Ceramides and metabolic profiles of patients with acute coronary disease: a cross-sectional study.

Metabolic Syndrome (MS) is a rapidly growing medical problem worldwide and is characterized by a cluster of age-related metabolic risk factors. The presence of MS increases the likelihood of developing atherosclerosis and significantly raises the morbidity/mortality rate of acute coronary syndrome (ACS) patients. Early detection of MS is crucial, and biomarkers, particularly blood-based, play a vital role in this process. This cross-sectional study focused on the investigation of certain plasma ceramides (Cer14:0, Cer16:0, Cer18:0, Cer20:0, Cer22:0, and Cer24:1) as potential blood biomarkers for MS due to their previously documented dysregulated function in MS patients. A total of 695 ACS patients were enrolled, with 286 diagnosed with MS (ACS-MS) and 409 without MS (ACS-nonMS) serving as the control group. Plasma ceramide concentrations were measured by LC-MS/MS assay and analyzed through various statistical methods. The results revealed that Cer18:0, Cer20:0, Cer22:0, and Cer24:1 were significantly correlated with the presence of MS risk factors. Upon further examination, Cer18:0 emerged as a promising biomarker for early MS detection and risk stratification, as its plasma concentration showed a significant sensitivity to minor changes in MS risk status in participants. This cross-sectional observational study was a secondary analysis of a multicenter prospective observational cohort study (Chinese Clinical Trial Registry, https://www.who.int/clinical-trials-registry-platform/network/primary-registries/chinese-clinical-trial-registry-(chictr), ChiCTR-2200056697), conducted from April 2021 to August 2022.

Clinical study on 2 types of intrauterine stents with different thickness and hardness in the treatment of moderate-to-severe intrauterine adhesions. / ä¸¤ç§ä¸åŒåŽšåº¦å’Œç¡¬åº¦çš„å®«è ”æ”¯æž¶æ²»ç–—ä¸­é‡åº¦å®«è ”ç²˜è¿žçš„ä¸´åºŠç ”ç©¶ï¼ˆè‹±æ–‡ï¼‰.

OBJECTIVES: Although hysteroscopic adhesiolysis (HA) is the main treatment for intrauterine adhesion (IUA), postoperative management of IUA remains challenging because there is no consensus on how to mitigate the high rate of postoperative adhesions reformation. This study aims to compare the effectiveness and safety of 2 types of intrauterine stents with different thickness and hardness in treating moderate-to-severe IUA. METHODS: A retrospective clinical study was conducted in the Third Xiangya Hospital of Central South University from November 2020 to July 2021. A total of 191 patients with moderate-to-severe IUA who received surgical treatment and placed intrauterine stents after HA to prevent recurrence of postoperative adhesions were included. According to the hardness and thickness of the intrauterine stents, the participants were divided into a case group (placed the novel thin intrauterine stent, n=62) and a control group (placed the conventional stent, n=129). After 2-3 menstrual cycles, a second-look hysteroscopy was performed, and the intrauterine stents were removed. The postoperative efficacy [the reduction of American Fertility Society (AFS) scores, the adhesions reformation rate, the changes in menstrual pattern, and the pregnancy rate during the follow-up], safety (the adverse events), and applicability (the difficulty of stent removal) were compared between the 2 groups. RESULTS: No significant differences in preoperative clinical characteristics were observed between the 2 groups (all P>0.05). The menstrual volume of all patients was increased after the treatment. The reduction of AFS scores and the menstruation recovery rate were not significantly different between the 2 groups (P=0.519 and P=0.272, respectively). Notably, there was no case of displacement in the case group, while the displacement rate of the control group was 2.3% (P=0.552). Moreover, there was no significant difference in abdominal pain or postoperative abnormal vaginal bleeding between the 2 groups (P=0.823 and P=0.851, respectively). However, the difficulty rate of removing the thinner stents was significantly lower than that of removing the traditional stent (21.0% vs 38.8%, P=0.014). During the follow-up for half a year of the postoperative period, the pregnancy rate did not differ significantly in the case and control groups (45.0% vs 34.6%, P=0.173). CONCLUSIONS: The novel intrauterine stent shows noninferior efficacy and had a good safety profile compared with conventional stents in treating moderate-to-severe IUA. Importantly, it was more convenient to be removed without increasing the rate of displacement and detachment. Therefore, it could reduce the amount of damage to the endometrium and has higher applicability than conventional stents.

Fertility-sparing treatment for cervical mullerian adenosarcoma: A case report and literature review. / å­å®«é¢ˆè ºè‚‰ç˜¤ä¿ç•™ç”Ÿè‚²åŠŸèƒ½æ²»ç–—1ä¾‹å¹¶æ–‡çŒ®å¤ä¹ .

Currently, whole uterus and bilateral tubal resection and oophorectomy is the main treatment of cervical mullerian adenosarcoma. However, young patients generally wish to retain reproductive function. The clinical data of a patient with cervical mullerian adenosarcoma, who underwent fertility preservation surgery were collected. A 13-year-old girl with abnormal vaginal bleeding and a 1.0 cm flocculent echogenicity in the lower part of the uterine cavity to the cervical canal and a cervical mass of about 61 mm×37 mm was found in the pelvic MRI. After initial diagnosis of dysfunctional uterine bleeding in adolescence and cervical blood clot, the patient was treated with artificial cycle treatment, but her symptoms did not improve. Then she was transferred to the Third Xiangya Hospital of Central South University for uninjured virgin membrane hysteroscopy and cervical mass electrotomy, but a few pedicles remained after the operation, and the pathology suggested a cervical mullerian adenosarcoma. Because the patient was young and had not yet given birth, she was treated with primary IAP regimen of chemotherapy and subcutaneously injected with gonadotropin-releasing hormone analogue (GNRH-A) once every 28 days (6 times in total) to protect the ovarian function. After the chemotherapy, she was treated with uninjured virgin membrane hysteroscopy and pedicle electrotomy of cervical mullerian adenosarcoma. After the operation, she received chemotherapy with IAP regimen for 5 times. After discharge, she was treated with megestrol 200 mg per day for 3 years. During 5 years of regular follow-up, no abnormality was seen. Cervical mullerian adenosarcoma in non-sexual women is easily misdiagnosed as ovulation dysfunction abnormal uterine bleeding. The necessity of hysteroscopy should be emphasized, and for patients with low-grade early-stage lesions who wish to retain fertility, local resection could be chosen, but attention is paid to lifelong follow-up to exclude long-term recurrence.

Development of a liquid chromatography-tandem mass spectrometry method for simultaneous determination of five isoflavonoids and seven neurochemicals in rat brain dialysate and its application to a pharmacological study.

Pueraria lobata is a medicinal plant widely used in traditional Chinese medicine. The total pueraria isoflavones have demonstrated positive effect against neurological disorders. In the present study, we first develop an ultra high performance liquid chromatography and tandem mass spectrometry method to quantify the multiple active pueraria isoflavonoids and neurochemical markers in brain dialysate to provide tools for further exploring the functional mechanism of pueraria isoflavones for neuroactivities. A phenomenex Luna C18 column (50 × 2.0 mm, 5 µm) was employed with acetonitrile/0.05% formic acid in water as the mobile phase for the separation of analytes. A mass spectrometer with electrospray ionization source in positive/negative ion switching mode was used for multiple reaction monitoring of the detected compounds. The method was validated and proved acceptable. The intra- and interday precision across quality control levels was within 13.87 for all analytes, whereas the deviation of assay accuracies ranged between 0.03 and 11.53%. The method was successfully applied to a pharmacological study of pueraria isoflavones in rat brain.

LncRNA-TCONS_00034812 in cell proliferation and apoptosis of pulmonary artery smooth muscle cells and its mechanism.

Long noncoding RNAs (lncRNAs) have been discovered to be playing important role in various biological processes. However, the contribution of lncRNAs to pulmonary artery hypertension (PAH) remains largely unknown. Pulmonary vascular remodeling is an important pathological feature of PAH, leading to increased vascular resistance and reduced compliance. Here, we investigated the biological role of lncRNAs in PAH. Differences in the lncRNAs and mRNAs between hypoxia PAH rats and normoxia rats were screened using microarray analysis. The results showed that 36 lncRNAs and 519 mRNAs were upregulated in the pulmonary arteries (PAs) of hypoxia PAH rats, whereas 111 lncRNAs and 246 mRNAs were downregulated. Expressions of the screened lncRNAs, including TCONS_00034812, were validated by real-time PCR. We revealed that the expression of TCONS_00034812 was significantly downregulated in PAs of PAH rats and hypoxia pulmonary artery smooth muscle cells (PASMCs). TCONS_00034812 knockdown promoted proliferation and inhibited apoptosis of PASMCs in vitro. Moreover, TCONS_00034812 regulated PASMCs function in vitro. We found that TCONS_00034812 increased the expression of transcription factors Stox1. TCONS_00034812 and Stox1 knockdown mediated PASMCs function through MAPK signaling. Our findings imply lncRNA as a critical regulator in PAH and demonstrate the potential of gene therapy and drug development for treating PAH. The present study reveals a novel mechano responsive lncRNA-TCONS_00034812, which modulates PASMCs proliferation and apoptosis, and participates in vascular remodeling during PAH.

Brain Pharmacokinetics and the Pharmacological Effects on Striatal Neurotransmitter Levels of Pueraria lobata Isoflavonoids in Rat.

Isoflavonoids are putatively active components of Pueraria lobata and has been demonstrated prominent neuro-protection effect against cerebrovascular disorders, hypertension or Parkinson's disease (PD). However, the molecular basis for the beneficial effect of Pueraria lobata on nervous systems has not been well revealed. The present study aims to assess striatum exposure to main active isoflavonoids and changes of striatal extracellular neurotransmitters levels in rat brain after intravenous administration of Pueraria lobata isoflavonoids extracts (PLF), to further elucidate its' substantial bases for neuro activities. Fifteen rats were divided into 3 groups (five rats in each group) to receive a dose of PLF at 80 or 160 mg/kg or normal saline (vehicle), respectively. An LC-MS/MS method was employed to determine the concentrations of five main isoflavonoids and multiple neurotransmitters in microdialysate from striatal extracellular fluid (ECF) of the rats. The exposed quantities of puerarin (PU), 3'-methoxypuerarin (MPU), daidzein-8-C-apiosyl-(1-6)-glucoside (DAC), and 3'-hydroxypuerarin (HPU) in striatum were dose-dependent. The content of daidzein (DAZ) was too low to be detected in all dialysate samples through the experiment. Optimal dose PLF (80 mg/kg) promoted DA metabolism and inhibited 5-HT metabolism. No obvious change in the level of GLu was determined. The concentration of GABA presented a temporary decline firstly and then a gradual uptrend followed by a further downtrend. Higher dose (160 mg/kg) PLF could enhance the metabolism of both DA and 5-HT, and lower the extracellular level of GLu, without changing GABA concentrations, which might result in alleviation on excitatory toxicity under conditions, such as ischemia. The results infer that different dose of PLF should be chosen to achieve appropriate neurochemical modulation effects under conditions, such as hypertension or ischemia/stroke. These findings may significantly contribute to a better understanding of the neuroprotective effect of Pueraria lobata and provide new insights into its application toward neuro-degenerative diseases in the future.

Long Non-Coding RNA MEG3 Downregulation Triggers Human Pulmonary Artery Smooth Muscle Cell Proliferation and Migration via the p53 Signaling Pathway.

BACKGROUND/AIMS: Increasing evidence has demonstrated a significant role of long non-coding RNAs (lncRNAs) in diverse biological processes, and many of which are likely to have functional roles in vascular remodeling. However, their functions in pulmonary arterial hypertension (PAH) remain largely unknown. Pulmonary vascular remodeling is an important pathological feature of PAH, leading to increased vascular resistance and reduced compliance. Pulmonary artery smooth muscle cells (PASMCs) dysfunction is involved in vascular remodeling. Long noncoding RNAs are potential regulators of PASMCs function. Herein, we determined whether long noncoding RNA-maternally expressed gene 3 (MEG3) was involved in PAH-related vascular remodeling. METHODS: The arterial wall thickness was examined by hematoxylin and eosin (H&E) staining in distal pulmonary arteries (PAs) isolated from lungs of healthy volunteers and PAH patients. The expression level of MEG3 was analyzed by qPCR. The effects of MEG3 on human PASMCs were assessed by cell counting Kit-8 assay, BrdU incorporation assay, flow cytometry, scratch-wound assay, immunofluorescence, and western blotting in human PASMCs. RESULTS: We revealed that the expression of MEG3 was significantly downregulated in lung and PAs of patients with PAH. MEG3 knockdown affected PASMCs proliferation and migration in vitro. Moreover, inhibition of MEG3 regulated the cell cycle progression and made more smooth muscle cells from the G0/G1 phase to the G2/M+S phase and the process could stimulate the expression of PCNA, Cyclin A and Cyclin E. In addition, we found that the p53 pathway was involved in MEG3-induced smooth muscle cell proliferation. CONCLUSIONS: This study identified MEG3 as a critical regulator in PAH and demonstrated the potential of gene therapy and drug development for treating PAH.

Effect of cortex mori on pharmacokinetic profiles of main isoflavonoids from pueraria lobata in rat plasma.

ETHNOPHARMACOLOGICAL RELEVANCE: Radix pueraria (the root of pueraria lobata (Wild.) Ohwi.), which contains a class of isoflavonoids as the main active components, as well as cortex mori (the root bark of Morus alba L), which contains abundant active alkaloids, have been employed for the treatment of diabetes in traditional Chinese medicine for centuries. In previous studies, pharmacodynamic synergistic reactions have been observed in compatible application of pueraria lobata isoflavonoids extracts (PLF) and cortex mori alkaloids extracts (CME) for inhibiting α-glycosidase activity. It has also been demonstrated that PLF can effectively slow down the absorption of active alkaloid from CME, so as to produce a higher effective concentration in small intestine for depressing the elevation of postprandial blood glucose through inhibiting α-glycosidase activity. AIM OF THE STUDY: In this study, the hypoglycemic effect of PLF, CME or CME-PLF mixture (the mixture of CME and PLF at a ratio of 1:6.3) was further evaluated through in vivo glucose tolerance studies. And the effect of CME on pharmacokinetic profiles of main isoflavonoids from PLF in rat plasma was investigated to further underlie compatibility mechanism of the two herbs. MATERIALS AND METHODS: Four groups of rats received an oral dose of starch solution alone or simultaneously with drugs by gavage feeding. The blood samples were collected to determine glucose concentrations by glucose oxidase method. In addition, another two groups of rats were orally administered with PLF or CME-PLF. The plasma samples were collected and assayed using an LC/MS/MS method for comparatively pharmacokinetic studies of five main isoflavonoids. RESULTS: For starch loading, co-administration of CME-PLF resulted in more potent inhibition effects on glucose responses compared to those by CME or PLF in rat. The isoflavonoids from PLF were rapidly absorbed, presenting similarly low concentrations in plasma. When CME was added, the Cmax and AUC of all the five isoflavonoids were increased. A phenomenon of double peaks was found for all analysts. The elimination rates of all the detected isoflavonoids were also slowed down with extension of t1/2. CONCLUSIONS: CME has been found to increase the absorption and delay the elimination of main isoflavonoids from PLF, which might result in higher concentrations of circulating active compounds for anti diabetes.

Ganoderic Acid A Metabolites and Their Metabolic Kinetics.

Ganoderic acid A (GAA), a representative active triterpenoid from Ganoderma lucidum, has been reported to exhibit antinociceptive, antioxidative, cytotoxic, hepatoprotective and anticancer activities. The present study aims (1) to identify GAA metabolites, in vivo by analyzing the bile, plasma and urine after intravenous administration to rats (20 mg/kg), and in vitro by incubating with rat liver microsomes (RLMs) and human liver microsomes (HLMs); (2) to investigate the metabolic kinetics of main GAA metabolites. Using HPLC-DAD-MS/MS techniques, a total of 37 metabolites were tentatively characterized from in vivo samples based on their fragmentation behaviors. The metabolites detected in in vitro samples were similar to those found in vivo. GAA underwent extensive phase I and II metabolism. The main metabolic soft spots of GAA were 3, 7, 11, 15, 23-carbonyl groups (or hydroxyl groups) and 12, 20, 28 (29)-carbon atoms. Ganoderic acid C2 (GAC2) and 7ß,15-dihydroxy-3,11,23-trioxo-lanost-26-oic acid were two main reduction metabolites of GAA, and their kinetics followed classical hyperbolic kinetics. The specific isoenzyme responsible for the biotransformation of the two metabolites in RLMs and HLMs was CYP3A. This is the first report on the comprehensive metabolism of GAA, as well as the metabolic kinetics of its main metabolites.

Plasma and brain pharmacokinetics of ganoderic acid A in rats determined by a developed UFLC-MS/MS method.

Ganoderic acid A (GAA), an active triterpenoid of the traditional Chinese herbal medicine Lingzhi, has been reported to exhibit antinociceptive, antioxidative, and anti-cancer activities. The present study aims to establish a sensitive and rapid UPLC-MS/MS method for studying the plasma and brain pharmacokinetics of GAA in rats. The analytes were separated on a C18 column eluted with a gradient mobile phase consisting of acetonitrile and 0.1% aqueous formic acid at 0.3mL/min. The eluate was monitored by a mass detector using an MRM (m/z, 515.3-285.1) model in negative electrospray ionization. The calibration curve showed good linearity (r2>0.99), with limits of detection and quantification of 0.25 and 2.00 nmol/L, respectively. The intra- and inter-day precision and accuracy were less than 9.99% and ranged from 97.45% to 114.62%, respectively. The extraction recovery from plasma was between 92.89% and 98.87%. GAA was found to be stable in treated samples at room temperature (22°C) for 12h and in plasma at -20°C for 7d. The developed method was successfully applied to a pharmacokinetic study of GAA in rats. GAA could be rapidly absorbed into the circulation (Tmax, 0.15h) and eliminated relatively slowly (t1/2, 2.46h) after orally dosing, and could also be detected in the brain lateral ventricle (Tmax, 0.25h and t1/2, 1.40h) after intravenously dosing. The absolute oral bioavailability and brain permeability of GAA were estimated to be 8.68% and 2.96%, respectively.

Pharmacokinetic profiles of the five isoflavonoids from Pueraria lobata roots in the CSF and plasma of rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine Radix Puerariae, the roots of Pueraria lobata (Wild.) Ohwi., has been widely used for the treatment of cardiovascular and cerebrovascular diseases in China for centuries. Isoflavonoids are believed the active components of this herb. AIM OF THIS STUDY: The present study aims to investigate the brain penetration and pharmacokinetics of five active isoflavonoids in the ventricular CSF and plasma of rats after intravenous administration of a Pueraria isoflavonoids (PIF) extract, to better understand the active components of this herb for neuro-activities. MATERIAL AND METHODS: Under anesthesia condition, SD rats (n=6) were successively suffered two surgeries for implanting cannulas at lateral ventricle and right jugular vein for brain microdialysis and blood collection, respectively. After recovery, the rats received intravenous dose of PIF at 80mg/kg and the concentrations of puerarin (PU), 3'-methoxypuerarin (MPU), 3'-hydroxypuerarin (HPU), daidzein (DA) and daidzein-8-C-apiosyl-(1-6)-glycoside (DAC) in the ventricular dialysate and plasma samples were determined using a ultra-fast liquid chromatography tandem mass spectrometry method. RESULTS: Complete concentration versus time profiles of the five components in plasma and four components except for HPU in ventricular CSF were obtained. After dosing, the average C0 values of PU, MPU, DA, DAC and HPU in plasma were estimated 6.53, 13.72, 1.54, 15.84 and 86.07µg/mL, and PU, MPU, DA and DAC were rapidly penetrated to the brain and reached to their Cmax of 521.52, 415.00, 74.34 and 380.03ng/mL in CSF at about 0.5-0.8h, respectively. The elimination t1/2 of PU, DA and DAC in CSF and plasma were no significant difference, while the t1/2 of MPU in ventricular CSF was longer than that in plasma which may attributable to the different physiological environment of central and peripheral compartments. The brain penetration index (AUCCSF/AUCplasma) was found to be about 9.29, 7.25, 11.96, and 4.21% for PU, MPU, DA, and DAC respectively. CONCLUSION: PU, MPU, DA, DAC can quickly penetrate to the brain through the blood brain barrier (BBB) and might be responsible for the neuro-pharmacological activities of P. lobata.

Simultaneous determination of fraxin and its metabolite, fraxetin, in rat plasma by liquid chromatography-tandem mass spectrometry and its application in a pharmacokinetic study.

For the first time, a rapid and sensitive high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of fraxin and its metabolite, fraxetin, in rat plasma, using esculin as the internal standard (IS). The plasma samples were precipitated with methanol before separation on an Nova-Pak C18 column (150mm×3.9mm, 3µm) using a mobile phase consisting of 0.1% formic acid and methanol (55:45) at a flow rate of 0.8mL/min. The analytes were detected by multiple reaction monitoring in the negative ion mode with the mass transitions at m/z 368.9→ m/z 191.9 (fraxin), m/z 206.9→ m/z 191.8 (fraxetin) and m/z 339.0→ m/z 176.9 (esculin, IS). The results demonstrated that the calibration curves for both analytes have good linearity (r≥0.995) over a concentration range of 5.00-3000ng/mL. The assay was validated according to the regulatory bioanalytical guidelines and proved acceptable. The intra- and inter-day precisions (R.S.D.%) were within 10.9% for both analytes, whereas the deviation of assay accuracies (R.E.%) ranged from -5.3 to 1.0%. The method was successfully applied to a pharmacokinetic study after a single oral dose of fraxin at 50mg/kg to rats.

Structure-Dependent Spin Polarization in Polymorphic CdS:Y Semiconductor Nanocrystals.

Searching for the polymorphic semiconductor nanocrystals would provide precise and insightful structure-spin polarization correlations and meaningful guidance for designing and synthesizing high spin-polarized spintronic materials. Herein, the high spin polarization is achieved in polymorphic CdS:Y semiconductor nanocrystals. The high-pressure polymorph of rock-salt CdS:Y nanocrystals has been recovered at ambient conditions synthesized by the wurtzite CdS:Y nanocrystals as starting material under 5.2 GPa and 300 °C conditions. The rock-salt CdS:Y polymorph displays more robust room-temperature ferromagnetism than wurtzite sample, which can reach the ferromagnetic level of conventional semiconductors doped with magnetic transition-metal ions, mainly due to the significantly enhanced spin configuration and defect states. Therefore, crystal structure directly governs the spin configuration, which determines the degree of spin polarization. This work can provide experimental and theoretical methods for designing the high spin-polarized semiconductor nanocrystals, which is important for applications in semiconductor spintronics.

Synthesis of ZnO nanosheet arrays with exposed (100) facets for gas sensing applications.

ZnO nanosheet (NS) arrays have been synthesized by a facile ultrathin liquid layer electrodeposition method. The ion concentration and electrode potential play important roles in the formation of ZnO NS arrays. Studies on the structural information indicate that the NSs are exposed with (100) facets. The results of Raman and PL spectra indicate that there existed a large amount of oxygen vacancies in the NSs. The gas sensing performances of the ZnO NS arrays are investigated: the ZnO NS arrays exhibited high gas selectivity and quick response/recovery for detecting NO2 at a low working temperature. High binding energies between NO2 molecules and exposed ZnO(100) facets lead to large surface reconstructions, which is responsible for the intrinsic NO2 sensing properties. In addition, the highly exposed surface and a large amount of oxygen vacancies existing in the NSs also make a great contribution to the gas sensing performance.

Metabolism profiles of nuciferine in rats using ultrafast liquid chromatography with tandem mass spectrometry.

Nuciferine (NF) is one of the main aporphine alkaloids existing in the traditional Chinese medicine Folium Nelumbinis (lotus leaves). Modern pharmacological studies have demonstrated that NF has a broad spectrum of bioactivities, such as anti-HIV and anti-hyperlipidemic effects, and has been recommended as a leading compound for new drug development. However, the metabolites and biotransformation pathway of NF in vivo have not yet been comprehensively investigated. The present study was performed to identify the metabolites of NF for exploring in vivo fates. Rat plasma and urine samples were collected after oral administration and prepared by liquid-liquid extraction with ethyl acetate. A method based on ultrafast liquid chromatography with tandem mass spectrometry was applied to identify the metabolites. Q1 (first quadrupole) full scan combined with a multiple reaction monitoring (MRM) survey scan were used for the detection of metabolites. MRM-information-dependent acquisition of enhanced product ions was used for the structural identification of detected metabolites. A total of 10 metabolites were identified, including phase I (demethylation, oxidation and dehydrogenation) and phase II (glucuronidation, sulfation and glutathione) biotransformation products. Demethylation is the main metabolic pathway of NF in the body. These results can help in improving understanding of the disposition and pharmacological mechanism of NF in the body. Copyright © 2016 John Wiley & Sons, Ltd.

Lotus Leaf Alkaloid Extract Displays Sedative-Hypnotic and Anxiolytic Effects through GABAA Receptor.

Lotus leaves have been used traditionally as both food and herbal medicine in Asia. Open-field, sodium pentobarbital-induced sleeping and light/dark box tests were used to evaluate sedative-hypnotic and anxiolytic effects of the total alkaloids (TA) extracted from the herb, and the neurotransmitter levels in the brain were determined by ultrafast liquid chromatography-tandem mass spectrometry. The effects of picrotoxin, flumazenil, and bicuculline on the hypnotic activity of TA, as well as the influence of TA on Cl(-) influx in cerebellar granule cells, were also investigated. TA showed a sedative-hypnotic effect by increasing the brain level of γ-aminobutyric acid (GABA), and the hypnotic effect could be blocked by picrotoxin and bicuculline, but could not be antagonized by flumazenil. Additionally, TA could increase Cl(-) influx in cerebellar granule cells. TA at 20 mg/kg induced anxiolytic-like effects and significantly increased the concentrations of serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), and dopamine (DA). These data demonstrated that TA exerts sedative-hypnotic and anxiolytic effects via binding to the GABAA receptor and activating the monoaminergic system.

An LC-MS/MS method for simultaneous determination of three Polygala saponin hydrolysates in rat plasma and its application to a pharmacokinetic study.

ETHNOPHARMACOLOGICAL RELEVANCE: Radix Polygala has a long history of use as a sedative in traditional Chinese medicine and its major ingredients are saponins, which are recognized effective in memory improvement but highly toxic to gastricintestinal mucosa. Polygala saponin hydrolysates (PSH), an alkaline hydrolysis product and also the intestinal metabolites of the saponins, exhibited stronger effects in improving memory of mice and had less toxicity than its original saponins. The present study aims to develop a sensitive LC-MS/MS method for simultaneously determining PSH three major active components, 3,4,5-trimethoxycinnamylic acid (TMCA), p-methoxycinnamylic acid (PMCA) and tenuifolin (TF), in rat plasma and apply the method to a pharmacokinetic study. MATERIALS AND METHODS: The acidic plasma (100µl) was treated by liquid-liquid extraction with ethyl acetate and reconstituted sample was analyzed on a C18 column eluted with acetonitrile-water (50:50) containing 0.2% formic acid at 0.4ml/min. The mass detection in negative electrospray ionization was used. The ion pairs for multiple reaction monitoring were set at m/z 237.0/103.0, 177.0/116.6 and 679.5/425.3 for TMCA, PMCA and TF, respectively. Their pharmacokinetic profiles were studied in rats after intravenous and oral dose of PSH at 20 and 100mg/kg, respectively. RESULTS: The calibration curves had good linearity (r(2)>0.99) for TMCA, PMCA and TF within the tested concentration ranges. The limits of detection and quantification were 1, 10, 0.5ng/ml and 10.0, 20.0, 1.0ng/ml, respectively. The intra-day and inter-day precisions were less than 18.9% and accuracies between 93.2% and 113.3%, and the extraction recovery ranged from 91.2% to 112.1% for all analytes. The pharmacokinetic study showed that TMCA, PMCA and TF could be rapidly absorbed into the circulation and reached their peak concentrations at about 9.1, 9.0 and 24.0min, respectively. TF had a lower oral bioavailability (2.0%) than TMCA (90.1%) and PMCA (96.5%), but it remained in the body much longer (t1/2, λz, 4.8h, oral dose) than TMCA (0.6h) and PMCA (0.9h). CONCLUSIONS: A sensitive LC-MS/MS method was developed and applied to a pharmacokinetic study of TMCA, PMCA and TF of PSH in rats. The three components are proved to be bio-available active components of PSH and might display their in vivo pharmacological activities at different levels and different time periods after oral administration.

Identification and characterization of potent CYP2D6 inhibitors in lotus leaves.

ETHNOPHARMACOLOGICAL RELEVANCE: The herb of lotus (Nelumbo nucifera) leaves is a commonly used traditional Chinese herbal medicine that is utilized for the treatment of sunstroke, to assuage thirst, and to cure both diarrhea and fever in China. Modern pharmacological studies have demonstrated that the herb exhibits various pharmacological effects, such as anti-hyperlipidemia, anti-obesity, anti-oxidant, anti-HIV, anti-microbial, and anti-hypoglycemic activities. Currently, the herb is becoming more popular in China as a "tea drink" or as a main ingredient of some herbal formulations, which implies that the herb and/or its products are now more likely to be concurrently administered with conventional medicines for losing body weight and reducing blood lipids. However, its potential inhibitory effect on human cytochrome P450 (CYP) has not been systemically investigated to date. The present study was performed to assess the potential inhibitory effects of lotus leaf alcoholic extract (LAE), its major fractions, and its main compounds on five CYP isoenzymes (CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) in vitro. MATERIAL AND METHODS: Five probe substrates were incubated with human liver microsomes in the presence or absence of the LAE, the alkaloid fraction (AF), the flavonoid fraction (FF), or the individual aporphine alkaloids, namely, nuciferine (NF), N-nornuciferine (N-NF), and 2-hydroxy-1-methoxyaporphine (HMA). After the incubation, the relative metabolites of the substrates were analyzed using LC-MS/MS. RESULTS: The results showed that the LAE strongly inhibited CYP2D6 with an IC50 value of 12.05µg/mL and weakly inhibited other isoenzymes. In addition, FF was found to weakly inhibit CYP2D6, whereas AF exerted a markedly higher inhibitory effect on CYP2D6 activity with an IC50 value of 0.96µg/mL. The three aporphine alkaloids isolated from the AF (NF, N-NF, and HMA) significantly inhibited CYP2D6 with IC50 values of 3.78, 3.76, and 3.15µM, respectively. Their Lineweaver-Burk plots and Dixon plots showed that NF, N-NF, and HMA competitively inhibited CYP2D6 activity with Ki values of 1.88, 2.34, and 1.56µM, respectively. CONCLUSION: The study revealed that the alkaloid compounds in lotus leaves exert a potent inhibitory effect on CYP2D6 isoenzyme. The possible drug interactions of the leaves and their preparations with conventional medicines should thus be taken into account.

Pharmacokinetic mechanism of enhancement by Radix Pueraria flavonoids on the hyperglycemic effects of Cortex Mori extract in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Diabetes mellitus, characterized by abnormal blood glucose evaluation, is a serious chronic disease. In the treatment of the disease, α-glycosidase inhibitors play an important role for controlling the postprandial blood glucose level. Cortex Mori, a traditional Chinese herbal medicine, has a long history of use for the treatment of headaches, cough, edema and diabetes. Modern pharmacological studies have shown that the herb has beneficial effects on the suppression of postprandial blood glucose levels by inhibiting α-glycosidase activity in the small intestine. 1-Deoxynojirimycin (DNJ), the main active ingredient of this herb, is recognized as a potent α-glycosidase inhibitor. Our previous studies have shown that the hypoglycemic effect of Cortex Mori extract (CME) was significantly improved when giving CME in combination with Radix Pueraria flavonoids (RPF). In the present study, the pharmacokinetics and intestinal permeability of DNJ were comparatively investigated in rats after being given orally or by intestinal perfusion with CME alone or in CME-RPF pairs, to explore the mechanism of this synergistic effect. MATERIALS AND METHODS: The role of RPF on the plasma and urine concentrations of DNJ from CME orally administered was investigated. Four groups of rats received a single oral dose of either CME or CME-RPF, at DNJ equivalent doses of 20 and 40mg/kg, respectively. After dosing, plasma and urine were collected and assayed by LC/MS/MS. In addition, another two groups of rats were used for small intestinal perfusion with CME or CME-RPF at DNJ concentration of 10µM. RESULTS: Compared to the data when dosing with CME alone, the Cmax of DNJ were decreased from 5.78 to 2.94µg/ml (p<0.05) and 10.66 to 5.35µg/ml (p<0.01); Tmax were delayed from 0.40 to 0.55h and 0.35 to 0.50h (p<0.05); and MRT were significantly prolonged from 1.14 to 1.72h (p<0.05) and 0.95 to 1.62h (p<0.01), after dosing with CME-RPF at DNJ doses of 20 and 40mg/kg, respectively. In addition, the urinary recovery of DNJ over the first 4h after dosing significantly decreased from 48.76% to 33.86%. Effective permeability (Peff) of DNJ was decreased from 7.53×10(-3) to 3.09×10(-3)cm/s (p<0.05) when RPF was added to CME, when it was evaluated using the rat intestinal perfusion model. CONCLUSIONS: All the above results demonstrate that RPF was able to suspend and delay the absorption of DNJ, but did not affect the total amount of DNJ in the body. The resulting higher concentration of DNJ in the small intestine produced a relatively stronger effect of depressing the elevation of the postprandial blood glucose level. These findings support the important role of RPF in the application of CME on blood glucose control.

Different pharmacokinetics of the two structurally similar dammarane sapogenins, protopanaxatriol and protopanaxadiol, in rats.

Dammarane Sapogenins (DS), with main ingredients of protopanaxatriol (PPT, 33%) and protopanaxadiol (PPD, 16%), is an alkaline hydrolyzed product of ginsenosides and had significant activities in improving learning and memory and decreasing chemotherapy-induced myelosuppression. In the present study, the pharmacokinetics and oral bioavailabilities of PPT and PPD were investigated when a single dose of DS was administrated orally (75 mg/kg) and intravenously (i.v., 30 mg/kg) to rats. Their in vitro stabilities in the GI tract were also investigated. PPT and PPD concentrations were measured by LC-MS. The results showed that PPT was eliminated rapidly from the body with an average t1/2, λz value of 0.80 h and CL of 4.27 l/h/kg after i.v. administration, while PPD was eliminated relatively slowly with a t1/2, λz of 6.25 h and CL of 0.98l/h/kg. After oral administration, both PPD and PPT could be absorbed into the body, but their systemic exposures were quite different. PPT was absorbed into the body quickly, with a Tmax of 0.58 h and a Cmax of 0.13 µg/ml, while PPD was absorbed relatively slowly with a Tmax of 1.82 h and a Cmax of 1.04 µg/ml. The absolute bioavailabilities of PPT and PPD were estimated as 3.69% and 48.12%, respectively. The stability test found that PPT was instable in the stomach with 40% degradation after 4h incubation at 37°C, both in pH1.2 buffer and in the stomach content solution. The instability in the stomach might be one of the reasons for PPT's poor bioavailability.