Lindenane sesquiterpenoid dimers from Chloranthus japonicus improve LDL uptake by regulating PCSK9 and LDLR.

UPLC-TOF-MS/MDF directed phytochemical research of Chloranthus japonicus led to the isolation of 46 lindenane sesquiterpenoid dimers, which included 13 new analogs. Their structures with absolute configurations were elucidated by analysis of spectroscopic data. Fourteen compounds with ester chains significantly decreased PCSK9 protein level in medium of HepG2 cells, especially for compounds 14 and 29 (5 µM) with inhibition rates of 69.0% and 72.8%, respectively. Compound 14 in HepG2 cells was evaluated via DiI-LDL uptake assays and found to increase LDL uptake by upregulating LDLR mRNA and protein level. Meanwhile, 14 decreased the secretion of PCSK9 protein in medium and downregulated intracellular PCSK9 protein and mRNA level. The discovery of these natural small molecule compounds provides a novel structure basis for design PCSK9 regulators, making them a promising lead for development of new lipid-lowering agents.

Calysepins I-VII, Hexasaccharide Resin Glycosides from Calystegia sepium and Their Cytotoxic Evaluation.

Seven new hexasaccharide resin glycosides, named calysepins I-VII (1-7), with 27-membered rings, were obtained from the aerial parts of Calystegia sepium. Their structures with absolute configuration were established on the basis of spectroscopic data interpretation analysis and the use of chemical methods. They were defined as hexasaccharides composed of one d-quinovose, four d-glucose, and one l-rhamnose unit, and their sugar moieties were partially acylated by (2S)-methylbutanoic acid in 1-7 and (2R,3R)-nilic acid in 1-5 and 7, which mainly differed at the positions of acylation. Additionally, calysepin IV (4) exhibited cytotoxicity against A549 cells with an IC50 value of 5.2 µM.

Anti-inflammatory sesquiterpene dimers and diterpenes from the aerial part of Inula japonica.

Phytochemical investigation on the 95% alcohol extract of the aerial part of Inula japonica led to the isolation of three new compounds, inulanolides F-G (1-2) and 17α-carboxaldehyde-ent-kaur-18-oic acid (3), together with four known compounds (4-7). The structures of new compounds were elucidated by using spectroscopic data. Most of the isolated compounds showed significant anti-inflammatory activities.

Production and Structural Diversification of Withanolides by Aeroponic Cultivation of Plants of Solanaceae: Cytotoxic and Other Withanolides from Aeroponically Grown Physalis coztomatl.

Withanolides constitute one of the most interesting classes of natural products due to their diversity of structures and biological activities. Our recent studies on withanolides obtained from plants of Solanaceae including Withania somnifera and a number of Physalis species grown under environmentally controlled aeroponic conditions suggested that this technique is a convenient, reproducible, and superior method for their production and structural diversification. Investigation of aeroponically grown Physalis coztomatl afforded 29 withanolides compared to a total of 13 obtained previously from the wild-crafted plant and included 12 new withanolides, physacoztolides I-M (9-13), 15α-acetoxy-28-hydroxyphysachenolide C (14), 28-oxophysachenolide C (15), and 28-hydroxyphysachenolide C (16), 5α-chloro-6ß-hydroxy-5,6-dihydrophysachenolide D (17), 15α-acetoxy-5α-chloro-6ß-hydroxy-5,6-dihydrophysachenolide D (18), 28-hydroxy-5α-chloro-6ß-hydroxy-5,6-dihydrophysachenolide D (19), physachenolide A-5-methyl ether (20), and 17 known withanolides 3-5, 8, and 21-33. The structures of 9-20 were elucidated by the analysis of their spectroscopic data and the known withanolides 3-5, 8, and 21-33 were identified by comparison of their spectroscopic data with those reported. Evaluation against a panel of prostate cancer (LNCaP, VCaP, DU-145, and PC-3) and renal carcinoma (ACHN) cell lines, and normal human foreskin fibroblast (WI-38) cells revealed that 8, 13, 15, and 17-19 had potent and selective activity for prostate cancer cell lines. Facile conversion of the 5,6-chlorohydrin 17 to its 5,6-epoxide 8 in cell culture medium used for the bioassay suggested that the cytotoxic activities observed for 17-19 may be due to in situ formation of their corresponding 5ß,6ß-epoxides, 8, 27, and 28.

Anti-Fibrosis Effects of Magnesium Lithospermate B in Experimental Pulmonary Fibrosis: By Inhibiting TGF-ßRI/Smad Signaling.

Pulmonary fibrosis is a severe and irreversible interstitial pulmonary disease with high mortality and few treatments. Magnesium lithospermate B (MLB) is a hydrosoluble component of Salvia miltiorrhiza and has been reported to have antifibrotic effects in other forms of tissue fibrosis. In this research, we studied the effects of MLB on pulmonary fibrosis and the underlying mechanisms. Our results indicated that MLB treatment (50 mg/kg) for seven days could attenuate bleomycin (BLM)-induced pulmonary fibrosis by reducing the alveolar structure disruption and collagen deposition in the C57 mouse model. MLB was also found to inhibit transforming growth factor-beta (TGF-ß)-stimulated myofibroblastic transdifferentiation of human lung fibroblast cell line (MRC-5) cells and collagen production by human type II alveolar epithelial cell line (A549) cells, mainly by decreasing the expression of TGF-ß receptor I (TGF-ßRI) and regulating the TGF-ß/Smad pathway. Further studies confirmed that the molecular mechanisms of MLB in BLM-induced pulmonary fibrosis mice were similar to those observed in vitro. In summary, our results demonstrated that MLB could alleviate experimental pulmonary fibrosis both in vivo and in vitro, suggesting that MLB has great potential for pulmonary fibrosis treatment.

A novel bile acid analog, A17, ameliorated non-alcoholic steatohepatitis in high-fat diet-fed hamsters.

Being endocrine signaling molecules that regulate lipid metabolism and affect energy balance, bile acids are potential drug candidates for non-alcoholic steatohepatitis (NASH). Obeticholic acid (OCA) could improve NASH accompanied by significant side effects. Therefore, it is worthwhile to develop safer and more effective bile acid analogs. In this study, a new bile acid analog A17 was synthesized and its potential anti-NASH effects were assessed in vitro and in vivo. The impact of A17 on steatosis was investigated in the rat primary hepatocytes challenged with oleic acid. It was found that A17 alleviated lipid accumulation by reducing fatty acid (FA) uptake and promoting FA oxidation. The reduction of FA uptake came from inhibiting fatty acid translocase (Cd36) expression. The promotion of FA oxidation came from stimulating the phosphorylation of adenosine monophosphate (AMP)-activated protein kinase alpha (AMPKα). In addition, A17 reduced lipopolysaccharide-induced inflammation in Raw264.7 cells by activating Takeda G protein-coupled receptor 5 (TGR5). In in vivo study, male Golden Syrian hamsters were fed with high fat (HF) diet and then treated with 50 mg/kg/d A17 for 6 weeks. A17 lowered the lipid profiles and liver enzyme levels in serum and improved liver pathological conditions with less side effects compared with OCA. Further studies confirmed that the molecular mechanisms of A17 in vivo were similar to those in vitro. In conclusion, a novel bile acid analog A17 was identified to ameliorate NASH in HF-fed hamsters. The potential mechanisms could be contributed to reducing FA uptake, stimulating FA oxidation and relieving inflammation.

Lipid-Lowering Activities of Cucurbitacins Isolated from Trichosanthes cucumeroides and Their Synthetic Derivatives.

In the ongoing efforts to discover natural cholesterol-lowering compounds, dihydrocucurbitacin B, isolated from Trichosanthes cucumeroides roots, was found to promote LDL uptake by upregulating LDLR protein in a PCSK9-dependent process. In this study, an in-depth investigation of T. cucumeroides roots afforded 27 cucurbitacins (1-27), including seven new cucurbitacins (1-7), and their structures were elucidated by spectroscopic data analyses. In order to gain insight into their structure-activity relationship, cucurbitacin derivatives (B1-11 and DB1-11) were synthesized. Evaluation of lipid-lowering activities of these cucurbitacins by an LDL uptake assay in HepG2 cells revealed that most of the compounds improved the LDL uptake rate, among which hexanorisocucurbitacin D (6) and isocucurbitacin D (21) exhibited the highest activities (rates of 2.53 and 2.47, respectively), which were comparable to that of the positive control, nagilactone B (rate of 2.07). According to a mechanistic study by Western blot analysis, compounds 6 and 21 dose-dependently increased LDLR protein levels and reduced PCSK9 protein levels, representing promising new lipid-lowering drug candidates.

23,24-Dihydrocucurbitacin B promotes lipid clearance by dual transcriptional regulation of LDLR and PCSK9.

23,24-Dihydrocucurbitacin B (designated as C95 in this article) is a cucurbitane triterpenoid that has been shown to possess a variety of pharmacological activities, such as anti-inflammatory and anti-HIV-1 activities etc. In this study, we investigated the effects of 23,24-dihydrocucurbitacin B on lipid regulation. We showed that 23,24-dihydrocucurbitacin B (1-5 µM) dose-dependently promoted DiI-LDL uptake in HepG2 cells by upregulating low-density lipoprotein receptor (LDLR) protein. In HepG2 cells, 23,24-dihydrocucurbitacin B (1-10 µM) dose-dependently enhanced LDLR promoter activity by elevating the mature form of SREBP2 (sterol regulatory element binding protein 2) protein levels on one hand, and inhibited PCSK9 (proprotein convertase subtilisin/kexin type 9) promoter activity by attenuating HNF1α (hepatocyte nuclear factor-1α) protein levels in nuclei on the other hand. Consequently, the expression of LDLR protein markedly increased, whereas the PCSK9-mediated LDLR protein degradation decreased. In a high-cholesterol LVG golden Syrian Hamster model, administration of 23,24-dihydrocucurbitacin B (30 mg · kg-1⋅ d-1, intragastric, for 3 weeks) significantly decreased the serum LDL-cholesterol (LDL-C) levels. PCSK9 protein levels in the serum and liver tissues were significantly decreased, whereas LDLR protein levels in liver tissues were significantly increased in the treated animals as compared with the control animals. In conclusion, our study demonstrates for the first time that 23,24-dihydrocucurbitacin B exhibits dual transcriptional regulation of LDLR and PCSK9 in HepG2 cells by increasing SREBP2 protein levels and decreasing HNF1α protein levels in the nuclei. These results propose a new strategy to simultaneously manage LDLR and PCSK9 protein expression and provide a promising lead compound for drug development.

Teratopyrones A-C, Dimeric Naphtho-γ-Pyrones and Other Metabolites from Teratosphaeria sp. AK1128, a Fungal Endophyte of Equisetum arvense.

Bioassay-guided fractionation of a cytotoxic extract derived from a solid potato dextrose agar (PDA) culture of Teratosphaeria sp. AK1128, a fungal endophyte of Equisetum arvense, afforded three new naphtho-γ-pyrone dimers, teratopyrones A-C (1-3), together with five known naphtho-γ-pyrones, aurasperone B (4), aurasperone C (5), aurasperone F (6), nigerasperone A (7), and fonsecin B (8), and two known diketopiperazines, asperazine (9) and isorugulosuvine (10). The structures of 1-3 were determined on the basis of their spectroscopic data. Cytotoxicity assay revealed that nigerasperone A (7) was moderately active against the cancer cell lines PC-3M (human metastatic prostate cancer), NCI-H460 (human non-small cell lung cancer), SF-268 (human CNS glioma), and MCF-7 (human breast cancer), with IC50s ranging from 2.37 to 4.12 µM while other metabolites exhibited no cytotoxic activity up to a concentration of 5.0 µM.

Efficient ligand discovery from natural herbs by integrating virtual screening, affinity mass spectrometry and targeted metabolomics.

Although natural herbs have been a rich source of compounds for drug discovery, identification of bioactive components from natural herbs suffers from low efficiency and prohibitive cost of the conventional bioassay-based screening platforms. Here we develop a new strategy that integrates virtual screening, affinity mass spectrometry (MS) and targeted metabolomics for efficient discovery of herb-derived ligands towards a specific protein target site. Herb-based virtual screening conveniently selects herbs of potential bioactivity whereas affinity MS combined with targeted metabolomics readily screens candidate compounds in a high-throughput manner. This new integrated approach was benchmarked on screening chemical ligands that target the hydrophobic pocket of the nucleoprotein (NP) of Ebola viruses for which no small molecule ligands have been reported. Seven compounds identified by this approach from the crude extracts of three natural herbs were all validated to bind to the NP target in pure ligand binding assays. Among them, three compounds isolated from Piper nigrum (HJ-1, HJ-4 and HJ-6) strongly promoted the formation of large NP oligomers and reduced the protein thermal stability. In addition, cooperative binding between these chemical ligands and an endogenous peptide ligand was observed, and molecular docking was employed to propose a possible mechanism. Taken together, we established a platform integrating in silico and experimental screening approaches for efficient discovery of herb-derived bioactive ligands especially towards non-enzyme protein targets.

Magnesium lithospermate B ameliorates microcirculation perfusion in rats by promoting vascular NO production via activating the PI3K/AKT pathway.

Microcirculation morphologically refers to the blood flow in vessels of less than 150 µm in diameter, including arterioles, capillaries and venules, which provides nutrients and removes metabolic byproducts within tissues. Microcirculation dysfunction is involved in the pathological progress of many diseases, such as obesity, hypertension, and insulin resistance. In this study we investigated the effects of magnesium lithospermate B (MLB), an active compound of the traditional Chinese medicine Slavia miltiorrhiza, on the microcirculation dysfunction in rats and the underlying molecular mechanisms. The effects of MLB on microcirculation were assessed in vivo by measuring the hindlimb blood perfusion in dextran-induced microcirculation dysfunction rats and mesentery blood flow in anesthetized rats. We demonstrated that administration of MLB restored the impaired rat hindlimb blood flow and promoted the mesenteric micoperfusion in vivo. We further revealed in these two animal models that MLB treatment significantly increased the production of total nitrite in vascular tissues (mesentery, aorta, and heart), which was confirmed in human microvascular endothelial cells (HMEC-1) treated with MLB in vitro. Moreover, we showed that MLB treatment significantly increased the phosphorylation of endothelium nitric oxide synthase (eNOS) via inducing AKT phosphorylation in vivo and in vitro. Co-administration of the eNOS inhibitor L-NAME (20 mg/kg) abolished the protective effects of MLB against dextran-induced microcirculation dysfunction in rats, whereas pretreatment with PI3K inhibitor LY294002 (10 µM) prevented eNOS activation in MLB-treated HMEC-1 cells. Our results suggest that MLB can restore the microcirculation dysfunction via activating eNOS, and in turn enhancing the vascular nitric oxide production, which is medicated by MLB-caused activation of the PI3K/AKT pathway.

Magnesium lithospermate B protects the endothelium from inflammation-induced dysfunction through activation of Nrf2 pathway.

Magnesium lithospermate B (MLB) is an active component of Salvia miltiorrhiza Radix, a traditional Chinese herb used in treating cardiovascular diseases. In this study, we investigated the protective effects of MLB against inflammation-induced endothelial dysfunction in vitro and in vivo, and the underlying mechanisms. Endothelial dysfunction was induced in human dermal microvascular endothelial cells (HMEC-1) in vitro by lipopolysaccharide (LPS, 1 µg/mL). We showed that pretreatment with MLB (10-100 µM) dose-dependently inhibited LPS-induced upregulation of inflammatory cytokines ICAM1, VCAM1, and TNFα, which contributed to reduced leukocytes adhesion and attenuation of endothelial hyperpermeability in HMEC-1 cells. SD rats were injected with LPS (10 mg/kg, ip) to induce endothelial dysfunction in vivo. We showed that pretreatment with MLB (25-100 mg/kg, ip) dose-dependently restored LPS-impaired endothelial-dependent vasodilation in superior mesenteric artery (SMA), attenuated leukocyte adhesion in mesenteric venules and decreased vascular leakage in the lungs. We further elucidated the mechanisms underlying the protective effects of MLB, and revealed that MLB pretreatment inhibited NF-κB activation through inhibition of IκBα degradation and subsequent phosphorylation of NF-κB p65 in vitro and in vivo. In HMEC-1 cells, MLB pretreatment activated the nuclear factor erythroid-2-related factor 2 (Nrf2) pathway. Knockdown of Nrf2 with siRNA abolished the inhibitory effects of MLB on IκBα degradation and ICAM1 up-regulation, which were mimicked by PKC inhibition (Gö6983) or PI3K/Akt inhibition (LY294002). In summary, our results demonstrate that MLB inhibits NF-κB activation through PKC- and PI3K/Akt-mediated Nrf2 activation in HMEC-1 cells and protects against LPS-induced endothelial dysfunction in murine model of acute inflammation.

Pd-Catalyzed Acetoxylation of γ-C(sp3)-H Bonds of Amines Directed by a Removable Bts-Protecting Group.

Pd-catalyzed acetoxylation of γ-C(sp3)-H bonds directed by Bts-protected amines using inexpensive PhI(OAc)2 as oxidant is reported. The Bts-protecting group is easily introduced and removed under mild conditions. This protocol provides an important strategy for the construction of γ-hydroxyl amine derivatives.

Pd-catalyzed intramolecular C(sp2)-H amination of phenylalanine moieties in dipeptides: synthesis of indoline-2-carboxylate-containing dipeptides.

A palladium-catalyzed intramolecular C(sp2)-H amination of phenylalanine moieties in dipeptides is described. By this protocol, a series of indoline-2-carboxylate-containing dipeptides were synthesized from dipeptides. The N-protected amino acid moiety within the peptide is used as an intrinsic bidentate directing group. This intramolecular C-H amination reaction provides an appealing strategy for the post-synthetic modification of peptides.

Crassins A-H, Diterpenoids from the Roots of Croton crassifolius.

Phytochemical investigation of the 70% acetone extract of Croton crassifolius roots afforded eight new diterpenoids, crassins A-H (1-8), and 19 known compounds. The structures of the new compounds were determined by spectroscopic methods, and their absolute configurations were determined by electronic circular dichroism, single-crystal X-ray diffraction analysis, comparison with literature data, and biogenetic considerations. Crassins A (1) and B (2) are new ring B-rearranged diterpenoids, whereas crassin C (3) is a new ring A-rearranged diterpenoid. Crassin H (8) exhibited selective cytotoxicity against A549 cells (IC50 5.2 µM) compared with HL-60 cells (IC50 11.8 µM). The known compound chettaphanin-II exhibited moderate activity against the A549 and HL-60 cell lines (IC50 8.4 and 10.5 µM, respectively).

Withanolides from Aeroponically Grown Physalis peruviana and Their Selective Cytotoxicity to Prostate Cancer and Renal Carcinoma Cells.

Investigation of aeroponically grown Physalis peruviana resulted in the isolation of 11 new withanolides, including perulactones I-L (1-4), 17-deoxy-23ß-hydroxywithanolide E (5), 23ß-hydroxywithanolide E (6), 4-deoxyphyperunolide A (7), 7ß-hydroxywithanolide F (8), 7ß-hydroxy-17-epi-withanolide K (9), 24,25-dihydro-23ß,28-dihydroxywithanolide G (10), and 24,25-dihydrowithanolide E (11), together with 14 known withanolides (12-25). The structures of 1-11 were elucidated by the analysis of their spectroscopic data, and 12-25 were identified by comparison of their spectroscopic data with those reported. All withanolides were evaluated for their cytotoxic activity against a panel of tumor cell lines including LNCaP (androgen-sensitive human prostate adenocarcinoma), 22Rv1 (androgen-resistant human prostate adenocarcinoma), ACHN (human renal adenocarcinoma), M14 (human melanoma), SK-MEL-28 (human melanoma), and normal human foreskin fibroblast cells. Of these, the 17ß-hydroxywithanolides (17-BHWs) 6, 8, 9, 11-13, 15, and 19-22 showed selective cytotoxic activity against the two prostate cancer cell lines LNCaP and 22Rv1, whereas 13 and 20 exhibited selective toxicity for the ACHN renal carcinoma cell line. These cytotoxicity data provide additional structure-activity relationship information for the 17-BHWs.

Identification of Cyclic Depsipeptides and Their Dedicated Synthetase from Hapsidospora irregularis.

Seven cyclic depsipeptides were isolated from Hapsidospora irregularis and structurally characterized as the calcium channel blocker leualacin and six new analogues based on the NMR and HRESIMS data. These new compounds were named leualacins B-G. The absolute configurations of the amino acids and 2-hydroxyisocaproic acids were determined by recording the optical rotation values. Biological studies showed that calcium influx elicited by leualacin F in primary human lobar bronchial epithelial cells involves the TRPA1 channel. Through genome sequencing and targeted gene disruption, a noniterative nonribosomal peptide synthetase was found to be involved in the biosynthesis of leualacin. A comparison of the structures of leualacin and its analogues indicated that the A2 and A4 domains of the leualacin synthetase are substrate specific, while A1, A3, and A5 can accept alternative precursors to yield new molecules.

Multidrug resistance-reversal effects of resin glycosides from Dichondra repens.

Investigation of hydrophobic extract of Dichondra repens (Convolvulaceae) led to the isolation of three new resin glycosides dichondrins A-C (1-3), and three known resin glycosides cus-1, cus-2, and cuse 3. All the isolated resin glycosides with an acyclic core were evaluated for their multidrug resistance reversal activities, and the combined use of these compounds at a concentration of 25µM increased the cytotoxicity of vincristine by 1.03-1.78-fold.

Directing-group-assisted copper-catalyzed oxidative esterification of phenols with aldehydes.

A directing-group-assisted copper-catalyzed oxidative esterification of phenols with aldehydes using TBHP as an oxidant was described. This methodology which showed the advantages of base, ligand free, short routes and functional group tolerance could be used as an alternative protocol for the classical esterification reactions.

Ruthenium-catalyzed oxidative coupling of 2-aryl-4-quinazolinones with olefins: synthesis of pyrrolo[2,1-b]quinazolin-9(1H)-one motifs.

A ruthenium-catalyzed oxidative coupling of 2-aryl-quinazolinones with olefins via C-H bond activation followed by an intramolecular aza-Michael reaction is described. This strategy allows the direct and efficient construction of pyrrolo[2,1-b]quinazolin-9(1H)-one scaffolds.