Identification of diterpenoids from Salvia castanea Diels f. tomentosa Stib and their antitumor activities.

Four new diterpenoid tropolones, salvirrddones A-D (1-4), and four new icetexanes, salvirrddices A-D (9-12), along with thirteen new 11,12-seco-norabietane diterpenoids, salvirrddnor A-M (14-24, 31, 32) and sixteen known compounds (5-8, 13, 25-30, 33-37), were isolated from the roots and rhizomes of Salvia castanea Diels f. tomentosa Stib. Their structures were elucidated by comprehensive spectroscopic analyses, quantum chemical calculations, and X-ray crystallography. Structurally, compounds 1-8 represent a class of rare natural products featuring a unique cyclohepta-2,4,6-trienone moiety with diterpenoid skeletons. Bioassays showed that only diterpenoid tropolones 3, 5, 6, and 7 exhibited significant activity against several human cancer cell lines with IC50 values ranging from 3.01 to 11.63 µM. Additionally, 3 was shown to inhibit Hep3B cell proliferation, block the G0/G1 phase of the cell cycle, induce mitochondrial dysfunction and oxidative stress, promote apoptosis, as well as inhibit migration and invasion in vitro. Meanwhile, 3 demonstrated anti-proliferative, pro-apoptotic, and migration-inhibitory effects in the Hep3B xenograft zebrafish model in vivo. Network pharmacological analysis and molecular docking results suggested that 3 may treat hepatocellular carcinoma (HCC) through the PI3K-Akt signaling pathway, as well as by binding PARP1 and CDK2 targets. Overall, the present results extremely expand the repertoire of diterpenoids from natural products and may provide a novel chemical scaffold for the discovery of new antitumor drugs.

Characterization of the ADP-ß-D-manno-heptose biosynthetic enzymes from two pathogenic Vibrio strains.

ADP-activated ß-D-manno-heptoses (ADP-ß-D-manno-heptoses) are precursors for the biosynthesis of the inner core of lipopolysaccharide in Gram-negative bacteria. Recently, ADP-D-glycero-ß-D-manno-heptose (ADP-D,D-manno-heptose) and its C-6'' epimer, ADP-L-glycero-ß-D-manno-heptose (ADP-L,D-manno-heptose), were identified as potent pathogen-associated molecular patterns (PAMPs) that can trigger robust innate immune responses. Although the production of ADP-D,D-manno-heptose has been studied in several different pathogenic Gram-negative bacteria, current knowledge of ADP-ß-D-manno-heptose biosynthesis in Vibrio strains remains limited. Here, we characterized the biosynthetic enzymes of ADP-D,D-manno-heptose and the epimerase that converts it to ADP-L,D-manno-heptose from Vibrio cholerae (the causative agent of pandemic cholera) and Vibrio parahaemolyticus (non-cholera pathogen causing vibriosis with clinical manifestations of gastroenteritis and wound infections) in comparison with their isozymes from Escherichia coli. Moreover, we discovered that ß-D-mannose 1-phosphate, but not α-D-mannose 1-phosphate, could be activated to its ADP form by the nucleotidyltransferase domains of bifunctional kinase/nucleotidyltransferases HldEVC (from V. cholerae) and HldEVP (from V. parahaemolyticus). Kinetic analyses of the nucleotidyltransferase domains of HldEVC and HldEVP together with the E. coli-derived HldEEC were thus carried out using ß-D-mannose 1-phosphate as a mimic sugar substrate. Overall, our works suggest that V. cholerae and V. parahaemolyticus are capable of synthesizing ADP-ß-D-manno-heptoses and lay a foundation for further physiological function explorations on manno-heptose metabolism in Vibrio strains. KEY POINTS: • Vibrio strains adopt the same biosynthetic pathway as E. coli in synthesizing ADP-ß-D-manno-heptoses. • HldEs from two Vibrio strains and E. coli could activate ß-D-mannose 1-phosphate to ADP-ß-D-mannose. • Comparable nucleotidyltransfer efficiencies were observed in the kinetic studies of HldEs.

(±)-Salvicatone A: A Pair of C27-Meroterpenoid Enantiomers with Skeletons from the Roots and Rhizomes of Salvia castanea Diels f. tomentosa Stib.

(±)-Salvicatone A (1), a C27-meroterpenoid featuring a unique 6/6/6/6/6-pentacyclic carbon skeleton with a 7,8,8a,9,10,10a-hexahydropyren-1 (6H)-one motif, was isolated from the roots and rhizomes of Salvia castanea Diels f. tomentosa Stib. Its structure was characterized by comprehensive spectroscopic analyses along with computer-assisted structure elucidation, including ACD/structure elucidator and quantum chemical calculations with 1H/13C NMR and electronic circular dichroism. Biogenetically, compound 1 was constructed from decarboxylation following [4 + 2] Diels-Alder cycloaddition reaction between caffeic acid and miltirone analogue. Bioassays showed that (-)-1 and (+)-1 inhibited nitric oxide production in lipopolysaccharide-induced RAW264.7 macrophage cells with an IC50 value of 6.48 ± 1.25 and 15.76 ± 5.55 µM, respectively. The structure-based virtual screening based on the pharmacophores in ePharmaLib, as well as the molecular docking and molecular dynamics simulations study, implied that (-)-1 and (+)-1 may potentially bind to retinoic acid receptor-related orphan receptor C to exert anti-inflammatory activities.

Competitive CatSper Activators of Progesterone from Rhynchosia volubilis.

The root Rhynchosia volubilis was widely used for contraception in folk medicine, although its molecular mechanism on antifertility has not yet been revealed. In human sperm, it was reported that the cation channel of sperm, an indispensable cation channel for the fertilization process, could be regulated by various steroid-like compounds in plants. Interestingly, these nonphysiological ligands would also disturb the activation of the cation channel of sperm induced by progesterone. Therefore, this study aimed to explore whether the compounds in R. volubilis affect the physiological regulation of the cation channel of sperm. The bioguided isolation of the whole herb of R. volubilis has resulted in the novel discovery of five new prenylated isoflavonoids, rhynchones A - E (1:  - 5: ), a new natural product, 5'-O-methylphaseolinisoflavan (6: ) (1H and 13C NMR data, Supporting Information), together with twelve known compounds (7:  - 18: ). Their structures were established by extensive spectroscopic analyses and drawing a comparison with literature data, while their absolute configurations were determined by electronic circular dichroism calculations. The experiments of intracellular Ca2+ signals and patch clamping recordings showed that rhynchone A (1: ) significantly reduced cation channel of sperm activation by competing with progesterone. In conclusion, our findings indicat that rhynchone A might act as a contraceptive compound by impairing the activation of the cation channel of sperm and thus prevent fertilization.

A novel ent-pimarane-type diterpenoid from Sigesbeckia glabrescens with anti-inflammatory activity.

A novel ent-pimarane-type diterpenoid, sigesbeckia J (1), along with two known diterpenoids, siegesbeckia acid (2) and ent-18-acetoxy-16R,17-dihydroxykauran-19-oic acid (3), were isolated from the aerial parts of Sigesbeckia glabrescens Makino. Their chemical structures were elucidated based on extensive spectroscopic interpretation. The absolute configuration of ent-pimarane-type diterpenoid (1) was determined by comparing experimental and calculated ECD spectra. Compared with the positive control minocycline (IC50 32.84 µM), compound 1 exhibited moderate cell growth anti-inflammatory activities in vitro by testing their inhibition of LPS-induced NO production in BV2 microglial cells, with IC50 value of 58.74 µM.

[A new norsesquiterpenoid from Schisandra chinensis].

This study investigated the chemical components from the leaves and stems of Schisandra chinensis. Three norsesquiterpenoids were isolated from S. chinensis by various column chromatographies(silica gel, Sephadex LH-20, and MCI), reversed-phase medium-pressure preparative, and semi-preparative high-performance liquid chromatography(HPLC). Their structures were identified based on physicochemical properties, mass spectrometry(MS), nuclear magnetic resonance(NMR), ultraviolet(UV), and electro-nic circular dichroism(ECD) as(3R,4R,5R,6S,7E)-3,4,5,6-tetrahydroxy-7-megastigmen-9-one(1),(3S,5R,6R,7E)-3,5,6-trihydroxy-7-megastigmen-9-one(2), and(3S,4R,9R)-3,4,9-trihydroxymegastigman-5-ene(3). Compound 1 was a new compound, and its absolute configuration was determined by ECD. Compounds 2 and 3 were isolated from the Schisandra plant for the first time.

Alkaloids of Delphinium grandiflorum and their implication to H2O2-induced cardiomyocytes injury.

Three new diterpenoid alkaloids (1-3), and eight known alkaloids (4-11) were isolated from the aerial parts of Delphinium grandiflorum. Grandifline A (1) represents an unprecedented diterpenoid alkaloid ring system featuring a C-7NC17 hemiaminal moiety and a lactone fragment through the linkage of C-17OC19 unit. And we named this newly-discovered class of rearranged C19-diterpenoid alkaloid scaffold as grandiflodines (B-12). Grandifline B (2) is the first naturally-occurring 7,17-secolycoctonine diterpenoid alkaloid with a C-7OC17 unit forming a hemiacetal. Their structures were elucidated via spectroscopic data and single-crystal X-ray diffraction analysis. The protective effects of compounds 1-11 on H2O2-induced cardiomyocytes injury were assayed. And compounds 6 and 10 showed significant protective effects, with IC50 values of 1.881 ± 0.680 µM and 1.904 ± 0.750 µM, respectively. Further, compound 6 could reduce oxidative damage by inhibiting cell death via the AMPK/AKT/mTOR signaling pathway in H2O2-induced H9C2 cells.

Sesquiterpene Lactones from Sigesbeckia glabrescens Possessing Potent Anti-inflammatory Activity by Directly Binding to IKKα/ß.

Chromatographic fractionation of Sigesbeckia glabrescens led to the identification of 10 new sesquiterpene lactones, named siegesbeckialides I-O (1-7) and glabrescones A-C (8-10), along with 14 known analogues. An anti-inflammatory activity assay showed that siegesbeckialide I (1) most potently inhibited LPS-induced NO production in RAW264.7 murine macrophages. Furthermore, siegesbeckialide I suppressed the protein expression of iNOS and COX2, as well as the release of PGE2, IL-1ß, IL-6, and TNF-α in LPS-stimulated RAW264.7 cells. Mechanistically, siegesbeckialide I directly binds to inhibitors of IKKα/ß and suppresses their phosphorylation. This leads to the inhibition of IKKα/ß-mediated phosphorylation and degradation of inhibitor α of NF-κB (IκBα), as well as the activation of NF-κB signaling.

Euphorfistrines A-G, cytotoxic and AChE inhibiting triterpenoids from the roots of Euphorbia fischeriana.

Seven new triterpenoids including two cycloartanes (1-2), a lanostane (3), a tirucallane (4), a dammarane (5), an ursane (6), and an oleanane (7), along with nineteen known triterpenoids (8-26), have been obtained from the roots of Euphorbia fischeriana. Their structures were established by NMR, HRESIMS, single-crystal X-ray diffraction analysis, Mosher's method, NMR calculations, ECD analysis, and comparison with structurally related known analogues. Among them, compounds 1 and 8 were a pair of cycloartane-type triterpenoids epimers. Our bioassays have established that compounds 1-5 and 10 displayed moderate cytotoxic effects, and the structure-activity relationships of cycloartane-type triterpenoids (CTTs) were further examined. Notably, some triterpenoids displayed moderate inhibitory effects against AChE by an in vitro screened experiment. Triterpenoid 7 (Euphorfistrine G, ETG) displayed the potent inhibitory effect with IC50 = 2.45 and Ki = 2.30 µM (inhibition kinetic). And, in silico docking analyses have been performed to investigate the inhibitory mechanism of compound 7.

Baphicacanthcusines A-E, Bisindole Alkaloids from the Leaves of Baphicacanthus cusia (Nees) Bremek.

Four pairs of stereoisomeric indole alkaloids, (±)-baphicacanthcusines A-D (1-4), and one new indole alkaloid, baphicacanthcusine E (5), together with nine known compounds were identified from the leaves of Baphicacanthus cusia. (±)-1 and -2 possess an unprecedented skeleton in which two indole moieties are bridged by a phenylpropane unit. (±)-3 represents the first natural dispiro-oxazolidinone bisoxindoles. The absolute configurations in 1-5 were assigned based on quantum chemical calculations, including the calculated chemical shift with DP4plus analysis, the calculated optical rotation values, and the calculated electronic circular dichroism spectra. A plausible biosynthetic pathway for 1-5 was proposed. Compounds (±)-1, (-)-2, and 11 exhibited cytotoxicity against MCF-7 cells with IC50 values of 20.0-78.5 µM.

ent-Pimarane diterpenoids from Siegesbeckia glabrescens with anti-inflammatory activity.

Nine new ent-pimarane-type diterpenoids, siegesbeckia A-I (1-9), together with four known analogues ent-3α,15,16,19-tetrahydroxypimar-8(14)-ene (10), 15,16-dihydroxypimar-8(14)-en-3-one (11), 14ß,16-epoxy-7-pimarene-3α,15ß-diol (12) and darutigenol (13), were obtained from the aerial parts of Siegesbeckia glabrescens Makino. The structures of these compounds were elucidated by the interpretation of HRESIMS, 1D NMR and 2D NMR data. Their configurations were determined by ECD analysis and the structure of compound 1 was confirmed by X-ray crystallography. Putative biosynthetic pathways were proposed for 1-13. The anti-inflammatory effects of the compounds were evaluated by testing their inhibition of LPS-induced NO production in BV2 microglial cells. The results revealed that new compounds 2, 6 and 8 exhibited potent inhibitory activities with IC50 values of 33.07, 42.39 and 63.26 µM, which compared well with the positive control minocycline (IC50 32.84 µM).

Two new alcohol glycosides from the roots of Paeonia intermedia C. A. Meyer.

Two new alcohol glycosides, 1-O-ß-d-glucopyranosyl-deoxypaeonisuffrone (1) and 9-O-ß-d-apiofuranoyl-(1→6)-ß-d-glucopyranosyl-xanthoarnol (2), together with eight known compounds (3-10), have been isolated from the dried roots of Paeonia intermedia C. A. Meyer. Their structures were mainly elucidated on the basis of ESIMS, one- and two-dimensional NMR techniques. Antibacterial activities of compounds 1-10 were evaluated, and compounds 9 and 10 showed antibacterial activities against Staphylococcus argenteus CMCC26003 and Escherichia coli CMCC44103. [Formula: see text].

Effects of Deacidification on Composition of Schisandra chinensis Ethanolic Extract and Studies on Acute Toxicity in Mice.

Schisandra chinensis Fructus (SCF), a well-known traditional medicinal material, is a rich source of dibenzocyclooctene type lignans and polyphenols, which are important ingredients in SCF and show various activities. SCF also contains about 18% organic acids, mainly citric acid, which makes the fruit and extract taste extremely sour and limited its application in beverages or food industries. In the present study, a chemical deacidification method was applied to defatted and non-defatted ethanol extract of SCF, and the effects on organic acid, lignans, and phenolic compounds were evaluated. Free radical scavenging activity and acute toxicity in mice before and after deacidification were also compared. Our results demonstrated that chemical deacidification significantly decreased the contents of organic acid and lignan compounds and markedly improves the safety of the ethanol extract of SCF, which will facilitate the comprehensive utilization of SCF extract in food and beverage industries.

Screening of an Endophyte Transforming Polydatin to Resveratrol from Reynoutria Japonica Houtt and the Optimization of Its Transformation Parameters.

Resveratrol showed various kinds of bioactivities, such as antioxidant, antimicrobial, anticancer effects and, therefore, has been used widely as an important ingredient in medication, healthy foods and cosmetics. However, in nature, resveratrol usually exists at low content and more often exists as polydatin. Therefore, it becomes important to find the cost-effective and environmental-friendly way to transform polydatin to resveratrol. In this study, endophytes were isolated from the rhizome tissue of Reynoutria japonica and screened for transforming polydatin to resveratrol using reversed-phase high-performance liquid chromatography (RP-HPLC) and confirmed by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) spectroscopy. A bacterium identified as Bacillus aryabhattai using 16S rRNA phylogenetic tree analysis showed highest transformation rate. The transforming conditions were optimized including substrate concentration, substrate addition time, culture temperature and inoculation ratio. Our results demonstrated that the bacteria isolated from R. japonica rhizome tissue showed high activity in transforming polydatin into resveratrol. Crude extract of R. japonica root and rhizome (RJE) was also tested as substrate and it was found that the transformation was significantly inhibited at 10.0 mg/mL RJE. Emodin at equivalent concentration of 10.0 mg/mL RJE showed no inhibition activity, and glucose content in RJE was trace and far from enough to exhibit the inhibitory activity. Successive solvent partition followed by an inhibition activity assay revealed that the ethyl acetate fraction showed the main inhibition activity. However, due to the coexistence of polydatin and compounds with inhibitory activity, the concentration of RJE can only be used at limited concentration as substrate.

Comparative Studies on Polysaccharides, Triterpenoids, and Essential Oil from Fermented Mycelia and Cultivated Sclerotium of a Medicinal and Edible Mushroom, Poria Cocos.

Poria cocos, an important medicinal and edible fungus, is well known in East Asia. The main active components are water-soluble polysaccharides (WPS) and triterpenoids. Due to the growing market demand, long cultivation period, and consumption of pine trunk during cultivation, alternative methods for producing P. cocos or its active components should be investigated. In this study, WPS, triterpenoids, monosaccharide composition, and essential oil in fermented mycelia and cultivated sclerotium were analyzed using UV spectrophotometry, HPLC, pre-column derivatization, and HS-GC/MS, respectively. Our results showed that the WPS and triterpenoids in mycelia are several times higher than those in sclerotium. Among the 62 compounds identified by HS-GC/MS analysis from the essential oil obtained from the fermentation media and a fresh external layer, the two main fragrances in common were linalool and methyl phenylacetate. Our results suggested that it is applicable to produce polysaccharides and triterpenoids by the fermentation of P. cocos, and a strategy to improve triterpenoid production in the fermentation process was proposed.

Constituents from the Seeds of Sophora Alopecuroides L.

Three new isoflavone glucosides, kudonol A-C (1-3), two new ester derivatives of phenylpropanoid, kudolignan A and B (4-5) and five known compounds, (-)-maackiain (6), neoliquiritin (7), methyl 4-coumarate (8), methyl ferulate (9) and (+)-wikstromol (10), were isolated from an extract of dried seeds of the traditional Chinese medicinal plant Sophora alopecuroides L. Their structures were established by NMR and HRESIMS data analyses. The monosaccharide part's configuration of isoflavone glucosides was confirmed by acid hydrolysis and analyzed by a JAsco OR-4090 chiral detector, comparing it to standard substance D-glucose. The cytotoxicity effects against HeLa, Hep3B, MCF-7 and H1299 cells were tested by CCK-8 assay.

A sensitive UPLC-MS/MS method for simultaneous determination of polyphenols and theaflavins in rat plasma: Application to a pharmacokinetic study of Da Hong Pao tea.

A method based on ultra-performance liquid chromatography-tandem mass spectrometry has been developed for the rapid and simultaneous determination of five catechins and four theaflavins in rat plasma using ethyl gallate as internal standard. The pharmacokinetic profiles of these compounds were compared after oral administration of five kinds of Da Hong Pao tea to rats. Biosamples processed with a mixture of ß-glucuronidase and sulfatase were extracted with ethyl acetate-isopropanol. Chromatographic separation was achieved by gradient elution using 10 mm HCOONH4 solution and methanol as the mobile phase. Analytes were detected using negative ion electrospray ionization in multiple reaction monitoring mode. The lower limits of quantification were 1.0, 0.74 and 0.5 ng/mL for theaflavins, two catechins and three catechins, respectively. The validation parameters were well within acceptable limits. The average half-lives (t1/2 ) in blood of the reference solution group was much shorter than those of tea samples. The values of AUC0-t and Cmax of the polyphenols and theaflavins exhibited linear pharmacokinetic characteristics which were related to the dose concentration.

Paris saponin VII induces cell cycle arrest and apoptosis by regulating Akt/MAPK pathway and inhibition of P-glycoprotein in K562/ADR cells.

Paris saponinVII (PSVII) is a steroidal saponin isolated from the roots and rhizomes of Trillium tschonoskii Maxim. We found that PSVII could inhibit the growth of adriamycin-resistant human leukemia cells (K562/ADR) in a dose-dependent manner. Furthermore, the molecular mechanism underlying the cytotoxicity and downregulation of P-glycoprotein (P-gp) expression by PSVII was clarified. PSVII significantly suppressed cell proliferation by cell cycle arrest in the G0/G1 phase, which was associated with an obvious decrease in cyclin B1/D1 and CDK2/4/6 protein expression. Moreover, PSVII could attenuate mitochondrial membrane potential, increase the expression of apoptosis-related proteins, such as Bax and cytochrome c, and decrease the protein expression levels of Bcl-2, caspase-9, caspase-3, PARP-1, and p-Akt. We also found that JNK, ERK1/2, and p38 were regulated by PSVII in K562/ADR cells. And further studies indicated that the decrease in the reactive oxygen species level inhibited intrinsic P-gp expression. Therefore, PSVII-induced apoptosis in K562/ADR cells was associated with Akt/MAPK and the inhibition of P-gp. In addition, PSVII induced a robust autophagy in K562/ADR cells as demonstrated by the degradation of LC3-I. These results provide a biochemical basis for possible clinical applications of PSVII in the treatment of leukemia.

Chemical characteristics of the fungus Ganoderma lucidum and their inhibitory effects on acetylcholinesterase.

The chemical characteristic of a well-known folk medicine Ganoderma lucidum has been investigated by a series of chromatographic technologies, which displayed the presences of 45 lanostane type triterpenoids, including two new nor-lanostane triterpenoids (40, 41). Their structures were identified on the basis of spectroscopic data analysis (UV, IR, HRESIMS, 1D, and 2D NMR). Notably, some triterpenoids displayed moderate inhibitory effects against AChE (acetylcholinesterase) by an in vitro screened experiment. Triterpenoid 2 displayed the potent inhibitory effect with IC50 10.8 and Ki 14.95 µM (inhibition kinetic). The preliminary SAR has been discussed by the docking analyses between ganoderic acids (1, 2) and AChE.

Diterpenoids from the roots of Euphorbia fischeriana and their inhibitory effects on α-glucosidase.

Chemical investigation has been performed on the roots of Euphorbia fischeriana, a traditional Chinese medicine. Three diterpenoids were obtained using various chromatographic techniques, and their structures were determined by spectroscopic data including HRESIMS, 1D NMR, 2D NMR, ECD, and calculated ECD, which gave two new diterpenoids, daphnane type (1) and ent-pimarene type (3). Additionally, the isolated compounds (1-3) displayed moderate inhibitory effects against α-glucosidase in an in vitro bioassay.