Mengchun Cheng

Synergistic inhibitory effect of Icariside II with Icaritin from Herba Epimedii on pre-osteoclastic RAW264.7 cell growth

Increasing evidence shows the therapeutic superiority of herbal extracts in comparison to isolated single constituents. One of the reasons may be attributed to the synergy effect of compound combinations. Flavonoids from Herba Epimedii have been shown to have therapeutic effect against bone loss. Our previous study showed that Icariside II inhibited pre-osteoclast RAW264.7 growth. The aim of this study was to investigate whether the activity of Icariside II is synergized by other components of Herba Epimedii. The inhibitory activity of Icariside II was significantly enhanced in the presence of the extract of Herba Epimedii (EHE) at the ratio of 1:1, 1:5 and 1:10. Icaritin, another flavonoid constituent, was shown here to inhibit RAW264.7 growth in a dose-dependent manner. Further, we found that Icariside II, together with Icaritin, synergistically inhibited RAW264.7 growth. The synergistic effect is significant when the ratio of Icariside II and Icaritin was 10:1, 5:1, 1:1, 1:2, and 1:5, respectively. In conclusion, Icaritin were an active component. The inhibitory activity of Icariside II on pre-osteoclast RAW264.7 growth was synergized by Icaritin, which maybe contribute to the efficiency of Herba Epimedii extract on curing bone-related diseases, such as osteoporosis.

Functional analysis of cultured neural cells for evaluating cold/cool- and hot/warm-natured Chinese herbs.

Recently, modern scientific research has been required to understand pharmacological basis of traditional Chinese medicine (TCM) theory based on the ancient clinical experience, and to investigate the molecular mechanisms of action of Chinese herbs. Here, 20 Chinese herbs, classified into 4 properties (hot, warm, cold and cool) of TCM, were analyzed for their ability to exhibit antioxidant action, to enhance glucose uptake by murine microglia N9 cells, and to influence neurotransmitter norepinephrine (NE) release from rat pheochromocytoma PC12 cells. We found a generally protective effect of both hot/warm-natured and cold/cool-natured herbs against H(2)O(2)-induced N9 cell death, partially by elevating superoxide dismutase (SOD) activity. Glucose uptake was elevated after treatment with some hot/warm-natured herbs. In addition, most herbs with hot/warm nature tended to stimulate NE release, while such stimulatory effect was not observed in the herbs with cold/cool nature. Two cold/cool-natured herbs, Rhizoma coptidis and Radix scutellariae, even significantly suppressed the release. These results suggest that the distinct abilities of Chinese herbs to regulate neural cell functions appear to be correlated with their natures identified in traditional TCM theory, and may be a useful guide for their utility in neural degenerative diseases.

Comparative pharmacokinetics of gastrodin in rats after intragastric administration of free gastrodin, parishin and Gastrodia elata extract.

ETHNOPHARMACOLOGICAL RELEVANCE Gastrodia elata Blume, a traditional Chinese herb, was widely used against convulsant, vertigo, paralysis, epilepsy, tetanus, asthma and immune dysfunctions. Gastrodin is one of the major bioactive components of G. elata and it is known for its anticonvulsive, anti-inflammatory, antiepileptic and neuroprotective effects. MATERIALS AND METHODS An ultra high performance liquid chromatography-fluorescence detection (UHPLC-FLD) method was developed to determine gastrodin in rat plasma. Gastrodin and Thiamphenicol (internal standard, IS) were extracted from rat plasma by immediately protein precipitation. The pharmacokinetics of gastrodin in rats by following differently administered types was studies: intragastric administration of gastrodin (100mg/kg), parishin (116 mg/kg, with the same mole of gastrodin moiety) and G. elata extract (2.3g/kg, with the same mole of gastrodin moiety). Non-compartmental pharmacokinetic profiles were constructed using the software of WinNonlin (Phoenix, version 6.3), and the pharmacokinetic parameters were compared using unpaired Students t-test. RESULTS The results showed that the pharmacokinetic parameters, including Cmax, Tmax, AUC0-∞, t1/2, MRT, Vd, CL, were quite different among the three types of gastrodin administration. The administration of parishin and G. elata extract, which either could convert to gastrodin in vivo or contained free gastrodin and abundant gastrodin conjugates, gave rise to higher elimination half-life (t1/2) and mean residence time (MRT) values for gastrodin compared to free gastrodin administered. CONCLUSION The comparison of the pharmacokinetics of gastrodin among three different administered types of gastrodin in rats suggested that administration of parishin or G. elata extract in clinic may result in a longer duration time of action than that of the administration of free gastrodin. The results may provide some guidance for the clinical applications of parishin and G. elata.

Rapid and sensitive analysis of parishin and its metabolites in rat plasma using ultra high performance liquid chromatography-fluorescence detection.

A simple, rapid and sensitive ultra high performance liquid chromatography with fluorescence detection (UHPLC-FLD) method was developed and validated for quantification of parishin and its metabolites in rats. Plasma samples were prepared by protein precipitation and then analyzed using UHPLC-FLD system. Repeated optimization showed that parishin and its metabolites, including gastrodin, p-hydroxybenzyl alcohol, parishin B and parishin C, could be sensitively detected based on the autofluorescence when excitation and emission wavelengths were set at 225nm and 295nm, respectively. The limit of detections (LODs) of GAS, HBA, PB, PC and PA reached 0.6, 0.8, 1, 1 and 1ng/mL, respectively. The linearity for all targets was within the range 2.5-5000ng/mL and the correlation coefficient (r2) was larger than 0.999. Importantly, our method was almost free from matrix effects and the recoveries were higher than 80%. Additionally, our method also had high precision and accuracy for all analytes, presenting RSDs and REs within ±6% and ±14%, respectively. Finally, the validated UHPLC-FLD method was successfully applied for studying the pharmacokinetics of parishin following intragastrically administration in rats.

Metabolomic profiling of emodin-induced cytotoxicity in human liver cells and mechanistic study

Emodin is one of the most representative natural anthraquinone polyphenols and the liver is one of the major target organs for drug-induced toxicology. The hepatocyte is frequently affected due to its role in emodin metabolism and accumulation. Although the hepatotoxicity of emodin has been reported, its toxicological mechanism is still unclear. The purpose of the present study was to evaluate the cytotoxicity of emodin in cultured human normal liver cells (L-02), to investigate the toxicity-related metabolic pathways and to predict the possible toxicity mechanism. Cell viability was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cytotoxicity tests demonstrated a concentration-dependent toxic effect of emodin on L-02 cells. Cells were treated for 48 h with low, medium and high doses of emodin, respectively, and then subjected to metabolomics analysis using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS). Intracellular metabolomics analysis revealed that emodin significantly disturbed cellular glutathione and fatty acid metabolism. In addition, an emodin–cysteine adduct was identified in cell culture medium, and its level increased with increasing concentrations of emodin. The possible relationship among metabolic disorders, adduct formation and emodin hepatotoxicity was also discussed. This study provides new insight into the cytotoxicity of emodin on metabolic pathways in human liver cells.

Analysis of the metabolic profile of parishin by ultra-performance liquid chromatography/quadrupole-time of flight mass spectrometry

Parishin is a dominant active ingredient originating from Gastrodia elata Blume, and has good neuroprotective effects against brain disorders. In the present study, the metabolic profile of parishin by in vitro and in vivo experiments was investigated using ultra-high performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry (UHPLC/Q-TOF MS) combined with an automated MS(E) technique. By comparison with reference compounds, accurate mass measurement, the characteristic fragmentation patterns of the parent drug parishin and gastrodin and relevant bio-transformation knowledge, 14 metabolites (seven hydrolyzates and seven derivatives of gastrodin) were detected and identified in rat plasma and urine after intragastric administration of parishin, including processes of hydrolyzation, oxidation, sulfation and glucuronidation. According to the proposed metabolic pathways of parishin, in vitro hydrolytic experiments and metabolic study of gastrodin in rat plasma, it can be inferred that parishin mainly functions as a prodrug and undergoes hydrolysis before being absorbed into the blood. The hydrolyzate, mainly gastrodin, was involved in further metabolism, which was responsible for pharmacological activities of parishin. In conclusion, this work provides valuable information on parishin metabolism using a rapid and reliable UHPLC/Q-TOF MS method, which could be widely used for the metabolic investigation of natural product.

Identification of major metabolites in rat urine and plasma of N6-(4-hydroxybenzyl) adenine riboside by LC/MS/MS

N(6) -(4-hydroxybenzyl) adenine riboside, a novel neuroprotective compound found in Gastrodia elata at trace level, is regarded as a potential drug for the treatment of neural degenerative disease. To understand the metabolism of this compound, the metabolites in rat urine and plasma of N(6) -(4-hydroxybenzyl) adenine riboside were analyzed by HPLC-ESI-MS/MS after oral administration of this compound. Beside the parent compound, six phase I metabolites and four phase II metabolites in urine were detected by scanning all possible metabolites in extracted ion chromatograms mode. By comparing their product ion spectra and retention times with those of parent compound, these metabolites were identified and proved to be mainly formed via hydrolysis or hydroxylation in phase I, N-sulfation or N-glucuronidation in phase II or their combinations. Similarly, the parent compound, one phase I metabolite and two phase II metabolites were also identified in rat plasma. Therefore, the in vivo metabolic pathways of N(6) -(4-hydroxybenzyl) adenine riboside in rat were proposed.

Rhizoma coptidis and berberine-induced activation of murine microglia N9 cells

AIMS OF THE STUDY To investigate the effect of water extract of Rhizoma coptidis (WEC) and berberine on the activation of murine microglia N9 cells and corresponding mechanism related to mitochondria. MATERIALS AND METHODS Phagocytic activity of murine microglia N9 cells was measured by neutral red staining method after the cells were treated with various concentrations of WEC and alkaloids for 24h. Flow cytometric analysis was performed to determine the level of intracellular ROS, Ca(2+), and mitochondrial transmembrane potential (Delta psi) after 87 microg/ml of WEC and 12.4 microg/ml of berberine treatment. Global changes of gene expression in WEC- and berberine-treated N9 cells were measured using cDNA microarray. RESULTS WEC and berberine, but not palmatine and jatrorrhizine, enhanced phagocytic activity of murine N9 cells in a dose-dependent manner. Both of WEC and berberine stimulated free radical generation, enhanced mitochondrial Delta psi and induced gene expression of Ndufab1, Cox6a2 and Atp5a1. However, a more significant phagocytic effect was observed for WEC. WEC, but not berberine, increased intracellular Ca(2+) concentration. The gene expression of Atp5c1 was selectively up-regulated by WEC, while three genes of Uqcrq, Cox8b, and Atp5g2 were induced by berberine. CONCLUSIONS WEC and berberine activated murine microglia N9 cells by the regulation of mitochondrial function and mitochondria-related signal molecules. The action of WEC is stronger than that of berberine, indicating that the effect of WEC is ascribed partially, but not totally, to berberine.

Cytotoxic biflavones from Stellera chamaejasme.

Bioassay-guided phytochemical studies on Stellera chamaejasme led to the isolation of two new biflavones, chamaejasmenin E (1) and chamaejasmin D (2), together with ten known compounds. The structures of new compounds were elucidated by extensive spectroscopic analyses and their absolute configurations on 2, 3, 2″ and 3″ were confirmed by TDDFT quantum chemical calculated ECD spectra combined with experimental ECD spectra. All isolated biflavones were evaluated for their cytotoxic activities against Bel-7402 and A549 tumor cell lines, and sikokianin D (3) was found to possess the most potential cytotoxic activities against both the two cell lines with IC50 values of 1.29 ± 0.21 and 0.75 ± 0.25 μM, respectively. Moreover, some structure-function relationships of these bioflavones for cytotoxic activities were explored and summarized.