Salvianolic acid B inhibits myocardial I/R-induced ROS generation and cell apoptosis by regulating the TRIM8/GPX1 pathway.

CONTEXT: Salvianolic acid B (SalB) can attenuate myocardial ischemia/reperfusion (I/R) injury, but the mechanisms are not entirely known. OBJECTIVE: Our study investigates if SalB protects cardiomyocytes against I/R injury by regulating Tripartite motif (TRIM) protein. MATERIALS AND METHODS: AC16 cardiomyocytes were treated with I/R, and then with SalB (10, 25 and 50 µM) for 24 h, while control cells were cultured under normal conditions. Female Sprague-Dawley rats were subjected to I/R injury, and then intravenously injected with 20, 40, or 60 mg/kg SalB or saline, as a control, rats received sham operation and saline injection. RESULTS: Upon treatment, apoptotic rate, reactive oxygen species (ROS), and malondialdehyde (MDA) were increased 10-, 3.8-, and 1.3-fold, respectively, while superoxide dismutase (SOD) activity was reduced by 62.1% compared to control cells. I/R treatment elevated the mRNA and protein expression of TRIM8. SalB treatment remarkably abolished the above-mentioned effects of I/R treatment. TRIM8 knock-down could partially alleviate I/R-induced myocardial injury. TRIM8 overexpression promoted cardiomyocyte injury, which was alleviated by SalB. Moreover, TRIM8 negatively regulated protein expression of antioxidant enzyme glutathione peroxidase 1 (GPX1). TRIM8 protein interacted with GPX1 and TRIM8 overexpression promoted GPX1 ubiquitnation. GPX1 knock-down abolished the protective effects of SalB on I/R-injured cardiomyocytes. Our in vivo experiments confirmed the effects of SalB on I/R-induced myocardial injury. DISCUSSION AND CONCLUSIONS: SalB protected cardiomyocytes from I/R-induced apoptosis and oxidative stress in vitro and in vivo, which was partly mediated by the TRIM8/GPX1 axis. This suggests that down-regulation of TRIM8 expression may ameliorate I/R-induced myocardial injury.

Interleukin-33 regulates the endoplasmic reticulum stress of human myometrium via an influx of calcium during initiation of labor.

Background: Inflammation is currently recognized as one of the major causes of premature delivery. As a member of the interleukin-1ß (IL-1ß) family, interleukin-33 (IL-33) has been shown to be involved in normal pregnancy as well as a variety of pregnancy-related disorder. This study aims to investigate the potential function of IL-33 in uterine smooth muscle cells during labor. Methods: Myometrium samples from term pregnant (≥37 weeks gestation) women were either frozen or cells were isolated and cultured. Immunohistochemistry and western blotting were used to assess the distribution of IL-33. Cultured cells were incubated with lipopolysaccharide (LPS) to mimic inflammation as well as in the presence of 4µ8C (IRE1 inhibitor III) to block endoplasmic reticulum (ER) stress and BAPTA-AM, a calcium chelator. Results: LPS reduced the expression of nuclear IL-33 in a time-limited manner and induced ER stress. However, knockdown of IL-33 increased LPS-induced calcium concentration, ER stress and phosphorylation of nuclear factor kappa-B (NF-κB), and P38 mitogen-activated protein kinase (P38 MAPK). In addition, siRNA IL-33 further stimulates LPS enhanced cyclooxygenase-2 (COX-2) expression via NF-κB and p38 pathways. IL-33 expression was decreased in the nucleus with the onset of labor. LPS-induced ER stress and increased expression of the labor-associated gene, COX-2, as well as IL-6 and IL-8 in cultured myometrial cells. IL-33 also increased COX-2 expression, but after it was knocked down, the stimulating effect of LPS on calcium was enhanced. 4µ8C also inhibited the expression of COX-2 markedly. The expression of calcium channels on the membrane and intracellular free calcium ion were both increased which was accompanied by phosphorylated NF-κB and p38. Conclusions: These data suggest that IL-33 may be involved in the initiation of labor by leading to stress of the ER via an influx of calcium ions in human uterine smooth muscle cells. Funding: This study was supported by grants from the National Natural Science Foundation of China (No. 81300507).

Decorin-Mediated Inhibition of Human Trophoblast Cells Proliferation, Migration, and Invasion and Promotion of Apoptosis In Vitro.

Preeclampsia (PE) is a unique complication of pregnancy, the pathogenesis of which has been generally accepted to be associated with the dysfunctions of extravillous trophoblast (EVT) including proliferation, apoptosis, and migration and invasion. Decorin (DCN) has been proved to be a decidua-derived TGF-binding proteoglycan, which negatively regulates proliferation, migration, and invasiveness of human extravillous trophoblast cells. In this study, we identified a higher expression level of decorin in severe PE placentas by both real-time reverse transcription-polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC). And an inhibitory effect of decorin on proliferation, migration, and invasion and an enhanced effect on apoptosis in trophoblast cells HTR-8/SVneo and JEG-3 were validated in vitro. Also the modulations of decorin on trophoblast cells' metastasis and invasion functions were detected through regulating the matrix metalloproteinases (MMP2 and MMP9). Thus, we suggested that the contribution of decorin to the modulation of trophoblast cells might have implications for the pathogenesis of preeclampsia.

Resveratrol inhibits trophoblast apoptosis through oxidative stress in preeclampsia-model rats.

Resveratrol has been shown to be a therapeutic agent for cardiovascular disorders by maintaining a lower redox level in vivo through its anti-oxidant properties. Resveratrol can prevent cells from p53- and reactive oxygen species-dependent apoptosis induced by interleukin-1b. We identified an inhibitory effect of resveratrol against oxidative stress and apoptosis using the TUNEL assay in NG-Nitro-l-arginine methyl ester-induced preeclampsia in rats. To investigate a possible association between resveratrol and the apoptosis caused by oxidative stress in vitro, assays for superoxide dismutase and malondialdehyde as well as flow cytometric analyses were conducted in HTR-8/SVneo cells after hypoxic treatment with or without resveratrol for 24 h. These data suggest that resveratrol significantly opposes the effects of oxidative stress in vivo and in vitro.

High-performance liquid chromatography-electrospray ionization tandem mass spectrometry for metabolism study of timosaponin AIII.

A sensitive method based on high-performance liquid chromatography-electrospray ionization tandem mass spectrometry was developed for the determination of timosaponin AIII (TA3) and its in vivo and in vitro metabolites. The rat plasma, urine, feces and tissue samples were collected after oral administration of TA3 at a single dose of 300 mg/kg. TA3 was incubated into artificial gastric juice and artificial intestinal juice. The in vivo and in vitro samples were purified by using liquid-liquid extraction. The structures of metabolites were elucidated by comparing their molecular weights, retention times and tandem mass spectrometric spectra with those of the parent drug. As a result, four metabolites (deglycosylated TA3, two hydroxylated TA3 and timosaponin BII) and the parent drug were found in in vivo and in vitro samples. In addition to the parent drug, one, one and two metabolites were identified in heart, urine and feces, respectively. Only the parent drug was detected in plasma, liver and kidney. One hydroxylation metabolite and TA3 were identified from incubation samples with AGJ, whereas two hydroxylation metabolites and TA3 were detected from the incubation with AIJ. This is the first systematic metabolism study of TA3. The biotransformation pathways of TA3 primarily included deglycosylation, hydroxylation and glycosylation.

Analysis and detection of the chemical constituents of Radix Polygalae and their metabolites in rats after oral administration by ultra high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry.

Radix Polygalae (RP), the dried root of Polygala tenuifolia Willd., is a well-known traditional Chinese medicine to mediate sedative, antipsychotic, cognitive improving, neuroprotective, and anti-inflammatory therapeutic effects on the central nervous system. In this work, ultra high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UHPLC/ESI-Q-TOF-MS/MS) was established for the separation and characterization of the chemical constituents in Radix Polygalae and their metabolites in rat plasma and urine after oral administration. Samples were separated on an Agilent Zorbax Eclipse Plus-C18 column (100mm×2.1mm, 1.8µm) with 0.1% formic acid aqueous solution and acetonitrile as the mobile phase under gradient conditions. Overall, 50 compounds were characterized from the RP, 9 of which are to our knowledge reported for the first time. In vivo, 10 components and 2 metabolites were observed in rat plasma, and 27 components and 7 metabolites were detected in rat urine. The results from this work improve our understanding on the chemical constituents of RP and their metabolic profiling.

Analysis of multiple constituents in Cong-Ming-Tang, a Chinese herbal formula for the treatment of amnesia, by high-performance liquid chromatography with quadrupole time-of-flight mass spectrometry.

INTRODUCTION: Cong-Ming-Tang (CMT), named smart-soup in English, is a well-known traditional Chinese medicine formula for the treatment of amnesia in China. However, the isolation, purification and identification procedures of the major bioactive constituents in CMT are difficult and time consuming. OBJECTIVE: To establish a rapid and sensitive high-performance liquid chromatography/electrospray ionisation with quadrupole time-of-flight tandem mass spectrometry (HPLC-QTOF/MS/MS) method that could be applied to rapidly separate and identify the major bioactive constituents in CMT. METHODS: Methanolic extract of CMT was used for HPLC-QTOF/MS/MS analysis. Separation was performed on an Agilent Poroshell 120 EC- C18 column (2.7 ×100 mm .i.d., 2.7 µm) with 0.1% formic acid aqueous solution and acetonitrile as the mobile phase under gradient conditions. Both positive and negative ion modes were employed. RESULTS: This analytical tool allowed the identification of 55 compounds from CMT formulae by comparing their retention times and MS spectra with those of authentic compounds or literature data in both positive and negative ion modes, including 4 xanthone C-glycosides, 4 sucrose esters, 11 oligosaccharide multi-esters,15 triterpene saponins, 15 triterpene acids, 2 lignans and 4 phenylpropanoids. Onjisaponin MF was tentatively elucidated as a new triterpene saponin based on the summarised fragmentation rules. CONCLUSION: HPLC-QTOF/MS/MS provides a new powerful approach to identify the major chemical constituents in CMT rapidly and accurately. This study proposed a series of potential bioactive components without preparative isolation from the crude extract of CMT and indicated that the HPLC-QTOF/MS/MS method also can be a promising tool for the analysis of other traditional Chinese medicines.

[Effect of transforming growth factor ß1 on the expression of matrix metalloproteinase 9, tissue inhibitor of metalloproteinase 1 and nuclear factor kappa B signalling pathway in the human amniotic cells WISH].

OBJECTIVE: To investigate the effect of transforming growth factor ß1 (TGF-ß1) on the expression of matrix metalloproteinase 9 (MMP-9), tissue inhibitor of metalloproteinase 1 (TIMP-1), nuclear factor kappa B (NF-κB) and the possible signalling pathways in human amniotic cells WISH. METHODS: The WISH cell line was cultured. WISH cells were added with TGF-ß1 of different concentrations (0, 2, 10 and 20 ng/ml, respectively) for 24 hours. Then, reverse transcription (RT) PCR and western blotting were used to analyze the protein and mRNA expression of TIMP-1 and MMP-9; and the expression of NF-κB was analyzed by western blot. RESULTS: (1) The profile of TIMP-1 mRNA (0.413 ± 0.036, 0.623 ± 0.058, 1.392 ± 0.124, 1.387 ± 0.102) in WISH cells elevated when the concentration of TGF-ß1 increased (0, 2, 10, 20 ng/ml). In accordance with TIMP-1 mRNA, the expression of TIMP-1 also elevated with the increase of TGF-ß1 (0.357 ± 0.031, 0.596 ± 0.048, 1.243 ± 0.097 and 1.359 ± 0.121, respectively). And when 2, 10 or 20 ng/ml of TGF-ß1 was added, the TIMP-1 mRNA and protein were significantly higher than the TIMP-1 mRNA and protein when no TGF-ß1 was added (P < 0.05). (2) In contrast with TIMP-1, MMP-9 mRNA (1.325 ± 0.056, 0.987 ± 0.081, 0.610 ± 0.034, 0.347 ± 0.023) in WISH cells decreased when the concentration of TGF-ß1 increased (0, 2, 10, 20 ng/ml). The MMP-9 protein (1.119 ± 0.064, 1.008 ± 0.052, 0.578 ± 0.041, 0.401 ± 0.015) also decreased with the increase of TGF-ß1. And when 2, 10 or 20 ng/ml of TGF-ß1 was added, the MMP-9 mRNA and protein were significantly lower than the MMP-9 mRNA and protein when no TGF-ß1 was added (P < 0.05). (3) The NF-κB protein (1.423 ± 0.065, 1.116 ± 0.045, 0.796 ± 0.041, 0.359 ± 0.021) was significantly reduced with the increase of TGF-ß1 (0, 2, 10, 20 ng/ml;P < 0.05). CONCLUSIONS: The mRNA and protein expression of TIMP-1 decreased when TGF-ß1 was low in WISH cells, whereas those of MMP-9 elevated when TGF-ß1 was low. The unbalance of TIMP-1 and MMP-9 was related to the pathology of the premature rupture of membrane. And the NF-κB singalling pathway might be an important mechanism in the regulation of TIMP-1 and MMP-9 system.

In vivo metabolism study of vaccarin in rat using HPLC-LTQ-MSn.

In order to illustrate the main biotransformation pathways of vaccarin in vivo, metabolites of vaccarin in rats were identified using a specific and sensitive high-performance liquid chromatography-electrospray ionization linear ion trap mass spectrometry (LTQ XL™) method. The rats were administered a single dose (200 mg/kg) of vaccarin by oral gavage. By comparing their changes in molecular masses (ΔM), retention times and spectral patterns with those of the parent drug, the parent compound and six metabolites were found in rat urine after oral administration of vaccarin. The parent compound and five metabolites were detected in rat plasma. In heart, liver and kidney samples, respectively, one, four and three metabolites were identified, in addition to the parent compound. Three metabolites, but no trace of parent drug, were found in the rat feces. This is the first systematic metabolism study of vaccarin in vivo. The biotransformation pathways of vaccarin involved methylation, hydroxylation, glycosylation and deglycosylation.

Metabolism and pharmacokinetics of mangiferin in conventional rats, pseudo-germ-free rats, and streptozotocin-induced diabetic rats.

To clarify the role of the intestinal flora in the absorption and metabolism of mangiferin and to elucidate its metabolic fate and pharmacokinetic profile in diabetic rats, a systematic and comparative investigation of the metabolism and pharmacokinetics of mangiferin in conventional rats, pseudo-germ-free rats, and streptozotocin (STZ)-induced diabetic rats was conducted. Forty-eight metabolites of mangiferin were detected and identified in the urine, plasma, and feces after oral administration (400 mg/kg). Mangiferin underwent extensive metabolism in conventional rats and diabetic rats, but the diabetic rats exhibited a greater number of metabolites compared with that of conventional rats. When the intestinal flora were inhibited, deglycosylation of mangiferin and sequential biotransformations would not occur. Pharmacokinetic studies indicated a 2.79- and 2.35-fold increase in the plasma maximum concentration and the area under the concentration-time curve from 0 to 24 h of mangiferin in diabetic rats compared with those for conventional rats, whereas no significant differences were observed between conventional rats and pseudo-germ-free rats. Further real-time quantitative reverse transcription-polymerase chain reaction results indicated that the multidrug resistance (mdr) 1a level in the ileum increased, whereas its level in the duodenum and the mdr1b mRNA levels in the duodenum, jejunum, and ileum decreased in diabetic rats compared with those in conventional rats. With regard to the pseudo-germ-free rats, up-regulated mdr1a mRNA levels and down-regulated mdr1b mRNA levels in the small intestines were observed. The diabetic status induced increased UDP-glucuronosyltransferase (UGT) 1A3, UGT1A8, UGT2B8, and sulfotransferase (SULT) 1A1 mRNA levels and decreased catechol-O-methyltransferase (COMT), UGT2B6, UGT2B12, and SULT1C1 mRNA levels. These results might partially explain the different pharmacokinetic and metabolic disposition of mangiferin among conventional and model rats.

Metabolism studies of casticin in rats using HPLC-ESI-MS(n).

Casticin (3',5-dihydroxy-3, 4',6,7-tetramethoxyflavone) has been revealed to possess various kinds of pharmacological activities, including immunomodulatory, anti-hyperprolactinemia, anti-tumor and neuroprotetective activities. In order to gain an understanding of the biotransformation of casticin in vivo, a systematic method based on liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS(n) ) was developed to identify the metabolites of casticin in rats after oral administration of single dose of casticin at 200 mg/kg. By comparing their changes in molecular masses (ΔM), retention times and spectral patterns with those of the parent drug, the parent compound and 25 metabolites were identified in rat plasma, urine and six selected tissues. This is the first systematic metabolism study of casticin in vivo. The results indicated that methylation, demethylation, glucuronidation and sulfation were the main biotransformation pathways of casticin in vivo.

Bioactivity-guided fractionation and analysis of compounds with anti-influenza virus activity from Gardenia jasminoides Ellis.

Bioassay-guided fractionation of extracts from Fructus Gardeniae led to analysis of its bioactive natural products. After infection by influenza virus strain A/FM/1/47-MA in vivo, antiviral activity of the extracts were investigated. The target fraction was orally administered to rats and blood was collected. High-performance liquid chromatography coupled with photo diode array detector and electrospray ion trap multiple-stage tandem mass spectrometry was applied to screen the compounds absorbed into the blood. A structural characterization based on the retention time, ultraviolet spectra, parent ions and fragmentation ions was performed. Thirteen compounds were confirmed or tentatively identified. This provides an accurate profile of the composition of bioactive compounds responsible for the anti-influenza properties.

In vivo metabolism study of polygalic acid in rat using HPLC-ESI-MSn.

A very simple and direct method has been established for the determination of polygalic acid and its metabolites in rat urine based on HPLC coupled with electrospray ionization multi-stage tandem mass spectrometry (HPLC-ESI-MS(n)). The rats were administered a single dose (100 mg/kg) of polygalic acid by oral gavage. The urine samples were collected and purified through a C(18) solid-phase extraction cartridge, and then these pretreated samples were injected into a reversed-phase C(18) column with a gradient elution program, whereas acetonitrile-0.5% aqueous formic acid was used as mobile phase and detected by an on-line MS/MS system. As a result, the parent drug and its four metabolites were identified and characterized in rat urine for the first time by comparing their changes in molecular mass (ΔM), retention times and full-scan MS(n) spectra with those of the parent drug. A possible metabolic pathway of polygalic acid was investigated and proposed. More importantly, the results demonstrated that the newly developed method (HPLC-ESI-MS(n)) was sensitive, simple and suitable for the determination of polygalic acid and its metabolites in biological samples.

[Effect of epidermal growth factor on the expression of matrix metalloproteinase-9 and the signalling pathways involved in the trophoblast cell line JEG-3].

OBJECTIVE: To investigate the effect of epidermal growth factor (EGF) on the expression of Matrix metalloproteinase-9 (MMP-9) and the signalling pathways involved in the trophoblast cell line JEG-3. METHODS: The JEG-3 trophoblast cell line was used in this study. (1) JEG-3 cells were cultured with various concentrations of EGF (0, 1, 10, 20 ng/ml) for 24 hours and the expression of MMP-9 was tested by western blotting and reverse transcription PCR (RT-PCR). (2) Western blotting and RT-PCR were also used to investigate the expression of MMP-9 expression after incubation for 0, 4, 12 and 24 hours with EGF treatment (10 ng/ml) in JEG-3 cells. (3) According to the different added ingredients, JEG-3 cells were divided into some groups: control group (without EGF), EGF group (exposure to 10 ng/ml EGF), EGF+ inhibitors group (exposure to 10 ng/ml EGF+ 20 ng/ml SB203580 or exposure to 10 ng/ml EGF+ 10 ng/ml U0126), inhibitors group (exposure to 20 ng/ml SB203580 or exposure to 10 ng/ml U0126). Western blotting were used to investigate the expression levels of MMP-9, nuclear factor kappa B (NF-κB), p38MAPK, phospho-p38MAPK (p-p38MAPK), extracellular-signal regulated kinase (ERK) and phospho-ERK (p-ERK) protein in JEG-3 cells after incubation for 24 hours. RESULTS: (1) The profiles of MMP-9 mRNA were increased by various concentrations of EGF (0, 1, 10, 20 ng/ml) in JEG-3 cells after 24 h-culture. The expression of MMP-9 mRNA in JEG-3 cells exposure at 1 ng/ml of EGF (0.567±0.056), 10 ng/ml of EGF (1.392±0.133), 20 ng/ml of EGF (1.971±0.067) were significantly higher respectively (P<0.05), compared with 0 ng/ml of EGF treatment (0.166±0.015). Similarly, MMP-9 mRNAs were also increased with the increasing incubation time. Compared to EGF (10 ng/ml) stimulation for 0 h (0.253±0.044), the MMP-9 mRNA profiles were 0.470±0.026, 1.061±0.115, 1.453±0.180 for 4, 12 and 24 hours, respectively (P<0.05). (2) In accordance to the mRNA profiles, the expression of MMP-9 protein was also increased by different concentrations of EGF (0, 1, 10, 20 ng/ml) in JEG-3 cells after 24 h-culture. The abundance of MMP-9 protein in the three groups was 0.043±0.012, 0.085±0.008, 0.142±0.015, with a significantly higher expression, compared with 0 ng/ml of EGF treatment (0.004±0.001, P<0.05) respectively. Similarly, MMP-9 proteins were also increased with the increasing incubation time. Compared to EGF (10 ng/ml) stimulation for 0 h (0.030±0.009), the profiles of MMP-9 protein were 0.137±0.010, 0.240±0.010, 1.240±0.061 for 4, 12 and 24 hours, respectively (P<0.05). (3) Both p38MAPK and ERK signalling pathways were activated by EGF in JEG-3 cells. The expression of p-p38MAPK was significantly higher (without or with 10 ng/ml EGF, 234.1±4.1 vs. 260.9±2.5, P<0.05), however, the p38MAPK inhibitor SB203580 markedly suppressed the increase in p-p38MAPK content induced by EGF (227.9±2.4 vs. 260.9±2.5, P<0.05). Similarly, the expression of p-ERK was significantly higher with EGF treatment (812.2±3.5) vs. without EGF group (453.4±5.8) (P<0.05), while the ERK inhibitor U0126 significantly inhibited the increased p-ERK content in response to EGF treatment (71.0±1.2 vs. 812.2±3.5, P<0.05). (4) The p38MAPK inhibitor SB203580 significantly reduced the expression of EGF-induced MMP-9 (0.645±0.270 vs. 1.476±0.452, P<0.05) and NF-κB (0.530±0.026 vs. 0.959±0.017, P<0.05). (5) The ERK inhibitor U0126 also significantly reduced the expression of EGF-induced MMP-9 (0.623±0.030 vs. 2.112±0.056, P<0.05) and NF-κB (0.325±0.082 vs. 0.939±0.153, P<0.05). CONCLUSION: EGF induced the expression of MMP-9 in a time and dose-dependant manner in JEG-3 cells. EGF enhanced MMP-9 expression through the activation of p38MAPK and ERK signalling pathways in JEG-3 cells.

Complete NMR spectral assignments of two new iridoid diastereoisomers from the flowers of Plumeria rubra L. cv. acutifolia.

Two new iridoid diastereoisomers (1, 2), together with five known compounds, were isolated from the flowers of Plumerian rubra L. cv. acutifolia. Their structures were elucidated by the means of in-depth spectroscopic and mass-spectrometric analyses, particularly 1D and 2D NMR spectroscopy.

Identification of major xanthones and steroidal saponins in rat urine by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry technology following oral administration of Rhizoma Anemarrhenae decoction.

Rhizoma Anemarrhenae (Zhimu in Chinese), the dried rhizome of Anemarrhena asphodeloides Bge. (Fam. Liliaceae), is a well-known traditional Chinese medicinal herb and has been used clinically in China for centuries to cure various diseases. However, like other traditional Chinese medicines, the effective constituents of this medicine, especially the assimilation and metabolites in vivo, which are very important to show their effects, have not been systematically studied. In this paper, solid-phase extraction and liquid chromatography-atmospheric pressure chemical ionization mass spectrometry technologies were used to study the constituents absorbed into rat urine and their metabolites after oral administration of Rhizoma Anemarrhenae decoction. A total of 11 compounds, including two xanthones, three of their metabolites and six steroidal saponins, were identified in rat urine sample. They were neomangiferin (1), glucuronide and monomethyl conjugate of mangiferin (2), mangiferin (3), monomethyl conjugate of mangiferin (4), dimethyl conjugate of mangiferin (5), timosaponin N or timosaponin E1 (6), timosaponin BII (7), timosaponin BIII (8), anemarrhenasaponin I or anemarrhenasaponin II (9), timosaponin AII (10) and timosaponin AIII (11). The results would efficaciously narrow the potentially active compounds range in Rhizoma Anemarrhenae decoction, and pave a helpful way for follow-up mechanism of action research.

Identification of major alkaloids and steroidal saponins in rat serum by HPLC-diode array detection-MS/MS following oral administration of Huangbai-Zhimu herb-pair Extract.

Huangbai-Zhimu herb-pair (HBZMHP) is a widely used Chinese traditional medicine formula in treating various diseases; however, its active components have remained unknown. In this paper, serum chemistry and combined high-performance liquid chromatography (HPLC), diode-array detection and mass-spectrometry (MS) techniques were used to study the constituents of HBZMHP extract absorbed into rat serum after oral administration. A total of nine characteristic HPLC peaks in the TIC chromatograms were identified as magnoflorine (1), menisperine (2), palmatine (3), berberine (4), timosaponin N or timosaponin E1 (5), timosaponin D (6), timosaponin BIII, anemarsaponin C or xilingsaponin B (7) timosaponin BII (8) and timosaponin AIII (9). All of the identified peaks were constituents of HBZMHP extract. The results narrow the range of active compounds to be found in HBZMHP extract, and pave the way for the follow-up action mechanism research.

Cytotoxic triterpenoid saponins from Vaccaria segetalis.

By the guidance of bioassay, one new cytotoxic triterpenoid saponin, 3-O-[beta-D-galactopyranosyl-(1-->2)-beta-D-glucuronopyranosyl] quillaic acid 28-O-beta-D-glucopyranosyl-(1-->3)-beta-D-xylopyranosyl-(1-->4)-alpha-L-rhamnopyranosyl-(1-->2)-[beta-D-fucopyranosyl-(1-->4)]-beta-D-fucopyranoside (1), and five known cytotoxic triterpenoid saponins, vaccaroside E (2), vaccaroside G (3), vaccaroside B (4), segetoside H (5) and segetoside I (6), were isolated from Vaccaria segetalis. Their structures were established on the basis of ESI-MS, IR, extensive NMR ((1)H NMR, (13)C NMR, TOCSY, (1)H-(1)H COSY, DEPT, HMQC, HMBC and ROESY) analyses, chemical degradation, and by comparing with previously reported data. Compounds 1-6 showed moderate cytotoxic activities against LNcap, P-388 and A-549 cell lines with IC(50) values in the range 0.1-12.9 microM.

Identification and determination of four metabolites of mangiferin in rat urine.

Four metabolites of mangiferin were firstly isolated and identified from rat urine. The structures of the four metabolites were determined to be 1,3,7-trihydroxyxanthone (M-1), 1,3,6,7-tetrahydroxyxanthone (M-2), 1,3,6-trihydroxy-7-methoxyxanthone (M-3) and 1,7-dihydroxyxanthone (M-4), respectively. A simple and specific analytical method for determination of the four metabolites in rat urine was developed by high performance liquid chromatography (HPLC). Quercetin was employed as an internal standard. The correlation coefficients of the calibration curves were higher than 0.997, both intra- and inter-day precision of four metabolites were determined and their R.S.D. did not exceed 10%. The accuracy and linear range had been investigated in detail. The cumulative urinary excretions of the four metabolites were measured and the possible metabolic pathway of the metabolites was discussed.

LC-MS characterization of efficacy substances in serum of experimental animals treated with Sophora flavescens extracts.

Anti-DHBV (duck hepatitis B virus) activity was found in the aqueous extracts of Sophora flavescens Ait. in vivo. Liquid chromatography/electrospray ionization ion trap mass spectrometry was applied to characterize the components in duck serum after oral administration of S. flavescens extract. Oxymatrine (1), sophoranol (2), sophoridine (3) and matrine (4) were identified in the serum. Further research on the four compounds was evaluated for their antiviral activity against HBV (hepatitis B virus) in cell culture. The results suggested that oxymatrine, sophoranol and matrine were the efficacy substances for anti-HBV activity in aqueous extracts of S. flavescens Ait.