyphal formation were regulated by 6.

Divergent Total Synthesis of Euphoranginol†C, Euphoranginone†D, ent-Trachyloban-3ß-ol, ent-Trachyloban-3-one, Excoecarin†E, and ent-16α-Hydroxy-atisane-3-one.

A divergent synthetic approach to biogenetically related diterpenoids such as ent-kauranes, ent-trachylobanes, ent-beyerane, and ent-atisane has been developed. The unified synthetic route involves the De Mayo reaction to rapidly generate the bicyclo[3.2.1]-octane moiety of ent-kaurane. The key reactions also include bioinspired nucleophilic cyclopropanation generating the [3.2.1.02,7 ]-tricyclic core of ent-trachylobane and regioselective cyclopropane fragmentation furnishing ent-beyerane and ent-atisane through the nucleophilic attack and protonation of the cyclopropane ring. This strategy enables the asymmetric total syntheses of six diterpenoids from the commercially available geraniol.

Botrysphin D attenuates arsenic-induced oxidative stress in human lung epithelial cells via activating Nrf2/ARE signaling pathways.

Oxidative stress is one of the main pathogenesis for many human diseases. Nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway plays a key role in regulating intracellular antioxidant responses, and thus activation of Nrf2/ARE signaling pathway is a potential chemopreventive or therapeutic strategy to treat diseases caused by oxidative damage. In the present study, we have found that treatment of Beas-2B cells with botrysphins D (BD) attenuated sodium arsenite [As (III)]-induced cell death and apoptosis. Meanwhile, BD was able to upregulate protein levels of Nrf2 and its downstream genes NQO1 and γ-GCS through inducing Nrf2 nuclear translocation, enhancing protein stability, and inhibiting ubiquitination. It was also found that BD-induced activation of the Nrf2/ARE pathway was regulated by PI3K, MEK1/2, PKC, and PERK kinases. Collectively, BD is a novel activator of Nrf2/ARE pathway, and is verified to be a potential preventive agent against oxidative stress-induced damage in human lung tissues.

Prenylated Bibenzyls from the Chinese Liverwort Radula constricta and Their Mitochondria-Derived Paraptotic Cytotoxic Activities.

Nine new prenylated bibenzyls, radstrictins A-I (1-9), and 11 known congeners were obtained from the Chinese liverwort Radula constricta. Their structures were identified by analysis of HRMS, NMR, and electronic circular dichroism data. Radstrictins A-F (1-6) were isolated as a racemate or scalemic mixtures. All the isolated compounds were subjected to cytotoxicity assessment. Methyl 2,4-dihydroxy-3-(3-methyl-2-butenyl)-6-phenethylbenzoate (10) exhibited significant activity against human lung cancer cell lines A549 and NCI-H1299 with IC50 values of 6.0 and 5.1 µM, respectively. Further research revealed that cell death triggered by 10 occurred via mitochondria-derived paraptosis.

Heptaketides from an Endolichenic Fungus Biatriospora sp. and Their Antifungal Activity.

Twelve new heptaketides, biatriosporins A-L (1-12), biatriosporin M (13) (a ramulosin derivative), and 19 known compounds (14-32) were isolated from the endolichenic fungus Biatriospora sp. (8331C). The structures of these compounds were determined by analyzing MS and NMR data. The absolute configurations of compounds 1, 2, 7, and 9 were determined by single-crystal X-ray diffraction analysis, whereas compound 10 was deduced with Mosher's method. Four of the compounds were active in an antifungal assay. The most potent compound, compound 4, also sensitized clinically derived azole-resistant Candida albicans strains to fluconazole (FLC). A mechanistic investigation revealed that 4 inhibited the function of efflux pumps and reduced the transcriptional expression of the efflux-pump-related genes CDR1 and CDR2.

ent-Eudesmane-Type Sesquiterpenoids from the Chinese Liverwort Chiloscyphus polyanthus var. rivularis.

Seven new ent-eudesmane-type sesquiterpenoids (1-7) and six known analogues (8-13) were isolated from the Chinese liverwort Chiloscyphus polyanthus var. rivularis. Their structures were determined from analysis of MS and NMR spectroscopic data and single-crystal X-ray diffraction. A cytotoxic evaluation showed that compound 1 exhibited weak inhibitory activity against the A549 cancer cell line with an IC50 value of 27.7 µM.

Four New Kaurane Diterpenoids from the Chinese Liverwort Jungermannia comata Nees.

In our continuing program to find new bioactive compounds from the Chinese liverworts, four new kaurane-type diterpenoids, (6ß)-kaur-16-ene-6,9-diol (1), (6ß,12ß)-kaur-16-ene-6,9,12-triol (2), (6ß)-kaur-16-ene-5,6,9-triol (3), and kaur-16-ene-9,19-diol (4), have been isolated from the Chinese liverwort Jungermannia comata Nees. Five known kaurane-type diterpenoids (5 - 9) and four known trachylobane-type diterpenoids (10 - 13) were also obtained. The structures of the new compounds were established unequivocally on the basis of spectroscopic data. The absolute configuration of compound 1 was established by comparing experimental and calculated electronic circular dichroism spectra.

Three new drimane-type sesquiterpenoids, chaetothyrins A-C, from an endolichenic fungus Chaetothyriales sp.

Three new drimane-type sesquiterpenoids chaetothyrins A-C (1-3), were isolated from an endolichenic fungus Chaetothyriales sp. (4341B). Their structures were determined by analysis of MS and NMR data as well as single-crystal X-ray diffraction analysis.

Polyacetylated labdane-type diterpenoids, ptychantins P-R from Chinese liverwort Ptychanthus striatus.

Three new polyacetylated labdane diterpenoids ptychantins P-R (1-3) and four known compounds (4-7) were isolated from an EtOH extract of the Chinese liverwort Ptychanthus striatus (Lehm. & Lindenb.) Nees. Their structures were established by extensive analysis of spectroscopic data (IR, UV, HRESIMS, 1D NMR, and 2D NMR).

Notolutesins A-J, dolabrane-type diterpenoids from the Chinese liverwort Notoscyphus lutescens.

Ten new dolabrane-type diterpenoids, notolutesins A-J (1-10), were isolated from the Chinese liverwort Notoscyphus lutescens, along with four known compounds. The structures of the new compounds were established on the basis of extensive spectroscopic data, and that of 1 was confirmed by single-crystal X-ray crystallography. The absolute configuration of 1 was determined by comparing its experimental and calculated electronic circular dichroism spectra. All of the isolates were evaluated for their cytotoxicity against a small panel of human cancer cell lines, and compound 1 exhibited an IC50 value of 6.2 µM against the PC3 human prostate cancer cell line.

Secondary metabolites from the Chinese liverwort Cephaloziella kiaeri.

Sixteen new clerodane diterpenoids, cephaloziellins A-P (1-16), and two known analogues (17 and 18) were isolated from an EtOH extract of the Chinese liverwort Cephaloziella kiaeri. The structures of the new compounds were elucidated from extensive spectroscopic data (IR, UV, HRESIMS, 1D NMR, and 2D NMR), and the structures of 5, 9, and 15 were confirmed by single-crystal X-ray diffraction analyses. The absolute configurations of all new compounds were established by comparing experimental and calculated electronic circular dichroism spectra.

cis-clerodane diterpenes from the liverwort Scapania ciliata.

Chemical investigation of the Chinese liverwort Scapania ciliata led to the isolation of four new cis-clerodane lactones, named ciliatolides A-D (1-4, resp.), among which compound 1 was found to be a tetranorclerodanoid. Their structures were determined by extensive analysis of spectroscopic data, and, in the case of compound 1, together with a single-crystal X-ray diffraction analysis. The absolute configurations were established by analysis of the CD spectra and by quantum-chemical CD calculations. The cytotoxicities of compounds 1-4 were preliminarily tested against the PC3 and MCF-7 cell lines.