Ethyl Lithospermate Reduces Lipopolysaccharide-Induced Inflammation through Inhibiting NF-κB and STAT3 Pathways in RAW 264.7 Cells and Zebrafish.

OBJECTIVE: To explore the anti-inflammatory effects of ethyl lithospermate in lipopolysaccharide (LPS)-stimulated RAW 264.7 murine-derived macrophages and zebrafish, and its underlying mechanisms. METHODS: 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) assays were performed to investigate the toxicity of ethyl lithospermate at different concentrations (12.5-100 µ mol/L) in RAW 264.7 cells. The cells were stimulated with LPS (100 ng/mL) for 12 h to establish an inflammation model in vitro, the production of pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor α (TNF-α) were assessed by enzyme linked immunosorbent assay (ELISA). Western blot was used to ascertain the protein expressions of signal transducer and activator of transcription 3 (STAT3), nuclear factor kappa B (NF-κB) p65, phospho-STAT3 (p-STAT3, Tyr705), inhibitor of NF-κB (IκB) α, and phospho-I κB α (p-IκB α, Ser32), and confocal imaging was used to identify the nuclear translocation of NF-κB p65 and p-STAT3 (Tyr705). Additionally, the yolk sacs of zebrafish (3 days post fertilization) were injected with 2 nL LPS (0.5 mg/mL) to induce an inflammation model in vivo. Survival analysis, hematoxylin-eosin (HE) staining, observation of neutrophil migration, and quantitative real-time polymerase chain reaction (qRT-PCR) were used to further study the anti-inflammatory effects of ethyl lithospermate and its probable mechanisms in vivo. RESULTS: The non-toxic concentrations of ethyl lithospermate have been found to range from 12.5 to 100 µ mol/L. Ethyl lithospermate inhibited the release of IL-6 and TNF-α(P<0.05 or P<0.01), decreased IκBα degradation and phosphorylation (P<0.05) as well as the nuclear translocation of NF-κB p65 and p-STAT3 (Tyr705) in LPS-induced RAW 264.7 cells (P<0.01). Ethyl lithospermate also decreased inflammatory cells infiltration and neutrophil migration while increasing the survival rate of LPS-stimulated zebrafish (P<0.05 or P<0.01). In addition, ethyl lithospermate also inhibited the mRNA expression levels of of IL-6, TNF-α, IκBα, STAT3, and NF-κB in LPS-stimulated zebrafish (P<0.01). CONCLUSION: Ethyl lithospermate exerts anti-Inflammatory effected by inhibiting the NF-κB and STAT3 signal pathways in RAW 264.7 macrophages and zebrafish.

Salvianolic acids from Salvia miltiorrhiza Bunge and their anti-inflammatory effects through the activation of α7nAchR signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Cardiovascular disease (CVD) is a serious disease with a high incidence rate and mortality. Inflammation is closely related to the occurrence of CVDs. As an essential medicine of promoting blood circulation and removing blood stasis in China, Salvia miltiorrhiza Bunge (Danshen) is widely used to treat CVDs due to its anti-inflammatory and cardiovascular protective effects. Salvianolic acids are the most abundant component in the water extract of S. miltiorrhiza, which has a significant effect on the treatment of CVDs. However, due to the complex composition of salvianolic acids, the active molecules and their underlying mechanisms have not been fully explored. AIM OF THIS STUDY: The present study aims to isolate and identify salvianolic acids from Danshen with anti-inflammatory activity and explore the potential mechanisms of isolates. METHODS: The structures of isolated salvianolic acids were elucidated by UV, IR, NMR, MS and electronic circular dichroism (ECD) calculations. Then anti-inflammatory activities of isolates were screened out by the zebrafish inflammation models. The most active compound was further used to explore the anti-inflammatory mechanisms on LPS-stimulated RAW 264.7 cells. The key inflammatory cytokines IL-6 and TNF-α were measured by enzyme-linked immunosorbent assay (ELISA). The protein expression levels of STAT3, p-STAT3 (Tyr705), NF-κB p65, IκBα, p-IκBα (Ser32) and α7nAchR were determined by Western blotting. The nuclear translocation of p-STAT3 (Tyr705) and NF-κB p65 was evaluated by immunofluorescence assays. Finally, the in vivo anti-inflammatory mechanisms were investigated by observation of neutrophil migration, H&E staining, survival analysis and quantitative PCR (Q-PCR) in LPS-microinjected zebrafish. RESULTS: Two new and four known compounds were isolated from Danshen. Among them, isosalvianolic acid A-1 (C1) and ethyl lithospermate (C5) inhibited neutrophil migrations in three zebrafish inflammation models and C1 with the best activities decreased the secretion of IL-6 and TNF-α and inhibited the expression level of p-IκBα (Ser32) in LPS stimulated RAW 264.7 cells. In addition, C1 also reduced the nuclear translocation of NF-κB p65 and p-STAT3 (Tyr705). Moreover, C1 significantly upregulated the protein expression of α7nAchR, and the knockdown of α7nAchR counteracted the effects of C1 on the production of IL-6 and TNF-α and the expression levels of p-STAT3 (Tyr705), NF-κB p65 and p-IκBα (Ser32). In vivo experiments, C1 decreased the migration and infiltration of inflammatory cells, increased the survival ratio and inhibited the mRNA level of IL-6, TNF-α, STAT3, NF-κB and IκBα in LPS-microinjected zebrafish. CONCLUSION: Two new and four known compounds were isolated from Danshen. Among them, C1 exerted anti-inflammatory activities by activating α7nAchR signaling and subsequently inhibiting STAT3 and NF-κB pathways. This study provided evidence for the clinical application of Danshen and contributed to the development of C1 as a novel in the treatment of cardiovascular disease.

Isoquinoline alkaloids from Corydalis edulis Maxim. Exhibiting insulinotropic action.

Eleven undescribed isoquinoline analogues, namely edulisines A-K, along with sixteen known alkaloids, were isolated from the whole plants of Corydalis edulis. The structures of the isolated alkaloids were established on the basis of extensive spectroscopic data (1D and 2D NMR, UV, IR, and HRESIMS). Their absolute configurations were determined by single-crystal X-ray crystallographic analysis and ECD. Compounds (+)-1 and (-)-1 are a pair of undescribed isoquinoline alkaloids bearing a unique coupled pattern of coptisine and ferulic acid via Diels-Alder [4 + 2] cycloaddition, while compounds (+)-2 and (-)-2 feature benzo [1,2-d:3,4-d]bis [1,3]dioxole moiety. Compounds (+)-2, (-)-2, (-)-5, 10, 13, 15, 20, 22, and 23 significantly triggered the secretion of insulin in the HIT-T15 cells at a concentration of 40 µM.

Alkaloids with acetylcholinesterase inhibitory activity from Corydalis racemosa (Thunb.) Pers.

Two new isoquinoline alkaloids (1 and 2) along with fourteen known alkaloids (3-16) were isolated from Corydalis racemosa (Thunb.) Pers. Their structures were elucidated by analyzing spectroscopic and spectrometric data (NMR, UV, IR, and MS) and comparing their spectroscopic, spectrometric and physicochemical data with the values archived in the literature. The absolute configurations of new compounds were determined via X-ray crystallographic assay and electronic circular dichroism calculations. Acetylcholinesterase (AChE) inhibitory activity of all compounds was evaluated. Compounds 5, 6, 9, 11, and 12 exhibited inhibitory activity against AChE with IC50 values ranged from 10.2 to 63.4 µM.

Pre-clinical pharmacological profile of QF-036, a potent HIV-1 maturation inhibitor.

QF-036 is an HIV-1 maturation inhibitor in pre-clinical development, and its antiviral activity against a laboratory HIV-1 strain and two drug-resistant strains was determined in the C8166 line. QF-036 was also subjected to absorption, distribution and metabolism (ADM) assessment in vitro, and pharmacokinetic profiles were evaluated in rats and monkeys. The 50% effective concentrations (EC50 ) of QF-036 against the three strains were 20.36 nM, 0.39 µM and 2.11 nM, respectively, demonstrating better antiviral potential than the first-generation antiviral maturation inhibitor bevirimat. QF-036 demonstrated moderate cell permeability, high plasma protein binding ability and good metabolic stability in vitro. After oral QF-036 administration to rats and monkeys, both species exhibited moderate bioavailability, and the plasma drug exposure increased in an approximately dose-proportional manner. When administered orally (30 mg/kg) to monkeys, the QF-036 plasma concentration (Cmax ) peaked at 3671 ng/mL (4.82 µM), 12 to 2410 times higher than the EC50 of laboratory or resistant HIV-1 strains. Moreover, the plasma concentration of QF-036 at 12 hours after administration was 263 ng/mL (0.35 µM), which approximately matched the highest EC50 value of the three test strains. The favourable viral inhibitory activity and pharmacokinetic properties provide critical support for QF-036 as a promising anti-HIV therapeutic candidate.

[Two new phenylpropanoid amide glycosides from whole plants of Corydalis racemosa].

Two new phenylpropanoid amide glycosides and ten analogues were isolated from the CH_2Cl_2 layer of 95% ethanol extract of the whole plants of Corydalis racemosa by using various chromatographic techniques, including silica gel, Sephadex LH-20, ODS column chromatographies, and semi-preparative HPLC. Their structures were identified on the basis of physicochemical properties, MS, NMR, and IR spectroscopic data as N-cis-sinapoyltyramine-4'-O-ß-glucoside(1), N-cis-sinapoyl-3-methoxytyramine-4'-O-ß-glucoside(2), N-cis-sinapoyltyramine(3), N-cis-feruloyltyramine(4), N-trans-cinnamoyltyramine(5), N-trans-feruloylphenethylamine(6), N-trans-p-methoxycinnamoyl-3-hydoxyoctopamine(7), N-cis-feruloyl-3-methoxytyramine(8), N-trans-feruloyltyramine(9), N-trans-feruloyl-3-methoxytyramine(10), N-trans-sinapoyltyramine(11), and N-trans-p-coumaroyltyramine(12). Compounds 1 and 2 are new compounds. Compounds 3-7 are obtained from the plants of Papaveraceae for the first time, and compounds 8-12 are firstly isolated from C. racemosa.

Single- and repeated-dose toxicity studies on the novel HIV maturation inhibitor QF-036 in Sprague-Dawley rats.

QF-036 is a novel human immunodeficiency virus (HIV) maturation inhibitor that is a lupine triterpenoid derivative. The objective of this study was to evaluate the safety of QF-036. A single oral toxicity and a 4-week repeated oral toxicity were investigated in Sprague-Dawley (SD) rats. The single oral toxicity study of QF-036 in SD rats showed that no mortality or visible pathological changes were noted at doses of 100, 300, and 1000 mg/kg. QF-036 exhibited a non-linear toxicokinetic profile over the dose range of 100-1000 mg/kg in the single dose study, and a saturation trend appeared at doses of 100 and 300 mg/kg. In the 4-week oral toxicity and toxicokinetic study, SD rats were given 0, 50, 100, and 200 mg/kg QF-036 once daily for 4 weeks, followed by a 4-week recovery period. No mortality or significant effects on food consumption, body weight, or behavior were observed. In addition, there were no test article-related changes in hematology, clinical biochemistry and histopathology. The no observed adverse effect level (NOAEL) was 200 mg/kg. The toxicokinetic study demonstrated a dose-dependent increase in the systemic exposure to QF-036 after 4 weeks of oral administration. There were no marked sex differences or drug accumulation observed for repeated doses of QF-036.

[A new nor-sesquiterpene glycoside from Corydalis edulis].

This study is to investigate the chemical constituents from the whole plant Corydalis edulis. The chemical constituents were separated and purified by macroporous resin D101, silica gel, Sephadex LH-20, ODS, and semi-preparative HPLC. Their structures were determined by physicochemical properties and spectroscopic data. Four compounds were isolated from the dichloromethane and water extracts of the whole plant C. edulis, and identified as 6'-ß-D-xylosylicariside B2(1),(3S,5R,6S,7E)-5,6-epoxy-3-hydroxy-7-megastigmen-9-one(2), loliolide(3), and 5,5'-dimethoxybiphenyl-2,2'-diol(4), respectively. Compound 1 is a new compound, of which the absolute configuration was established by electronic circular dichroism(ECD) calculations. Compound 4 is obtained from the plants of Papaveraceae family for the first time. Compounds 2 and 3 are firstly isolated from the Corydalis genus.

[Cytogenetic and clinical study of myeloid leukemia].

OBJECTIVE: To explore the clinical cytogenetic features and prognosis of myeloid leukemia patients. METHODS: Bone marrow direct method and/or 24h culture without phytohaemagglutimin(PHA) were used to prepare the chromosomes and karyotype analysis was performed with R-banding and G-banding techniques. RESULTS: Among 420 patients with acute myeloid leukemia (AML), 223 cases were found to exhibit clonal chromosome abnormalities, accounted for 53.1%. t(8; 21), t(15; 17), inv(16)and del(11) were specifically associated with M2b, M3, M4Eo and M5 respectively. Out of 158 patients with chronic myeloid leukemia (CML), 96.8% (153/158) were found to exhibit clonal chromosome abnormalities. T(9;22) was specifically associated with CML and some cases of M0, M1 and M2. In these myeloid leukemia cases, there were 18 cases (AML 13 cases, CML 15 cases) without clonal chromosome abnormalities, accounted for 3.1% (18/578) and this phenomenon agreed with the diagnose of clinical signs, marrow morphology and immunology incompletely. CONCLUSION: Karyotype analysis was not only helpful to the diagnose and differential diagnose of myeloid leukemia, but also an important standard of the remission, relapse and therapeutic effect of myeloid leukemia. Chromosome analysis can be made exactly with the probe and FISH technique on the basic of chromosome karyotype analysis.