Ion-pair compounds of diacerein for enhancing skin permeability in vitro: the compatibility-permeability relationship of counter ion and diacerein.

This research aimed to investigate how the relationship between counter ion and diacerein (DCN) exerts an effect on the skin penetration of DCN ion-pair compounds. After the ion-pair compounds were formed by DCN and organic amines with different functional groups, the hydrogen bond of these compounds was confirmed by Fourier-transform infrared (FTIR) spectroscopy and molecular docking. The skin of porcine ears was employed to conduct the in vitro skin penetration, DCN - triethanolamine was the most potential candidate with the Q24h of 7.89 ± 0.38 µg/cm2 among organic amines with different functional groups. Whereas among the homologous fatty amine, the most permeable compound was DCN - lauryl amine with the Q24h of 11.28 ± 0.48 µg/cm2. Molecular simulation was employed to explore the relationship between counter ion and DCN. It was revealed by the bind energy curve that DCN had the strongest compatibility with triethanolamine among organic amines and laurylamine (N12) among fatty amines. It was amazingly found that the in vitro permeation fluxes of DCN ion-pair compounds would increase with enhancing the compatibility of counter ion and DCN. These findings broadened our understanding of how the relationship between drug and counter ion affects the skin penetration of ion-pair compounds.

Exploring the mechanism of Buxue Yimu Pill on hemorrhagic anemia through molecular docking, network pharmacology and experimental validation.

Buxue Yimu Pill (BYP) is a classic gynecological medicine in China, which is composed of Angelica sinensis (Oliv.) Diels, Leonurus japonicus Houtt, Astragalus membranaceus (Fisch.) Bunge, Colla corii asini and Citrus reticulata Blanco. It has been widely used in clinical therapy with the function of enriching Blood, nourishing Qi, and removing blood stasis. The current study was designed to determine the bioactive molecules and therapeutic mechanism of BYP against hemorrhagic anemia. Herein, GC-MS and UPLC/Q-TOF-MS/MS were employed to identify the chemical compounds from BYP. The genecards database (https: //www.genecards.org/) was used to obtain the potential target proteins related to hemorrhagic anemia. Autodock/Vina was adopted to evaluate the binding ability of protein receptors and chemical ligands. Gene ontology and KEGG pathway enrichment analysis were conducted using the ClusterProfiler. As a result, a total of 62 candidate molecules were identified and 152 targets related to hemorrhagic anemia were obtained. Furthermore, 34 active molecules and 140 targets were obtained through the virtual screening experiment. The data of molecular-target (M-T), target-pathway (T-P), and molecular-target-pathway (M-T-P) network suggested that 32 active molecules enhanced hematopoiesis and activated the immune system by regulating 57 important targets. Pharmacological experiments showed that BYP significantly increased the counts of RBC, HGB, and HCT, and significantly down-regulated the expression of EPO, IL-6, CSF3, NOS2, VEGFA, PDGFRB, and TGFB1. The results also showed that leonurine, leonuriside B, leosibiricin, ononin, rutin, astragaloside I, riligustilide and levistolide A, were the active molecules closely related to enriching Blood. In conclusion, based on molecular docking, network pharmacology and validation experiment results, the enriching blood effect of BYP on hemorrhagic anemia may be associated with hematopoiesis, anti-inflammation, and immunity enhancement.

miR-665 is downregulated in glioma and inhibits tumor cell proliferation, migration and invasion by targeting high mobility group box 1.

Glioma is the most common brain tumor in adults. microRNAs (miRNAs/miRs) play an essential role in tumor development and progression. The present study aimed to investigate the potential clinical significance and function of miR-665 in glioma. Reverse transcription-quantitative PCR analysis was used to detect the expression of miR-665 in glioma tissues and cells. Survival curves were constructed using the Kaplan-Meier method. Cox regression analysis was performed to investigate the prognostic significance of miR-665. Cell Counting Kit-8 and Transwell assays were used to evaluate the role of miR-665 in glioma. Bioinformatics analysis and Dual-luciferase reporter assays were used to predict the putative direct targets of miR-665. Western blotting was used to evaluate the activity of the Wnt/ß-catenin pathway. The relative expression of miR-665 was decreased in glioma tissues and cells and this downregulation was significantly associated with the Karnofsky performance scale score and World Health Organisation grade. Patients with glioma with low miR-665 expression had a shorter overall survival time compared with the high expression group. Besides, overexpression of miR-665 suppressed the proliferation, migration and invasion of glioma cells, while knockdown of miR-665 promoted these cellular behaviors. High mobility group box (HMGB)1 was a direct target of miR-665. It was also demonstrated that miR-665 may suppress glioma progression by targeting HMGB1 and inhibiting the Wnt/ß-catenin pathway. Taken together, these data suggested that miR-665 may have a tumor suppressor role in glioma by targeting HMGB1. Therefore, miR-665 may be a novel prognostic biomarker and the miR-665/HMGB1 axis may be a novel therapeutic target for the treatment of glioma.

Ion-Pair Compounds of Strychnine for Enhancing Skin Permeability: Influencing the Transdermal Processes In Vitro Based on Molecular Simulation.

This research aimed to explore how Strychnine (Str) ion-pair compounds affect the in vitro transdermal process. In order to prevent the influence of different functional groups on skin permeation, seven homologous fatty acids were selected to form ion-pair compounds with Str. The in vitro permeation fluxes of the Str ion-pair compounds were 2.2 to 8.4 times that of Str, and Str-C10 had the highest permeation fluxes of 42.79 ± 19.86 µg/cm2/h. The hydrogen bond of the Str ion-pair compounds was also confirmed by Fourier Transform Infrared (FTIR) Spectroscopy, Nuclear Magnetic Resonance (NMR) Spectroscopy and molecular simulation. In the process of molecular simulation, the intercellular lipid and the viable skin were represented by ceramide, cholesterol and free fatty acid of equal molar ratios and water, respectively. It was found by the binding energy curve that the Str ion-pair compounds had better compatibility with the intercellular lipid and water than Str, which indicated that the affinity of Str ion-pair compounds and skin was better than that of Str and skin. Therefore, it was concluded that Str ion-pair compounds can be distributed from the vehicle to the intercellular lipid and viable skin more easily than Str. These findings broadened our knowledge about how Str ion-pair compounds affect the transdermal process.

Mechanisms of Triptolide-Induced Hepatotoxicity and Protective Effect of Combined Use of Isoliquiritigenin: Possible Roles of Nrf2 and Hepatic Transporters.

Triptolide (TP), the main bioactive component of Tripterygium wilfordii Hook F, can cause severe hepatotoxicity. Isoliquiritigenin (ISL) has been reported to be able to protect against TP-induced liver injury, but the mechanisms are not fully elucidated. This study aims to explore the role of nuclear transcription factor E2-related factor 2 (Nrf2) and hepatic transporters in TP-induced hepatotoxicity and the reversal protective effect of ISL. TP treatment caused both cytotoxicity in L02 hepatocytes and acute liver injury in mice. Particularly, TP led to the disorder of bile acid (BA) profiles in mice livers. Combined treatment of TP with ISL effectively alleviated TP-induced hepatotoxicity. Furthermore, ISL pretreatment enhanced Nrf2 expressions and nuclear accumulations and its downstream NAD(P)H: quinine oxidoreductase 1 (NQO1) expression. Expressions of hepatic P-gp, MRP2, MRP4, bile salt export pump, and OATP2 were also induced. In addition, in vitro transport assays identified that neither was TP exported by MRP2, OATP1B1, or OATP1B3, nor did TP influence the transport activities of P-gp or MRP2. All these results indicate that ISL may reduce the hepatic oxidative stress and hepatic accumulations of both endogenous BAs and exogenous TP as well as its metabolites by enhancing the expressions of Nrf2, NQO1, and hepatic influx and efflux transporters. Effects of TP on hepatic transporters are mainly at the transcriptional levels, and changes of hepatic BA profiles are very important in the mechanisms of TP-induced hepatotoxicity.

The Ethanol Extract of Licorice (Glycyrrhiza uralensis) Protects against Triptolide-Induced Oxidative Stress through Activation of Nrf2.

To investigate the potential role of nuclear factor erythroid 2-related factor 2 (Nrf2) in licorice ethanol extract (LEE) against triptolide- (TP-) induced hepatotoxicity, HepG2 cells were exposed to LEE (30, 60, and 90 mg·L-1) for 12 h and then treated with TP (50 nM) for 24 h. Besides, an acute liver injury model was established in ICR mice by a single dose of TP (1.0 mg·kg-1, i.p.). Relevant oxidant and antioxidant mediators were analyzed. TP led to an obvious oxidative stress as evidenced by increasing levels of ROS and decreasing GSH contents in HepG2 cells. In vitro results were likely to hold true in in vivo experiments. LEE protected against TP-induced oxidative stress in both in vitro and in vivo conditions. Furthermore, the decreased level of Nrf2 in the TP-treated group was observed. The mRNA levels of downstream genes decreased as well in ICR mice liver, whereas they increased in HepG2 cells. In contrast, LEE pretreatment significantly increased the level of Nrf2 and its downstream genes. LEE protects against TP-induced oxidative stress partly via the activation of Nrf2 pathway.

A Novel Brucine Gel Transdermal Delivery System Designed for Anti-Inflammatory and Analgesic Activities.

The seeds of Strychnosnux-vomica L., as a traditional Chinese medicine, have good anti-inflammatory and analgesic activities. However, it usually leads to gastrointestinal irritation and systemic toxicity via oral administration. In the study, it was discovered that a novel gel transdermal delivery system contained brucine, the main effective component extracted from Strychnosnux-vomica. Results showed that the brucine gel system inhibited arthritis symptoms and the proliferation of the synoviocytes in the rat adjuvant arthritis model, which indicated its curative effect for rheumatoid arthritis. Meanwhile, it significantly relieved the xylene-induced ear edema in the mouse ear swelling test, which manifested its anti-inflammatory property. Moreover, the brucine gel eased the pain of paw formalin injection in the formalin test, which demonstrated its analgesic effects. In addition, the brucine significantly inhibited lipopolysaccharide (LPS)-induced Prostaglandin E2 (PGE2) production without affecting the viability of cell in vitro anti-inflammatory test, which proved that its anti-inflammatory and analgesic actions were related to inhibition of prostaglandin synthesis. It is suggested that the brucine gel is a promising vehicle for transdermal delivery on the treatment of inflammatory disease.

Dynamic detection of non-protein-bound strychnine and brucine in rabbit muscle and synovial fluid after topical application of total Strychnos alkaloid patches.

Semen Strychni, a known toxic drug in Chinese pharmacopoeia, is notable for its therapeutic effects on local muscle and joint pain. However, oral administration can be risky. Topically administered drugs accumulate in the topical muscles and knee joints without any major increase in plasma levels; only non-protein-bound drugs in the biological fluids of target tissues are effective for therapeutic effects. A sensitive and rapid ultra performance liquid chromatography - mass spectrometry (UPLC-MS) method coupled with a microdialysis technique was developed to determine the non-protein-bound strychnine (Str) and brucine (Bru) in rabbit muscle and synovial fluid microdialysate. The UPLC separation was carried out using a 1.7µm BEH C18 column (50 mm × 2.1 mm) with a mobile phase consisting of methanol: water (29.5:70.5, v/v) with 0.1% formic acid and 20 mM ammonium acetate in water. The method was validated at concentrations ranging from 0.58 ng/ml to 467.20 ng/ml for Str and from 0.42 ng/ml to 422.40 ng/ml for Bru. Intra-day and inter-day accuracy ranged from 99.1% to 103.2% for Str and from 95.8% to 108.8% for Bru with intra-day and inter-day precision within 9.7%. The proposed method was successfully applied to determine non-protein-bound Str and Bru, and the analysates concentration remained stable in rabbit muscle and synovial fluid after topical application of total Strychnos alkaloid patches, which indicated that total Strychnos alkaloid patches could substitute for the traditional oral administration of Semen Strychni.

HPLC-DAD method for comprehensive quality control of Semen Strychni.

CONTEXT: Semen Strychni is the seed of Strychnos nux-vomica L. (Loganiaceae). Its quality control procedure remains an issue since previous reports only focused on Strychnos alkaloids. To the best of our knowledge, chlorogenic acid (a phenolic acid) and loganin (an iridoid glycoside) are selected for the first time as marker constituents of quality control for Semen Strychni because of their bioactive activity correlating with therapeutic effects. OBJECTIVE: This study aimed to develop a simple and comprehensive quantity control method for Semen Strychni. MATERIALS AND METHODS: The optimal ultrasonic extraction procedure was carried out for 45 min using 50% aqueous methanol with 1% formic acid. The satisfactory chromatographic separation was achieved on an Ultimate LP-C18 column with gradient elution using acetonitrile and water containing 30 mmol/L ammonium acetate and 1% formic acid. The high performance liquid chromatography method with diode array detector was validated for linearity, limit of detection and quantification (LOQ), precision, repeatability, accuracy and stability. RESULTS: All the calibration curves showed good linearity (r(2) ≥ 0.999). The LOQ values for chlorogenic acid, loganin, strychnine, brucine, strychnine N-oxide and brucine N-oxide were 0.54, 0.83, 0.48, 0.50, 0.52 and 0.54 µg/mL, respectively. The method was reproducible with good accuracy in the range 95.6-104.4% and relative standard deviation (RSD) values less than 4.55%. The method was then applied to determine the components of the seed coat, seed leaf, endosperm and whole seed of Semen Strychni. CONCLUSION: This newly established method is validated as a simple and practical tool for authentication and quality control of Semen Strychni.

Quantitative analysis of olanzapine in rat brain microdialysates by HPLC-MS/MS coupled with column-switching technique.

A rapid and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method coupled with column-switching technique was developed for the determination of olanzapine in rat brain microdialysates. A C8 guard column was used to desalt the samples before analytical separation on a C18 column and detection with tandem mass spectrometry. The mobile phase consisted of methanol/acetonitrile/water (v/v/v, 22.5/22.2/55) was used for desalting and the mobile phase consisted of methanol/acetonitrile/water (v/v/v, 43/43/14) was for analytical separation, water in both mobile phases contained 0.1% ammonium acetate. The lower limit of quantification (LLOQ) for olanzapine was 0.085 ng/ml. The method was linear from LLOQ to 34 ng/ml with a coefficient of determination >0.998. Intra- and inter-day accuracy and precision were determined with variability less than 13.24% (R.S.D). This sensitive method was successfully applied to quantify the concentration of olanzapine in rat brain microdialysates. With this study, the effect of the alcohol extract of Schisandra sphenanthera Rehd. et Wils on the concentration of olanzapine in brain was investigated.