A hyper-bilirubin cell model was established for its relevance to the pathological state of jaundice in human. This model was used to screen for the pharmacological components of Yin-Zhi-huang (YZH). Total bilirubin, indirect bilirubin in cells, and direct bilirubin in extracellular fluid were quantified after HepaRG cells were incubated with serum from rats injected with multiple components of YZH. Cellular uptake was determined by dynamic multiple reaction monitoring (DMRM) using LC-MS/MS. We found that the stable hyper-bilirubin HepaRG cell model could be established by incubating cells with 40 μg·mL^-1 bilirubin and 50 μg·mL^-1 probenecid. When the hyper-bilirubin cell model was incubated with serum from rats of YZH injection, there were 52.4% and 60.1% decrease in intercellular total bilirubin and indirect bilirubin, respectively, and 52.5% increase in extracellular direct bilirubin. Using DMRM mode, 53 components could be determined, and 8 potential bioactive candidates were identified from the serum. This method could be used to screen for bioactive metabolites of YZH. This strategy is simple, highly active, sensitive and specific, providing a new method for high throughput screening of therapeutic or toxic metabolites from traditional Chinese medicine. The regulations of Ethics Committee in the First Hospital of Lanzhou University were abided in the rat experiment of this study.

Danhong injection (DHI) and ceftriaxone sodium were used in combination based on their experimental uses in clinic. This study was designed to investigate the impact of ceftriaxone on pharmacokinetics and pharmacodynamics of the phenolic acids from DHI. After administration of DHI for 7 d, ceftriaxone (CFTX) was combined with DHI for the next 7 d in adult male Sprague-Dawley (SD) rats. All the drugs were administered through caudal vein. UHPLC-TQ-MS was applied in determining the plasma concentration of p-coumaric acid (p-CA), salvianolic acid D (SaD), rosmarinic acid (RA) and salvianolic acid B (SaB). The pharmacokinetic parameters of the combination group or the Danhong injection alone group were calculated by statistical moment method, C_max and the average of the area under the curve AUC_0-t using 90% confidence interval of the bioequivalence and bioavailability degree module in DAS 3.2.8 statistic software. The results showed that C_max of p-CA, SaD, RA and SaB were unqualified within 90% confidence intervals for bioequivalence statistics. And the results showed that AUC_0-t of SaD, RA and SaB within 90% confidence intervals for bioequivalence statistics were unqualified. There were no significant difference in the t_max (P>0.05). The results of anticoagulation in vivo showed that the international normalized ratio (INR), prothrombin time (PT), thrombin time (TT) and activated partial thromboplastin time (APTT) were significantly increased when combined with CFTX (P < 0.05 or P < 0.01). The results in antithrombotic effects revealed that the thromboxane B₂ (TXB₂) level in serum was significantly decreased (P < 0.01) in the combination group compared with Danhong injection alone. However, there was no significant difference in antiplatelet effects. These results suggest that CFTX may enhance the anticoagulation and antithrombotic effects of DHI through altering pharmacokinetics and pharmacodynamics in SD rats.

The study was conducted to characterize the pharmacokinetics, distribution, metabolism and excretion of CHMFL-FLT3-122 after a single oral dose of 50 mg·kg^-1[¹⁴C] labeled CHMFL-FLT3-122 in rats. Isotope tracing techniques were used to analyze drug concentration and identify the distribution of drugs in tissues and metabolites in biological samples. The experiments were approved by the Animal Ethics Committee of XenoBiotic Laboratories-China, Inc. The absolute bioavailability in male and female rats were 45.83% and 50.92% respectively. The parent drug and its metabolites were extensively distributed in the stomach, intestine, liver and lung, and were eliminated completely in 48 h. The majority of radioactivity was excreted through the feces at 92.34% of the dose with a small fraction through urine at 3.99% of the dose. The parent drug was the most significant circulating component, representing 49.23% and 70.65% over the 0-48 h collection time interval in the plasma of male and female. Two major metabolites, M553 (sulfate conjugate) and M457 (N-dealkyl product), were identified in plasma. Metabolites of CHMFL-FLT3-122, including ten phase Ⅰ and four phase Ⅱ metabolites, were identified. The metabolic pathways of CHMFL-FLT3-122 were proposed as N-dealkylation, oxidation, amide hydrolysis, sulfate conjugation, and glucuronic conjugation.

In ischemic stroke, increased level of neuronal complex of nitric oxide synthase (nNOS)-postsynaptic density protein-95 (PSD-95) plays an important role in neuronal damage. We aimed to establish a screening model to identify compounds capable of uncoupling nNOS interaction with PSD-95. In this model, human embryonic kidney-293T (HEK-293T) cells were transfected with either pCDH-Flag-nNOS or pcDNA3.1-PSD-95 plasmid to obtain the protein of Flag-nNOS or PSD-95. Incubating Flag-nNOS with PSD-95 causes formation of the nNOS-PSD-95 complex. ZL006, a known uncoupler of nNOS-PSD-95 interaction, can disturb the interaction between Flag-nNOS and PSD-95, serving as a positive control. The method coupling antibodies to magnetic beads with glutaraldehyde was used to decrease the cost and increase the efficiency. To establish that our model is suitable for selecting nNOS-PSD-95 uncouplers, we evaluated the ability of IC87201, another reported uncoupler of nNOS-PSD-95 interaction, and structural analogs of ZL006. IC87201 and one structure analog of ZL006 showed uncoupling effect, supporting that our model can be used to select different types uncoupler blocking nNOS-PSD-95 interaction.

To investigate the effect of Sishen Wan (SSW) on intestinal flora in diarrhea-predominant irritable bowel syndrome (IBS-D) rats and explore the efficacy of this regiment for improving IBS-D, we divided 45 SPF male SD rats randomly into control, disease, SSW, Ershen Wan (ESW) and Wuweizasan (WWZS) groups. The spleen-kidney-yang deficiency type IBS-D rat model was prepared by a composite factor and administered for 14 days. After collecting the feces of the rats, total DNA was extracted from the stool samples. Primers were designed based on the 16S r RNA V3 to V4 regions of the bacteria, and used for high-throughput sequencing with the Illumina Miseq platform. We found that SSW can effectively reduce the diarrhea index (P < 0.05) and reduce the high sensitivity of intestinal tract (P < 0.05) of IBS-D rats. The principal component analysis (PCA), principal co-ordinates analysis (PCoA) and non-metric multidimensional scale analysis (NMDS) based on the Beta diversity distance showed that there were significant differences in the composition of the gut microbiota among the five groups (P < 0.05). The disease group has the lowest in abundance, uniformity and diversity of gut microbiota. Compared with the control group, the disease group showed a significant increase in Proteobacteria, Actinobacteria, Veillonococcus and Mycoplasma (P < 0.05), but a significant reduction in Pleaverella (P < 0.05). Compared with the disease group, SSW administration caused significant reduction in the Proteobacteria and Mycoplasma (P < 0.05), but significant increases of Clostridium, Turicibacter and Romboutsia (P < 0.05). Our study shows that SSW has the potential as a therapeutic regiment for treatment of IBS-D due to partial regulation of the intestinal flora. In addition, there is a synergy between ESW and WWZS.

Using the idiosyncratic lipopolysaccharide (LPS)-mediated hepatotoxicity model as a positive control, liver injury induced by Cortex Dictamni aqueous extract (AE) or Cortex Dictamni ethanol extracts (EE) was evaluated. Idiosyncratic hepatotoxicity model was established in rats[Institutional Animal Care and Use Committee (IACUC)-2018-008] by injecting LPS at a dosage of 2.8 mg·kg^-1. Rats were randomly divided into 10 groups. The plasma levels of liver function biomarkers such as alanine transaminase (ALT), aspartate aminotransferase (AST) were measured. Histological changes (HE staining), hepatocellular apoptosis and the content of cytokines of liver were measured. Network pharmacology was used to analyze the relationship between chemical components and immunity in Cortex Dictamni. Compared with the control group, the doses (25, 50 g·kg^-1) of AE or EE had no significant changes in ALT, AST and liver pathology (P>0.05). The doses of 4.2 g·kg^-1 of AE or EE+LPS groups exhibited an elevation in ALT, AST and serum cytokines (P < 0.01). Disorder of liver lobular arrangement and irregular island-like or massive necrosis of liver cells were observed in these groups. Network pharmacology shows that Cortex Dictamni may directly or indirectly participate in the process of immunomodulation. We found that Cortex Dictamni regulated 15 core targets and affected 19 pathways, including apoptosis, TNF-α, NF-kappa B signaling pathways. These results suggest that Cortex Dictamni can induce idiosyncratic hepatotoxicity and the water extract can induce more serious liver injury then ethanol extract of Cortex Dictamni. These findings provide a reference for elucidating the idiosyncratic hepatotoxicity induced by Cortex Dictamni.

The combination of ginkgo ketoester tablet-donepezil (GD) is a popular combination commonly used in clinic for the treatment of Alzheimer's disease. To evaluate the learning and memory improving ability of different proportions of the two drugs. We optimized the ratio of GD for treatment of dementia using a mouse model. Dementia was induced by multiple neuronal damages in mice. The experimental protocols were approved by the Animal Experimental Ethical Committee of Nanjing University of Chinese Medicine and all the procedures were strictly conducted in accordance with ethical principle of animal use and care. Morris water maze, brain hematosylin-eosin staining and the changes of the neurotransmitters and related enzymes in the plasma or brain tissues were tested to determine the effect of GD on dementia mice. The results showed that the dementia mice were significantly different from the normal group in terms of behavior, pathological sections and related indicators. Compared to the dementia mice, partial administration groups could improve learning and memory ability as well as indexes in the blood and brain tissues. Both the principal component analysis and multi-attribute comprehensive index methods were used to comprehensively evaluate the total effect of GD on anti-dementia. The results showed that the combination of two drugs at the dose of 0.5 to 1 times was in a dose-effect relationship, and the dose of 1 (the clinical equivalent) had the best treatment effect. Then based on the optimal dose, GD 1:1 had best effect, which was consistent with the clinical use of two drugs. This provides scientific basis for more effective application of the compatibility between ketoester tablet and donepezil for modern clinic medicine.

A LC-MS/MS method for quantification of norfloxacin in human plasma had been developed. This method was applied to the pharmacokinetics study of norfloxacin in the human. The plasma sample was precipitated by methanol and ciprofloxacin was used as the internal standard (IS). Chromatographic separation was performed on a Symmetry® C₁₈ column(100 mm×4.6 mm, 5 μm). Mobile phase contains 0.3% formic acid and 5% methanol in deionized water at a flow rate of 0.45 mL·min^-1. Norfloxacin and ciprofloxacin (IS) were ionized with an ESI source and operated in positive ion mode. The detected ions were m/z 320.3→302.1 (norfloxacin), m/z 332.3→314.1 (ciprofloxacin). This LC-MS/MS method yielded a linearity over the range of 10-1 000 ng·mL^-1 with the lower limit of quantitation (LLOQ) of 10 ng·mL^-1. The intra and inter-assay precisions (RSD%) were within the range of 2.64%-7.23% and the accuracy (RE%) was less than ±5.00%. The pharmacokinetic parameters t_max, C_max, AUC_0-t, and t_1/2 were 1.28±0.364 h, 627±171 ng·mL^-1, 2 938±850 h·ng·mL^-1, and 6.01±1.36 h, respectively. This LC-MS/MS method was proven simple, sensitive, rapid and suitable for pharmacokinetics study of norfloxacin in the human and Approved by the Medical Ethics Committee of Liuzhou Workers' Hospital.

The molecular identification of Ophiocordyceps sinensis and its adulterants was carried out by real-time fluorescent PCR with TaqMan probe. Genomic DNA was extracted from 100 samples of Ophiocordyceps sinensis and its adulterants. MEGA 7.0 software was used for comparative analysis to define the variable sites between Ophiocordyceps sinensis and its adulterants according to the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA). A set of specific primers and TaqMan probe were designed using Primer Premier 6.0 software, and sensitivity and specificity studies were performed on two different real-time fluorescent PCR systems (Genesig q16 and Bio-Rad CFX96). The sensitivity study showed that the detectable DNA template concentration of Ophiocordyceps sinensis for the real-time fluorescent PCR was 0.016 ng·μL^-1 in the Bio-Rad CFX96 system and 15.527 ng·μL^-1 in the Genesig q16 system, respectively. Meanwhile, this method had good specificity for Ophiocordyceps sinensis on Genesig q16 and Bio-Rad CFX96 systems, so Ophiocordyceps sinensis could be clearly distinguished from Ophiocordyceps nutans, Cordyceps gunnii, Cordyceps militaris, Cordyceps cicadae, Cordyceps liangshanensis, Cordyceps gracilis. Our results indicate that real-time fluorescent PCR with TaqMan probe can be used to accurately identify Ophiocordyceps sinensis from its adulterants. This provides a technical method that has wide applications for market management and quality control of Chinese materia medica.

Protein-protein interaction (PPI) plays an important role in many steps of the human immunodeficiency virus (HIV) life cycle. Targeting these protein-protein interactions, especially the interaction between the virus with host cells, can provide new insights into the development of HIV inhibitors with novel mechanisms of action. Herein, we review the latest discoveries of PPI inhibitors based on the mechanisms of action of various protein-protein interactions with specific research examples.