Our previous report showed that polyacetylene compound, 1-Heptadecene-11, 13-diyne-8, 9, 10-triol (PA) from the root of Cirsium japonicum var. ussuriense has anti-inflammatory activity. In this study we investigated the role of the PA as inhibitor of caspase-1, which converts prointerleukin-1beta (proIL-1beta) to active IL-1beta and is activated by inflammasome involved in the inflammatory process. We tested the effect of PA on the production of pro-inflammatory cytokines, IL-1beta in murine macrophage cell line, RAW264.7. PA inhibited lipopolysaccharide (LPS)-induced IL-1beta production by macrophages at a dose dependent manner. PA also suppressed the activation of caspase-1. The mRNA level of ASC (apoptosis-associated spec-like protein containing a CARD), an important adaptor protein of inflammasome, was decreased in the PA treated group. Therefore our results suggest that the anti-inflammatory effect of PA is due to inhibit the caspase-1 activation.
Cell Line ; Cirsium ; Cytokines ; Macrophages ; Polyacetylenes ; RNA, Messenger

OBJECTIVETo develop a green and rapid method for extraction of lobetyolin from C. pilosula.

METHODExtraction of lobetyolin from C. pilosula with supercritical carbon dioxide in the presence of ethanol was studied. The effects of pressure, temperature, volume of cosolvent and extraction time on efficiency and their interactive relationships were discussed, based on central composite design and response surface methodology (RSM).

RESULTThe key effect factor was volume of cosolvent. The extraction yield of lobetyolin was 0.078 6 mg x g(-1) when C. pilosula (40-60 mesh) was extracted at 30 MPa, 60 degrees C and 2 L x min(-1) (as CO2 in normal pressure and temperature) for 100 minutes with supercritical CO2 and 1 mL x min(-1) ethanol as dynamic cosolvent.

CONCLUSIONThis result is better than that obtained from traditional method. Therefore, the optimized process is valuable for extraction of lobetyolin from C. pilosula.

Carbon Dioxide ; chemistry ; Chemical Fractionation ; methods ; Codonopsis ; chemistry ; Drugs, Chinese Herbal ; chemistry ; Ethanol ; chemistry ; Polyacetylenes ; chemistry

Here, we designed to examine the anti-inflammatory effects on RAW264.7 cells and the immunosuppressive effects by evaluating interleukin-2 (IL-2) production in Jurkat T cells using a MeOH extract of Panax notoginseng roots. The results showed that the MeOH extract inhibited the synthesis of nitric oxide (NO) in a dose-dependent manner (IC₅₀ value of 7.08 µg/mL) and displayed effects on T cell activation at a concentration of 400 µg/mL. In efforts to identify the potent compounds, bioactivity-guided fractionation of the MeOH extract and chemical investigation of its active CH₂Cl₂-, EtOAc-, and butanol-soluble fractions led to the successful isolation and identification of eleven compounds, including two polyacetylenes (1, 2), a steroid saponin (3), seven dammarane-type ginsenosides (4 – 10), and an oleanane-type ginsenoside (11). Among them, compound 11 was isolated from this plant for the first time. Compound 2 exhibited potent inhibitory effects on NO synthesis and an immunosuppressive effect with IC₅₀ values of 2.28 and 65.57 µM, respectively.
Ginsenosides ; Interleukin-2 ; Nitric Oxide ; Panax notoginseng ; Panax ; Plants ; Polyacetylenes ; Saponins ; T-Lymphocytes

OBJECTIVETo develop an HPLC method for determination of two polyacetylenes, lobetyolin and lobetyolinin, in Herba Lobeliae Chinensis.

METHODC18 column was used with the mobile phase consisted of acetonitrile and water. Linear gradient elution from 10% to 40% acetonitrile in 25 min was applied, at the flow rate of 1.0 mL x min(-1), the detection wavelength was at 267 nm.

RESULTLower contents of lobetyolin and lobtyolinin were found in collected samples of Herba Lobeliae Chinensis. The highest amounts of lobetyolin and lobetyolinin were found to be 0.461 and 0.436 mg x g(-1) in a sample procured from Hong Kong. However, there were no lobetyolin and lobetyolinin in some of the samples.

CONCLUSIONA simple and effective HPLC method to analyze the two polyacetylenes in Herba Lobeliae Chinensis was established. It could be applied for the quality control of this herb.

Chromatography, High Pressure Liquid ; methods ; Lobelia ; chemistry ; Molecular Structure ; Plants, Medicinal ; chemistry ; Polyacetylenes ; analysis ; chemistry ; Quality Control ; Reproducibility of Results

OBJECTIVETo determine lobetyolin in the root of Codonopsis tangshen from the various cultivation areas.

METHODA Supelco Discovery C18 Column (4.6 mm x 250 mm, 5 microm) was used with acetonitrile-0.5% acetic acid in water (20:80) as the mobile phase and UV detection was at 268 nm.

RESULTTwenty-four batches of the samples were analyzed. The content of lobetyolin ranged from 0.0403-0.9667 mg x g(-1).

CONCLUSIONThe method was simple, reproducible and reliable. It can be used to control the quality of C. tangshen.

China ; Chromatography, High Pressure Liquid ; methods ; Codonopsis ; chemistry ; Drugs, Chinese Herbal ; chemistry ; Plant Roots ; chemistry ; Polyacetylenes ; analysis ; Quality Control

AIMTo compare two methods, the total radioactivity assay (RA method) and the radioactivity assay after separation with high performance liquid chromatography (HPLC-RA method).

METHODS125I-Lidamycin was prepared by Iodogen method and separated by size exclusive high performance liquid chromatography. The pharmacokinetic parameters of lidamycin were assayed by two methods after intravenous injection to mice at the dose of 100 microg x kg(-1), and compared by statistical analysis.

RESULTSThe pharmacokinetic parameters (Vd, T1/2alpha, T1/2beta, K21, K10, K12, AUC and CL) showed significant difference between the two methods (P < 0.05).

CONCLUSIONThe HPLC-RA method was better than the RA method to determine unchanged 125I-lidamycin.

Aminoglycosides ; blood ; pharmacokinetics ; Animals ; Antibiotics, Antineoplastic ; blood ; pharmacokinetics ; Area Under Curve ; Chromatography, High Pressure Liquid ; methods ; Enediynes ; Female ; Iodine Radioisotopes ; Isotope Labeling ; Male ; Mice ; Sensitivity and Specificity

AIMTo investigate the chemosensitivity to lidamycin (C-1027) in mdr1 gene overexpressing cancer cell lines established by drug induction and by gene-transfection.

METHODSDNA was cloned by RT-PCR and then eukaryotic expressing recombinant plasmid pcDNA3. 1/mdrl was constructed. Using Lipofectamine 2000, a strain of stably transfected human hepatoma cancer cells, HepG2/mdrl, was obtained. The mdr1 mRNA level, P-glycoprotein (P-gp) level and the activity of P-gp to extrude drugs in cancer cells were determined by RT-PCR, immunofluorescence analysis and rhodamine 123 efflux assay. The chemosensitivity of cancer cells with low or high mdr1 expression to lidamycin and other antitumor drugs was tested by MTT assay.

RESULTSThe mdr1 mRNA and P-gp levels in KBv200, MCF-7/ADR, and stably transfected HepG2/mdr1 cells were much higher than that in respective parent KB, MCF-7 and HepG2 cells. The IC50 values of lidamycin for KBv200, MCF-7/ADR and HepG2/mdrl cells were (0.24 +/- 0.20) nmol x L(-1), (0.028 +/- 0.011) nmol x L(-1), and (0.020 +/- 0.011) nmol x L(-1), respectively. Compared with parental cells, the values of resistant fold for KBv200, MCF-7/ADR and HepG2/mdr1 cells to lidamycin were 6.8, 1.6 and 1.3 fold; to adriamycin were 37.2, 181.3 and 8.8 fold; to taxol were 336.8, 49.2 and 40.3 fold, respectively.

CONCLUSIONLidamycin is highly active to multidrug resistant cancer cells. The chemosensitivity of those resistant cancer cells to lidamycin is approximately at the similar level as that of parent cancer cells.

ATP-Binding Cassette, Sub-Family B, Member 1 ; analysis ; genetics ; Aminoglycosides ; pharmacology ; Antibiotics, Antineoplastic ; pharmacology ; Cell Line, Tumor ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; Enediynes ; pharmacology ; Genes, MDR ; Humans ; Neoplasms ; drug therapy ; pathology ; Transfection

AIMTo investigate the synergetic effect and the mechanism of antitumor action of the antibiotic lidamycin in combination with cisplatin in vitro.

METHODSCytotoxicity of the drugs was measured by clonogenic assay. Chromatin condensation was observed by co-staining with fluorescent dyes, Hoechst 33342 and propidium iodide. Apoptotic sub-G1 was detected by flow cytometry and DNA ladder was observed using agarose gel electrophoresis. Bcl-2 protein level was detected by Western blot assay.

RESULTSBy using clonogenic assay, lidamycin in combination with cisplatin was found to have synergetic effects on the proliferation of human hepatoma BEL-7402 cells. The data showed that BEL-7402 cells treated with cisplatin and lidamycin in combination produced internucleosomal DNA fragmentation analysed by agarose gel electrophoresis. The results of flow cytometry showed that cisplatin and lidamycin administrated in combination showed no obvious change in G1 phase distribution compared with single treatment. However, this combination reduced the S phase arrest and reversed the reduction of G2/M phase induced by single treatment. The results also showed that there was 11.3% or 9.37% of cells undergoing apoptosis in BEL-7402 cells treated with cisplatin or lidamycin, respectively, while it showed 32.4% of apoptotic cells in combination treatment. Cisplatin, lidamycin and combination of cisplatin and lidamycin was shown to induce typical chromatin condensation in BEL-7402 cells. The study showed that 0.5 mumol.L-1 cisplatin or 1 x 10(-4) mumol.L-1 lidamycin alone decreased Bcl-2 protein level, while lidamycin in combination with cisplatin strongly inhibited expression of Bcl-2 proteins in BEL-7402 cells.

CONCLUSIONThe results suggest that lidamycin enhancement of cisplatin-induced apoptosis associates with decrease of Bcl-2 protein expression, which may be useful for cancer chemotherapy.

Aminoglycosides ; pharmacology ; Antibiotics, Antineoplastic ; pharmacology ; Antineoplastic Agents ; pharmacology ; Apoptosis ; Carcinoma, Hepatocellular ; pathology ; Cisplatin ; pharmacology ; Drug Synergism ; Enediynes ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; S Phase ; drug effects ; Tumor Cells, Cultured

AIMA bioassay method was established for the determination of active concentrations of lidamycin and studied its pharmacokinetics in mice and dogs.

METHODSCytotoxicity of lidamycin in vitro was used to determine drug serum concentrations in vivo.

RESULTSValidity of methodology met the requirements of pharmacokinetic study. The concentration-time profile in mice after iv lidamycin of 100, 50 and 10 microg x kg(-1) was best fitted with 2-compartmental model with T1/2alpha and T1/2beta of 0.77-1.8 min and 5.6-7.2 min, respectively. The AUC were 2851.3, 887.8 and 166.4 microg x min x L(-1), respectively and increased with dose nonlinearly. There were similar trends between AUC and the potency of tumor growth inhibition. After iv lidamycin of 12 microg x kg(-1) in dogs, the concentrations of lidamycin decreased rapidly and the AUC was 16 microg x min x L(-1), which were lower and quicker than those in mice. The levels in serum after second administration at day 15, were lower than those of the first.

CONCLUSIONActive concentrations and pharmacokinetics of lidamycin were obtained by bioassay method successfully. There are species differences and single and multi-dosing differences in the pharmacokinetics of lidamycin.

Aminoglycosides ; blood ; pharmacokinetics ; pharmacology ; Animals ; Antibiotics, Antineoplastic ; blood ; pharmacokinetics ; pharmacology ; Area Under Curve ; Biological Assay ; Dogs ; Enediynes ; Female ; Humans ; Injections, Intravenous ; KB Cells ; metabolism ; Liver Neoplasms ; pathology ; Male ; Mice ; Neoplasm Transplantation ; Sarcoma 180 ; pathology ; Species Specificity

Lidamycin (LDM) is a potent antitumor antibiotic. Previous studies have shown that LDM could inhibit proliferation and migration in endothelial cells. In the present report, the effect of LDM on angiogenesis of zebrafish embryo was studied. The results showed that treatment of zebrafish embryos with LDM resulted in significant inhibition of angiogenesis. Morphological observation, quantitative endogenous alkaline phosphatase (EAP) assay, alkaline phosphatase staining, and transgenic zebrafish assay were performed to evaluate vascular development defects in zebrafish. The results indicated that after the zebrafish embryos were exposed to LDM, angiogenesis defects of zebrafish embryos were observed, including pericardial edema, reduced numbers of circulating red blood cells, suppression of zebrafish vessel growth, and absences of SIV (subintestinal vein). The expression of VEGF was detected by RT-PCR assay, quantitative reverse transcriptase real-time PCR (qRT-PCR) assay and Western blotting analysis. The results revealed that LDM could inhibit the expression of VEGF protein, while the expression of mRNA was not significantly affected. The study suggests that LDM could inhibit the zebrafish embryo angiogenesis by down-regulation ofVEGF expression.
Aminoglycosides ; pharmacology ; Animals ; Animals, Genetically Modified ; embryology ; genetics ; physiology ; Antibiotics, Antineoplastic ; pharmacology ; Down-Regulation ; Embryo, Nonmammalian ; drug effects ; Enediynes ; pharmacology ; Neovascularization, Physiologic ; drug effects ; genetics ; RNA, Messenger ; metabolism ; Vascular Endothelial Growth Factor A ; genetics ; metabolism ; Zebrafish ; embryology ; genetics ; physiology