Nonaqueous capillary electrophoresis is used for the determination of the contents of diosgenin and ruscogenin in Radix Ophiopogonis. The operating buffer was composed of 20 mmol x L(-1) Na2B4O7-HCl (pH 7.61) in 70% methanol. The applied voltage was 25 kV and detection potential was at +0.70 V. With these conditions, the components were successfully separated. The content of diosgenin in Radix Ophiopogonis was 0.018 mg x g(-1) and ruscogenin was 0.008 mg x g(-1). The average recoveries of diosgenin and ruscogenin were 102% and 99.2%, respectively. A new method of the quality control of diosgenin and ruscogenin in Radix Ophiopogonis is provided.
Diosgenin ; analysis ; Electrophoresis, Capillary ; methods ; Ophiopogon ; chemistry ; Plant Roots ; chemistry ; Plants, Medicinal ; chemistry ; Quality Control ; Spirostans ; analysis

AIM: To investigate the chemical constituents of Dioscorea zingiberensis C. H. Wright. METHODS: The compounds were isolated by various chromatographic techniques, and the structures of the new steroidal saponins were elucidated by extensive 1D- and 2D-NMR, MS, and IR spectral analysis. RESULTS: The 70% EtOH extract of the rhizomes of Dioscorea zingiberensis afforded two new steroidal saponins, zingiberenosides A (1) and B (2), along with eight known analogues, 3β, 26-dihydroxy-25(R)-furosta-Δ(5, 20(22))-diene-3-O-α-L- rhamnopyranosyl-(1→2)-O-β-D-glucopyranoside (3), methyl parvifloside (4), deltoside (5), methyl deltoside (6), zingiberensis new saponin (7), deltonin (8), progenin III (9) and diosgenin-diglucoside (10). CONCLUSION Two new steroidal saponins were isolated from Dioscorea zingiberensis and their structures determined.
Dioscorea ; chemistry ; Diosgenin ; chemistry ; isolation & purification ; Molecular Structure ; Plant Extracts ; chemistry ; Rhizome ; chemistry ; Saponins ; chemistry ; isolation & purification ; Spirostans ; chemistry ; isolation & purification ; Steroids ; chemistry ; isolation & purification

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*

A competitive enzyme-linked immunosorbent assay (ELISA) was developed to determine ruscogenin (RUS) by using the monoclonal antibody (McAb). The monoclonal antibody against RUS, secreted from the established hybridoma cell lines, was identified as being of the IgG1 isotype. The McAb exhibited high specificity to RUS, showing a very slight cross reactivity with diosgenin (15.7%), and no cross-reactivity to sarsasapogenin, diammonium glycyrrhizinate, oleanolic acid and notoginsenoside R1. The established ELISA, at an IC50 value of 157.55 ng·mL(-1) and a detection limit (IC20) of 20.57 ng·mL(-1), was compared with HPLC analyses, and a good correlation between ELISA and HPLC-ELSD analyses of RUS in the extract of Radix Ophiopogonis was obtained. The experimental data indicated that the ELISA method exhibits more advantages over HPLC-ELSD, such as low detection limit, high specificity, low background, and no requirement for sample pre-treatment, and is more suitable for the determination of natural components in Chinese traditional medicines and in biological samples for pharmacokinetic studies.
Antibodies, Monoclonal ; analysis ; Drugs, Chinese Herbal ; analysis ; Enzyme-Linked Immunosorbent Assay ; methods ; Sensitivity and Specificity ; Spirostans ; analysis

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 study the accumulation of active ingredients, the absorption and transformation of N, P and K in Anemarrhena asphodeloides and provide basis for determination of the harvest time and fertilizing.

METHODSamples were collected in different phrases and the weight of dry matter, the content of N, P and K of different organs and the content of sarsasapogenin were determined.

RESULTAbsorption of N, P and K started by the root and rhizoma after July. At the end of August, the N and K of the aerial part transfered largely into rhizome. The content of sarsasapogenin in rhizome was the highest in early spring.

CONCLUSIONAdditional fertilizer is helpful to increase the yield in July of the second year after the transplantation. The quality is the best when harvest in early spring.

Absorption ; Anemarrhena ; metabolism ; Fertilizers ; Nitrogen ; pharmacokinetics ; Phosphorus ; pharmacokinetics ; Plant Components, Aerial ; metabolism ; Plant Roots ; metabolism ; Plants, Medicinal ; metabolism ; Potassium ; pharmacokinetics ; Rhizome ; metabolism ; Seasons ; Spirostans ; metabolism

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