To explore the effect of ophiopogonin D on main fatty acid metabolic enzymes in human cardiomyocyte AC-16,so as to provide reference for cardiovascular protection mechanism and safe clinical application of Ophiopogon japonicus.CCK-8 (cell counting kit-8) was used to detect the effect of different concentrations of ophiopogonin D on the viability of cardiomyocytes.Meanwhile,the effect of different concentrations of ophiopogonin D on the morphology and quantity of cardiomyocytes was observed under microscope.The effect of ophiopogonin D on the mRNA expression of CYP2J2,CYP4F3,CYP4A11,CYP4A22 and CYP4F2 in cardiomyocytes was detected by RT-PCR.Western blot was used to detect the protein expression of CYP4F3 in different concentrations of ophiopogonin D.Compared with the control group,low-concentration ophiopogonin D had no effect on the viability of cardiomyocytes.However,ophiopogonin D with a concentration of higher than 20μmol·L~(-1)could promote the viability.Under the microscope,ophiopogonin D with a concentration of below 100μmol·L~(-1)had no significant effect on the morphology and number of cardiomyocytes.RT-PCR results showed that compared with the control group,5μmol·L~(-1)ophiopogonin D could slightly up-regulate mRNA expressions of CYP2J2 and CYP4F3,while high-concentration ophiopogonin D (10 and 20μmol·L~(-1)) could significantly induce mRNA expressions of CYP2J2and CYP4F3 in a dose-dependent manner (P<0.05).The same concentration of ophiopogonin D had a little effect on the mRNA expressions of CYP4A11,CYP4A22 and CYP4F2.Western blot results showed that 20μmol·L~(-1)ophiopogonin D could significantly induce the protein expression of CYP4F3 in a dose-dependent manner (P<0.05).Based on the above results,ophiopogonin D (less than100μmol·L~(-1)) has no effect on the viability of AC-16 cardiomyocytes.Ophiopogonin D (less than 100μmol·L~(-1)) can selectively induce the expressions of CYP2J2 and CYP4F3,regulate the metabolic pathway of fatty acid signaling molecules,and thus protecting the cardiovascular system.
Fatty Acids ; Humans ; Myocytes, Cardiac ; Saponins/pharmacology* ; Spirostans/pharmacology*

This study is aimed to investigate the intervention effect and possible mechanism of ophiopogonin D( OPD) in protecting cardiomyocytes against ophiopogonin D'( OPD')-induced injury,and provide reference for further research on toxicity difference of saponins from ophiopogonins. CCK-8 assay was used to evaluate the effect of OPD and OPD' on cell viability. The effect of OPD on OPD'-induced cell apoptosis was measured by flow cytometry. Morphologies of endoplasmic reticulum were observed by endoplasmic reticulum fluorescent probe. PERK,ATF-4,Bip and CHOP mRNA levels were detected by Real-time quantitative polymerase chain reaction( PCR) analysis. ATF-4,phosphorylated PERK and e IF2α protein levels were detected by Western blot assay. RESULTS:: showed that treatment with OPD'( 6 μmol·L-1) significantly increased the rate of apoptosis; expressions of endoplasmic reticulum stress related genes were increased. The morphology of the endoplasmic reticulum was changed. In addition,different concentrations of OPD could partially reverse the myocardial cell injury caused by OPD'. The experimental results showed that OPD'-induced myocardial toxicity may be associated with the endoplasmic reticulum stress,and OPD may modulate the expression of CYP2 J3 to relieve the endoplasmic reticulum stress caused by OPD'.
Apoptosis ; Cardiotonic Agents ; pharmacology ; Cells, Cultured ; Endoplasmic Reticulum Stress ; drug effects ; Humans ; Myocytes, Cardiac ; drug effects ; Saponins ; pharmacology ; Spirostans ; pharmacology

OBJECTIVETo investigate the effects of ophiopogonin D on human breast cancer MCF-7 cells.

METHODSCell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and colony formation experiments. Cell cycle was measured with cell cycle flow cytometry and a living cell assay. Apoptosis and terminal deoxynucleoitidyl transferase-mediated dUTP nick end labeling assays were performed to detect the apoptosis of MCF-7 cells induced by ophiopogonin D. Finally, Western blotting was used to explore the mechanism.

RESULTSExposure of cells to ophiopogonin D resulted in marked decreases in viable cells and colony formation with a dose-dependent manner. Treatment of these cells with ophiopogonin D also resulted in cell cycle arrest at the G(2)/M phase, and increased apoptosis. Mechanistically, ophiopogonin D-induced G(2)/M cell cycle arrest was associated with down-regulation of cyclin B1. Furthermore, activation of caspase-8 and caspase-9 was involved in ophiopogonin D-induced apoptosis.

CONCLUSIONThe data suggested that ophiopogonin D inhibits MCF-7 cell growth via the induction of cell cycle arrest at the G(2)/M phase.

Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; G2 Phase Cell Cycle Checkpoints ; drug effects ; Humans ; M Phase Cell Cycle Checkpoints ; drug effects ; MCF-7 Cells ; Saponins ; pharmacology ; Spirostans ; pharmacology

This study aimed to investigate the effect and mechanism of ophiopogonin D (OP-D) on Ang Ⅱ-induced HUVECs apoptosis, in order to provide a reliable basis for the safety and efficacy of traditional Chinese medicines. The effect of Ang Ⅱ on survival and total proteins content of HUVECs were measured by MTT and Western blotting. The effect of OP-D on Ang Ⅱ-induced lactate dehydrogenase (LDH) release rate in HUVECs was measured by enzyme standard instrument. The effects of OP-D and 11,12-EET on phosphorylation of JNK/c-Jun induced by Ang Ⅱ were measured by Western blot and RT-PCR with the help of JNK specific inhibitor SP600125 and CYP450 isozymes selective inhibitor 6-(2-propargyloxyphenyl) hexanoic acid (PPOH). The cell apoptosis was assayed by flow cytometry. According to the results, different doses of Ang Ⅱ had no significant effect on cell survival; treatment with Ang Ⅱ at 1×10⁻⁶ mol·L⁻¹ could increase the release of LDH (<0.001), improve the JNK and c-Jun phosphorylation levels(<0.01, <0.001), increase the expression of caspase-3(<0.01), and promote the apoptosis of HUVECs(<0.001). The phosphorylation of JNK and c-Jun could be inhibited by the pre-treatment with SP600125, 11,12-EET and OP-D. Pre-treatment with OP-D could significantly reduce the release of LDH induced by Ang Ⅱ stimulation, decrease the expression of caspase-3, and diminish the apoptosis of cells. The protective effect of OP-D was suppressed, when being pretreated with PPOH. The experimental results showed that the apoptosis of HUVECs induced by Ang Ⅱ may be associated with JNK/c-Jun signaling pathway. OP-D-mediated CYP2J2 expression increased 11,12-EET levels, and could remarkably resist Ang Ⅱ-induced injury and apoptosis of cells, which is associated with the maintenance of endothelium homeostasis.
Angiotensin II ; Apoptosis ; Arachidonic Acids ; metabolism ; Cells, Cultured ; Cytochrome P-450 Enzyme System ; metabolism ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; Humans ; Phosphorylation ; Saponins ; pharmacology ; Signal Transduction ; Spirostans ; pharmacology

An unusual novel C27-steroidal glycoside sulfate was isolated from the underground organs of Liriope graminifolia (Linn.) Baker with three known compounds. Their chemical structures were determined by spectral analysis, including HR-MS, 1D and 2D NMR as (25S)-ruscogenin 1-sulfate-3-O-alpha-L-rhamnopyranoside (1), (25S)-ruscogenin 1-O-beta-D-xylopyranosyl-3-O-alpha-L-rhamnopyranoside (2), hesperidin (3), and 4', 7-dihydroxy-5-methoxyflavanone (4). Compound 1 has cytotoxic activities against K562 and HL60 cells with IC50 values of 18.6 microg x mL(-1) and 16.5 microg x mL(-1), respectively.
Antineoplastic Agents, Phytogenic ; chemistry ; isolation & purification ; pharmacology ; Cell Proliferation ; drug effects ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; pharmacology ; Glycosides ; chemistry ; isolation & purification ; pharmacology ; HL-60 Cells ; Hesperidin ; chemistry ; isolation & purification ; pharmacology ; Humans ; Inhibitory Concentration 50 ; K562 Cells ; Liriope Plant ; chemistry ; Plant Tubers ; chemistry ; Plants, Medicinal ; chemistry ; Spirostans ; chemistry ; isolation & purification ; pharmacology

OBJECTIVETo develop an HPLC-ELSD method for the determination of 25 (R, S) ruscogenin 1-O-[beta-D-glucopyranosyl (1 --> 2)] [beta-D-xylopyranosyl (1 --> 3)] beta-D-fucopyranoside in the tuberous roots of Liriope muscari from different habitats and different harvest time.

METHODA Shimadzu C18 column (4.6 mm x 150 mm, 5 microm) with a solvent system consisting of acetonirile-water (46: 54) was used, and detected by ELSD. The temperature of drift tube was 94 degrees C and the nebulizer nitrogen flow rate was 2.5 L x min(-1).

RESULTThe calibration curve of 25 (R, S) ruscogenin 1-O-[beta-D-glucopyranosyl (1 --> 2)] [beta-D-xylopyranosyl (1 --> 3)] beta-D-fucopyranoside showed good linearity in the range of 1.02-12.228 microg and the average recovery was 100.80%, with RSD of 1.8%. 10 batches of L. muscari from different habitats were analyzed, and the contents were 0.25% - 0.41%. The contents of 15 batches from different harvest time were 0.13%-0.38%.

CONCLUSIONThe method is simple, rapid and sensitive, and can be used for determination of 25 (R, S) ruscogenin 1-O-[beta-D-glucopyranosyl (1 --> 2)] [beta-D-xylopyranosyl (1 --> 3)] beta-D-fucopyranoside in L. muscari. It provides the valuable basis for quality assessment of L. muscari.

Chromatography, High Pressure Liquid ; methods ; Ecosystem ; Liliaceae ; chemistry ; Liriope Plant ; chemistry ; Magnetic Resonance Spectroscopy ; methods ; Molecular Sequence Data ; Molecular Structure ; Plant Preparations ; analysis ; chemistry ; pharmacology ; Plant Roots ; chemistry ; physiology ; Plant Structures ; chemistry ; Saponins ; chemistry ; Spirostans ; analysis ; chemistry ; pharmacology ; Triterpenes ; isolation & purification

This study aimed to examine whether ophiopogonin D (OP-D) is capable of protecting cardiomyocytes against DOX-induced injury and the mechanisms involved. H9c2 cells were cultured. MTT assay was used to evaluate cell viability and toxicity. Mito-tracker as fluorescence probe was used to measure ROS content raised from mitochondria. The mRNA and protein expression of ATF6alpha, GRP78 and CHOP were analyzed using real-time PCR and Western blotting, respectively. The results showed that a significant endoplasmic reticulum stress (ERS) was induced upon exposure of H9c2 cells to DOX as indicated by the increase in the expression of ERS related proteins, which was paralleled with the accumulation of reactive oxygen species (ROS) and decrease in the viability of H9c2 cells. Whereas, DOX-induced ROS accumulation and up-regulation of ERS related proteins were partially abolished by pretreatment with OP-D. Consequently, a DOX-induced ERS was mitigated by application of OP-D. Similarly, DOX-induced decrease in cell viability was partially attenuated by either inhibiting CHOP or pretreatment with N-acetylcysteine (NAC), an antioxidant. Moreover, cardiac ultrastructural abnormalities seen in mouse receiving DOX injections were obviously ameliorated by pretreatment of OP-D. Taken together, the present study proved that OP-D protects cardiomyocytes against DOX-induced injury, at least in part, through reducing ROS accumulation and alleviating ERS.
Acetylcysteine ; Activating Transcription Factor 6 ; metabolism ; Animals ; Antioxidants ; Cell Line ; Cell Survival ; Doxorubicin ; adverse effects ; Endoplasmic Reticulum Stress ; drug effects ; Heat-Shock Proteins ; metabolism ; Mice ; Mitochondria ; metabolism ; Myocytes, Cardiac ; drug effects ; Rats ; Reactive Oxygen Species ; metabolism ; Saponins ; pharmacology ; Spirostans ; pharmacology ; Transcription Factor CHOP ; metabolism ; Up-Regulation

This study is to investigate the antitumor activity of ophiopogonin B (OP-B). MTT assay, flow cytometric analysis, acridine orange staining, Lyso-Tracker Red staining and HeLa-GFP-LC3 transfect cells assay were used to detect the proliferation activity, apoptosis and autophagy of HeLa cells. The results showed that OP-B exerted potent antiproliferative activity on HeLa cells, the cell growth inhibition effect of OP-B was not due to apoptosis and OP-B could induce autophagy of HeLa cells. OP-B also induced the protein expression up-regulation of Beclin-1 and promoted LC3 I transformation LC3 II, which were representative proteins of autophagy. Furthermore, 3-MA, an inhibitor of autophagy, not only inhibited OP-B-mediated autophagy but also almost completely reversed the antiproliferative effect of OP-B, suggesting that the growth inhibition effect of OP-B was autophagy dependent. Western blotting demonstrated that OP-B inhibited the phosphorylation of Akt and its' downstream vital protein, such as mTOR and p70S6K. In addition, OP-B also induced the protein expression up-regulation of PTEN, which is a negative regulation protein for Akt/mTOR signaling pathway. However, OP-B did not affect the protein expression of total Akt. Collectively, the antitumor effects of OP-B were autophagy-dependent via repression Akt/mTOR signaling pathway. Therefore, OP-B is a prospective inhibitor of Akt/mTOR and may be used as an alternative compound to treat cervical carcinoma.
Adenine ; analogs & derivatives ; pharmacology ; Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; metabolism ; Autophagy ; drug effects ; Beclin-1 ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; HeLa Cells ; Humans ; Membrane Proteins ; metabolism ; Microtubule-Associated Proteins ; metabolism ; Ophiopogon ; chemistry ; PTEN Phosphohydrolase ; metabolism ; Phosphorylation ; Plants, Medicinal ; chemistry ; Proto-Oncogene Proteins c-akt ; metabolism ; Ribosomal Protein S6 Kinases, 70-kDa ; metabolism ; Saponins ; pharmacology ; Signal Transduction ; drug effects ; Spirostans ; pharmacology ; TOR Serine-Threonine Kinases ; metabolism ; Up-Regulation