Shuping Li

Metabolites identification of harmane in vitro/in vivo in rats by ultra-performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry

Harmane, a β-carboline alkaloid with a wide spectrum of pharmacological activities, is naturally present in the human diet, in numerous foodstuffs and in hallucinogenic plants such as Peganum harmala, Banisteriopsis caapi and Tribulus terrestris. However, the precise metabolic fate of harmane remains unknown. In order to know whether harmane is extensively metabolized, a rapid and sensitive method using ultra-performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC/ESI-QTOF-MS) was used to analyze the metabolic profile of harmane in vitro and in vivo in rats. A total of 21 metabolites were identified from the rat liver microsomes and rat liver S9 (9), rat urine (11), feces (16), bile (16), and plasma (10) after a single oral administration of harmane using MetaboLynx™ and MassFragment ™ software tools. It indicated that the biliary and faecal clearance were the major excretion routes for harmane as well as its metabolites. The specific CLogP values combined with different acidic and alkaline mobile phase were helpful and useful for distinguishing N-oxidation and monohydroxylation metabolites. The metabolic transformation pathways of harmane included monohydroxylation, dihydroxylation, N-oxidation, O-glucuronide conjugation, O-sulphate conjugation, and glutathione conjugation. In conclusion, this study showed an insight into the metabolism of harmane.

Rapid and sensitive detection of the inhibitive activities of acetyl- and butyryl-cholinesterases inhibitors by UPLC-ESI-MS/MS

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are legitimate therapeutic targets for Alzheimers disease. The classical approach for screening potential AChE/BChE inhibitors was developed by Ellman. However, the background color of compounds or plant extracts remained uncertain and frequently interfered with the detection of the secondary reaction, thereby easily yielding false positive or false negative results. Rapid, selective, and sensitive ultra-performance liquid chromatography combined with electrospray ionization tandem mass spectrometry method was developed and used for the detection of AChE and BChE inhibition by directly determining the common product, choline (Ch). Proper separation was achieved for choline and chlormequat (internal standard) within 1.2min via isocratic elution (0.1% fromic acid:methanol=98:2) on an HSS T3 column following a simple precipitation of proteins for sample treatment. The relative standard deviations of the intra- and inter-day precisions were below 7.34 and 9.09%, respectively, whereas the mean accuracy for the quality control samples was 100.31±10.93%. The method exhibited the advantages of small total reaction volume (100μL), short analysis time (1.2min), high sensitivity (LOQ of 0.036μM for Ch), and low cost (little consumption enzymes of 0.0035 and 0.008unitmL(-1) for AChE and BChE, and substrates of 5.505 and 7.152μM for ACh and BCh in individual inhibition, respectively), and without matrix effect (90.00-105.03%). The developed method was successfully applied for detecting the AChE and BChE inhibitive activities for model drugs, including galanthamine, tacrine, neostigmine methylsulfate, eserine, as well as β-carboline and quinazoline alkaloids from Peganum harmala.

Inhibitive activities detection of monoamine oxidases (MAO) A and B inhibitors in human liver MAO incubations by UPLC–ESI-MS/MS

A sensitive UPLC-ESI-MS/MS method was developed for determining and screening of inhibitors of monoamine oxidase (MAO) by using mix MAO enzymes prepared from human liver. 5-Hydroxytryptamine (5-HT) and 2-phenethylamine (2-PEA) were used as substrates for MAO-A and MAO-B in incubations, and 5-hydroxyindole-3-acetic acid (5-HIAA) and phenylacetic acid (PAA) resulting from 5-HT and 2-PEA were used as markers to evaluate inhibitive activities of test compounds on MAO-A and MAO-B. Proper separation was achieved for positive multiple reaction monitoring of 5-HIAA (m/z 192.1→146.1) and negative multiple reaction monitoring PAA (m/z 135.0→91.0) via isocratic elution (0.1% fromic acid:acetonitrile=60:40) on a HSS T3 column following a simple precipitation of proteins for sample treatment. The relative standard deviations of intra- and inter-day precisions were ranged from 1.74% to 6.76% and 0.77% to 9.35%. The mean accuracies for the quality control samples were 101.37±6.60% and 101.39±2.85%, respectively. This method exhibited characteristics of small total reaction volume (100μl), short analysis time (3.5min), highly sensitivity, low cost and without matrix effect (103.56±2.33% to 112.63±8.57% for 5-HIAA and 105.68±8.75% to 112.76±4.67% for PAA). The developed method was successfully applied for detection of the MAO-A and MAO-B inhibitive activities by model drugs, including pargyline, clorgyline, as well as β-carboline alkaloids from Peganum harmala.

In vivo evaluation of the antitussive, expectorant and bronchodilating effects of extract and fractions from aerial parts of Peganum harmala linn

ETHNOPHARMACOLOGICAL RELEVANCE Aerial parts of Peganum harmala Linn (APP) is used as traditional medical herb in Uighur medicine in China, and it is traditionally used for treatment of cough and asthma.The aim of the present study is to evaluate the antitussive, expectorant and bronchodilating effects of extract and fractions (alkaloids and flavonoids) from APP, and to support its folk use with scientific evidence, and lay a foundation for its further researches. MATERIALS AND METHODS APP was extracted with 50% ethanol by reflux, extracts were concentrated in vacuum to afford total extract of APP (EXT). EXT was separated to provide alkaloid fraction (ALK) and flavonoid fraction (FLA) by macroporous resin. Antitussive evaluations were carried out with cough models in mice and guinea pigs induced by ammonia liquor, capsaicin, and citric acid. Phenol red secretion experiments in mice were performed to evaluate the expectorant activity. Bronchodilating activities were evaluated with a bronchoconstrictive challenge induced by acetylcholine chloride and histamine in guinea pigs. RESULTS In all the three antitussive tests, the EXT and ALK could significantly inhibit the frequency of cough, and prolong the cough latent period in animals. High dose of EXT (1650 mg/kg) and ALK (90 mg/kg) in mice and in guinea pigs created therapeutic activities as good as that of codeine phosphate (30 mg/kg). EXT could significantly increase phenol red secretion in mice for 0.64, 1.08 and 1.29 fold averagely at dosages of 183, 550, and 1650 mg/kg, ALK for 0.63, 0.96, 1.06 fold averagely at dosages of 10, 30, and 90 mg/kg, and ammonium chloride (1500 mg/kg, standard expectorant drug) for 0.97 fold, comparing with control group. Aminophylline could dramatically prolong the preconvulsive time for 162.28% in guinea pigs, EXT for 67.34%, 101.96% and 138.00% at dosages of 183, 550, and 1650 mg/kg, ALK for 55.47%, 97.74% and 126.77% at dosages of 10, 30, and 90 mg/kg, and FLA for 84.69%, 95.94% and 154.52% at dosages of 10, 30, and 90 mg/kg, comparing with pretreatment. CONCLUSIONS APP is an effective traditional folk medicine for the treatment of cough with potent antitussive, expectorant and bronchodilating activities. The alkaloid fraction is proved to be the most effective components of APP. The alkaloids from APP may be valuable lead compounds for drug development of respiratory diseases.

Interspecies metabolic diversity of harmaline and harmine in in vitro 11 mammalian liver microsomes.

The β-carboline alkaloids harmaline and harmine are widely present in hallucinogenic plants with great potential for treating depression, Parkinsons disease, and Alzheimers disease. The present study was to elucidate metabolic difference of harmaline and harmine in 11 mammalian liver microsomes in order to quantitate species-specific metabolic profiles. Using the probe substrate reaction, the enzymatic activities for 8 CYP450 isozymes of 11 liver microsomes were characterized. Combining ultra performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC-ESI-Q/TOF-MS) and ultra performance liquid chromatography combined with electrospray ionization quadrupole tandem mass spectrometry (UPLC-ESI-MS/MS) methods, 18 metabolites for harmaline and 11 for harmine were identified. The metabolism patterns differences of them presented discrepancy in the quality and quantity of metabolites. It was notable that O-sulfate conjugation was detected in all species except sheep. The intrinsic clearance CLint, LM values for the metabolites harmine and harmol in rabbits (37.5 and 42.4 μL/min/mg) were higher than those in other animals, while dogs (16.2 and 16.7 μL/min/mg) and humans (16.0 and 16.3 μL/min/mg) exhibited similar in vitro metabolic clearance. These observations suggested that harmaline and harmine were rapidly metabolized in liver microsomes of rat, mouse, and rabbit; moderately metabolized in human and dog; while weakly metabolized in sheep. Comprehensive analysis of the metabolism indicated that dogs and humans showed considerable similarity in the elimination of parent drugs, metabolic profiles, and catalytic processes. To summarize, these findings illustrated that in vitro studies of harmaline and harmine metabolic profiles in different species are helpful for the proper selection and interpretation of animal models for pharmacological and toxicological evaluation, and will ultimately provide useful guidance for the development of β-carboline alkaloids. Copyright

Simultaneous determination of vasicine and its major metabolites in rat plasma by UPLC-MS/MS and its application to in vivo pharmacokinetic studies

An efficient and sensitive ultra-performance liquid chromatography-tandem mass spectrometry method has been developed and validated to simultaneously determine and quantify vasicine (VAS) and its major metabolites including vasicinone (VAO), 9-oxo-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-3-yl hydrogen sulfate (VAOS), 1,2,3,9-tetrahydro-pyrrolo[2,1-b]quinazolin-3-yl hydrogen sulfate (VASS), 1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-3-β-D-glucuronide (VASG), vasicinol (VASL) and vasicinolone (VAOL) using pseudoephedrine as the internal standard in rat plasma. The chromatographic separation was conducted on a HSS T3 column (100 mm × 2.1 mm, 1.8 μm) with the gradient elution using a mobile phase of methanol–0.1% formic acid in water at a flow rate of 0.4 mL min−1 for 7 min. The tandem mass spectrometric detection was conducted using multiple reaction monitoring (MRM) by positive electrospray ionization (ESI). The corresponding lower limits of quantitation (LLOQ) of the method were 0.73, 0.80, 0.75, 0.80, 0.82, 0.87, 0.82 ng mL−1 for VAO, VAOS, VASS, VASG, VAS, VASL and VAOL, respectively. The within- and between-run precision for all analytes were less than 7.66% and 12.30%, respectively. The recovery for all analytes was between 85.89% and 114.58%, and the matrix effects for all analytes were not observed. By the UPLC-MS/MS method, the relative quantitation of five metabolites of 9-oxo-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazolin-3-β-D-glucuronide (VAOG), hydroxylation–acetylation products of VAS (HVAS1 and HVAS2) and methylation–acetylation products of VAS (MVAS1 and MVAS2) were achieved by standard curves derived from the urine sample with treatment by VAS as a reference substance, in which the considerable target metabolites were included. This method was successfully applied to pharmacokinetic studies of VAS and its metabolites in rats. The activity of the components in plasma after intravenous administration of VAS (2 mg kg−1) was evaluated by in vitro anti-butyrylcholinesterase assays. The results indicated that in vivo butyrylcholinesterase inhibitive activities were mainly due to the different concentrations of prototype VAS and a few other metabolites.

Anti-amnesic effect of extract and alkaloid fraction from aerial parts of Peganum harmala on scopolamine-induced memory deficits in mice

ETHNOPHARMACOLOGICAL RELEVANCE Aerial parts of Peganum harmala Linn (APP) is used as traditional medical herb for treatment of forgetfulness in Uighur medicine in China. But, the active ingredients and underlying mechanisms are unclear. AIM OF THE STUDY The present study was undertaken to investigate the improvement effects of extract and alkaloid fraction from APP on scopolamine-induced cognitive dysfunction and to elucidate their underlying mechanisms of action, and to support its folk use with scientific evidence, and lay a foundation for its further researches. MATERIALS AND METHODS The acetylcholinesterase (AChE) inhibitory activities of extract (EXT), alkaloid fraction (ALK) and flavonoid fraction (FLA) from APP were evaluated in normal male C57BL/6 mice. The anti-amnesic effects of EXT and ALK from APP were measured in scopolamine-induced memory deficits mice by the Morris water maze (MWM) tasks. The levels of biomarkers, enzyme activity and protein expression of cholinergic system were determined in brain tissues. RESULTS The AChE activity was significantly decreased and the content of neurotransmitter acetylcholine (ACh) was significantly increased in normal mice cortex and hippocampus by treatment with donepezil at dosage of 8mg/kg, EXT at dosages of 183, 550, 1650mg/kg and ALK at dosages of 10, 30, 90mg/kg (P<0.05), and the AChE activity and the content of ACh were not significantly changed in cortex and hippocampus after treatment with FLA at dosages of 10, 30, 90mg/kg (P>0.05). In the MWM task, scopolamine-induced a decrease in both the swimming time within the target zone and the number of crossings where the platform had been placed were significantly reversed by treatment with EXT at dosages of 550, 1650mg/kg and ALK at dosages of 30, 90mg/kg (P<0.05). Moreover, the activity and protein expression of AChE was significantly decreased and the content of neurotransmitter ACh was significantly increased in cerebral cortex of scopolamine-induced mice by treatment with EXT at dosages of 183, 550, 1650mg/kg and ALK at dosages of 10, 30, 90mg/kg (P<0.05), compared with scopolamine-treated group. CONCLUSIONS EXT and ALK from APP exert beneficial effect on learning and memory processes in mice with scopolamine-induced memory impairment. APP is an effective traditional folk medicine and the ALK fraction is proved to be the main effective components for the treatment of forgetfulness. The ALK may be valuable source for lead compounds discovery and drug development for treatment of memory impairment such as in Alzheimers disease.

Exposure Characteristics of the Analogous β-Carboline Alkaloids Harmaline and Harmine Based on the Efflux Transporter of Multidrug Resistance Protein 2

Harmaline and harmine occur naturally in plants and are distributed endogenously in human and animal tissues. The two β-carboline alkaloids possess potential for treating Alzheimers disease, Parkinsons disease, depression and other central nervous system diseases. However, studies have showed that the two compounds have similar structures but with quite different bioavailability. The aim of this study was to elucidate the exposure difference and characterize the in vitro transport, metabolism, and pharmacokinetic properties of harmaline and harmine. The results showed that the harmaline and harmine transport across the Caco-2 and MDCK cell monolayers was varied as the time, concentration, pH and temperature changed. The absorption of harmaline and harmine was significantly decreased when ES (OATPs inhibitor), TEA (OCTs/OCTNs substrate), NaN3 (adenosine triphosphate inhibitor), or sodium vanadate (ATPase Na+/K+-dependent inhibitor) was added. However, when given MK571 and probenecid (the typical MRP2 inhibitor), the PappAB of harmine was increased (1.62- and 1.27-folds), and the efflux ratio was decreased from 1.59 to 0.98 and from 1.59 to 1.19, respectively. In addition, the uptake ratio of harmine at 1 μM was >2.65 in the membrane vesicles expressing human MRP2. Furthermore, harmine could slightly up-regulate the expression of MRP2, which implying harmine might be the substrate of MRP2. Particularly, the CLint-value for harmine was ~1.49-folds greater than that of harmaline in human liver microsomes. It was worth noting that the F-value of harmine was increased 1.96-folds after harmine co-administration with probenecid. To summarize, comprehensive analysis indicated that harmaline and harmine were absorbed by transcellular passive diffusion and a pH- and Na+-dependent mechanism might be mediated by OATPs and OCTs/OCTNs. MRP2 but MDR1 or BCRP might be involved in the transport of harmine. Furthermore, harmine was more unstable and easily metabolized than harmaline. All these findings suggested that harmine not only appears be an MRP2 substrate, but also possesses weak metabolic stability, and eventually leads to a low oral bioavailability. Taken together, the elucidated absorption, transport, metabolism as well as pharmacokinetic characteristics of harmaline and harmine provide useful information for designing delivery systems, pharmacological applications and avoiding drug-drug interactions.

Analogous β-Carboline Alkaloids Harmaline and Harmine Ameliorate Scopolamine-Induced Cognition Dysfunction by Attenuating Acetylcholinesterase Activity, Oxidative Stress, and Inflammation in Mice

The analogous β-carboline alkaloids, harmaline (HAL) and harmine (HAR), possess a variety of biological properties, including acetylcholinesterase (AChE) inhibitory activity, antioxidant, anti-inflammatory, and many others, and have great potential for treating Alzheimer’s disease (AD). However, studies have showed that the two compounds have similar structures and in vitro AChE inhibitory activities but with significant difference in bioavailability. The objective of this study was to comparatively investigate the effects of HAL and HAR in memory deficits of scopolamine-induced mice. In the present study, mice were pretreated with HAL (2, 5, and 10 mg/kg), HAR (10, 20, and 30 mg/kg) and donepezil (5 mg/kg) by intragastrically for 7 days, and were daily intraperitoneal injected with scopolamine (1 mg/kg) to induce memory deficits and then subjected to behavioral evaluation by Morris water maze. To further elucidate the underlying mechanisms of HAL and HAR in improving learning and memory, the levels of various biochemical factors and protein expressions related to cholinergic function, oxidative stress, and inflammation were examined. The results showed that HAL and HAR could effectively ameliorate memory deficits in scopolamine-induced mice. Both of them exhibited an enhancement in cholinergic function by inhibiting AChE and inducing choline acetyltransferase (ChAT) activities, and antioxidant defense via increasing the antioxidant enzymes activities of superoxide dismutase and glutathione peroxidase, and reducing maleic diadehyde production, and anti-inflammatory effects through suppressing myeloperoxidase, tumor necrosis factor α, and nitric oxide as well as modulation of critical neurotransmitters such as acetylcholine (ACh), choline (Ch), L-tryptophan (L-Trp), 5-hydroxytryptamine (5-HT), γ-aminobutyric acid (γ-GABA), and L-glutamic acid (L-Glu). Furthermore, the regulations of HAL on cholinergic function, inflammation, and neurotransmitters were more striking than those of HAR, and HAL manifested a comparable antioxidant capacity to HAR. Remarkably, the effective dosage of HAL (2 mg/kg) was far lower than that of HAR (20 mg/kg), which probably due to the evidently differences in the bioavailability and metabolic stability of the two analogs. Taken together, all these results revealed that HAL may be a promising candidate compound with better anti-amnesic effects and pharmacokinetic characteristics for the treatments of AD and related diseases.

Stereoselective glucuronidation metabolism, pharmacokinetics, anti-amnesic pharmacodynamics, and toxic properties of vasicine enantiomers in vitro and in vivo

ABSTRACT Vasicine (VAS) is a potential natural cholinesterase inhibitor for treatment of Alzheimers disease. Due to one chiral centre (C‐3) presenting in molecule, VAS has two enantiomers, d‐vasicine (d‐VAS) and l‐vasicine (l‐VAS). The study was undertaken to investigate the stereoselective glucuronidation metabolism, pharmacokinetics, anti‐amnesic effect and acute toxicity of VAS enantiomers. In results, the glucuronidation metabolic rate of l‐VAS was faster than d‐VAS in human liver microsomes and isoenzymes tests, and it was proved that the UDP‐glucuronosyltransferase (UGT) 1A9 and UGT2B15 were the major metabolic enzymes for glucuronidation of l‐VAS, while only UGT1A9 for d‐VAS, which take responsibility of the significantly less metabolic affinity of d‐VAS than l‐VAS in HLM and rhUGT1A9. The plasma exposure of d‐VAS in rats was 1.3‐fold and 1.6‐fold higher than that of l‐VAS after intravenous and oral administration of d‐VAS and l‐VAS, respectively. And the plasma exposure of the major glucuronidation metabolite d‐VASG was one of tenth of l‐VASG or more less, no matter by intravenous or oral administration. Both d‐VAS and l‐VAS were exhibited promising acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities, and the BChE inhibitory activity of d‐VAS with IC50 of 0.03 ± 0.001 &mgr;M was significantly stronger than that of l‐VAS with IC50 of 0.98 ± 0.19 &mgr;M. The molecular docking results indicated that d‐VAS and l‐VAS could bind to the catalytic active site (CAS position) either of human AChE and BChE, and the BChE combing ability of d‐VAS (the score of GBI/WAS dG −7.398) was stronger than that of l‐VAS (the score of GBI/WAS dG −7.135). Both d‐VAS and l‐VAS could improving the learning and memory on scopolamine‐induced memory deficits in mice. The content of acetylcholine (ACh) after oral administration d‐VAS increased more than that of l‐VAS in mice cortex, through inhibiting cholinesterase (ChE) and increasing choline acetyltransferase (ChAT). In addition, the LD50 value of d‐VAS (282.51 mg·kg−1) was slight lower than l‐VAS (319.75 mg·kg−1). These results indicated that VAS enantiomers displayed significantly stereoselective metabolic, pharmacokinetics, anti‐amnesic effect and toxic properties in vitro and in vivo. The d‐VAS might be the dominant configuration for treating Alzheimers disease. Graphical abstract Figure. No Caption available.