Opposite trends of glycosides and alkaloids in Dendrobium nobile of different age based on UPLC-Q/TOF-MS combined with multivariate statistical analyses.

INTRODUCTION: Alkaloids and glycosides are the active ingredients of the herb Dendrobium nobile, which is used in traditional Chinese medicine. The pharmacological effects of alkaloids include neuroprotective effects and regulatory effects on glucose and lipid metabolism, while glycosides improve the immune system. The pharmacological activities of the above chemical components are significantly different. In practice, the stems of 3-year-old D. nobile are usually used as the main source of Dendrobii Caulis. However, it has not been reported whether this harvesting time is appropriate. OBJECTIVE: The aim of this study was to compare the chemical characteristics of D. nobile in different growth years (1-3 years). METHODS: In this study, ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q/TOF-MS) was employed to analyze the constituents of D. nobile. The relative abundance of each constituent was analyzed with multivariate statistical analyses to screen the characteristic constituents that contributed to the characterization and classification of D. nobile. Dendrobine, a component of D. nobile that is used for quality control according to the Chinese Pharmacopoeia, was assayed by gas chromatography. RESULTS: As a result, 34 characteristic constituents (VIP > 2) were identified or tentatively identified as alkaloids and glycosides based on MS/MS data. Moreover, the content of alkaloids decreased over time, whereas the content of glycosides showed the opposite trend. The absolute quantification of dendrobine was consistent with the metabolomics results. CONCLUSION: Our findings provide valuable information to optimize the harvest period and a reference for the clinical application of D. nobile.

Zuotai (ß-HgS)-containing 70 Wei Zhen-Zhu-Wan differs from mercury chloride and methylmercury on hepatic cytochrome P450 in mice.

Background: Zuotai (mainly ß-HgS)-containing 70 Wei-Zhen-Zhu-Wan (70W, Rannasangpei) is a famous Tibetan medicine for treating cardiovascular and gastrointestinal diseases.  We have shown that 70W protected against CCl 4 hepatotoxicity.  CCl 4 is metabolized via cytochrome P450 (CYP) to produce reactive metabolites. Whether 70W has any effect on CYPs is unknown and such effects should be compared with mercury compounds for safety evaluation.   Methods: Mice were given clinical doses of 70W (0.15-1.5 g/kg, po), Zuotai (30 mg/kg, po), and compared to HgCl 2 (33.6 mg/kg, po) and MeHg (3.1 mg/kg, po) for seven days. Liver RNA and protein were isolated for qPCR and Western-blot analysis. Results: 70W and Zuotai had no effects on hepatic mRNA expression of Cyp1a2, Cyp2b10, Cyp3a11, Cyp4a10 and Cyp7a1, and corresponding nuclear receptors [aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), pregnane X receptor (PXR), peroxisome proliferator-activated receptor-α (PPARα); farnesoid X receptor (FXR)]. In comparison, HgCl 2 and MeHg increased mRNA expression of Cyp1a2, Cyp2b10, Cyp4a10 and Cyp7a1 except for Cyp3a11, and corresponding nuclear receptors except for PXR. Western-blot confirmed mRNA results, showing increases in CYP1A2, CYP2B1, CYP2E1, CYP4A and CYP7A1 by HgCl 2 and MeHg only, and all treatments had no effects on CYP3A. Conclusions: Zuotai and Zuotai-containing 70W at clinical doses had minimal influence on hepatic CYPs and corresponding nuclear receptors, while HgCl 2 and MeHg produced significant effects.  Thus, the use of total Hg content to evaluate the safety of HgS-containing 70W is inappropriate.

Identification of Pyrrolizidine Alkaloids in Senecio Plants by Liquid Chromatography-Mass Spectrometry.

Pyrrolizidine alkaloids (PAs) are considered as the major constituents that cause hepatoxicity in Senecio plants. PAs can be found in about 3%-5% of the world's flowering plants. Nowadays, the identification method of PAs by separation and preparation was too slow and lacked effective power. A rapid method to identify PAs in plants must be developed. Based on the fragmentation regularity, the hepatoxic PAs and nonhepatoxic PAs were characterized by liquid chromatography-mass spectrometry (LC-MS). The detailed structures of PAs in five Senecio plants were identified based on tandem mass spectrometry (MS/MS) spectrum and chemical research information. In the present study, some new fragmentation regularities of PAs have been found, such as product ions at m/z 122, m/z 140 and m/z 124, m/z 142, which have been discovered as the characteristic fragments of lactone and mono-esterase type of saturated PAs, respectively. Moreover, two product ions at m/z 120 and m/z 138 have been reported as the characteristic fragments of unsaturated PAs. Some of them were found in Senecio species for the first time, and some of them may be new nature product or even new compound. Finally, we classified these plants into five categories based on PAs which were identified in the present study; the result corresponded with the classification by morphology. In addition, we have found some constituents that have odd molecular weight number only in Senecio species but not in Ligularia species; the detailed structures of these non-PAs constituents need penetrating study. LC-MS was rapid and sensitive method for detecting and identifying PAs in plants. Pyrrolizidine alkaloids were the toxiferous constituent of Senecio plants. In this study, we found that PAs can be used as the characteristic constituent of Senecio species.

Identification of the UDP-glucuronosyltransferase isozyme involved in senecionine glucuronidation in human liver microsomes.

Senecionine (SEN) is a representative of the hepatotoxic pyrrolizidine alkaloids. Although phase I metabolism for cytochrome P450-mediated metabolic activation of SEN was investigated extensively, phase II metabolism for glucuronidation of this compound has not been investigated until now. In our present study, one unique glucuronidation product of SEN in human liver microsomes (HLMs) was identified as SEN N-glucuronide using an authentically synthesized product for which the structure was identified via (1)H and (13)C NMR analysis. Subsequently, kinetics indicated that SEN N-glucuronidation followed the typical Michaelis-Menten model and only one major isozyme participated in it. Finally, this isozyme was demonstrated to be UDP-glucuronosyltransferase (UGT) 1A4, with the direct evidence that recombinant UGT1A4 exhibited predominant and exclusive activity on SEN N-glucuronidation. This result was confirmed by other experiments including chemical inhibition by selective inhibitors and a correlation study between activities of SEN N-glucuronidation and various UGT isozymes. The exclusive role of UGT1A4 on SEN N-glucuronidation was strengthened additionally by its inhibitory kinetic study in which the selective inhibitor of UGT1A4 showed a similar inhibition pattern and K(i) values in both HLM and recombinant UGT1A4 systems. Because UGT2B10 activity failed to correlate with SEN N-glucuronidation in HLMs from 10 individuals, it was impossible for UGT2B10 to play an important role in this metabolism.

Glucuronidation, a new metabolic pathway for pyrrolizidine alkaloids.

Pyrrolizidine alkaloids (PAs) possess significant hepatotoxicity to humans and animals after metabolic activation by liver P450 enzymes. Metabolism pathways of PAs have been studied for several decades, including metabolic activation, hydroxylation, N-oxidation, and hydrolysis. However, the glucuronidation of intact PAs has not been investigated, although glucuronidation plays an important role in the elimination and detoxication of xenobiotics. In this study, PAs glucuronidation was investigated, and three important points were found. First, we demonstrated that senecionine (SEN)-a representative hepatotoxic PA-could be conjugated by glucuronic acid via an N-glucuronidation reaction catalyzed by uridine diphosphate glucuronosyl transferase in human liver microsomes. Second, glucuronidation of SEN was catalyzed not only by human but also other animal species and showed significant species differences. Rabbits, cattle, sheep, pigs, and humans showed the significantly higher glucuronidation activity than mice, rats, dogs, and guinea pigs on SEN. Kinetics of SEN glucuronidation in humans, pigs, and rabbits followed the one-site binding model of the Michaelis-Menten equation, while cattle and sheep followed the two-sites binding model of the Michaelis-Menten equation. Third, besides SEN, other hepatotoxic PAs including monocrotaline, adonifoline, and isoline also underwent N-glucuronidation in humans and several animal species such as rabbits, cattle, sheep, and pigs.

Characterization of nuciferine metabolism by P450 enzymes and uridine diphosphate glucuronosyltransferases in liver microsomes from humans and animals.

AIM: to characterize the metabolism of nuciferine by P450 enzymes and uridine diphosphate glucuronosyltransferase (UGT) in liver microsomes from humans and several other animals including rats, mice, dogs, rabbits and monkeys. METHODS: nuciferine was incubated with both human and animal liver microsomal fractions containing P450 or UGT reaction components. Ultra performance liquid chromatography coupled with mass spectrometry was used to separate and identify nuciferine metabolites. Chemical inhibition was used to identify the involved isozymes. Species difference of nuciferine metabolism in human and various animals were investigated in the liver microsomal incubation system. RESULTS: among the nuciferine metabolites detected and identified, seven were catalyzed by P450 and one by UGT. Ketoconazole inhibited the formation of M292, M294 and M312. Furafylline, 8-methoxypsoralen and quercetin inhibited the formation of M282. Hecogenin showed a significant inhibitory effect on nuciferine glucuronidation. While the P450-catalyzed metabolites showed no species differences, the glucuronidation product was only detected in microsomes from humans and rabbits. CONCLUSION: the isozymes UGT 1A4, CYP 3A4, 1A2, 2A6 and 2C8 participated in the hepatic metabolism of nuciferine. Based on the observed species-specific hepatic metabolism of nuciferine, rats, mice, dogs and even monkeys are not suitable models for the pharmacokinetics of nuciferine in humans.

Detection of chlorite, chlorate and perchlorate in ozonated saline.

Medical ozone is used to treat various diseases, including numerous pathologies associated with chronic pain. Chronic pain may be treated by systemic administration of ozone, with ozonated autohemotherapy (OAH) being the commonly used method. In the clinic, intravenous infusion of ozonized saline has been used to treat various diseases. Compared with OAH, ozonized saline infusion is less technically demanding and causes minimal damage to veins. However, it has been indicated that ozone may oxidize saline and generate toxic substances, and therefore, the safety of ozone treatment has been questioned. In the present study, the potential chemical compounds produced from ozone and saline, including chlorite, chlorate and perchlorate, were examined at various time-points with ion chromatography-mass spectrometry (IC-MS). A control group (pure oxygen group) and an ozone group were included in the present study. Two subgroups were included within each group: A saline bottle (made from polypropylene) subgroup and an ozone-resistant blood transfusion bag [made from medical polyvinyl chloride, di(2-ethyl) hexyl phthalate plasticized] subgroup. For the ozone group, 100 ml saline and 100 ml medical ozone at various concentrations (20, 40 or 60 µg/ml in pure oxygen) were injected into the saline bottle or blood bag, and for the control group, 100 ml of pure oxygen was injected into the saline bottle or blood bag. The presence and the content of chlorite, chlorate and perchlorate were determined at different time-points (3, 6 and 15 days after mixing) by IC-MS. Chlorate was detected in the ozone groups at three time-points and its content increased as the ozone concentration and the reaction time increased. Under the same conditions (the same ozone concentration and the same incubation time), the chlorate content (0.90±0.14-7.69±0.48 µg/l) in the blood bag subgroup was significantly lower than that in the saline bottle subgroup (45.23±6.14-207.6±15.63 µg/l). However, chlorite and perchlorate were not detected at any time-point in the two groups. In addition, in the control group (pure oxygen group), chlorite, chlorate and perchlorate were not detected at any time-point. These results indicate that ozone reacts with saline to produce chlorate. Ozone may also react with the polypropylene saline bottle to increase the chlorate content in the bottled solution. Due to a lack of toxicology studies of chlorate in blood, it remains elusive whether ozonated saline and chlorate at the range of 0.90±0.14-7.69±0.48 µg/l has any toxic effects. The potential toxicity of chlorate should be considered when ozonated saline is used for clinical infusions.

Characterization of cardamonin metabolism by P450 in different species via HPLC-ESI-ion trap and UPLC-ESI-quadrupole mass spectrometry.

AIM: To characterize the metabolism of cardamonin by the P450 enzymes in human and animal liver microsomes. METHODS: Cardamonin was incubated with both human and animal liver microsomal incubation systems containing P450 reaction factors. High performance liquid chromatography coupled with ion trap mass spectrometry was used to identify the metabolites. Serial cardamonin dilutions were used to perform a kinetic study in human liver microsomes. Selective inhibitors of 7 of the major P450 isozymes were used to inhibit cardamonin hydroxylation to identify the isozymes involved in cardamonin metabolism. The cardamonin hydroxylation metabolic capacities of human and various other animals were investigated using the liver microsomal incubation system. RESULTS: Two metabolites generated by the liver microsome system were detected and identified as hydroxylated cardamonin. The Km and Vmax values for cardamonin hydroxylation were calculated as 32 micromol/L and 35 pmol x min(-1) x mg(-1), respectively. Furafylline and clomethiazole significantly inhibited cardamonin hydroxylation. Guinea pigs showed the highest similarity to humans with respect to the metabolism of cardamonin. CONCLUSION: CYP 1A2 and 2E1 were identified as the P450 isozymes involved in the metabolism of cardamonin in human liver microsomes. Furthermore, our research suggests that guinea pigs could be used in the advanced pharmacokinetic studies of cardamonin in vivo.

Sodium ferulate inhibits myocardial hypertrophy induced by abdominal coarctation in rats: Involvement of cardiac PKC and MAPK signaling pathways.

Sodium ferulate (SF) is the sodium salt of ferulic acid which is an active ingredient of Radix Angelica Sinensis and Ligusticum chuanxiong hort. Here, we investigated SF inhibition in a rat model of myocardial hypertrophy induced by coarctation of the abdominal aorta. Following coarctation, rats were given SF (20, 40, and 80 mg/kg/day) for 25 consecutive days. We characterized myocardial hypertrophy using myocardial hypertrophic parameters, histopathology, and gene expression of atrial natriuretic factor (ANF) -a gene related to myocardial hypertrophy. We detected the levels of angiotensin II (Ang II) and endothelin-1 (ET-1), protein kinase C beta (PKC-ß), Raf-1, extracellular regulated protein kinase 1/2 (ERK1/2), and mitogen-activated protein kinase phosphatase-1 (MKP-1) in myocardium. Notably, coarctation of the abdominal aorta increases myocardial hypertrophic parameters, cardiac myocyte diameter, the concentration of Ang II and ET-1 in myocardium, and gene expression of ANF. SF significantly ameliorates myocardial hypertrophy caused by coarctation of the abdominal aorta; reduces concentrations of Ang II and ET-1; suppresses the overexpression of ANF, PKC-ß, Raf-1, and ERK1/2; and increases the expression of MKP-1. These results indicate that SF alleviates myocardial hypertrophy induced by coarctation of the abdominal aorta, and these protective effects could be related to the inhibition of PKC and mitogen-activated protein kinase (MAPK) signaling pathways.

Dendrobium nobile Lindl. Alkaloids Decreases the Level of Intracellular ß-Amyloid by Improving Impaired Autolysosomal Proteolysis in APP/PS1 Mice.

As the major degradation pathway for long-lived proteins and organelles, macroautophagy is a decisive factor for the survival and longevity of cells. The existing evidence indicates that the disruption of substrate proteolysis in autolysosomes is the main mechanism underlying autophagy failure in Alzheimer's disease (AD). Thus, the restoration of normal lysosomal proteolysis and autophagy efficiency is a novel therapeutic strategy in the treatment of AD. In this study, 9-month-old APPswe/PS1ΔE9 transgenic (APP/PS1) mice were administered Dendrobium nobile Lindl. alkaloids (DNLA, 40 and 80 mg/kg) or Metformin (80 mg/kg), and age-matched wild-type mice were administered an isovolumic vehicle orally once a day for 4 months. The results demonstrated that DNLA significantly improved learning and memory function in APP/PS1 transgenic mice in the Morris water maze. Furthermore, DNLA could increase the expression of the v-ATPase A1 subunit to facilitate lysosomal acidification, prompt the dissociation of the cation independent-mannose-phosphate receptor from cathepsin (cat) D, promote the proteolytic maturation of cat D, increase the degradation of accumulated autophagic vacuoles (AVs) and ß-amyloid (Aß) contained in the AVs, and alleviate neuronal and synaptic injury. These findings demonstrate that DNLA improves learning and memory function in APP/PS1 mice, and the mechanisms appear to be due to the promotion of intracellular Aß degradation by increasing the protein level of v-ATPase A1 and then improving autolysosomal acidification and proteolysis.

Protective effects of Dendrobium nobile Lindl. alkaloids on amyloid beta (25-35)-induced neuronal injury.

Dendrobium nobile Lindl. alkaloids (DNLA), the active ingredients of a traditional Chinese medicine Dendrobium, have been shown to have anti-oxidative effects, anti-inflammatory action, and protective effect on neurons against oxygen-glucose deprivation. However, it is not clear whether DNLA reduces amyloid-beta (Aß)-induced neuronal injury. In this study, cortical neurons were treated with DNLA at different concentrations (0.025, 0.25, and 2.5 mg/L) for 24 hours, followed by administration of Aß25-35 (10 µM). Aß25-35 treatments increased cell injury as determined by the leakage of lactate dehydrogenase, which was accompanied by chromatin condensation and mitochondrial tumefaction. The damage caused by Aß25-35 on these cellular properties was markedly attenuated when cells were pretreated with DNLA. Treatment with Aß25-35 down-regulated the expressions of postsynaptic density-95 mRNA and decreased the protein expression of synaptophysin and postsynaptic density-95, all changes were significantly reduced by pretreatment of cells with DNLA. These findings suggest that DNLA reduces the cytotoxicity induced by Aß25-35 in rat primary cultured neurons. The protective mechanism that DNLA confers on the synaptic integrity of cultured neurons might be mediated, at least in part, through the upregulation of neurogenesis related proteins synaptophysin and postsynaptic density-95.

Dendrobium nobile Lindl alkaloid, a novel autophagy inducer, protects against axonal degeneration induced by Aß25-35 in hippocampus neurons in vitro.

AIMS: Axonal degeneration is a pathological symbol in the early stage of Alzheimer's disease (AD), which can be triggered by amyloid-ß (Aß) peptide deposition. Growing evidence indicates that deficit of autophagy eventually leads to the axonal degeneration. Our previous studies have shown that Dendrobium nobile Lindl alkaloid (DNLA) had protective effect on neuron impairment in vivo and in vitro; however, the underlying mechanisms is still unclear. METHODS: We exposed cultured hippocampus neurons to Aß25-35 to investigate the effect of DNLA in vitro. Axonal degeneration was evaluated by immunofluorescence staining and MTT assay. Neurons overexpressing GFP-LC3B were used to measure the formation of autophagosome. Autophagosome-lysosome fusion, the lysosomal pH, and cathepsin activity were assessed to reflect autophagy process. Proteins of interest were analyzed by Western blot. RESULTS: DNLA pretreatment significantly inhibited axonal degeneration induced by Aß25-35 peptide in vitro. Further studies revealed DNLA treatment increased autophagic flux through promoting formation and degradation of autophagosome in hippocampus neurons. Moreover, enhancement of autophagic flux was responsible for the protective effects of DNLA on axonal degeneration. CONCLUSIONS: DNLA prevents Aß25-35 -induced axonal degeneration via activation of autophagy process and could be a novel therapeutic target.

Dendrobium alkaloids prevent Aß25-35-induced neuronal and synaptic loss via promoting neurotrophic factors expression in mice.

BACKGROUND: Neuronal and synaptic loss is the most important risk factor for cognitive impairment. Inhibiting neuronal apoptosis and preventing synaptic loss are promising therapeutic approaches for Alzheimer's disease (AD). In this study, we investigate the protective effects of Dendrobium alkaloids (DNLA), a Chinese medicinal herb extract, on ß-amyloid peptide segment 25-35 (Aß25-35)-induced neuron and synaptic loss in mice. METHOD: Aß25-35(10 µg) was injected into the bilateral ventricles of male mice followed by an oral administration of DNLA (40 mg/kg) for 19 days. The Morris water maze was used for evaluating the ability of spatial learning and memory function of mice. The morphological changes were examined via H&E staining and Nissl staining. TUNEL staining was used to check the neuronal apoptosis. The ultrastructure changes of neurons were observed under electron microscope. Western blot was used to evaluate the protein expression levels of ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), and brain-derived neurotrophic factor (BDNF) in the hippocampus and cortex. RESULTS: DNLA significantly attenuated Aß25-35-induced spatial learning and memory impairments in mice. DNLA prevented Aß25-35-induced neuronal loss in the hippocampus and cortex, increased the number of Nissl bodies, improved the ultrastructural injury of neurons and increased the number of synapses in neurons. Furthermore, DNLA increased the protein expression of neurotrophic factors BDNF, CNTF and GDNF in the hippocampus and cortex. CONCLUSIONS: DNLA can prevent neuronal apoptosis and synaptic loss. This effect is mediated at least in part via increasing the expression of BDNF, GDNF and CNTF in the hippocampus and cortex; improving Aß-induced spatial learning and memory impairment in mice.