Complex components of Shengmai formula interact with organic cation transporter 2 (OCT2) in MDCK cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Shengmai formula (SMF) is a well-known Chinese herbal compound preparation, which is utilized extensively for the treatment of myocardial ischemia, arrhythmia and other life-threatening conditions. Our previous researches have shown that some of the active ingredients in SMF can interact with organic anion transport polypeptide 1B1 (OATP1B1), breast cancer resistance protein (BCRP) and organic anion transporter 1 (OAT1), etc. Organic cation transporter 2 (OCT2) is a highly expressed uptake transporter in the kidney, and its interaction with the major active components in SMF remains unclear. AIM OF THE STUDY: We purposed to explore OCT2-mediated interactions and compatibility mechanisms of the main active compounds in SMF. MATERIALS AND METHODS: Fifteen active ingredients of SMF, including ginsenoside Rb1, Rd, Re, Rg1, Rf, Ro and Rc, methylophiopogonanone A and B, ophiopogonin D and D', schizandrin A and B, schizandrol A and B, were selected to investigate OCT2-mediated interactions in Madin-Darby cacine kidney (MDCK) cells stably expressing OCT2. RESULTS: Among the above 15 main active components, only ginsenosides Rd, Re and schizandrin B could significantly inhibit the uptake of 4-(4-(dimethylamino)styryl)-N-methyl pyridiniumiodide (ASP+), a classical substrate of OCT2. Ginsenoside Rb1 and methylophiopogonanone A can be transported by MDCK-OCT2 cells, and their uptake was significantly reduced when OCT2 inhibitor decynium-22 was added. Ginsenoside Rd could remarkably reduce the uptake of methylophiopogonanone A and ginsenoside Rb1 by OCT2, ginsenoside Re only decreased the uptake of ginsenoside Rb1, while schizandrin B had no effect on the uptake of both. CONCLUSIONS: OCT2 mediates the interaction of the major active components in SMF. Ginsenosides Rd, Re and schizandrin B are the potential inhibitors of OCT2, while ginsenosides Rb1 and methylophiopogonanone A are the potential substrates of OCT2. There is an OCT2-mediated compatibility mechanism among these active ingredients of SMF.

A high-resolution mass spectrometry-based methodology for characterization and identification of methylophiopogonanone B metabolites from cryopreserved hepatocytes and liver microsomes.

Methylophiopogonanone B (MOB), one of the homoisoflavonoids isolated from Ophiopogon japonicus, has been demonstrated to possess antioxidative and antitumor activities. The aim of this work was to investigate the metabolism of MOB using liver microsomes and hepatocytes. MOB was individually incubated with rat, monkey, and human hepatocytes to generate the metabolites. To investigate the bioactivation pathways, MOB was incubated with liver microsomes in the presence of glutathione (GSH). All the metabolites were detected and identified using LC with a quadrupole Orbitrap mass spectrometer. Under the current conditions, nine metabolites were identified in hepatocyte incubations. Of these metabolites, M7 derived from hydroxylation was identified as the most abundant metabolite in hepatocyte incubation. MOB was metabolized via demethylation, hydroxylation, and glucuronidation. In liver microsomes, five GSH conjugates were detected and identified. MOB was subjected to bioactivation through demethylation yielding M9, which further formed quinone-methide and ortho-quinone intermediates, followed by GSH conjugation. This work is the first to study the metabolism of MOB, which will help us understand its disposition and efficacy.

Interaction of the main active components in Shengmai formula mediated by organic anion transporter 1 (OAT1).

ETHNOPHARMACOLOGICAL RELEVANCE: Shengmai formula (SMF) is a classical traditional Chinese medicine prescription, which is widely used in the treatment of cardiovascular and cerebrovascular diseases. Our previous studies have demonstrated that some components in SMF can interact with each other through breast cancer resistance protein, sodium taurocholate co-transporting polypeptide, organic anion transporting polypeptide 1B1 and 1B3. Organic anion transporter 1 (OAT1) is highly expressed in kidney, mediating the elimination of many endogenous and exogenous substances. However, the interaction between the main active components in SMF and OAT1 is not clear. AIM OF THE STUDY: This study aimed to investigate the interactions of the major bioactive components in SMF mediated by OAT1. MATERIALS AND METHODS: Four main fractions, namely, ginseng total saponins (GTS), ophiopogon total saponins (OTS), ophiopogon total flavonoids (OTF), fructus schisandrae total lignans (STL), and 12 active components, namely, ginsenoside Rg1, Re, Rd and Rb1, ophiopogonin D and D', methylophiopogonanone A and B, schizandrol A and B, schizandrin A and B, were selected to explore the interactions of SMF with OAT1 using cell and rat models. RESULTS: The above four main fractions in SMF all exhibited inhibitory effects on the uptake of 6-carboxyfluorescein (6-CF), a classic substrate of OAT1. Among the 12 main effective components, only ginsenoside Re, Rd, and methylophiopogonanone A showed inhibition of 6-CF uptake. Additionally, we found that schizandrin B was transported by HEK293-OAT1 cells, and schizandrin B uptake was markedly inhibited by GTS, OTS, OTF, ginsenoside Re, Rd, and methylophiopogonanone A. In rats, ginsenoside Re, Rd, and methylophiopogonanone A jointly increased the AUC(0-t), AUC(0-∞), and Cmax of schizandrin B, but they decreased its clearance in plasma and excretion in urine. CONCLUSIONS: Ginsenoside Re, Rd, and methylophiopogonanone A were the potential inhibitors of OAT1, and may interact with some drugs serving as OAT1 substrates clinically. Schizandrin B was a potential OAT1 substrate, and its OAT1-mediated transport was inhibited by ginsenoside Re, Rd, and methylophiopogonanone A. OAT1-mediated interactions of the main active components in SMF can be regarded as one of the important compatibility mechanisms of traditional Chinese medicine preparations.

Modulation of transporter activity of OATP1B1 and OATP1B3 by the major active components of Radix Ophiopogonis.

Radix Ophiopogonis is often an integral part of many traditional Chinese formulas, such as Shenmai injection used to treat cardio-cerebrovascular diseases. This study aimed to investigate the influence of the four active components of Radix Ophiopogonis on the transport activity of OATP1B1 and OATP1B3. The uptake of rosuvastatin in OATP1B1-HEK293T cells were stimulated by methylophiopogonanone A (MA) and ophiopogonin D' (OPD') with EC50 calculated to be 11.33 ± 2.78 and 4.62 ± 0.64 µM, respectively. However, there were no remarkable influences on rosuvastatin uptake in the presence of methylophiopogonanone B (MB) or ophiopogonin D (OPD). The uptake of atorvastatin in OATP1B1-HEK293T cells can be increased by MA, MB, OPD and OPD' with EC50 calculated to be 6.00 ± 1.60, 13.64 ± 4.07, 10.41 ± 1.28 and 3.68 ± 0.85 µM, respectively. The uptake of rosuvastatin in OATP1B3-HEK293T cells was scarcely influenced by MA, MB and OPD, but was considerably increased by OPD' with an EC50 of 14.95 ± 1.62 µM. However, the uptake of telmisartan in OATP1B3-HEK293T cells was notably reduced by OPD' with an IC50 of 4.44 ± 1.10 µM, and barely affected by MA, MB and OPD. The four active components of Radix Ophiopogonis affect the transporting activitives of OATP1B1 and OATP1B3 in a substrate-dependent manner.

Comparison on homoisoflavones extracted from Ophiopogon japonicus in Sichuan and Zhejiang and their anti-oxidative activity / 中草药

Objective: To compare the contents of three homoisoflavones and their anti-oxidative activity of Ophiopogon japonicus in Sichuan (OJS) and Zhejiang (OJZ). Methods: The determination was performed on ACQUITYTM UPLC BEH C18 column (100 mm×2.1 mm, 1.7 μm), the Ultra Performance Liquid Chromatography-Photo-Diode Array (UPLC-PDA) technology was applied, and the mobile phase consisted of acetonitrile and 0.2% formic acid aqueous solution (55:45) with gradient elution program, flow rate was 0.20 mL/min with 2 μL of sample quantity at 296 nm, and the anti-DPPH radical efficiency of water extract from Ophiopogonis Radix were evaluated by UV-photometer. Results: The average values of methylophiopogonone A, methylophiopogonanone A, and methylophiopogonanone B were (3.06±0.54), (40.10±5.63), and (29.51±5.06) μg/g in OJS, respectively; while those in OJZ were (9.22±3.52), (106.63±27.56), and (256.97±61.79) μg/g, separately. The IC50 value was 16.59 mg/mL in OJS, while that in OJZ was 14.48 mg/mL. The IC50 value of positive control VC was 7.06 μg/mL. Conclution: Compared with OJS, the contents of homoisoflavones in OJZ are higher, and the anti-radical efficiency of water extract from OJZ is stronger. It provides the basis for the quality evaluation and geo-authentic research of Ophiopogonis Radix.

Determination of eight active components in Xuanmai Ganjie Granules by UPLC-MS/MS / 中草药

Objective: To develop a UPLC-MS/MS method for simultaneously determining harpagide, liquiritin, harpagoside, platycodin D, ammonium glycyrrhetate, ophiopogonin D, methylophiopogonanone A, and methylophiopogonanone B in Xuanmai Ganjie Granules (composed with Scrophulariae Radix, Ophiopogonis Radix, Glycyrrhizae Radix et Rhizoma, and Platycodonis Radix) from different pharmaceutical companies. Methods: The chromatographic separation was achieved on Phenomenex Kenetix C18 column (50 mm × 2.1 mm, 5 μm) by using a mobile phase consisted of acetonitrile and 0.1% formic acid water at the flow rate of 0.3 mL/min for gradient elution. Simultaneous monitoring of positive and negative ions and multiple reaction monitoring (MRM) scan mode were applied to the quantification of the components in Xuanmai Ganjie Granules; Sample volume was 5 μL. Results: There was good linearity between the absorption peak area and the concentration for harpagide, liquiritin, harpagoside, platycodin D, ammonium glycyrrhetate, ophiopogonin D, methylophiopogonanone A, and methylophiopogonanone B in the ranges of 9-2250, 8-2000, 3.4-850, 96-24000, 12.4-3100, 3.6-1900, 1.7-425, and 1.5-375 ng/mL, respectively. The average recoveries were ranged from 97.2% to 102.8% (RSD ≤ 2.7%). The contents of harpagide, liquiritin, harpagoside, platycodin D, ammonium glycyrrhetate, ophiopogonin D, methylophiopogonanone A, and methylophiopogonanone B in eight batches of samples were in the ranges of 32.8-107.6, 54.8-178.0, 14.6-70.7, 31.2-280.0, 106.4-287.9, 0.1-0.6, 0.01-0.07, and 0.03-0.17 μg/g, respectively. Conclusion: The developed method is simple, effective, and credible for determining the eight components in Xuanmai Ganjie Granules. It provides more helpful information for the comprehensive quality evaluation of Xuanmai Ganjie Granules.

Simultaneous determination of seven components in Shenmai Injection by HPLC-MS/MS / 中草药

Objective: To develop a liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for simultaneously analyzing seven components (ginsenosides Rg1, ginsenosides Re, ginsenosides Rb1, ophiopogonin D, ophiopogonin D', methylophiopogonanone A, and methylophiopogonanone B) in Shenmai Injection. Methods: Multiple reaction monitoring (MRM) scan mode was used for the quantification of five saponins and two flavones. The seven constituents were separated within 15 min on a Phenomenex Luna C18 column (150 mm × 2.1 mm, 5 μm) using a mobile phase consisted of acetonitrile and 0.03% acetic acid water solution with gradient elution. Results: The linear relationships between the concentration and peak areas of the seven target components were ginsenoside Rg1 Y=15.6 X + 1.63 × 104, ginsenoside Re Y=14 X + 5.36 × 103, ginsenoside Rb1 Y=2.46 X + 4.74 × 103, ophiopogonin D Y=11 X + 9.73 × 103, ophiopogonin D' Y=5.56 X + 1.64 × 103, methylophiopogonanone A Y=3.58 × 103 X + 2.33 × 104, and methylophiopogonanone B Y=4.87 × 103 X + 2.72 × 104, respectively. The precisions, repeatabilities, and stabilities of the method were good for the seven components. The average recovery ranged from 95.3%-104.3%, and the precision in terms of RSD was less than 2.4%. Conclusion: The method is rapid and reliable for the determination of the seven constituents in Shenmai Injection. Among these constituents, ophiopogonin D, ophiopogonin D', methylophiopogonanone A, and methylophiopogonanone B are quantified in the Shenmai Injection for the first time.

Determination of methylophiopogonanones A and B in Radix Ophiopogonis and its extracts by HPLC / 中草药

Objective To determine the contents of methylophiopogonanone A (MOA) and methylophiopogonanone B (MOB) in Radix Ophiopogonis and its extracts. Methods An HPLC-UV method was used for determining the contents of MOA and MOB in all samples. Analytical column was Kromasil C18 (250 mm?4.6 mm, 5 ?m). Mobile phase was acetonitrle-water (55∶45) and detection wavelength was 298 nm. The flow rate of mobile phase was 1 mL/min, and temperature was 30 ℃. Results The contents of MOA in Radix Ophiopogonis cropped in Sichuan and Zhejiang Provinces were 0.004 0%-0.009 6%, 0.006 7%-0.013 4%, and the contents of MOB were 0.002 1%-0.006 2%, 0.015 9%-0.028 2%, respectively. The contents of MOA in the extract of Radix Ophiopogonis cropped in Sichuan and Zhejiang Provinces were 0.007 5%-0.008 8%, 0.011 2%-0.012 6%, and the contents of MOB were 0.003 8%-0.005 1%, 0.020 7%-0.023 8%, respectively. Conclusion The contents of MOA and MOB in Radix Ophiopogonis cropped in Zhejiang Province and its extracts are more than those in Sichuan Province and its extrouts. The method can be used for the purpose of the quality control of Radix Ophiopogonis and its extracts.