Oxytrodiflavanone A and Oxytrochalcoflavanones A,B: New Biflavonoids from Oxytropis chiliophylla.

Three previously undescribed biflavonoids, oxytrodiflavanone A (1), and oxytrochalcoflavanones A,B (2,3), were isolated from the aerial part of Oxytropis chiliophylla, together with their putative biosynthetic monomers, i.e., (2S)-5,7-dihydroxyflavanone (4), (2S)-7-hydroxyflavanone (5), and 2',4'-dihydroxychalcone (6). The structures of these compounds were elucidated by a combination analysis of spectroscopic data. The cytotoxic activities of all the isolated compounds against PC-3 human prostate cancer cell line are also presented.

Structurally Diverse Cytotoxic Dimeric Chalcones from Oxytropis chiliophylla.

Ten isomeric cyclobutane- and cyclohexene-containing chalcone dimers, oxyfadichalcones A-G, were isolated from the aerial parts of Oxytropis chiliophylla. These included six new compounds and three pairs of enantiomers that are being reported from natural sources for the first time. The relative configurations were elucidated by spectroscopic data analysis, while the absolute configurations were determined by comparing the experimental and calculated electronic circular dichroism spectra. Quantitative LC-MS analysis of the main dimers from different parts of the plant revealed their characteristic accumulation in the viscous secretion and provided supporting evidence for the hypothesized photochemical biosynthesis. In addition, the cytotoxic activities of all isolates against the PC-3 human prostate cancer cell line are reported.

New Potential Biomarker for Methasterone Misuse in Human Urine by Liquid Chromatography Quadrupole Time of Flight Mass Spectrometry.

In this study, methasterone urinary metabolic profiles were investigated by liquid chromatography quadrupole time of flight mass spectrometry (LC-QTOF-MS) in full scan and targeted MS/MS modes with accurate mass measurement. A healthy male volunteer was asked to take the drug and liquid-liquid extraction was employed to process urine samples. Chromatographic peaks for potential metabolites were hunted out with the theoretical [M - H](-) as a target ion in a full scan experiment and actual deprotonated ions were studied in targeted MS/MS experiment. Fifteen metabolites including two new sulfates (S1 and S2), three glucuronide conjugates (G2, G6 and G7), and three free metabolites (M2, M4 and M6) were detected for methasterone. Three metabolites involving G4, G5 and M5 were obtained for the first time in human urine samples. Owing to the absence of helpful fragments to elucidate the steroid ring structure of methasterone phase II metabolites, gas chromatography mass spectrometry (GC-MS) was employed to obtain structural information of the trimethylsilylated phase I metabolite released after enzymatic hydrolysis and the potential structure was inferred using a combined MS method. Metabolite detection times were also analyzed and G2 (18-nor-17ß-hydroxymethyl-2α, 17α-dimethyl-androst-13-en-3α-ol-ξ-O-glucuronide) was thought to be new potential biomarker for methasterone misuse which can be detected up to 10 days.

Analysis on dynamic changes of etizolam and its metabolites and exploration of its development prospect using UPLC-Q-exactive-MS.

As one of the most widely abused designer benzodiazepines in the world, etizolam has been found in many cases in many countries. In this study, UPLC-Q-Exactive-MS was used for the first time to establish a dynamic change model of etizolam and its metabolites in rats. Compared with previous studies, the detection sensitivity and reproducibility of the instrument were higher. In the experiment, we optimized the traditional pharmacokinetic model based on Gauss function. According to the significant difference of etizolam in the plasma elimination phase of rats, a new pharmacokinetic model based on Lorentz function was established to describe the dynamic changes of etizolam more rigorously, which made the error effects lower and the accuracy of the pharmacokinetic parameters was improved. At the same time, the pharmacokinetic parameters of etizolam were compared with four other designer benzodiazepines reported in previous studies in rats, and we found the direct reason for the popularity of etizolam in the NPS market and explored the future development of etizolam for the first time. In addition, 21 metabolites were found through rat experiments to effectively detect etizolam abuse for a long time, of which 4 metabolites had the longest detection window and could be used as long-acting metabolites for experiments, which greatly prolongs the detection window and extends the time range in which etizolam was detected in real cases. This study is the first to conduct a systematic and comprehensive study on the metabolism and pharmacokinetics of etizolam and find out the direct reason for the prevalence of etizolam abuse, and we also discuss the development trend of etizolam in the future market of new psychoactive substances, which is beneficial for forensic experts to assess the trend of drug abuse and can provide reference for relevant drug control and drug treatment.

Metabolic profiling of a new synthetic cannabinoid receptor agonist, ADMB-FUBIATA, with human liver microsomes, human primary hepatocytes and human recombinant CYP450 enzymes using LC-quadrupole-orbitrap MS.

A novel synthetic cannabinoid receptor agonist (SCRA), ADMB-FUBIATA, featuring an acetamide-linked structure, has emerged on the illicit drug market. To provide dependable verification of its consumption and identify reliable biomarkers, we investigated an in vitro metabolism study of ADMB-FUBIATA incubated with human primary hepatocytes (HPHs) for the first time and correlated our findings with those from human liver microsomes (HLMs). In this work, ADMB-FUBIATA (10 µM) was incubated with HLM and HPH for 1 and 5 h, respectively, and then subjected to LC-quadrupole-orbitrap MS. A total of 25 metabolites across 8 metabolic pathways were identified after incubation with HLM and HPH, respectively. Monohydroxylation and N-dealkylation were the major metabolic pathways, and formation to ketone was first identified. In addition, the metabolism of ADMB-FUBIATA were found to be mediated by multiple CYP450 enzymes, predominantly CYP2C19, 2D6, and 3A4. This research also initially characterized the fragmentation patterns of the metabolites of ADMB-FUBIATA, elaborating on their structural relationship with ADMB-FUBIATA analogs. To effectively monitor ADMB-FUBIATA abuse, metabolites M4 and M1 were proposed as reliable biomarkers by cross-validating the HLM and HPH incubation results.

A LC-MS/MS method for determination of clenbuterol enantiomers in animal tissues and its application to the enantioselective distribution in Bama mini-pigs.

OBJECTIVES: To establish and validate a simple and reliable analytical method for separation and determination of clenbuterol enantiomers (R-(-)-clenbuterol & S-(+)-clenbuterol) in animal tissues, and apply it to the enantioselective distribution of clenbuterol in Bama mini-pigs. METHODS: A LC-MS/MS analytical method was developed and validated in positive multiple reaction monitoring mode with electrospray ionization. After perchloric acid deproteinization, samples were pretreated only by one step liquid-liquid extraction using tert-butyl methyl ether under strong alkaline condition. Teicoplanin was used as chiral selector and 10 mM ammonium formate methanol solution was used as mobile phase. The optimized chromatographic separation conditions were completed in 8 min. Two chiral isomers in 11 edible tissues from Bama mini-pigs were investigated. RESULTS: R-(-)-clenbuterol and S-(+)-clenbuterol can be baseline separated and accurately analyzed with a linear range of 5-500 ng/g. Accuracies ranged from -11.9-13.0% for R-(-)-clenbuterol and -10.2-13.2% for S-(+)-clenbuterol, intra-day and inter-day precisions were between 0.7 and 6.1% for R-(-)-clenbuterol and 1.6-5.9% for S-(+)-clenbuterol. R/S ratios in edible tissues of pigs were all significantly lower than 1. CONCLUSIONS: The analytical method has good specificity and robustness in determination of R-(-)-clenbuterol and S-(+)-clenbuterol in animal tissues, and can be used as a routine analysis method for food safety and doping control. There is a significant difference in R/S ratio between pig feeding tissues and pharmaceutical preparations (racemate with R/S ratio of 1), which makes it possible to identify the source of clenbuterol in doping control and investigation.

Metabolic Profile Analysis of Designer Benzodiazepine Etizolam in Zebrafish and Human Liver Microsomes.

As one of the most widely abused designer benzodiazepines worldwide, Etizolam is characterized by its high addiction potential, low production cost, and difficulty in detection. Due to the rapid metabolism of Etizolam in the human body, the probability of detecting the Etizolam parent drug in actual case samples by forensic personnel is low. Therefore, without detecting the parent drug, analysis of Etizolam metabolites can help forensic personnel provide references and suggestions on whether the suspect has taken Etizolam. This study simulates the objective metabolic process of the human body. It establishes a zebrafish in vivo metabolism model and a human liver microsome in vitro metabolism model to analyze the metabolic characteristics of Etizolam. A total of 28 metabolites were detected in the experiment, including 13 produced in zebrafish, 28 produced in zebrafish urine and feces, and 17 produced in human liver microsomes. The UPLC-Q-Exactive-MS technology was used to analyze the structures and related metabolic pathways of Etizolam metabolites in zebrafish and human liver microsomes, and a total of 9 metabolic pathways were identified, including monohydroxylation, dihydroxylation, hydration, desaturation, methylation, oxidative deamination to alcohol, oxidation, reduction acetylation, and glucuronidation. Among them, metabolites involving hydroxylation reactions (including monohydroxylation and dihydroxylation) accounted for 57.1% of the total number of potential metabolites, indicating that hydroxylation may be the major metabolic pathway of Etizolam. Based on the response values of each metabolite, monohydroxylation (M1), desaturation (M19), and hydration (M16) were recommended as potential biomarkers for Etizolam metabolism. The experimental results provide reference and guidance for forensic personnel in identifying Etizolam use in suspects.

Gas chromatography properties and mass spectrometry fragmentation of anabolic androgenic steroids in doping control.

Aim: This study aimed to investigate the gas chromatographic properties and mass spectrometric fragmentations of anabolic androgenic steroids (AASs) after trimethylsilylated derivatization. Materials & methods: A total of 113 AASs were analyzed through gas chromatography-mass spectrometry in the full-scan mode. Results: New fragmentation pathways yielding m/z 129, 143 and 169 ions were analyzed. Based on the characteristics of the A-ring, seven classes of drugs were identified and analyzed. Conclusion: The fragmentation pathway of a new classification of 4-en-3-hydroxyl was reported for the first time. The relationship between the chemical structures of AASs and their retention time, along with their molecular ion peak abundance, was also reported herein for the first time.

Detection of 20 endogenous anabolic steroid esters with Girard's Reagent P derivatization in dried blood spots using UPLC-Q-Orbitrap-MS.

The esters of endogenous anabolic steroids are the most frequently used doping agents for prolonging the half-life of exogenously ingested endogenous anabolic steroids. As a cost- and time-saving matrix, dried blood spots (DBSs) are valuable for directly detecting endogenous anabolic steroid esters in blood and for providing conclusive evidence of their abuse. In this study, a method for simultaneous detection of 20 endogenous anabolic steroid esters in DBSs based on ultra-high performance liquid chromatography-quadrupole-Orbitrap mass spectrometry (UPLC-Q-Orbitrap-MS) with parallel reaction monitoring (PRM) was developed and validated, and 10 of these esters were analyzed in DBSs for the first time. This method analyzes the greatest number and types of endogenous anabolic steroid esters of any current method using DBSs. Girard's Reagent P (GRP) was used for the derivatization of endogenous anabolic steroid esters in a DBS matrix for the first time, and the conditions of the derivatization reaction were optimized to achieve a higher sensitivity compared to previous methods. Selectivity, limit of detection (LOD), extraction recovery, precision (intra- and inter-), matrix effects, and carry-over were analyzed to validate the method. The LODs were lower and the recoveries were higher than those of previous studies. The relative standard deviation of the intraday precision was below 20% and the interday precision was below 35%. A product ion analysis of GRP nandrolone ester, GRP boldenone ester, GRP dehydroepiandrosterone acetate, and GRP androstanolone ester derivatives was performed, and the structures of the fragment ions were proposed for the first time.

Identification and characterization of higenamine metabolites in human urine by quadrupole-orbitrap LC-MS/MS for doping control.

Higenamine is an alkaloid found in aconite, Annona squamosa, nanzhu (sacred bamboo), and other plants. It can be used to treat coughing, asthma, heart failure, and erectile dysfunction as well as aid in weight loss. It is also a banned substance in and out of competition as defined by the World Anti-Doping Agency (WADA). In this work, higenamine metabolic profiles were investigated in detail. Two healthy volunteers (one male and one female) took a higenamine tablet (5 mg), and urine samples were collected for two weeks. Solid-phase extraction (SPE) without enzymatic hydrolysis was used to clean the urine samples, and the urine extracts were then analyzed by liquid chromatography-quadrupole-orbitrap mass spectrometry (quadrupole-orbitrap LC-MS/MS) with accurate mass measurements. Higenamine and 32 metabolites were detected: 6 methylated, 10 sulfated and 16 glucuronidated metabolites. The chemical structures were elucidated by their fragmentation patterns, and accurate molecular formula determination was obtained for these newly reported metabolites. Three metabolic pathways containing methylation, glucuronidation, sulfation, and combinations of these were provided with methylation as the main metabolic pathway. The post-dose detection windows within urine of all 32 metabolites were compared with that of the parent drug, and a new potential biomarker (M7) was suggested for higenamine misuse. All urine samples were processed by two sample preparation methods: the dilute-and-shoot (DS) procedure and acid hydrolysis followed by SPE, and the time periods for a higenamine positive trails of two methods were compared. Although the DS method used to process the urine samples of athletes in the most of WADA-accredited laboratories to detect only free higenamine, acid hydrolysis followed by SPE is preferable and offers routine analysis to avoid false-negative results.

Simultaneous detection of 93 anabolic androgenic steroids in dietary supplements using gas chromatography tandem mass spectrometry.

In recent years, anabolic androgenic steroids (AASs) have been frequently detected as undeclared ingredients in dietary supplements, where the adverse analytical findings (AAFs) were obtained from analysis of athletes' urine samples after ingestion. In our present study, a GC-MS/MS method for simultaneous detection of 93 anabolic steroids was developed. The chromatographic and mass spectrometric conditions were optimized, and selective reaction monitoring (SRM) mode was adopted to obtain the necessary sensitivity. The whole sample analysis process was completed within 23 min, and the limit of detection (LOD) was 0.5-4 ng.g-1 for solid samples and 0.1-0.8 ng.mL-1 for liquid samples. This method was verified according to World Anti-Doping Agency (WADA) regulations. In addition, the method was found to be specific, accurate. The developed method was then applied to a routine analysis of more than 300 liquid and solid dietary supplements, and one testosterone-positive sample was found. Three suspected drugs, (4-hydroxyandrostenedione, DHEA, and 6-Br androstenedione) were found in three dietary supplements obtained from the Internet through the pretreatment method of this study. This study provides a high-throughput method for screening and monitoring the ingredients of supplements and their subsequent harm to public health.

Metabolic Profiles of 5F-MDMB-PICA in Human Urine, Serum and Hair Samples Using LC-Q Exactive HF-MS.

In 2020, 5F-MDMB-PICA (5F-MDMB-2201) was one of the most common synthetic cannabinoids (SCs) identified in drugs seized by the Beijing Drug Control Agency, and it was categorized as Schedule II by the United Nations Office on Drugs and Crime in March 2020. It is difficult to detect 5F-MDMB-PICA in biological matrices due to its fast metabolic rate in vivo. In this work, 5F-MDMB-PICA metabolic profiles were investigated by liquid chromatography--quadrupole exactive high field orbitrap mass spectrometer (LC-Q Exactive HF MS), with accurate mass measurements in human urine, serum and pubic hair. To obtain intact metabolites, solid-phase extraction for urine and serum and direct ultrasonic extraction for pubic hair were applied to clean the samples without enzymatic hydrolysis. The differences in 5F-MDMB-PICA metabolism in the three different matrices were compared for the first time to determine the best detection biomarkers for monitoring 5F-MDMB-PICA misuse. Urine samples were determined to be the preferred biological material for identifying 5F-MDMB-PICA abuse. Forty-seven intact metabolites were detected in human urine, the ester hydrolyzed with glucuronidated metabolite in urine samples can be used as the primary biomarker to identify drug misuse. Fifteen metabolites were found in serum samples. Ester hydrolysis was considered to be the major metabolic pathway, and a large number of metabolites were involved with it. Zero metabolites apart from the parent drug were detected in pubic hair samples. Twenty-eight new metabolites and their metabolic pathways were characterized and tentatively identified by LC-QE-HF-MS, and a new potential biomarker (M5 ester hydrolysis + propionic acid) was also identified.

Metabolic profiles and screening tactics for MDMB-4en-PINACA in human urine and serum samples.

MDMB-4en-PINACA (Methyl 3,3-dimethyl-2-[1-(pent-4-en-1-yl)-1H-indazole-3-carboxamido] butanoate) is a potent agonist of the CB1 receptor. In 2021, it was one of the most common synthetic cannabinoid receptor agonists (SCRAs) seized by the Beijing Drug Control Agency. MDMB-4en-PINACA can be hard to detect in biological specimens because of ester hydrolysis. In this work, a highly sensitive liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method was developed for the detection of MDMB-4en-PINACA metabolites in urine, serum, and hair samples. Metabolites from authentic samples were compared with those from human liver microsomes (HLMs) in vitro and in zebrafish in vivo. A total of 75 metabolites, including 44 previously unreported metabolites, were identified from urine samples. We found that 11 metabolic pathways were involved in MDMB-4en-PINACA metabolism, including acetylation, a novel metabolic pathway for SCRAs. Our results revealed that ester hydrolysis and hydroxylation were to the major metabolic pathways involved in MDMB-4en-PINACA metabolism. Using serum samples, we detected 9 metabolites along with the parent drug. Only the parent drug was detected using hair samples. The existence of ADB-4en-PINACA makes the currently used biomarkers for MDMB-4enPINACA not very specific for the intake of MDMB-4en-PINACA. Therefore, based on the identified metabolites and their structural features, we propose more sensitive screening tactics for MDMB-4en-PINACA using urine and serum samples.

Mass spectrometric characterization of toremifene metabolites in human urine by liquid chromatography-tandem mass spectrometry with different scan modes.

The metabolism and excretion of toremifene were investigated in one healthy male volunteer after a single oral administration of 120 mg toremifene citrate. Different liquid chromatographic/tandem mass spectrometric (LC/MS/MS) scanning techniques were carried out for the characterization of the metabolites in human urine for doping control purposes. The potential characteristic fragmentation pathways of toremifene and its major metabolites were presented. An approach for the metabolism study of toremifene and its analogs by liquid chromatography-tandem mass spectrometry was established. Five different LC/MS/MS scanning methods based on precursor ion scan (precursor ion scan of m/z 72.2, 58.2, 44.2, 45.2, 88.2 relative to five metabolic pathways) in positive ion mode were assessed to recognize the metabolites. Based on product ion scan and precursor ion scan techniques, the metabolites were proposed to be identified as 4-hydroxy-toremifene (m/z 422.4), 4'-hydroxy-toremifene (m/z 422.4), α-hydroxy-toremifene (m/z 422.4), 3,4-dihydroxy-toremifene (m/z 404.2), toremifene acid (m/z 402.2), 3-hydroxy-4-methoxy-toremifene (m/z 456.2), dihydroxy-dehydro-toremifene (m/z 440.2), 3,4-dihydroxy-toremifene (m/z 438.2), N-demethyl-4-hydroxy-toremifene (m/z 408.3), N-demethyl-3-hydroxy-4-methoxy-toremifene (m/z 438.3). In addition, a new metabolite with a protonated molecule at m/z 390.3 was detected in all urine samples. The compound was identified by LC/MS/MS as N-demethyl-4,4'-dihydroxy-tamoxifene. The results indicated that 3,4-dihydroxy-toremifene (m/z 404.2), toremifene acid (m/z 402.2) and N-demethyl-4,4'-dihydroxy-tamoxifene (m/z 390.3) were major metabolites in human urine.

Dried blood spots in doping analysis.

A series of dried blood spot (DBS) detection methods for doping agents have been developed in the last two decades. The DBS technique minimizes invasiveness and reduces storage and shipping costs. Recently, the World Anti-Doping Agency announced the use of DBS for the 2022 Beijing Winter Olympic Games and Paralympic Games owing to the advantages of the DBS application in routine doping control. Therefore the further development of detection methods for doping agents in DBS is important and urgent. This review summarizes five aspects of DBS application in doping analysis: sample collection, storage conditions, pretreatment, instrumentation and validation according to the Prohibited List issued by the World Anti-Doping Agency, and proposes some suggestions for future studies of DBS in doping analysis.

Enantiomeric analysis of clenbuterol in Chinese people by LC-MS/MS to distinguish doping abuse from meat contamination.

Aim: Follow-up investigations are often required for clenbuterol-positive cases. A method to distinguish doping abuse from meat contamination was developed. Materials & methods: A total of 26 volunteers were recruited to ingest clenbuterol contaminated-pork and clenbuterol tablets. Results: For 20 volunteers, after ingestion of contaminated-pork, R-(-)/S-(+)-clenbuterol ratio was <1.0, while the value was >1.0 after taking clenbuterol tablets. However, after taking clenbuterol tablets, some ratio points of the other six volunteers were between 0.9 and 1.0. A case of an abnormal cold and fever, which returned to normal after recovery, was also reported firstly. Conclusion: A change in R-(-)/S-(+)-clenbuterol was reported in the Chinese population initially. A ratio of 0.9 was recommended in doping related cases for the Chinese population.

Doping control analysis for adrafinil and its major metabolites in human urine.

A new and reliable two-step liquid chromatography/tandem mass spectrometry (LC/MS/MS) method in combination with gas chromatography/mass spectrometry (GC/MS) for the screening and confirmation of adrafinil and its major metabolites, modafinil and modafinil acid, in human urine has been developed and validated. The method involved reversed-phase C18 solid-phase extraction (SPE) cartridge extraction and MS analysis by means of LC/MS/MS and GC/MS. The study illustrated that the ESI capillary temperature played a key role in the formation of the protonated molecule. The limits of detection (LODs) of the developed method for the three compounds were lower than the minimum required performance limit (MRPL) of the World Anti-Doping Agency (WADA). The human urine samples obtained after the oral administration of modafinil and from the Beijing 2008 Olympic Games were analyzed by using the described method, which has also been successfully applied to routine analyses and the WADA Proficiency Test.

Characterization and tentative identification of new flunitrazepam metabolites in authentic human urine specimens using liquid chromatography-Q exactive-HF hybrid quadrupole-Orbitrap-mass spectrometry (LC-QE-HF-MS).

Flunitrazepam (FNZ) is a potent hypnotic, sedative, and amnestic drug used to treat severe insomnia. In our recent study, FNZ metabolic profiles were investigated carefully. Six authentic human urine samples were purified using solid phase extraction (SPE) without enzymatic hydrolysis, and urine extracts were then analyzed by liquid chromatography-Q exactive-HF hybrid quadrupole-Orbitrap-mass spectrometry (LC-QE-HF-MS), using the full scan positive ion mode and targeted MS/MS (ddms2) technique to make accurate mass measurements. There were 25 metabolites, including 13 phase I and 12 phase II metabolites, which were detected and tentatively identified by LC-QE-HF-MS. In addition, nine previously unreported phase II glucuronide conjugates and four phase I metabolites are reported here for the first time. Eight metabolic pathways, including N-reduction and O-reduction, N-glucuronidation, O-glucuronidation, mono-hydroxylation and di-hydroxylation, demethylation, acetylation, and combinations, were implicated in this work, and 2-O-reduction together with dihydroxylation were two novel metabolic pathways for FNZ that were identified tentatively. Although 7-amino FNZ is widely considered to be the primary metabolite, a previously unreported metabolites (M12) can also serve as a potential biomarker for FNZ misuse.

Analysis of AMB-FUBINACA Biotransformation Pathways in Human Liver Microsome and Zebrafish Systems by Liquid Chromatography-High Resolution Mass Spectrometry.

In this study, the metabolic profiles of a new illicit drug AMB-FUBINACA were investigated using both human liver microsome and zebrafish models. Liquid chromatography Q Extractive HF Hybrid Quadrupole-Orbitrap mass spectrometry (LC-QE-HF-MS) was employed to analyze the metabolic sites and pathways. AMB-FUBINACA was added to the in vitro liver microsome incubation model to simulate the metabolic processes in human body. The results showed that a total of 17 metabolites were generated in the human liver microsome model; the main metabolic pathways of the phase I metabolism included ester hydrolysis, methylation, ester hydrolysis combined with decarboxylation, hydroxylation, ester hydrolysis combined with indazole ring hydroxylation, etc. while glucuronidation served as the main metabolic pathway of the phase II metabolism. The zebrafish system produced a similar result with 16 of the same 17 metabolites identified. The phase I metabolites M3.1 (ester hydrolysis), M1.2 (alkyl chain hydrolysis) and the phase II metabolite M3.2 (M3.1 glucuronide) were recommended to be the potential poisoning markers.

Pedicularioside A from Buddleia lindleyana inhibits cell death induced by 1-methyl-4-phenylpyridinium ions (MPP+) in primary cultures of rat mesencephalic neurons.

Parkinson's disease is characterized by the progressive degeneration of midbrain dopaminergic neurons. Buddleia lindleyana is a traditional Chinese herb, commonly called Zui Yu Cao. The purification and identification of pedicularioside A and other phenylethanoid glycosides from this plant have been reported. However, their neuroprotective effects on the 1-methyl-4-phenylpyridinium ion (MPP(+))-induced death of rat mesencephalic neuron primary cultures and the precise mechanism of this protection remains unclear. We used the 3-(4, 5-dimethylthiozol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay for cellular growth to examine the effects of five phenylethanoid glycosides isolated from B. lindleyana, including pedicularioside A, leucosceptoside A, isoacteoside, acteoside, and arenariside, on the viability of mesencephalic neurons treated with MPP(+). Of the compounds tested, pedicularioside A exhibited the greatest degree of protection from MPP(+)-induced cell death. We also observed a marked increase in the number of tyrosine hydroxylase immunoreactive neurons. Pedicularioside A inhibited expression of the caspase-3 gene and cleavage of poly (ADP-ribose) polymerase (PARP) in cultures exposed to MPP(+). Our results suggest that pedicularioside A has a neuroprotective effect to improve the survival of mesencephalic neurons (dopaminergic neurons and non-dopaminergic neurons). The mode of action appears to be the inhibition of caspase-3 gene expression, thereby protecting mesencephalic neurons from MPP(+)-induced cell death.