Epimedium sagittatum inhibits TLR4/MD-2 mediated NF-κB signaling pathway with anti-inflammatory activity.

BACKGROUND: Epimedium sagittatum (Sieb.et Zucc.) Maxim., Ying-Yang-Huo in Chinese has been used as a traditional Chinese medicine and is deemed to "reinforce the kidney Yang". Previous studies showed that E. sagittatum could modulate the immune system and treat some chronic disease such as rheumatic arthritis, cardiovascular diseases and osteoporosis. The aim of this study is to evaluate the anti-inflammatory effects of ethyl acetate extracts (YYHs) of E. sagittatum and its mechanisms of action. METHODS: In order to explore the composition of YYHs, YYHs was analyzed using high performance liquid chromatography-mass spectrometry-mass spectrometry (HPLC-MS/MS) and in comparison with reference standards. Anti-inflammatory model was established in LPS-induced RAW264.7 cells. The levels of nitric oxide (NO) were measured with the Griess reagent. Production of tumor necrosis factor-alpha (TNF-α) and interleukin-2 (IL-2) were measured by enzyme-linked immunosorbent assays (ELISA). In addition, expression of p-p65 protein and TLR4/MD-2 complex was detected by western blots and flow cytometric, respectively. Nuclear factor kappa B (NF-κB) nuclear translocation was observed by fluorescence microscope. RESULTS: A total of eight compounds were identified, of which icariside II was the most abundant compound. YYHs (12.5-50 µg/mL) had no obvious cytotoxic effect on cells, and remarkably inhibited LPS-induced production of NO, TNF-α and IL-2 with a dose-dependent manner. Additionally, YYHs up-regulated expression of p-p65 and TLR4/MD-2 complex. Further research showed that YYHs significantly suppressed NF-κB p65 nuclear translocation. CONCLUSION: In brief, YYHs contributed to the inhibition of LPS-induced inflammatory response through the TLR4/MD-2-mediated NF-κB pathway and may be a potential choice to combat inflammation diseases. It includes a schema of pathways at the end of the paper.

[In vivo study of Chuankezhi metabolism in rat].

To study the metabolic products of main compounds of Chuankezhi injection in rat, 12 Sprague Dawley rats were classed into 2 groups, a blank control group and an intermuscular administration group, respectively. Rat feces and urine samples were collected from 0−24 h and 24−48 h after administration. All the samples were ultrasonically treated with methanol and then analyzed using LC-LTQ Orbitrap MSn. By comparison with the total ion chromatogram of samples from the blank control group, the metabolites in the samples of drug-treated group were screened. These metabolites were further analyzed by multistage product ion scanning and comparison of retention time with reference substances. As a result, a total of 12 flavonoid metabolites were tentatively identified from the rat feces and no metabolite was discovered in the rat urine. Epimedin C and icariin were detected in the rat blood samples after 30 min of administration, but their metabolites and other original flavones were not detected. Furthermore, no original flavones and their metabolites were detected in rat blood samples after 2 and 4 h of administration. The potential metabolism paths were further characterized and the principal in vivo transformation of flavones from Chuankezhi injection were deglycosylation, dehydration, methylation, oxidation and isomerization in rats.

[Simultaneous determination of 4 prenylflavonoids of Chuankezhi injection in rat plasma by LC-MS/MS].

A quantitative method for epimedin A, B, C and icariin in rat plasma was established using LC-MS/MS after intermuscular administration of Chuankezhi injection to rat. Chromatographic separation was performed on an Agilent Eclipse XDB-C(18) column (150 mm × 2.1 mm, 5.0 µm) at 40 ℃. Mobile phase consisted of acetonitrile-0.1% formic acid in water（35∶65）, and the flow rate was 0.22 m L·min(-1). The LC effluent was detected and analyzed using an ESI-triple quadrupole tandem mass spectrometer under the multiple reaction monitoring (MRM) in the negative ion mode. The plasma samples were treated with solid phase extraction prior to LC-MS/MS analysis. As a result, all of the four analytes displayed a good linearity over the concentration of 1-1 000 ng·mL(-1). The RSDs of intra-day and inter-day assays were less than 5.99% and 10.16%, respectively. The relative recovery of each analyte was between 88.1%-101.1% with RSD < 7.9% and the absolute recovery was between 72.0%-86.6%（RSD < 6.3%）. In conclusion, the established method shows good specificity, sensitivity and efficiency for quantifying the four flavonoid glycosides contained in rat plasma.

[Pharmacokinetics of epimedin A, B, C and icariin of Chuankezhi injection in rat].

To study pharmacokinetic characteristics of epimedin A, B, C and icariin after intermuscular administration of Chuankezhi injection to rat. The established RRLC-MS/MS method was applied for simultaneous determination of four analytes in rat plasma and calculating their pharmacokinetic parameters. As a result, each analyte showed a good linear relationship in the concentration range of 1-1 000 µg•L⁻¹.The intra-day precise was 96.9%-107.5% with RSD<5.99%, inter-day precise was 92.3%-105.0% with RSD<10.16%. The relative recovery of four analytes was 88.1%-101.1% with RSD<7.9% and their absolute recovery was 72.0%-86.6% with RSD<6.3%. After intermuscular administration of Chuankezhi injection, the plasma concentration of four flavonoid glycosides rapidly arose to peaks at about 10 min, and then quickly declined in rat. Tmax of epimedin A, B, C and icariin was 0.21, 0.19, 0.16 and 0.49 h, respectively, and their mean elimination half-life(t1/2z) was 0.60, 0.62, 0.47 and 0.49 h. The established method was validated to be sensitive, rapid and specific for determination of the four analytes. Serum concentration of 4 species of epimedium flavonoids in Chuankezhi injection was low, and their absorption and elimination seem quickly, displaying similar pharmacokinetic characteristics in this study.

[Simultaneous determination of 4 phenolic acids in cangerzi by ultra-performance liquid chromatography].

In this study, an analytical method was developed and used to quantify simultaneously protocatechuic acid, neochlorogenic acid, cryptochlorogenic acid and 1, 3-dicaffeoylquinic acid--four bioactive compounds contained in Fructus Xanthii using UPLC. The contents of four phenolic components of 28 batches of samples collected from different product areas and markets were determined and compared by means of this established method. The mobile phase was composed of methanol and water containing 0.1% phosphoric acid. Chromatography was monitored at dual-wavelengths--220 and 327 nm. Flow rate was 0.4 mL x min(-1) and column temperature was 35 degrees C. The correlation coefficient between concentration and chromatographic peak area of protocatechuic acid, neochlorogenic acid, cryptochlorogenic acid and 1, 3-dicaffeoylquinic acid was over 0.9999 in the range of 0.3570-35.70, 2.500-250.0, 1.060-106.1, 1.010-101.0 microg x mL(-1), respectively. The average recoveries of the four compounds were 97.68%, 99.55%, 97.92% and 100.4%, respectively. In conclusion, the established method can rapidly attain an accurate and reproducible result used to control the quality of Fructus Xanthii.

Quantitative and fingerprint analyses of Chinese sweet tea plant ( Rubus suavissimus S. Lee).

The quality of botanical food is increasingly assessed by the content of multiple bioactive compounds. This study reports, for the first time, an HPLC fingerprinting method for the quality evaluation of Rubus suavissimus leaves possessing multiple bioactivities. Five constituents, gallic acid, rutin, ellagic acid, rubusoside, and steviol monoside, were quantified and used in developing qualitative chromatographic fingerprints. The limits of detection and quantification ranged from 0.29 to 37.86 mug/mL. The relative standard deviations (RSDs) of intra- and interday precisions were no more than 3.14 and 3.01%, respectively. The average recoveries were between 93.1 and 97.5%. The developed method was validated in the analysis 14 leaf samples with satisfactory results. The contents of the five marker compounds accounted for an average of about 6% w/w with a variability of 16% among the 14 samples collected from a single site and year. Gallic acid was the least, whereas steviol monoside the most, variable compound among the 14 leaf samples. The characteristic compound rubusoside that is responsible for the sweet taste accounted for 5% of leaf weight. The validated method can now be used to quantitatively and qualitatively assess the quality of R. suavissimus leaves as traditional beverage or potential medicines.

Determination of ginsenoside-Rg(1) in human plasma and its application to pharmacokinetic studies following intravenous administration of 'Shenmai' injection.

'Shenmai' injection is derived from traditional Chinese medicine 'Shenmaisan' and made from Radix ginseng Rubra and Radix Ophiopogonis. Ginsenoside-Rg(1), as a major constituent of Radix ginseng Rubra, is considered responsible for the efficacy of this injection. A rapid, simple and accurate method has been established for determination of ginsenoside-Rg(1) in Shenmai injection and human plasma using LC-ESI-MS/MS, and to study the pharmacokinetics of Rg(1) in ten healthy volunteers after intravenous single dosing of 60 mL of Shenmai injection. Following solid-phase extraction (SPE), samples were separated on a C(18) column coupled with electrospray ionization mass spectrometry. The protonated analyte was quantified by multiple reaction monitoring (MRM) with a quadruple mass spectrometer in positive mode. Linearity was confirmed in the concentration range of 1 to 1000 ng/mL for Rg(1), and the lower limit of quantification (LLOQ, S/N > 10) was 1 ng/mL. The intraday and interday RSDs were within 15% and mean extraction recoveries ranged from 98.6% to 104.9%. The pharmacokinetics of Rg(1) in healthy volunteers conforms to the two-compartment open model. The main pharmacokinetics parameters were as follows: t(1/2beta), 2.09 +/- 1.89 h; CL, 0.03 +/- 0.01 L kg(-1) h(-1); AUC (0 approximately infinity), 124.4 +/- 35.9 2 ng mL(-1) h and AUC (0 approximately infinity), 127.9 +/- 37.2 ng mL(-1) h, respectively.

[HPLC fingerprinting of radix paeoniae alba].

To establish a sensitive and specific HPLC method for quality control of Radix Paeoniae Alba, HPLC method was applied for quality assessment of Radix Paeoniae Alba. HPLC analysis was performed on a Symmetry C18 column (250 mm x 4. 6 mm ID, 5 microm, Waters, USA). The mobile phase consisted of acetonitrile (solvent A) and water containing 0.1% (v/v) phosphoric acid (solvent B) at a constant flow rate of 0.8 mL x min(-1). An increasing linear gradient (v/v) of solvent A was used (t/min, % A): (0,10), (5,10), (25,15), (45, 22), (46, 65), (50, 80) and (60, 80). The column temperature was set at 25 degrees C. The chromatograms were monitored at 230 nm and the on-line UV spectra were recorded in the range of 190 - 400 nm. The HPLC chromatographic fingerprinting of Radix Paeoniae Alba, showing 11 characteristic peaks, was established from 28 lots of Radix Paeoniae Alba. The areas of main chromatographic peaks were found to complied with the following rule: paeoniflorin > 1, 2, 3, 4, 6-penta-O-galloyl-glucos > albiflorin > methyl gallate > other compounds. The chromatographic fingerprinting of Radix Paeoniae Alba with high specificity can be used to control its quality and assure lot-to-lot consistency.

[Rapid identification of compounds in cortex moutan by liquid chromatography-tandem mass spectrometry].

AIM: To analyze the chemical constituents of cortex moutan by liquid chromatography coupled with electrospray ionization mass spectrometry. METHODS: An on-line optimized HPLC-DAD/MS2 technique was employed. RESULTS: In the negative ion detection mode, 38 components such as monoterpene glucosides, galloylglucoses and acetophenones were isolated. Among them, over thirty compounds were identified, including paeonol, paeonilflorin, oxypaeoniflorin, benzoylpaeoniflorin, benzoyloxypaeoniflorin, galloylpaeoniflorin, galloyloxypaeoniflorin, mundanpioside A, C, D, E, H, etc. by the high performance liquid chromatography with diode-array detection in parallel with electrospray ionization and quadrupole-time of flight tandem mass spectrometry (HPLC-DAD/ESI-MS2). CONCLUSION: This method can be used to rapidly determine the constituents of cortex moutan.

Characterization of compounds in the Chinese herbal drug Mu-Dan-Pi by liquid chromatography coupled to electrospray ionization mass spectrometry.

Cortex Moutan is a well-known traditional Chinese medicine derived from Paeonia suffruticosa ANDREWS. However, root cortices of P. delavayi and P. decomposita also are used under the name of this drug in some regions such as Yunnan and Sichuan Provinces, respectively. In order to make a comparison of their chemical constituents, the compounds of the three Paeonia species were analyzed by high-performance liquid chromatography-diode array detection/electrospray ionization and quadrupole-time-of-flight tandem mass spectrometry (HPLC-DAD/ESI-MS2). A total of 50 compounds were observed in the 50% (v/v) methanolic extracts, including 17 monoterpenes, 14 galloyl glucoses, 10 acetophenones, 5 phenolic acids, 3 flavonoids and 1 triterpene. These chemical constituents were separated on a C18 column and identified or tentatively characterized based on UV spectra and MS fragmentation behavior. The chemical compositions of the three Paeonia species were found to have many differences. Paeonol was the predominant constituent of P. suffruticosa and P. decomposita, while P. delavayi contained albiflorin and more galloyl glucoses than the other two Paeonia species. Most of these identified compounds have been reported from P. delavayi and P. decomposita for the first time. The ESI-MS fragmentation behavior of monoterpene glycosides, acetophenones and galloyl glucoses was also investigated successively, and appropriate characteristic pathways were proposed. The large differences in chemical compounds among the three Paeonia species strongly encouraged further comparison of the bioactivities of these three species.

[Determination of ginsenoside Rd and its metabolites in rat urine by LC-MS].

AIM: To study the metabolic pathways of ginsenoside Rd in rats. METHODS: Urine samples were collected before and after 24 h of single oral administration of 150 mg and intravenous administration of 60 mg of ginsenoside Rd to six rats, separately. The samples were purified by SPE column and then were analyzed by liquid chromatography-ESI-mass spectrometry for putative metabolites. RESULTS: Parent drug and its seven metabolites were identified in rat urine based on comparing total ion chromatograms of the blank with the metalolic urine as well as mass spectra. Its main metabolic pathways and possible structures are elucidated. CONCLUSION: Oxidation, combination and deglucosylation were found to be the major metabolic pathway of ginsenoside Rd in rats.

[Studies on HPLC fingerprint of cortex moutan].

OBJECTIVE: To establish a HPLC fingerprint for quality evaluation of Cortex Moutan. METHOD: The HPLC chromatographic fingerprinting of 30 lots of Cortex Moutan was established and major peaks were identified by LC-MS and MS-MS. RESULT: The HPLC fingerprint of Cortex Moutan was established, showing 15 characteristic peaks. The areas of these peaks were found to complied with the following rule: paeonol > 1, 2, 3, 4, 6-penta-O-galloyl-glucos > methyl gallate > galloylpaeoniflorin > gallic acid > oxypaeoniflorin > other compounds. CONCLUSION: The chromatographic fingerprinting of Cortex Moutan with high specificity can be used to control its quality and monitor lot to lot consistency.

[Establishment of a method to detect multiple antennae carcinoembryonic antigen (MA-CEA) and MA-CEA level in the sera of patients with malignant tumors].

AIM: To establish a rapid quantitative method to detect the serum multiple antennae carcinoembryonic antigen (MA-CEA), and to measure the MA-CEA levels in the sera of patients with malignant tumors. METHODS: Neuraminidase (NMD) was used to digest the sialic acid at terminals of sugar chains of MA-CEA, and then the datura stramonium agglutinin (DSA) and anti-CEA antibody were used to set up streptavidin-biotin complex (ABC) system EIA (ABC-EIA) for detecting serum MA-CEA levels of 239 patients with carcinomas of lung, stomach, liver, colon and ovary. The serum CEA level was determined by ELISA. RESULTS: The level of serum MA-CEA was remarkably elevated in the CEA-positive patients with lung cancer. The positive rate of MA-CEA was 14.6% in 75 patients with lung cancers, and 47.3% in 19 CEA-positive patients with lung cancers. The MA-CEA was not detected in the sera of other tumor patients. CONCLUSION: We have developed a method for detection of MA-CEA levels, which lays the foundation for the differential diagnosis of lung cancer and for research on the other multiple antennae tumor markers.