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The combination of warfarin and compound Danshen dripping pill (CDDP) is helpful for patients with both coronary heart disease (CHD) and atrial fibrillation (AF). The main adverse drug reaction of warfarin is bleeding because of its narrow therapeutic index. The safety of a combination therapy with warfarin and CDDP is always a concern. Our previous research showed that the combination of warfarin and CDDP improved the quality of life for patients with both CHD and AF. This study describes the changes in dose and concentration of warfarin necessary and evaluates bleeding risk when warfarin is given concomitantly with CDDP. An ultra-performance liquid chromatography–MS/MS method with a chiral column was developed to assay the concentration of S-warfarin and R-warfarin in human plasma simultaneously. The method was applied to compare the concentration of warfarin in patients taking warfarin combined with CDDP and without CDDP. International normalized ratio (INR) values were monitored to evaluate bleeding risk. Paired t tests were then used to compare the dose and the concentration in 2 periods. Moreover, patients with VKORC1 , CYP2C9*3 , CYP4F2 , EPHX1 , and PROC gene polymorphisms were evaluated to determine interactions. The results indicate that the dose of warfarin had no significant change with or without CDDP. Also, the peak concentrations of S-warfarin and total warfarin were significantly different in CYP4F2 C/C patients, but there was no significant difference identified in other genetic groups. No bleeding occurred in the study. The dose of warfarin would be sustainable when combined with CDDP, because CDDP did not affect concentration of warfarin significantly in most patients and the change of INR was not significant. ChiCTR-ONRC-13003523. [ABSTRACT FROM AUTHOR]

In general, herbal medicines have been considered as safe by the general public, since they are naturally occurring and have been applied in treatment for over thousands of years. As the use of herbal medicine is rapidly increasing globally, the potential toxicity of herbal drugs, in particular drug-induced liver injury (DILI), has now become a serious medical issue. According to the literature, the authors analyzed and discussed the hepatotoxicity problem of Chinese herbal medicines (CHM), including global overview on herbal-induced liver injury (HILI), current research progress on toxic CHM, diagnosis and treatment of HILI, and modern approaches and technologies of study of hepatotoxicity. As to promote the recognition of HILI and tackle the issue, a guideline for the diagnosis and treatment of HILI has recently been drafted by Chinese scientists. As suggested by the guideline, the hepatotoxicity issue of CHM, as a matter of fact, is overestimated. Up to date, the investigation of hepatotoxicity of CHM is now booming with worldwide application of CHM. This review therefore provides useful information for investigating hepatotoxicity of herbal medicine and characterizing DILI caused by CHM. In addition, authors describe in which way further efforts should be made to study the rationale of CHM and liver injury. [ABSTRACT FROM AUTHOR]

The fruits of Forsythia suspensa (F. suspensa) have been used as a traditional Chinese medicine for 2000 years. Currently, the quality control of F. suspensa strictly follows the instructions of Chinese Pharmacopeia, which mainly controls the content of forsythoside A, phillyrin, and volatile oil. In this study, air pressure MALDI mass spectrometry imaging (AP‐MALDI MSI) was used to evaluate the quality of F. suspensa fruits and the distribution of dozens of active ingredients. The variation of active ingredients was measured for more than 30 batches of samples, regarding harvest time, cultivated environment, shelf‐life, and habitat. Fifty‐three active ingredients could be detected in F. suspensa fruits with AP‐MALDI MSI. Seven active ingredients were upregulated, four ingredients downregulated, and 15 ingredients did not change in ripe fruits. A sharp variation of active ingredients in late September was observed for the Caochuan fruits harvested in 2019, which is closely related to the appearance of the ginger color of the pericarp under the microscope observation. The microscope observation is a reliable way to classify ripe and green fruits instead of outlook. Just considering forsythoside A and phillyrin, it is found that wild fruits are better than cultivated fruits, but cultivated fruits have high contents of other ingredients. The shelf‐life of F. suspensa fruits is proposed to be 3 years, considering the 26 ingredients investigated. It was found that Luoning wild fruits are better than those from Caochuan with a new evaluation method. Mass spectrometry imaging is an easy, objective, and effective method to evaluate the quality of F. suspensa fruits. [ABSTRACT FROM AUTHOR]

Introduction: Abnormal lipid metabolism, one of the hallmarks in cancer, has gradually emerged as a novel target for cancer treatment. As organelles that store and release excess lipids, lipid droplets (LDs) resemble "gears" and facilitate cancer development in the body. Aim: This review discusses the life cycle of LDs, the relationship between abnormal LDs and cancer hallmarks, and the application of LDs in theragnostic and clinical contexts to provide a contemporary understanding of the role of LDs in cancer. Methods: A systematic literature search was conducted in PubMed and SPORTDiscus. Retrieve and summarize clinical trials of drugs that target proteins associated with LD formation using the Clinical Trials website. Create a schematic diagram of lipid droplets in the tumor microenvironment using Adobe Illustrator. Conclusion: As one of the top ten hallmarks of cancer, abnormal lipid metabolism caused by excessive generation of LDs interrelates with other hallmarks. The crosstalk between excessive LDs and intracellular free fatty acids (FFAs) promotes an inflammatory environment that supports tumor growth. Moreover, LDs contribute to cancer metastasis and cell death resistance in vivo. Statins, as HMGCR inhibitors, are promising to be the pioneering commercially available anti‐cancer drugs that target LD formation. [ABSTRACT FROM AUTHOR]

Plants make different defense mechanisms in response to different environmental stresses. One common way is to produce secondary metabolites. Temperature is the main environmental factor that regulates plant secondary metabolites, especially flavonoids and terpenoids. Stress caused by temperature decreasing to 4–10 °C is conducive to the accumulation of flavonoids and terpenoids. However, the accumulation mechanism under cold stress still lacks a systematic explanation. In this review, we summarize three aspects of cold stress promoting the accumulation of flavonoids and terpenoids in plants, that is, by affecting (1) the content of endogenous plant hormones, especially jasmonic acid and abscisic acid; (2) the expression level and activity of important transcription factors, such as bHLH and MYB families. This aspect also includes post-translational modification of transcription factors caused by cold stress; (3) key enzyme genes expression and activity in the biosynthesis pathway, in addition, the rate-limiting enzyme and glycosyltransferases genes are responsive to cold stress. The systematic understanding of cold stress regulates flavonoids, and terpenoids will contribute to the future research of genetic engineering breeding, metabolism regulation, glycosyltransferases mining, and plant synthetic biology. [ABSTRACT FROM AUTHOR]

Nephrotoxicity is the dose-limiting factor of cisplatin treatment. Magnesium isoglycyrrhizinate (MgIG) has been reported to ameliorate renal ischemia–reperfusion injury. This study aimed to investigate the protective effect and possible mechanisms of MgIG against cisplatin-induced nephrotoxicity from the perspective of cellular pharmacokinetics. We found that cisplatin predominantly accumulated in mitochondria of renal tubular epithelial cells, and the amount of binding with mitochondrial DNA (mtDNA) was more than twice that with nuclear DNA (nDNA). MgIG significantly lowered the accumulation of cisplatin in mitochondria and, in particular, the degree of target-binding to mtDNA. MgIG notably ameliorated cisplatin-induced changes in mitochondrial membrane potential, morphology, function, and cell viability, while the magnesium donor drugs failed to work. In a mouse model, MgIG significantly alleviated cisplatin-caused renal dysfunction, pathological changes of renal tubules, mitochondrial ultrastructure variations, and disturbed energy metabolism. Both in vitro and in vivo data showed that MgIG recovered the reduction of NAD+-related substances and NAD+-dependent deacetylase sirtuin-3 (SIRT3) level caused by cisplatin. Furthermore, SIRT3 knockdown weakened the protective effect of MgIG on mitochondria, while SIRT3 agonist protected HK-2 cells from cisplatin and specifically reduced platinum-binding activity with mtDNA. In conclusion, MgIG reduces the target-binding amount of platinum to mtDNA and exerts a protective effect on cisplatin-induced renal injury through SIRT3, which may provide a new strategy for the treatment of cisplatin-induced nephrotoxicity. [ABSTRACT FROM AUTHOR]

Acyl chlorides react rapidly with hydroxyl and amino groups in the absence of catalysts and therefore hold great promise for chiral mass spectrometry. Herein, a tandem mass spectrometry method based on derivatization with (−)‐camphanic acid chloride as a simple chiral probe was developed for the highly sensitive detection and quantitation of levodropropizine and its enantiomer, namely, dextrodropropizine. The diastereomeric derivatization products were quantified based on the relative abundances of their fragment ions produced upon collision‐induced dissociation in positive‐ion mode. The reactive site was elucidated using nuclear magnetic resonance spectroscopy and two‐dimensional total correlation spectroscopy, and the reaction mechanism was proved by stoichiometry studies. The degree of isomer recognition was investigated at different collision energies and determined as Rchiral ≈ 1.5. Thus, this study gives the way to the mass spectrometric identification and quantitation of levodropropizine and its enantiomer, and the developed method represents a new and practical technique for rapid and sensitive determination and quality control of enantiomers of chiral drugs. [ABSTRACT FROM AUTHOR]

Abstract: A series of derivatives of Matijing-Su (MTS, N-(N-benzoyl-l-phenylalanyl)-O-acetyl-l-phenylalanol) was synthesized and evaluated for their anti-hepatitis B virus (HBV) activities in 2.2.15 cells. The IC50 of compounds 9c (1.40μM), 9g (2.33μM) and 9n (2.36μM), etc. and the selective index of 9n (45.93) of the inhibition on the replication of HBV DNA were higher than those of the positive control lamivudine [41.59, (IC50: 82.42μM)]. Compounds 11d, 12a and 12e also exhibited significant anti-HBV activities. [Copyright &y& Elsevier]

Abstract: A simple, sensitive and selective liquid chromatography–electrospray mass spectrometric (LC–ESI-MS) method was developed and validated for simultaneous determination of strychnine and brucine in rat plasma, using tacrine as the internal standard (IS). Sample preparation involved a liquid–liquid extraction of the analytes with n-hexane, dichloromethane and isopropanol (65:30:5, v/v/v) from 0.1mL of plasma. Chromatographic separation was carried out on a Waters C18 column using a mobile phase of methanol–20mM ammonium formate–formic acid (32:68:0.68, v/v/v). Positive selected ion monitoring mode was used for detection of strychnine, brucine and the IS at m/z 335.2, m/z 395.2 and m/z 199.2, respectively. Linearity was obtained over the concentration range of 0.5–500ng/mL for strychnine and 0.1–100ng/mL for brucine. The lower limit of quantification was 0.5ng/mL and 0.1ng/mL for strychnine and brucine, respectively. The intra- and inter-day precision for both strychnine and brucine was less than 7.74%, and accuracy ranged from −4.38% to 2.21% at all QC levels. The method has been successfully applied to a pharmacokinetic study of processed Semen Strychni after oral administration to rats. [Copyright &y& Elsevier]

Abstract: A rapid, sensitive and accurate liquid chromatographic–tandem mass spectrometric method is described for the determination of metolazone in human blood. Metolazone was extracted from blood using ethyl acetate and separated on a C18 column interfaced with a triple quadrupole tandem mass spectrometer. The mobile phase consisting of a mixture of acetonitrile, 10mmol/l ammonium acetate and formic acid (60:40:0.1, v/v/v) was delivered at a flow rate of 0.5ml/min. Electrospray ionization (ESI) source was operated in positive ion mode. Selected reaction monitoring (SRM) mode using the transitions of m/z 366→ m/z 259 and m/z 321→ m/z 275 were used to quantify metolazone and the lorazepam (internal standard), respectively. The linearity was obtained over the concentration range of 0.5–500ng/ml for metolazone and the lower limit of quantitation (LLOQ) was 0.5ng/ml. For each level of QC samples, inter- and intra-run precision was less than 8.07 and 3.56% (relative standard deviation (RSD)), respectively, and the bias was within ±4.0%. This method was successfully applied to the pharmacokinetic study of metolazone formulation after oral administration to humans. [Copyright &y& Elsevier]

Acetyl-11-keto-β-boswellic acid (AKBA), a triterpenoid from Boswellia serrate, is regarded as an angiogenesis inhibitor. However, its toxicity is unknown. The aim of this study was to examine its developmental toxicity and cardiotoxicity. A developmental toxicity assay in zebrafish embryos/larvae from 4 to 96 hours post-fertilization (hpf) was performed and a cardiotoxicity assay was designed from 48 to 72 hpf. Markers of oxidative stress and related genes were selected to access the possible mechanisms. According to the results, AKBA induced pericardium edema, yolk-sac edema, abnormal melanin, spinal curvature, hatching inhibition and shortened body length. Further, increased SV-BA distance, reduced heart rate, increased pericardium area and decreased blood flow velocity were detected in AKBA treated groups. The inhibition of cardiac progenitor gene expression, such as Nkx2.5 and Gata4, may be related to cardiotoxicity. The activities of antioxidant enzymes were decreased and the content of MDA was increased. In addition, AKBA treatment decreased the expression levels of Mn-Sod, Cat, and Gpx. These results suggested that AKBA induced developmental toxicity and cardiotoxicity through oxidative stress. As far as we know, this is the first report on the toxicity of AKBA. It reminds us to pay attention to developmental toxicity and cardiotoxicity of AKBA. [ABSTRACT FROM AUTHOR]

Relinqing granules (RLQ) are being used alone or in combination with antibacterial drugs to treat urological disorders. This study investigates the pharmacokinetics of RLQ in humans and the potential for RLQ-perpetrated interactions on transporters. Twelve healthy subjects (six women and six men) participated to compare single- and multiple-dose pharmacokinetics of RLQ. In the single-dose study, all 12 subjects received 8 g of RLQ orally. After a 7-d washout period, the subjects received 8 g of RLQ for seven consecutive days (t.i.d.) and then a single dose. Gallic acid (GA) and protocatechuic acid (PCA) in plasma and urine samples were analysed using LC-MS/MS. The transfected cells were used to study the inhibitory effect of GA (50–5000 μg/L) and PCA (10–1000 μg/L) on transporters OAT1, OAT3, OCT2, OATP1B1, P-gp and BCRP. GA and PCA were absorbed into the blood within 1 h after administration and rapidly eliminated with a half-life of less than 2 h. The mean peak concentrations of GA (102 and 176 μg/L) and PCA (4.54 and 7.58 μg/L) were lower in males than females, respectively. The 24 h urine recovery rates of GA and PCA were about 10% and 5%, respectively. The steady-state was reached in 7 d without accumulation. GA was a potent inhibitor of OAT1 (IC50 = 3.73 μM) and OAT3 (IC50 = 29.41 μM), but not OCT2, OATP1B1, P-gp or BCRP. GA and PCA are recommended as PK-markers in RLQ-related pharmacokinetic and drug interaction studies. We should pay more attention to the potential for RLQ-perpetrated interactions on transporters. [ABSTRACT FROM AUTHOR]

Rotundic acid (RA), a plant-derived pentacyclic triterpene acid, has been reported to possess extensive pharmacological activities. The poor bioavailability limits its further development and potential clinic application. To clarify the potential mechanism for poor oral bioavailability. The single-dose pharmacokinetics of orally administered RA (10 mg/kg) in Sprague–Dawley rats without or with verapamil (25 or 50 mg/kg) were investigated. Additionally, MDCKII-MDR1 and Caco-2 cell monolayers, five recombinant human cytochrome P450 (rhCYP) enzymes (1A2, 2C8, 2C9, 2D6 and 3A4), and rat liver microsomes were also conducted to investigate its potential mechanism. Verapamil could significantly affect the plasma concentration of RA. Co-administered verapamil at 25 and 50 mg/kg, the AUC0–∞ increased from 432 ± 64.2 to 539 ± 53.6 and 836 ± 116 ng × h/mL, respectively, and the oral clearance decreased from 23.6 ± 3.50 to 18.7 ± 1.85 and 12.2 ± 1.85 L/h/kg, respectively. The MDCKII-MDR1 cell assay showed that RA might be a P-gp substrate. The rhCYPs experiments indicated that RA was mainly metabolized by CYP3A4. Additionally, verapamil could increase the absorption of RA by inhibiting the activity of P-gp, and slow down the intrinsic clearance of RA from 48.5 ± 3.18 to 12.0 ± 1.06 µL/min/mg protein. These findings indicated that verapamil could significantly affect the pharmacokinetic profiles of RA in rats. It was demonstrated that P-gp and CYP3A were involved in the transport and metabolism of RA, which might contribute to the low oral bioavailability of RA. [ABSTRACT FROM AUTHOR]

The threat of population exposure to ionizing radiation is increasing rapidly worldwide. Such exposure, especially at high-dose, is known to cause acute radiation syndrome (ARS). Hence, it is necessary to develop specific and sensitive biomarkers to accurately diagnose radiation injury and evaluate medical countermeasures. Caenorhabditis elegans (C. elegans), a model organism with a fine and sound olfactory system, was used to examine the odor of urine samples collected from irradiation-injured rats, and compared with those from un-irradiated control rats to investigate the 'special odor' of radiation injury. Subsequently, headspace SPME-GC-MS was applied for non-targeted metabolomic analysis of volatile organic compounds (VOCs) in urine, with the aim to discover changes of small molecule metabolites and identify odor biomarkers of irradiation injury. C. elegans showed significant attraction to the urine of total body irradiation (TBI) rats compared with control rats, indicating that irradiation injury can emit 'special odor' and the metabolites in urine VOCs were changed. Using metabolomics based on headspace SPME-GC-MS for metabolic profiles analysis, we screened 63 differentially expressed metabolites. Among them, 10 metabolites including p-Cresol with excellent diagnostic ability were identified as odor biomarkers according to receiver operating characteristic (ROC) curve analysis. This study confirmed the 'special odor' induced by irradiation injury, and identified biomarkers through urine VOCs analysis for the first time, which can provide a novel approach and insight to evaluate irradiation injury noninvasively, accurately and conveniently. [ABSTRACT FROM AUTHOR]

Background: Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by memory deficits and cognitive decline. Current drugs can only relieve symptoms, but cannot really cure AD. Cerebralcare Granule® (CG) is a Traditional Chinese medicine (TCM) containing a variety of biologically active compounds. In our previous studies, CG has shown a beneficial effect against memory impairment in mice caused by d-galactose. However, whether CG can be used as a complementary medicine for the treatment of AD remains unexplored. Here, we use a combination of CG and memantine hydrochloride (Mm) to treat Alzheimer-like pathology and investigate the effects and mechanisms in vivo. Methods: The histology of brain was examined with Hematoxylin–eosin (HE) staining, Golgi staining and Thioflavin S staining. ELISA was applied to assess the expression levels or activities of CAT, SOD, GSH-Px, MDA, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin (TBIL) in serum, as well as the levels of IL-6, IL-1β, and TNF-α in the mice brain. Western blotting was used to assess the expression of β-secretase (BACE1), amyloid precursor protein (APP), APPβ, APPα, synaptophysin (SYN), growth-associated protein 43 (GAP43), and postsynaptic density 95 (PSD95). Results: In the present study, the combination group (CG + Mm) significantly attenuated Alzheimer-like behavior without adverse effects in APP/PS1 mice, indicating its high degree of safety and efficacy after long-term treatment. CG + Mm reduced AD pathological biomarker Aβ plaque accumulation by inhibiting BACE1 and APP expression (P < 0.05 or P < 0.001). Besides, the combination group markedly inhibited the levels of IL-1β, IL-6, and TNF-α in hippocampus (P < 0.001), as well as activities of SOD, CAT, and GSH-Px in serum (P < 0.001). By contrast, the combination group improved synaptic plasticity by enhancing SYN, PSD95, and GAP43 expression. Conclusions: Taken together, these data supported the notion that CG combined with Mm might ameliorate the cognitive impairment through multiple pathways, suggesting that CG could play a role as complementary medicine to increase anti-AD effect of chemical drugs by reducing Aβ deposition, neuroinflammation, oxidative damage, and improving synaptic plasticity. [ABSTRACT FROM AUTHOR]

As a novel long-acting recombinant human insulin analogue, it is necessary to carry out the preclinical research for insulin LysArg. The purpose of this study was to characterise the pharmacokinetic, tissue distribution and excretion of insulin LysArg and provide a reference for its development. Three methods were used to measure the content of insulin LysArg in biological samples after a single subcutaneous administration in rats, including radioassay, radioassay after precipitation with TCA and separation by HPLC. After Subcutaneous administration of recombinant insulin LysArg 1, 2, 4 U/kg in rats, it showed both Cmax and AUC0–t were positively correlated with the dose. In the meanwhile, after a single subcutaneous administration of recombinant insulin LysArg at 2 U/kg in rats, the amount of radioactivity in most organs was highest at 1.5 h and then decreased gradually, no accumulation was found. The highest level of insulin LysArg was observed in the kidney. Like other macromolecules, insulin LysArg was mainly excreted from urine. The study fully illustrated the pharmacokinetic pattern of insulin LysArg, provided valuable informations to support its further development about safety and toxicology. [ABSTRACT FROM AUTHOR]

Inhalation of silicon dioxide (SD) results in pulmonary inflammatory responses and fibrosis. Isorhynchophylline (Isorhy) is the main alkaloid in the traditional Chinese herb Tripterygium wilfordii, which is reported to have anti-inflammatory activities in the nervous system. However, the effects of Isorhy on SD-induced pulmonary inflammation and fibrosis in mice are unknown. Male mice were exposed to a single dose of SD (2.5 mg/kg, intranasal inoculation) to induce pulmonary fibrosis (PF). The mice were woken up and immediately treated with Isorhy (20 mg/kg, intraperitoneal injection) for 14 or 42 days. The effects of Isorhy on inflammatory responses and lung fibrosis induced by SD were then investigated. After the 14-day treatment, there was a significant reduction in inflammatory cell infiltration in the lungs of mice, with reduced recruitment of inflammatory cells to the lungs. The concentration of pro-inflammatory factors in the bronchoalveolar lavage fluid was reduced, which alleviated inflammatory injury in the lung tissue. After the 42-day treatment, Isorhy alleviated inflammation and inhibited the release of fibrogenic factors in mice with PF. Isorhy also significantly reduced collagen deposition in the lung tissues of mice. Isorhy has the ability to reduce inflammatory responses and fibrosis associated with SD-induced acute lung injury. [ABSTRACT FROM AUTHOR]

The objective of this study was to clarify the species differences of pharmacokinetics of Y101 (N-[N-benzoyl-O-(2-dimethylaminoethyl)-l-tyrosyl]-l-phenylalaninol hydrochloride), a derivative of herbal ingredient with anti-HBV hepatitis activity, in rats, dogs, monkeys and humans. The metabolic stability and metabolite identification studies using liver microsomes in vitro, plasma protein binding using a rapid equilibrium dialysis in vitro, pharmacokinetic studies in vivo were carried out to evaluate the interspecies differences. The toxicokinetic study in monkeys was also investigated. The metabolic profiles were similar in monkeys and humans, which were significant different from rats and dogs in vitro. In vitro plasma protein binding showed no major differences between species with medium to high protein binding rates. After single oral dose to rats, dogs, and monkeys, the absolute oral bioavailability of Y101 was 44.9%, 43.1%, and 19.2%, respectively. There was no accumulation for Y101 toxicokinetics in monkeys after oral administration for 90 d. The metabolic profiles indicated monkey was the very animal model for preclinical safety evaluation of Y101. Our results have demonstrated the favorable pharmacokinetics profile of Y101, which supports the clinical trials in humans. [ABSTRACT FROM AUTHOR]

Aconite alkaloids are the main bioactive ingredients existing in Aconitum, for instance aconitine (AC), which exhibit potent analgesic, antirheumatic and other pharmacological effects. In this study, effects of long-term treatment with liquorice on pharmacokinetics of AC in rats were investigated. Pharmacokinetics of AC after oral administration of AC at 1.5 mg/kg either with pre-treatment of liquorice water extracts at 0.433 or 1.299 g/kg (crude drug), respectively, for one week or not were studied. Additionally, LS-180 cells and human primary hepatocytes were utilized to explore the potential effects of bioactive ingredients of liquorice on P-glycoprotein (P-gp) and Cytochromes P450 (CYPs), respectively. The results revealed that exposure of AC after pre-treatment with liquorice was altered remarkably. Area under the concentration-time curve (AUC) decreased from 161 ± 37.8 to 58.8 ± 8.97 and 44.7 ± 8.20 ng/mL*h, respectively. Similarly, Cmax decreased from 26.2 ± 5.19 to 11.8 ± 1.15 and 6.86 ± 0.600 ng/mL, respectively. In addition, expressions of CYPs of human primary hepatocytes were enhanced to various contents after induction. Moreover, accumulation of AC and hypaconitine (HA), not mesaconitine (MA) inside of LS-180 cells were reduced after pre-treatment by comparison with control. In conclusion, the exposure of AC in vivo declined after pre-treatment with liquorice extract, which may be highly associated with upregulated expression and/or function of CYPs and P-gp. [ABSTRACT FROM AUTHOR]

Context: Terrestrosin D (TED), from Tribulus terrestris L. (Zygophyllaceae), exhibits anti-tumour and anti-inflammatory activities. However, its effects on bleomycin (BLM)-induced pulmonary inflammation and the subsequent fibrotic changes remain unclear. Objective: To examine the anti-inflammatory and anti-fibrotic effects of TED against BLM in murine pulmonary tissues. Materials and methods: Male SPF mice received saline (control), TED (10 mg/kg), BLM (2.5 mg/kg), or BLM (2.5 mg/kg) + TED (10 mg/kg) group. BLM was administered as a single intranasal inoculation, and TED was intraperitoneally administered once daily. After 2 and 6 weeks of treatment, cell number and differentiation (Giemsa staining) and TNF-α, IL-6, IL-8, TGF-β1, and PDGF-AB levels (ELISA) were determined in the bronchoalveolar lavage fluid (BALF). Hydroxyproline (Hyp) content in the left pulmonary tissue was also determined (ELISA). The right pulmonary tissue was H&E-stained and assessed for the severity of pulmonary fibrosis using the Ashcroft scoring method. Compared with the BLM group, TED decreased inflammatory cell infiltration; number of macrophages (p < 0.05), neutrophils (p < 0.05), lymphocytes (p < 0.05); percentage of macrophages in the monocyte-macrophage system (p < 0.05), and levels of TNF-α (p < 0.01), IL-6 (p < 0.01), IL-8 (p < 0.05), TGF-β1 (p < 0.05), and PDGF-AB (p < 0.05) in the BALF. TED also reduced Hyp content (p < 0.05) in the pulmonary tissue and attenuated the BLM-induced deterioration in lung histopathology. Discussion and conclusions: TED can inhibit BLM-induced inflammation and fibrosis in the lungs of mice, which may be related to reduced inflammatory and fibrotic markers. These results could be further tested in humans through clinical studies. [ABSTRACT FROM AUTHOR]

Rationale: Shufeng Jiedu capsule (SFJDC), a prescription of traditional Chinese medicine, is mainly used for the treatment of acute upper respiratory tract infections. However, the bioactive components remain unclear, which partly limits its quality control and further development. This work aimed to carry out a study of plasma pharmacochemistry to identify the potential bioactive components of SFJDC. Methods: An effective approach based on a combination of ultra‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry (UPLC/QTOF‐MS) and multivariate statistical analysis was applied to comprehensively analyze the absorbed components and their metabolites in rat plasma after oral administration of SFJDC. After UPLC/QTOF‐MS detection, the differences between control and dosed plasma samples were distinguished by multivariate statistical analysis, and chromatographic signals of xenobiotic compounds were further extracted to identify structures. Results: A total of 46 SFJDC‐related xenobiotic compounds were identified as potential bioactive components in rat plasma. Among these, 27 absorbed prototype constituents were mainly flavonoids, anthraquinones, stilbenes, iridoids, lignans, naphthalenes, phenylethanoid glycosides and triterpenoid saponins. Especially for hastatoside, verbenalin, forsythoside A, phillyrin and emodin, they were closely related to the anti‐inflammatory effect of SFJDC. Conclusions: The absorbed components and metabolites of SFJDC in rat plasma were analyzed for the first time. This study will be conducive for ascertaining the quality markers of SFJDC for quality control and pharmacological mechanism research at the molecular level. [ABSTRACT FROM AUTHOR]

• Establishment and validation of an LC-MS/MS method to determine α -hydroxymetoprolol in CYP2D6 enzyme system. • Adoption of a new internal standard (dabigatran) instead of deuterium labeled α - hydroxymetoprolol. • Evaluation of four CYP2D6 variants activity (K m , V max , Cl int) towards metoprolol in vitro. Specific study about the effect of cytochrome P450 2D6 (CYP2D6) polymorphisms on the metabolism of clinic drugs is of great significance for drug safety investigation. Here, the interaction between CYP2D6 variants (*1, *2, *10, *39) and metoprolol (MET) was intensively researched in vitro from the aspect of drug-enzyme kinetic study. To obtain quantitative data, α -hydroxymetoprolol (main metabolite of MET) was selected as an ideal analyte and an LC–MS/MS method was adopted for sample determination. Firstly, by selecting suitable internal standard and optimizing separation condition, the LC–MS/MS method was established and validated. Then, the drug-enzyme incubation system was optimized by two parameters: incubation time and amount of enzyme. Lastly, the interaction between CYP2D6 allelic variants and MET was characterized by K m , V max and CL int. As a result, four CYP2D6 enzymes displayed diverse K m or V max towards MET and the values of CL int showed a wide range from 8.91 to 100%. Relative to CYP2D6*1 (CL int *1 = 100%), CYP2D6*2 demonstrated the second high catalytic activity (CL int *2/*1 = 74.87%) while CYP2D6*39 (CL int *39/*1 = 29.65%) and CYP2D6*10 (CL int *10/*1 = 8.91%) showed minimal catalytic activity. This comprehensive in vitro data suggested the prominent influence of CYP2D6 polymorphisms on the metabolism of MET, which could offer valuable information for personalized administration of MET in clinic. [ABSTRACT FROM AUTHOR]

Host-gut microbiota metabolic interactions are closely associated with health and disease. A manifestation of such co-metabolism is the vast structural diversity of bile acids (BAs) involving both oxidative stereochemistry and conjugation. Herein, we describe the development and validation of a LC-MS-based method for the analysis of human C24 BA metabolome in serum and urine. The method has high throughput covering the discrimination of oxidative stereochemistry of unconjugated species in a 15-min analytical cycle. The validated quantitative performance provided an indirect way to ascertain the conjugation patterns of BAs via enzyme-digestion protocols that incorporated the enzymes, sulfatase, β-glucuronidase, and choloylglycine hydrolase. Application of the method has led to the detection of at least 70 unconjugated BAs including 27 known species and 43 newly found species in the post-prandial serum and urine samples from 7 nonalcoholic steatohepatitis patients and 13 healthy volunteers. Newly identified unconjugated BAs included 3α, 12β-dihydroxy-5β-cholan-24-oic acid, 12α-hydroxy-3-oxo-5β-cholan-24-oic acid, and 3α, 7α, 12β-trihydroxy-5β-cholan-24-oic acid. High-definition negative fragment spectra of the other major unknown species were acquired to facilitate future identification endeavors. An extensive conjugation pattern is the major reason for the “invisibility” of the newly found BAs to other common analytical methods. Metabolomic analysis of the total unconjugated BA profile in combination with analysis of their conjugation patterns and urinary excretion tendencies have provided substantial insights into the interconnected roles of host and gut microbiota in maintaining BA homeostasis. It was proposed that the urinary total BA profile may serve as an ideal footprint for the functional status of the host-gut microbial BA co-metabolism. In summary, this work provided a powerful tool for human C24 BA metabolome analysis that bridges the gap between GC-MS techniques in the past age and LC-MS techniques currently prevailing in biomedical researches. Further applications of the present method in clinical, translational research, and other biomedical explorations will continue to boost the construction of a host-gut microbial co-metabolism network of BAs and thus facilitate the decryption of BA-mediated host-gut microbiota crosstalk in health and diseases.ᅟ [ABSTRACT FROM AUTHOR]

A simple, sensitive and selective LC-MS/MS method was developed for the quantitative analysis of liraglutide and validated in rat plasma. Human insulin was used as the internal standard. After a simple protein precipitation step, liraglutide was chromatographically separated using an InertSustain Bio C18 column with mobile phases comprising acetonitrile with 0.1% formic acid (A) and water with 0.1% formic acid (B). Detection was achieved using positive ion electrospray ionization in multiple-reaction monitoring (MRM) mode. Good linearity was observed in the concentration range 0.5–250 ng/mL (r 2  > 0.99). The intra- and inter-day precision values (expressed as relative standard deviation, RSD) of liraglutide ranged from 1.97–7.63% and 5.25–11.9, respectively. The accuracy (expressed as relative error, RE) ranged from −8.79–11.4%. Both the recovery and matrix effect were within acceptable limits. This method was successfully applied for the pharmacokinetics study of liraglutide in rats after subcutaneous administration. [ABSTRACT FROM AUTHOR]

A simple, sensitive and selective LC-MS/MS method for quantitative analysis of human insulin was developed and validated in dog plasma. Insulin glargine was used as the internal standard. After a simple step of solid-phase extraction, the chromatographic separation of human insulin was achieved by using InertSustain Bio C18 column with a mobile phase of acetonitrile containing 1% formic acid (A)—water containing 1% formic acid (B). The detection was performed by positive ion electrospray ionization in multiple-reaction monitoring (MRM) mode. Good linearity was observed in the concentration range of 1–1000 μIU/mL (r 2  > 0.99), and the lower limit of quantification was 1 μIU/mL (equal to 38.46 pg/mL). The intra- and inter-day precision (expressed as relative standard deviation, RSD) of human insulin were ≤12.1% and ≤13.0%, respectively, and the accuracy (expressed as relative error, RE) was in the range of −7.23–11.9%. The recovery and matrix effect were both within acceptable limits. This method was successfully applied for the pharmacokinetic study of human insulin in dogs after subcutaneous administration. [ABSTRACT FROM AUTHOR]

Benzbromarone is a uricosuric drug metabolized predominantly by cytochrome P450 2C9 from in vitro findings. Human CYP2C9 exhibits extensive genetic polymorphism and numbers of clinic studies have demonstrated that CYP2C9 genetic polymorphism has a significant influence on the pharmacokinetics of benzbromarone. But in vitro study on the interaction between CYP2C9 allelic isoforms and benzbromarone was rare. Here, an LC–MS/MS method was established and validated to determine the concentration of benzbromarone in different CYP2C9 enzyme incubation systems for the drug-enzyme interaction study. By selecting appropriate internal standard and optimizing separation system, including mobile phase, sample solvent and gradient elution condition, this LC–MS/MS method was developed with fine linearity (r 2 ≥ 0.996), good reproducibility (RSD ≤ 6.6%), high stability (92.37–114.67%), efficient recovery (91.23–109.82%) and acceptable matrix effect (110.54–115.31%). Based on this method, the interaction between 3 CYP2C9 allelic isoforms and benzbromarone was researched by kinetics parameters (K m , V max , Cl int ). As a result, CYP2C9*1 displayed the highest metabolic activity towards benzbromarone, CYP2C9*2 showed a little lower catalytic activity than CYP2C9*1 (relative clearance/*1 = 85.86%), CYP2C9*3 showed the lowest catalytic activity (relative clearance/*1 = 21.57%). The result illustrated that various CYP2C9 allelic isoforms showed different enzymatic activities towards benzbromarone, which could offer effective consultation for personalized administration in clinic. [ABSTRACT FROM AUTHOR]

Background Cold hyperalgesia is an intractable sensory abnormality commonly seen in peripheral neuropathies. Although glial cell line-derived neurotrophic factor family receptor alpha3 (GFRα3) is required for the formation of pathological cold pain has been revealed, potential transduction mechanism is poorly elucidated. We have previously demonstrated the contribution of enhanced activity of transient receptor potential melastatin 8 (TRPM8) to cold hyperalgesia in neuropathic pain using a rat model of chronic constriction injury (CCI) to the sciatic nerve. Recently, the enhancement of TRPM8 activity is attributed to the increased TRPM8 plasma membrane trafficking. In addition, TRPM8 can be sensitized by the activation of GFRα3, leading to increased cold responses in vivo. The aim of this study was to investigate whether GFRα3 could influence cold hyperalgesia of CCI rats via modulating TRPM8 expression and plasma membrane trafficking in dorsal root ganglion (DRG). Methods Mechanical allodynia, cold and heat hyperalgesia were measured on 1 day before CCI and the 1st, 4th, 7th, 10th and 14th day after CCI. TRPM8 total expression and membrane trafficking as well as GFRα3 expression in DRG were detected by immunofluorescence and western blot. Furthermore, GFRα3 small interfering RNA (siRNA) was intrathecally administrated to reduce GFRα3 expression in DRG, and the effects of GFRα3 knockdown on CCI-induced behavioral sensitization as well as TRPM8 total expression and membrane trafficking in both mRNA and protein levels were investigated, and the change in coexpression of TRPM8 with GFRα3 was also evaluated. Then, the effect of GFRα3 activation with artemin on pain behavior of CCI rats pretreated with the selective TRPM8 antagonist RQ-00203078 was observed. Results Here we found that TRPM8 total expression and plasma membrane trafficking as well as GFRα3 expression in DRG were initially increased on the 4th day after CCI, and maintained at the peak level from the 10th to the 14th day, which entirely conformed with the induction and maintenance of behavioral-reflex facilitation following CCI. The coexpression of TRPM8 with GFRα3, which was mainly located in peptidergic C-fibers DRG neurons, was also increased after CCI. Downregulation of GFRα3 protein in DRG attenuated CCI-induced cold hyperalgesia without affecting mechanical allodynia and heat hyperalgesia, and reduced the upregulations of TRPM8 total expression and plasma membrane trafficking as well as coexpression of TRPM8 with GFRα3 induced by CCI. Additionally, the inhibition of TRPM8 abolished the influence of GFRα3 activation on cold hyperalgesia after CCI. Conclusion Our results demonstrate that GFRα3 knockdown specially inhibits cold hyperalgesia following CCI via decreasing the expression level and plasma membrane trafficking of TRPM8 in DRG. GFRα3 and its downstream mediator, TRPM8, represent a new analgesia axis which can be further exploited in sensitized cold reflex under the condition of chronic pain. [ABSTRACT FROM AUTHOR]

Bile acids (BAs) are cholesterol metabolites with important biological functions. They undergo extensive host-gut microbial co-metabolisms during the enterohepatic circulation, creating a vast structural diversity and resulting in great challenges to separate and detect them. Based on the bioanalytical reports in the past decade, this work developed three chromatographic gradient methods to separate a total of 48 BA standards on an ethylene-bridged hybrid (BEH) C18 column and high-strength silica (HSS) T3 column and accordingly unraveled the factors affecting the separation and detection of them by liquid chromatography coupled with mass spectrometry (LC-MS). It was shown that both the acidity and ammonium levels in mobile phases reduced the electrospray ionization (ESI) of BAs as anions of [M−H], especially for those unconjugated ones without 12-hydroxylation. It was also found that the retention of taurine conjugates on the BEH C18 column was sensitive to the strength of formic acid and ammonium in mobile phases. By using the volatile buffers with an equivalent ammonium level as mobile phases, we comprehensively demonstrated the effects of the elution pH value on the retention behaviors of BAs on both the BEH C18 column and HSS T3 column. Based on the retention data acquired on a C18 column, we presented the ionization constants (p K ) of various BAs with the widest coverage beyond those of previous reports. When we made attempts to establish the structure-retention relationships (SRRs) of BAs, the lack of discriminative structural descriptors for BA stereoisomers emerged as the bottleneck problem. The methods and results presented in this work are especially useful for the development of reliable, sensitive, high-throughput, and robust LC-MS bioanalytical protocols for the quantitative metabolomic studies. [ABSTRACT FROM AUTHOR]

The gene for a protein domain, derived from a tumor marker, fused to His tag codons and under control of a T7 promotor was expressed in E. coli strain BL21 (DE3). The recombinant protein was purified from cell lysates through immobilized metal affinity chromatography and size-exclusion chromatography. A contaminating bacterial protein was consistently co-purified, even using stringent washing solutions containing 50 or 100 mM imidazole. Immunization of a dromedary with this contaminated protein preparation, and the subsequent generation and panning of the immune Nanobody library yielded several Nanobodies of which 2/3 were directed against the bacterial contaminant, reflecting the immunodominance of this protein to steer the dromedary immune response. Affinity adsorption of this contaminant using one of our specific Nanobodies followed by mass spectrometry identified the bacterial contaminant as FKBP-type peptidyl-prolyl cis - trans isomerase (SlyD) from E. coli . This SlyD protein contains in its C-terminal region 14 histidines in a stretch of 31 amino acids, which explains its co-purification on Ni-NTA resin. This protein is most likely present to varying extents in all recombinant protein preparations after immobilized metal affinity chromatography. Using our SlyD-specific Nb 5 we generated an immune-complex that could be removed either by immunocapturing or by size exclusion chromatography. Both methods allow us to prepare a recombinant protein sample where the SlyD contaminant was quantitatively eliminated. [ABSTRACT FROM AUTHOR]

[Display omitted] • Icaritin protects mice from MCAO/R injury by inhibiting neuroinflammation. • A photochemically induced coupling reaction system was used to explore the mechanism. • Cathepsin B is a key target for icaritin to inhibit neuroinflammation. Cerebral ischemic stroke is a major cause of mortality worldwide and severely affects the daily life of survivors. This study aimed to evaluate the therapeutic effects of icaritin (ICT) against cerebral ischemic stroke injury using a middle cerebral artery occlusion/reperfusion (MCAO/R) mice model, and investigate the pharmacological mechanisms via pharmacologically targeting molecular motor. Our results demonstrated that ICT significantly reduced the cerebral infarction size and brain edema of MCAO/R mice, and improved the neurological score at the same time. H&E and Nissl staining results confirmed that ICT alleviated the cerebral ischemia-induced damage in model mice and reduced the neuronal apoptosis caused by MCAO/R. More importantly, ICT promoted the expression of ZO1, Occludin and Claudin 5 by reducing the ratio of TIMP1/MMP2 and TIMP1/MMP9 in brain tissue to protect the blood–brain barrier. Further studies indicated that ICT inhibited the inflammatory level of brain tissue by regulating TLR4, MAPK and NLRP3 inflammasome signaling pathways. In combination with photochemically induced coupling reaction system, surface plasmon resonance, molecular docking and cellular thermal shift assay technology and Inhibitor Screening Kits, it was found that cathepsin B (CTSB) was the key target of ICT to attenuate neuroinflammation. To verify this conclusion, we inhibited the activity of CTSB in BV2 cells with CA-074 Me, and it demonstrated that ICT no longer had the inhibitory effect on glucose and oxygen deprivation-reoxygenation (OGD/R)-induced inflammation. When the mice were pretreated with CA-074 Me, ICT lost its protective effect on MCAO/R injury. Collectively, we reported ICT as a small-molecule targeting CTSB for anti-neuroinflammation. Morever, these findings broaden our knowledge of CTSB as a druggable target protein for neuroinflammation inhibition, and open a new avenue to novel therapy strategy for cerebral ischemia–reperfusion injury. [ABSTRACT FROM AUTHOR]

Bentysrepinine (Y101), a derivative of repensine (a compound isolated from Dichondrarepens Forst ), is a novel phenyalanine dipeptide inhibiting DNA-HBV and cccDNA activities and is currently under development for the treatment of hepatitis B virus (HBV)-infected hepatitis. Our previous study implied that there might be an existence of extensive metabolism of Y101 in rats. Therefore, it is necessary to perform metabolic profiling study to further evaluate its safety and drug-like properties. In this study, the metabolism of Y101 in rats was investigated by a convincible five-step strategy to characterize metabolites in plasma and that excreted into urine, bile and feces. The five-step strategy was realized by using an combined workflow on two different MS platforms, including various scan modes of liquid chromatography with hybrid quadruple-linear ion trap mass spectrometry (LC-QTRAP-MS/MS) and various post-acquiring data mining tools of liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (LC-QTOF-MS/MS). QTOF MS/MS was employed as a powerful complementary tool to enable high confidence of metabolites identification using its functions of accurate MS and MS/MS fragmentation. As a result, a total of 30 metabolites were detected, including 25 phase I and 5 phase II metabolites. Among them, four primary metabolites (M6–M9) were further identified by comparing with the authentic standards chemically synthesized. The possible metabolic pathways of Y101 in rats were proposed to be amide hydrolysis, monohydroxylation, dihydroxylation, N -oxidation, demethylation, methylation, glucosidation and glucuronidation. This is the first study of the metabolism of Y101 in rats. The five-step strategy was successfully used to systematically characterize metabolites of Y101 in rats, and it would be generally applied for metabolite identification of new drug candidate. [ABSTRACT FROM AUTHOR]

Silibinin, a natural flavanone, derived from the milk thistle plant ( Silybum marianum ), was illustrated for several medicinal uses such as liver-protective, anti-oxidant, anti-cancer, anti-inflammation and many other. However, silibinin has poor absorbance and bioavailability due to low water solubility, thereby limiting its clinical applications and therapeutic efficiency. To overcome this problem, the combination of silibinin with phosphatidylcholine (PC) as a formulation was used to enhance the solubility and bioavailability. The results indicated that silibinin-PC taken orally markedly enhanced bioavailability and therapeutic efficiency. In addition, a deeper understanding of the signaling pathways modulated by silibinin is important to realize its potential in developing targeted therapies against liver disorders and cancer. Silibinin has been shown to inhibit many cell signaling pathways in preclinical models, demonstrating promising effects against liver disorders and cancer through in vitro and in vivo studies. This review summarizes the pharmacokinetic properties, bioavailability, safety data, clinical activities and modulatory effects of silibinin in different cell signaling pathways against liver disorders and cancer. [ABSTRACT FROM AUTHOR]

Cerebralcare granule (CG) is a preparation of Traditional Chinese Medicine that widely used in China. It was approved by the China State Food and Drug Administration for treatment of headache and dizziness associated with cerebrovascular diseases. In the present study, we aimed to investigate whether CG had protective effect against d-galactose (gal)-induced memory impairment and to explore the mechanism of its action. d-gal was administered (100 mg/kg, subcutaneously) once daily for 8 weeks to induced memory deficit and neurotoxicity in the brain of aging mouse and CG (7.5, 15, and 30 g/kg) were simultaneously administered orally. The present study demonstrates that CG can alleviate aging in the mouse brain induced by d-gal through improving behavioral performance and reducing brain cell damage in the hippocampus. CG prevents aging mainly via suppression of oxidative stress response, such as decreasing NO and MDA levels, renewing activities of SOD, CAT, and GPx, as well as decreasing AChE activity in the brain of d-gal-treated mice. In addition, CG prevents aging through inhibiting NF-κB-mediated inflammatory response and caspase-3-medicated neurodegeneration in the brain of d-gal treated mice. Taken together, these data clearly demonstrates that subcutaneous injection of d-gal produced memory deficit, meanwhile CG can protect neuron from d-gal insults and improve memory ability. [ABSTRACT FROM AUTHOR]

A rapid and sensitive ultra fast liquid chromatography tandem mass spectrometry method (UFLC–MS/MS) was developed and validated for the simultaneous determination of six Saikosaponins (SSs) (SSa, SSb 1 , SSb 2 , SSd, SSc, SSf) of Bupleurum Dropping Pills (BDP) in rat plasma using chloramphenicol as the internal standard (IS). The SSs were separated using an ACQUITY UPLC ® BEH C 18 column (50 mm × 2.1 mm, 1.7 μm) and detection of these compounds were done by using a Qtrap 5500 mass spectrometer coupled with negative electrospray ionization (ESI) source under the multiple reaction monitoring (MRM) mode. According to regulatory guidelines, the established method was fully validated and results were showed within acceptable limits. The lower limit of quantifications (LLOQs) of all analytes were 0.2 ng/mL. The validated method was successfully applied into a pharmacokinetic study of orally administered BDP in rats. [ABSTRACT FROM AUTHOR]

Nine novel process impurities of N-[ N-benzoyl- O-(2-dimethylaminoethyl)- l-tyrosyl]- l-phenylalaninol ( Y101) observed during the laboratory optimization and later during its bulk synthesis are described in this article. The impurities were monitored by HPLC, and their structures were tentatively assigned on the basis of fragmentation patterns in LC−MS/MS and NMR spectroscopies. All of the impurities were synthesized, and their assigned constitutions were confirmed by co-injection in HPLC. In addition to the formation, synthesis, and characterization, the strategy for minimizing these impurities to a level accepted by the International Conference on Harmonisation (ICH) was also described. [ABSTRACT FROM AUTHOR]

Background The safe use of medications in pregnant females, their embryos and in offspring is important. The aim of the present study was to evaluate embryotoxicity of metformin ( MET) compared with other hypoglycemic drugs (rosiglitazone [ RSG] and glimepiride [ GLIM]), the anticancer drug 5-fluorouracil (5- FU), the anti-epileptic drug diphenylhydantoin ( DPH), the antibiotic penicillin G ( Pen G), and the cyclo-oxygenase ( COX)-2 inhibitor nimesulide ( NIM) in an embryonic stem cell test ( EST). Methods Differences in the expression of developmental marker genes following treatment with the test compounds during the course of differentiation (from embryonic stem cell D3 ( D3 cells) to myocardial cells) were determined using real-time quantitative polymerase chain reaction. In these studies, 5- FU was used as a positive control and Pen G was used as a negative control. The cytotoxicity of these drugs against D3 cells and 3 T3 fibroblasts was determined by the 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2 H-tetrazolium bromide ( MTT) assay. Embryotoxicity was classified according to the prediction model of EST. Results At concentrations >800 μg/mL MET had a greater cytotoxic effect on D3 cells than 3 T3 fibroblasts. At the highest concentration of MET (5 mg/mL), the cell viability of D3 cells and 3 T3 fibroblasts was <10% and >30%, respectively. The size of the embryonic body ( EB) differentiation area was almost the same over the concentration range 50-200 μg/mL MET, and there was no significant difference in EB differentiation area until a concentration of 400 μg/mL MET. At a concentration of 800 μg/mL MET, the size of EB outgrowth was significantly reduced. The same assays revealed GLIM, RSG, and NIM to be weakly embryotoxic substances. Conclusions Based on the EST, MET can be classified as a weakly embryotoxic substance, which suggests that it should be prescribed with caution to pregnant women with gestational diabetes. [ABSTRACT FROM AUTHOR]

An optimized synthetic method in high efficiency has been developed for the synthesis of lixivaptan from 2-nitrobenzyl bromide and pyrrole-2-carboxaldehyde. The byproducts among this procedure and an unknown impurity in crude product were investigated. The byproducts were speculated by 1H NMR or MS. The unknown impurity was characterized by 1H NMR, 13C NMR, and HRMS, confirming the structures as N-[3-chloro-4-(5 H-pyrrolo[2,1- c][1,4]benzodiazepine-10(11 H)-ylcarbonyl)phenyl]- N-(5-fluoro-2-methylbenzoyl)-5-fluoro-2-methylbenzamide. Afterwards, the impurity was synthesized to make comparisons. The target product lixivaptan was obtained with 47.6% overall yield and 99.93% purity. This cost-effective and environmentally friendly process is suitable for scale-up production. [ABSTRACT FROM AUTHOR]

The aim of this study was to develop a semi-mechanistic pharmacokinetic/pharmacodynamic (PK/PD) model to evaluate the in vitro synergy between vertilmicin and ceftazidime against Pseudomonas aeruginosa . The in vitro antimicrobial activity of vertilmicin alone was initially assessed by static and dynamic time–kill experiments against three bacterial strains, including MSSA, MRSA and P. aeruginosa . The combined killing effect with ceftazidime was then evaluated in a static time–kill study against P. aeruginosa . Vertilmicin displayed a concentration-dependent killing effect against the three bacterial strains, and its short half-life may possibly have a dramatic impact on antimicrobial activities. A two-compartment pharmacodynamic model consisting of drug-susceptible and -resistant compartments was developed to characterise the relationship between drug exposure and bacterial response for the time–kill curves from both monotherapy and combination therapy. Loewe additivity was incorporated into the pharmacodynamic model to describe the drug–drug interactive effect in the combination therapy. For monotherapy, the estimated EC 50 of the dynamic time–kill study against each strain was close to its MIC but was higher than that of the static time–kill study. The EC 50 of combination therapy was estimated at 2.67 mg/L compared with 4.54 mg/L in monotherapy, indicating an enhanced bactericidal capacity. The drug–drug interactive effect was not significantly synergistic but highly varied at each specific combination. Potential synergistic combinations could be screened using PK/PD modelling and simulation. These results demonstrated that PK/PD modelling provides an innovative approach to assist dose selection of combination vertilmicin and ceftazidime for future clinical study design. [ABSTRACT FROM AUTHOR]

Clevidipine, a vascular selective calcium channel antagonist of the dihydropyridine class, is rapidly metabolized by ester hydrolysis because of incorporation of an ester linkage into the drug molecule. To characterize its pharmacokinetic profiles in dogs, a simple, rapid and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for quantitation of clevidipine in dog blood. After one-step protein precipitation with methanol, the chromatographic separation was carried out on an Ecosil C 18 column (150 mm × 4.6 mm, 5 μm) with a gradient mobile phase consisting of methanol and 5 mM ammonium formate at a flow rate of 0.5 mL/min. The quantitation analysis was performed using multiple reaction monitoring (MRM) at the specific ion transitions of m / z 454.1 [M−H] − → m / z 234.1 for clevidipine and m / z 256.1 [M−H] − → m / z 227.1 for elofesalamide (internal standard) in the negative ion mode with electrospray ionization (ESI) source. This validated LC–MS/MS method showed good linearity over the range 0.5–100 ng/mL with the lower limit of quantitation (LLOQ) of 0.5 ng/mL together with the satisfied intra- and inter-day precision, accuracy, extraction recovery and matrix effect. Stability testing indicated that clevidipine in dog blood with the addition of denaturant methanol was stable on workbench for 1 h, at −80 °C for up to 30 days, and after three freeze–thaw cycles. Extracted samples were also observed to be stable over 24 h in an auto-sampler at 4 °C. The validated method has been successfully applied to a pharmacokinetic study of clevidipine injection to 8 healthy Beagle dogs following intravenous infusion at a flow rate of 5 mg/h for 0.5 h. [ABSTRACT FROM AUTHOR]

Context: Radix Aucklandiae, the dry rhizome of Aucklandia lappa Decne (Asteraceae), enjoyed traditional popularity for its antidiarrheal effects. Although there are many investigations on its chemical constituents and pharmacologic actions, few studies explaining its activity and mechanism in gastrointestinal disorders are available. Objective: In this paper, we focused on the effects of the methanol extract of R. Aucklandiae (RA ext) on gastrointestinal tract, so as to assess some of the possible mechanisms involved in the clinical treatment. Materials and methods: In vivo, in neostigmine-induced mice and normal mice, after intragastric administration, RA ext (100, 200, 300, and 400 mg/kg) was studied on gastrointestinal transit including gastric emptying and small intestinal motility. Meanwhile, in vitro, the effect of it (0.1, 0.2, 0.3, and 0.4 mg/mL) on the isolated tissue preparations of rat jejunum was also investigated , as well as costunolide and dehydrocostuslactone which were the main constituents. Results: In vivo, the gastric emptying increased and intestinal transit decreased after the administration of RA ext in normal mice. However, RA ext inhibited the gastric emptying and the intestinal transit throughout the concentrations in neostigmine-induced mice. In vitro, RA ext caused inhibitory effect on the spontaneous contraction of rat-isolated jejunum in a dose-dependent manner ranging from 0.1 to 0.4 mg/mL, and it also relaxed the acetylcholine chloride (Ach, 10−5 M), 5-hydroxytryptamine (5-HT, 200 μM)-induced, and K+ (60 mM)-induced contractions. RA ext shifted the Ca2+ concentration-response curves to right, similar to that caused by verapamil (0.025 mM). The Ca2+ concentration-response curves were shifted by costunolide (CO) (5.4, 8.1, and 10.8 μg/mL), dehydrocostuslactone (DE) (4.6, 6.9, and 9.2 μg/mL), costunolide-dehydrocostuslactone (CO-DE) (5.4-4.6, 8.1-6.9, and 10.8-9.2 μg/mL) to the right, similar to that caused by verapamil (0.01 mM). Discussion and conclusion: These results indicate that RA ext played a spasmolytic role in gastrointestinal motility, which is probably mediated through the inhibition of muscarinic receptors, 5-HT receptors, and calcium influx. The presence of cholinergic and calcium antagonist constituents may be the compatibility of CO and DE. All these results provide a pharmacological basis for its clinical use in the gastrointestinal tract. [ABSTRACT FROM AUTHOR]

Abstract: The effect of alkali-treatment for 0, 15, and 30 days at 35 °C on amylose content, morphological properties, crystalline properties, swelling power, solubility, absorbance spectra and in vitro digestibility of starches from Dioscorea zingiberensis, Dioscorea persimilis and Dioscorea opposita were investigated. The amylose content of all the three starches decreased after 15 days of alkaline treatment and then increased after 30 days of alkaline treatment. There were similar changes in relative crystallinity for D. zingiberensis and D. persimilis starches. It was observed that the three starches displayed a reduction trend in swelling power with a significant increase in solubility. Adhesion among some of the starch granules was observed after alkali-treatment for 30 days in D. zingiberensis and D. opposita starches, while D. persimilis starch showed some hollows on the granule surface. The rise in the absorbance ratio of 1047/1035 and 1047/1022 cm−1 from FT-IR was observed during alkali-treatment for D. persimilis and D. opposita starches. Alkali-treatment elevated the in vitro digestibility with resistant starch values climbing up from 50.16% to 64.95% and from 66.14% to 70.74% for D. zingiberensis and D. persimilis starches, respectively, but there was no significant change in resistant starch value for D. opposita starch. [Copyright &y& Elsevier]

Abstract: Ethnopharmacological relevance: Santalum album L., namely Sandalwood, honored as “Green Gold”, is a traditional Chinese herb which has the effects of anti-diarrhoeal and antibacterial activity. But there is limit scientific study on its activity and mechanism in gastrointestinal disorders. Materials and methods: in vivo, after intragastric administration, the methanol extract of Sandalwood (SE) (200, 400 and 800mg/kg) were studied in castor oil-induced diarrhoea mice. By the test of small intestinal hyperfunction induced by neostigmine, SE was studied on gastrointestinal transit including gastric emptying and small intestinal motility. Meanwhile, in vitro, the effects of SE (0.02, 0.05, 0.1, 0.2, 0.3, 0.4mg/mL) on the isolated tissue preparations of rat jejunum were also investigated. The rat jejunum strips were pre-contracted with acetylcholine (Ach; 10−6 M), 5-hydroxytryptamine (5-HT, 200μM) or potassium chloride (KCl; 60mM) and tested in the presence of SE. In addition, the possible myogenic effect was analyzed in the pretreatment of the jejunum preparations with SE or verapamil in Ca2+-free high-K+ (60mM) solution containing EDTA. Results: At doses of 200, 400 and 800mg/kg, SE showed significant anti-diarrhoeal activity against castor oil-induced diarrhoea as compared with the control. At the same doses, it also inhibited the gastric emptying and small intestinal motility in the mice of which small intestinal hyperfunction induced by neostigmine. It caused inhibitory effects on the spontaneous contraction of rat-isolated jejunum in dose-dependent manner ranging from 0.02 to 0.4mg/mL, and it also relaxed the Ach-induced, 5-HT-induced and K+-induced contractions. SE shifted the Ca2+ concentration–response curves to right, similar to that caused by verapamil (0.025mM). Conclusions: These findings indicated that SE played a spasmolytic role in gastrointestinal motility which was probably mediated through inhibition of muscarinic receptors, 5-HT receptors and calcium influx. All these results provide pharmacological basis for its clinical use in gastrointestinal tract. [Copyright &y& Elsevier]

Highlights: [•] RPS showed different metabolic alterations in normal and cancer mice. [•] RPS inhibited gluconeogenesis, alanine and glycine metabolism in cancer mice. [•] RPS inhibited glucose and valine to transform ketones in the normal mice. [Copyright &y& Elsevier]

Cardiac fibrosis is a pathogenic factor of many cardiovascular diseases (CVD), which seriously affects people's life and health, causing huge economic losses.Therefore, it is very significant to find an effective treatment for myocardial fibrosis. Adipokines are mainly derived from adipose tissue and have an prominent regulatory effect on glucose and lipid metabolism, inflammation, immune response and cardiovascular function. Adipose tissue is composed of a variety of cell types, including adipocytes, endothelial cells, macrophages and smooth muscle cells. Adipokines mainly include adiponectin, leptin, resistin, visfatin and omentin, which are synthesized and secreted by adipocytes. More and more evidence shows that adipokines can regulate the progress of cardiac fibrosis. This scientific review provides new ideas for targeting adipokines in the treatment of myocardial fibrosis and provides strategies for the development of new, safe, and effective pharmacological antagonists against myocardial fibrosis based on adipokines activity. [Display omitted] [ABSTRACT FROM AUTHOR]

Highlights: [•] RPS and cyclophosphamide both exhibited anti-cancer effect alone. [•] CYP2B6 and CYP3A4-mediated herb-drug interactions between RPS and other drugs. [•] RPS inhibited transference of cyclophosphamide into active and inactive compounds. [Copyright &y& Elsevier]

Abstract: Ethnopharmacological relevance: Radix Aucklandiae (RA), a well known traditional Chinese medicine, is widely used for treating various problems in digestive system. A selective and sensitive high-performance liquid chromatography coupled with mass spectrometry method was first developed and validated for simultaneous quantification of costunolide and dehydrocostuslactone in rat plasma with diazepam as internal standard after oral administration of RA extraction. Materials and methods: Plasma samples were extracted via solid-phase extraction and detected by multiple-reaction monitoring mode under positive electrospray. Chromatographic separation was accomplished on an Agilent C18 column (2.1mm×150mm, 5µm), with 0.1% formic acid and acetonitrile (1:1) as the mobile phase at a flow rate of 0.5mL/min. Results: The quantification was performed using the transitions of m/z 233/187 for costunolide, m/z 231/185 for dehydrocostuslactone and m/z 285/193 for diazepam, respectively. Calibration curves were linear over the concentration range of 0.7–769.7ng/mL for costunolide and 0.9–956.0ng/mL for dehydrocostuslactone. The intra-day and inter-day precisions (RSD%) for two compounds was less than 8.76% and 9.70% and the accuracy (RE%) range from 6.14% to 5.35%. The time to reach the maximum plasma concentration (T max ) was 10.46h for costunolide, 12.39h dehydrocostuslactone. The elimination half-time (t 1/2) of costunolide and dehydrocostuslactone was 5.54±0.81 and 4.32±0.71 (h). The AUC of costunolide and dehydrocostuslactone was 308.83 and 7884.51 respectively (ngh/mL). Conclusions: It was the first report for the study of pharmacokinetic profile of costunolide and dehydrocostuslactone in rat plasma after oral administration of RA extract. These results provided a meaningful basis for better understanding the absorption of traditional medicine, RA, and provide useful scientific data for clinical application. [ABSTRACT FROM AUTHOR]

Abstract: Polymeric micelles have been proven to be a promising nano-sized system for drug delivery. Understanding its in vivo behaviors at the whole body, tissue and cellular levels is critical for translating this drug delivery system into clinical practice. In this study, the 14.5nm micelles made of polyethylene glycol-phosphatidylethanolamine (PEG-PE) for delivery of doxorubicin and vinorelbine were investigated. Using confocal and two-photon microscopy imaging of live mice or tissue sections, we observed that after systemic administration, the fluorescently labeled PEG-PE micelles encapsulating doxorubicin migrated through blood vessels in entirety into the interstitial tissue, collected by lymphatic vessels, and accumulated in lymph nodes. Importantly, encapsulated drugs such as vinorelbine (Nanovin), preferentially accumulate in lymph nodes when compared to the free drugs. Moreover, the in vivo bioluminescent imaging showed that Nanovin significantly reduced lymph node metastasis rate (P <0.05) in 4T1-luc2 murine breast tumor bearing mice. Finally, we observed that Nanovin enhanced antitumor activity against primary tumors and lung metastases while having low toxicity in various 4 T1 tumor models. This study suggests that PEG-PE micelle is a promising drug delivery system for the treatment of lymphatic metastases, and may also have important applications in other lymphatic system-related diseases. [Copyright &y& Elsevier]

Abstract: Ethnopharmacological relevance: Cytotoxicity and hemolysis of saponins were the characteristic activities. Many researches have been devoted to the synthesis of saponins devoid of the hemolysis by structure-activity relationship studies. However, saponins contained in the plants were not fully researched on the two activities such as Parissteroidal saponins. Paris steroidal saponins as the active compounds of Paris plants have multiple effects.Materials and Methods: In the present study, we investigated the cytotoxicity and hemolytic activity of Paris plants collected from twelve places in China. The contents of nine known steroidal saponins in the plants were determined simultaneously using HPLC-ELSD method. The two activities are not related to the contents of saponins.Results: As the results of hemolysis of saponins shown, the hemolysis was positive related to the sugar chain. The membrane toxicity suggested that the integration of saponins with the membrane in erythrocyte was correlated to the kinds of saponins and the hemolysis of disosgenyl saponins were stronger than that of pennogenyl glycosides.Conclusions: These results provided information about the hemolysis and cytotoxicity of Paris steroidal saponins, which may be useful for their synthesis devoid of hemolysis toxicity. [Copyright &y& Elsevier]

Abstract: Rhizoma paridis saponins (RPS) played a good antitumor role in many clinical applications. However, low oral bioavailability limited its application. In this research, water extract of Curcuma (CW) significantly increased antitumor effect of Rhizoma paridis saponins (RPS). GC–MS was used to identify its polar composition. HPLC was applied for determination of the content of curcuminoids in CW. As a result, 47 analytes with 0.65% of curcuminoids were identified in CW. According to the in vivo anti-tumor data, the best proportion of curcuminoids in CW with RPS was 16:500 (w/w). Using this ratio, curcuminoids significantly increased absorption of RPS in the everted rat duodenum sac system. In addition, curcuminoids decreased the promotion of RPS on rhodamine 123 efflux. The effect of curcuminoids was similar to that of the P-gp inhibitor, cyclosporin A in combination with RPS. In conclusion, drug combination of water extract of Curcuma with RPS was a good method to increase the antitumor effect of RPS. This combination would be a potent anticancer agent used in the prospective application. [Copyright &y& Elsevier]

Context: Saponins are active compounds in natural products. Many researchers have tried to find the method for knowing their concentration in herbs. Some methods, such as solid-liquid extraction and solvent extraction, have been developed. However, the extraction methods of the steroidal saponins from Paris polyphylla Smith var. yunnanensis (Liliaceae) are not fully researched. Objective: To establish a simple extraction method for the separation of steroidal saponins from the rhizomes of P. polyphylla Smith var. yunnanensis. Materials and methods: Macroporous adsorption resins were used for the separation of steroidal saponins. To select the most suitable resins, seven kinds of macroporous resins were selected in this study. The static adsorption and desorption tests on macroporous resins were determined. Also, we optimized the temperature and the ethanol concentration in the extraction method by the contents of five kinds of saponins. Then, we compared the extraction method with two other methods. Results: D101 resin demonstrated the best adsorption and desorption properties for steroidal saponins. Its adsorption data fits best to the Freundlich adsorption model. The contents of steroidal saponins in the product were 4.83-fold increased with recovery yields of 85.47%. Discussion and conclusion: The process achieved simple and effective enrichment and separation for steroidal saponins. The method provides a scientific basis for large-scale preparation of steroidal saponins from the Rhizoma Paridis and other plants. [ABSTRACT FROM AUTHOR]