Amide alkaloids and neolignans from Piper hongkongense and their inhibitory activities of PCSK9 expression.

Four undescribed amide alkaloids hongkongensines A-C and 1-(1-oxo-6-hydroxy-2E,4E-dodecadienyl)-piperidine, five known amide alkaloids, and three known neolignans were isolated from the aerial part of Piper hongkongense. The planar structures of these compounds were determined by detailed analyses of HR-ESI-MS and NMR data. The absolute configurations of hongkongensines A-C were elucidated by single-crystal X-ray diffraction analysis and ECD calculations. Moreover, the inhibitory activities of PCSK9 expression in vitro for all compounds were assessed by PCSK9 AlphaLISA screening. Kadsurenone (10) displayed a significant inhibitory activity at 5 µM with an inhibition rate of 51.98%, compared with 55.55% of berberine (BBR 5 µM).

Dimeric ent-kauranoids isolated from Isodon japonica var. Glaucocalyx and their anti-inflammatory activities.

The phytochemical investigation of the aerial parts of Isodon japonica var. glaucocalyx afforded four undescribed (glaucocalyxin O-R, 1-4) and six known ent-kauranoids (5-10). Their structures were established using NMR and MS measurements. Compounds 1 and 2 are dimeric ent-kaurane-type diterpenoids. Moreover, the plausible biogenetic pathways for compounds 1 and 2 were proposed as Michael addition between two monomers. Eight compounds were assayed for their anti-inflammatory activity by evaluating NO production in LPS-induced RAW 267.4 cells, and compounds 7, 8 and 9 exhibited relatively remarkable anti-inflammatory activities at 10 µM.

Eighteen iridoids from the roots and rhizomes of Valeriana jatamansi and their protective effects against α-hemolysin.

Thirteen previously undescribed iridoids (1-13), together with five known iridoids (14-18) were isolated from the roots and rhizomes of Valeriana jatamansi Jones. Their structures with absolute configurations were elucidated by analysis of MS, NMR, optical rotation and their experimental and calculated electronic circular dichroism spectra. All of the isolated compounds were tested for their protective effects against α-hemolysin-induced cell death in A549 cells. Compounds 14, 16 and 17 showed moderate protective effects, and compounds 15 and 18 showed weak protective effects.

Five undescribed abietane-type diterpenes of Callicarpa macrophylla.

A phytochemical investigation of the medicinal plant Callicarpa macrophylla resulted in the characterization of two rare rearrangement abietane-type diterpenoids, macrophypene F-G (1-2), and three abietane diterpenoids, named macrophypene H-J (3-5). Additionally, five known diterpenoids (6-10) were identified. The structures of the newly discovered compounds were fully established through extensive analysis of HRESIMS, 1D and 2D NMR data. The absolute configurations of the isolated compounds were determined using CD comparison, chemical methods, and X-ray crystal diffraction experiments. Subsequently, all isolated diterpenoids were evaluated for their inhibitory effects on extracellular PCSK9 protein levels by PCSK9 AlphaLISA screening. Jiadfenoic acid B (6, 56.80% inhibition at 20 µM) and holophyllin F (10, 43.18% inhibition at 20 µM) significantly decreased PCSK9 protein levels in medium of HepG2 cells.

Five undescribed guanidine alkaloids from Plumbago zeylanica.

Five undescribed guanidine alkaloids, plumbagines HK (1-4) and plumbagoside E (5), as well as five known analogues (6-10) were isolated from the roots of Plumbago zeylanica. Their structures were established by extensive spectroscopic analyses and chemical methods. In addition, 1-10 were accessed their anti-inflammatory activities by measuring nitric oxide (NO) concentrations in LPS-induced RAW 264.7 cells. However, all compounds especially 1 and 3-5 could not inhibit the secretion of NO but significant increase the secretion of NO. The result reminded us that 1-10 may become potential novel immune potentiators.

Salvianolate ameliorates renal tubular injury through the Keap1/Nrf2/ARE pathway in mouse kidney ischemia-reperfusion injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Acute kidney injury (AKI) is a common clinical disease characterized by rapid loss of renal function. Salvianolate is a prescribed Chinese medicine derived from traditional Chinese medicine Salvia miltiorrhiza bunge that possesses many pharmacological effects, the active components extracted from Salvia miltiorrhiza bunge have been proved to protect ischemia-reperfusion (I/R)-AKI. AIM OF THE STUDY: This study aims to validate the therapeutic effect of SAL on I/R-AKI, and explore its potential pharmacological mechanism. MATERIALS AND METHODS: Mice were pretreated with/without salvianolate (10, 30, and 90 mg/kg) before renal ischemia-reperfusion operation. Serum creatinine, BUN, and H&E staining were performed to evaluate renal function. Immunofluorescence analysis was conducted to measure renal tubular injury including inflammatory factors and peroxide level. Apoptosis of the kidney tissues was determined by TUNEL assay. Keap1-Nrf2-ARE and apoptosis signaling pathways were measured by Western blot, RT-PCR, and YO-PRO-1 staining in kidneys or NRK52E cells. RESULTS: Pretreatment with SAL effectively alleviated renal function and ameliorated epithelial tubular injury, oxidative stress, and inflammatory response. Furthermore, the mechanistic study demonstrated that the SAL exerts anti-apoptotic effects through activation of the Keap1-Nrf2-ARE signaling pathway in renal tubular cells. CONCLUSION: These findings indicate the therapeutic benefit of salvianolate in the protection of renal injury from ischemia-reperfusion, and strengthen the evidence for the AKI treatment strategy by the anti-oxidative stress response, suggesting that SAL may be a potential agent for the treatment of AKI.

Neolinulicin A and B from Inula japonica and their anti-inflammatory activities.

A phytochemical study performed on Inula japonica led to isolation of a new 1,10-seco-sesquiterpene dimer Neolinulicin A (1) and 1,10-seco-sesquiterpene Neolinulicin B (2), together with nine known sesquiterpenes (3-11). Among them, Neolinulicin A (1), which has a new carbon skeleton, was a Diels-Alder [4 + 2] adduct of two sesquiterpene moieties. Their structures were established by extensive spectroscopic analysis. All of the isolated compounds showed inhibition of NO production in RAW 264.7 macrophages. The findings might supply information for the future design of anti-inflammatory agents from I. japonica.

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.

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.

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.

Indole alkaloid from Nauclea latifolia promotes LDL uptake in HepG2 cells by inhibiting PCSK9.

BACKGROUND: Proprotein convertase subtilisin/kexin type 9 (PCSK9) has been found to play a major role in atherosclerotic cardiovascular disease (ASCVD) by promoting hyperlipidemia. Its inhibition has therefore emerged as a viable drug target for improving the outcome of ASCVD. However, current monoclonal antibody PCSK9 inhibitors are considered cost ineffective and there is the need to discover new effective and cheaper small molecule alternatives. PURPOSE: The methanolic and ethanolic crude extracts of Nauclea latifolia have been shown to possess anti-hyperlipidemic activity, but the chemical component(s) responsible for this activity and the mechanism of action have remained unknown. The objective of this study was therefore to identify N. latifolia constituents with anti-hyperlipidemic activity and to investigate the inhibition of PCSK9 as a probable mechanism of action. METHOD: In the present study, compounds were isolated from the ethanolic extract of the stem of N. latifolia. The alkaloids were evaluated for their DiI-LDL uptake promoting activity in HepG2 cell. The most active compound was further assessed for its effect on low density lipoprotein receptor (LDLR) and PCSK9 protein expressions by western blot. RESULTS: 3R-3,14-dihydroangustoline (5), showed a relatively good activity in promoting LDL uptake (1.26-fold). It further increased LDLR protein expression and decreased the protein expression of PCSK9 in a dose dependent manner (1-50  µM). CONCLUSION: Alkaloids from N. latifolia may serve as a source of new PCSK9 inhibitors.

Cytotoxic and other withanolides from aeroponically grown Physalis philadelphica.

Eleven withanolides including six previously undescribed compounds, 16ß-hydroxyixocarpanolide, 24,25-dihydroexodeconolide C, 16,17-dehydro-24-epi-dioscorolide A, 17-epi-philadelphicalactone A, 16-deoxyphiladelphicalactone C, and 4-deoxyixocarpalactone A were isolated from aeroponically grown Physalis philadelphica. Structures of these withanolides were elucidated by the analysis of their spectroscopic (HRMS, 1D and 2D NMR, ECD) data and comparison with published data for related withanolides. Cytotoxic activity of all isolated compounds was evaluated against a panel of five human tumor cell lines (LNCaP, ACHN, UO-31, M14 and SK-MEL-28), and normal (HFF) cells. Of these, 17-epi-philadelphicalactone A, withaphysacarpin, philadelphicalactone C, and ixocarpalactone A exhibited cytotoxicity against ACHN, UO-31, M14 and SK-MEL-28, but showed no toxicity to HFF cells.

Jatrophane diterpenoids from Euphorbia helioscopia and their lipid-lowering activities.

The phytochemical study of Euphorbia helioscopia led to the isolation of 33 jatrophane diterpenoids (1-33), of which six (1-6) were new. This small jatrophane library was established to screen for potential lipid modulators. LDL-Uptake screening assay demonstrated that most of them improved LDL-Uptake rate in HepG2 cells, with compounds 16, 21 and 26 exhibiting more outstanding effects. Further exploration found that these three compounds could increase LDLR protein level in HepG2 cells dose-dependently. SAR studies suggested that substituted patterns of C-9, steric hindrance between C-14 and C-15, and the long conjugated fragment from C-5 to the carbonyl (C-9) were essential for the activity. Moreover, compound 21, a relatively abundant chemical in E. helioscopia, showed remarkable lipid-lowering effect in vivo, which makes it a promising lead for development of new lipid-lowering agents.

Serratene-type triterpenoids from Palhinhaea cernua.

Phytochemical investigation of the 95% ethanol extract of the whole plant of Palhinhaea cernua afforded five new serratene-type triterpenoids (1-5) together with four known analogues (6-9). The structures of the new compounds were elucidated by extensive spectroscopic analysis and X-ray crystallographic data. Compounds 1-3 triggered apoptosis of Hela-/- cell with EC50 values of 3.49 ±â€¯0.05, 0.28 ±â€¯0.13 and 7.98 ±â€¯0.03 µM, respectively.

Jatrophanes as promising multidrug resistance modulators: Advances of structure-activity relationships.

The phytochemical study of Euphorbia helioscopia afforded euphornin (1) in a large amount. Alkaline hydrolysis of 1 using potassium carbonate yielded the main product monodeacetyleuphornin (2), whose structural modification at 14-OH gave rise to 21 acylated derivatives euphornoate A-U (3-23). Thus, a mini compound library of jatrophanes was established to screen for MDR modulators. Biological studies clearly demonstrated the effect of C-14 pattern modification in MDR reversal activity and several compounds with RF values over 300 fold at 20 µM (6, 16, 20, 22, 23) were thought to be promising MDR modulators. The SARs are discussed, which reveal that introduction of an alkyl acyl group bearing 4 carbons at C-14 or an aryl acyl group with electron donating groups is favorable for the activity.

Resin glycosides from the seeds of Ipomoea muricata and their multidrug resistance reversal activities.

Resin glycosides represent an important chemotaxonomic marker of the Convolvulaceae family and possess multidrug resistance (MDR) reversal activity. In our recent study, nine previously undescribed resin glycosides, Calonyctins B-J, were isolated from the seeds of Ipomoea muricata. Their structures with the absolute configuration were established on the basis of comprehensive spectroscopic analysis and chemical methods. Among these, Calonyctins F-I possessed a skeleton in which the aglycone moiety and the oligosaccharide core were linked by a 3-hydroxy-2-methylbutanoic acid moiety to form a 25-membered macrocyclic structure. Calonyctins E, J, and muricatic acid C methyl ester were non-cytotoxic but enhanced the cytotoxicity of vincristine by 2.5-407.1 fold at 25 µM in KB/VCR cells. Calonyctin E was the most active one.

(19αH)-lupane and (9ßH)-lanostane triterpenes from Euphorbia helioscopia trigger apoptosis of tumor cell.

Chemical investigation of Euphorbia helioscopia resulted in the identification of seven new triterpenes named euphorbatrine A-G (1-7), including two (19αH)-lupane (1-2) and three (9ßH)-lanostane (4-6), which are very rare metabolites from plants. Their structures were elucidated by extensive spectroscopic analysis, X-ray crystallography and chemical methods. Compounds 5 and 6 triggered apoptosis of Hela-/- cell with EC50 values of 1.59±0.25 and 26.48±0.78µM, respectively.

Halimane-type diterpenoids from Vitex rotundifolia and their anti-hyperlipidemia activities.

Vitex rotundifolia is the variant of the traditional Chinese medicine (TCM) Vitex trifolia. Diterpenoids from V. trifolia have shown anti-hyperlipidemia activity. As part of a continuous research program of searching for anti-hyperlipidemia constituents from TCM, 95% alcohol extract of the fruits of V. rotundifolia was fully studied, and 18 diterpenoids were isolated, including eight previously undescribed compounds (viterofolins A-H). Among them, viterofolins A-B were previously undescribed rearranged halimane-type diterpenoids, viterofolins CH were previously undescribed halimane-type diterpenoids. These compounds were then firstly evaluated in lipid (Dil-LDL) uptake assay in HepG2 cells. Viterofolin H, (5S, 6R, 8R, 9R, 10S)-6-acetoxy-9-hydroxy-13 (14)-labden-16,15-olide and previtexilactone showed moderate activities in promoting LDL uptake (1.27-1.35 fold). This work laid the foundation for searching anti-hyperlipidemia natural products.