Folic acid-modified nonionic surfactant vesicles for gambogenic acid targeting: Preparation, characterization, and in vitro and in vivo evaluation.

The aim of present study was to develop folic acid (FA)-modified nonionic surfactant vesicles (NISVs, niosomes) as carrier systems for targeted delivery of gambogenic acid (GNA). The FA-GNA-NISVs exhibited a mean particle size of 180.77 ± 2.41 nm with a narrow poly dispersion index of 0.147 ± 0.08 determined by dynamic light scattering. Transmission electron microscopy also revealed that the FA-GNA-NISVs were spherical with double-layer structure. Entrapment efficiency (EE%) and zeta potential of the optimal FA-GNA-NISVs were 87.84 ± 1.06% and -37.33 ± 0.33 mV, respectively. Differential scanning calorimetry demonstrated that the GNA was in a molecular or amorphous state inside the FA-NISVs in vitro release profiles suggested that FA-GNA-NISVs could release GNA at a sustained manner, and less than 60% of GNA was released from the FA-NISVs within 12 hours of dialysis. in vivo pharmacokinetic results illustrated that FA-GNA-NISVs had considerably higher Cmax , area under curve (AUC0 - t ) and accumulation in lung. The cell proliferation study shown that the FA-GNA-NISVs significantly enhanced the in vitro cytotoxicity against A549 cells. Flow cytometry and fluorescence microscopy further demonstrated that the FA-GNA-NISVs increased apoptosis compared with nonmodified GNA-NISVs and free GNA. Moreover, FA-GNA-NISVs induced A549 cell apoptosis in a dose-dependent manner. In addition, cellular uptake assays showed a higher uptake of FA-GNA-NISVs than GNA-NISVs as well as free GNA. Taken together, it could be concluded that FA-GNA-NISVs were proposed as a novel targeting carriers for efficient delivering of GNA to cancers cells.

Multimodal Imaging Probe Development for Pancreatic ß Cells: From Fluorescence to PET.

Pancreatic ß cells are responsible for insulin secretion and are important for glucose regulation in a healthy body and diabetic disease patient without prelabeling of islets. While the conventional biomarkers for diabetes have been glucose and insulin concentrations in the blood, the direct determination of the pancreatic ß cell mass would provide critical information for the disease status and progression. By combining fluorination and diversity-oriented fluorescence library strategy, we have developed a multimodal pancreatic ß cell probe PiF for both fluorescence and for PET (positron emission tomography). By simple tail vein injection, PiF stains pancreatic ß cells specifically and allows intraoperative fluorescent imaging of pancreatic islets. PiF-injected pancreatic tissue even facilitated an antibody-free islet analysis within 2 h, dramatically accelerating the day-long histological procedure without any fixing and dehydration step. Not only islets in the pancreas but also the low background of PiF in the liver allowed us to monitor the intraportal transplanted islets, which is the first in vivo visualization of transplanted human islets without a prelabeling of the islets. Finally, we could replace the built-in fluorine atom in PiF with radioactive 18F and successfully demonstrate in situ PET imaging for pancreatic islets.

Preparation and preliminary pharmacokinetics study of GNA-loaded zein nanoparticles.

OBJECTIVES: Gambogenic acid (GNA), one of the main active ingredients isolated from Garcinia cambogia, has shown diverse antitumour activities. However, short biological half-life and low oral bioavailability severely limit its clinical application. Here, we developed GNA-loaded zein nanoparticles (GNA-ZN-NPs) based on phospholipid complex and zein nanoparticles to prolong the circulation time and enhance oral bioavailability of GNA. METHODS: The physicochemical properties of GNA-ZN-NP were characterized in details. The in vitro release profile, in vivo pharmacokinetic experiments and tissue distribution of GNA-ZN-NPs were also evaluated. KEY FINDINGS: The particle size, PDI and encapsulation efficiency of GNA-ZN-NPs were 102.90 nm, 0.027 and 76.35 ± 0.64%, respectively. The results of SEM, FTIR, DSC and XRD demonstrated that GNA-ZN-NPs were prepared successfully. The in vitro dissolution of GNA-ZN-NPs exhibited controlled release compared with raw GNA solution. The pharmacokinetic study showed that the AUC of GNA-ZN-NPs was significantly increased, and the t1/2 and MRT values of GNA-ZN-NPs were 3.21-fold and 2.19-fold higher than that of GNA solution. Tissue distribution results illustrated that GNA-ZN-NPs showed hepatic-targeting properties. CONCLUSION: GNA-ZN-NPs significantly enhanced the oral bioavailability and prolonged half-life of GNA, providing a promising oral drug delivery system to improve in vivo pharmacokinetic behaviour of GNA.

Self-assembled micelles based on gambogenic acid-phospholipid complex for sustained-release drug delivery.

Objective: To improve the water solubility and enhance the oral bioavailability of gambogenic acid (GNA). Methods: GNA-phospholipid complex (GNA-PLC) micelles were successfully prepared by anti-solvent method. Results: The encapsulation efficiency of GNA-PLC micelles can reach 99.33 % (w/w). The average particle size of the GNA-PLC micelles was 291.23 nm which was approximate agreed with the transmission electron microscopy (TEM). In vitro release profile showed the GNA-PLC and GNA-PLC micelles have significant sustained-release of GNA compared with crude GNA. Pharmacokinetic parameters indicated that the area under concentration-time curve (AUC0→t) of GNA in cases of GNA-PLC and GNA-PLC micelles are 2.04- and 3.92-fold higher than crude GNA, respectively. Conclusions: The better water solubility and higher bioavailability of GNA in GNA-PLC micelles with significant sustained-release of GNA endow the nanoparticle with great potential in GNA delivery system.

Altering Fundamental Trends in the Emission of Xanthene Dyes.

Fluorescent small molecules enable researchers and clinicians to visualize biological events in living cells, tissues, and organs in real time. Herein, the focus is on the structure and properties of the relatively rare benzo[ a]xanthenes that exhibit enhanced steric and electronic interactions due to their annulated structures. Three types of fluorophores were synthesized: (i) pH- and solvent-dependent seminaphthorhodafluors, (ii) pH- and solvent-independent seminaphthorhodafluors, and (iii) pH-independent but solvent-sensitive seminaphthorhodamines. The probes exhibited promising far-red to near-infrared (NIR) emission, large Stoke shifts, broad full width at half-maximum (fwhm), relatively high quantum yields, and utility in immunofluorescence staining. Deviation of the π-system from planarity due to changes in the fluorophore ionization state resulted in fluorescence properties that are atypical of common xanthene dyes.

Study of the Mechanisms by Which Aminoglycoside Damage Is Prevented in Chick Embryonic Hair Cells.

A major side effect of aminoglycoside antibiotics is mammalian hair cell death. It is thus intriguing that embryonic chick hair cells treated with aminoglycosides at embryonic day (E) 12 are insensitive to ototoxicity. To exclude some unknown factors in vivo that might be involved in preventing aminoglycoside damage to embryonic hair cells, we first cultured chick embryonic basilar papilla (BP) with an aminoglycoside antibiotic in vitro. The results indicated that the hair cells were almost intact at E12 and E14 and were only moderately damaged in most parts of the BP at E16 and E18. Generally, hair cells residing in the approximate and abneural regions were more susceptible to streptomycin damage. After incubation with gentamicin-conjugated Texas Red (GTTR), which is typically used to trace the entry route of aminoglycosides, GTTR fluorescence was not remarkable in hair cells at E12, was weak at E14, but was relatively strong in the proximal part of BP at E18. This result indicates that the amounts of GTTR that entered the hair cells are related to the degrees of aminoglycoside damage. The study further showed that the fluorescence intensity of GTTR decreased to a low level at E14 to E18 after disruption of mechanotransduction machinery, suggesting that the aminoglycoside entry into hair cells was mainly through mechanotransduction channels. In addition, most of the entered GTTR was not found to be colocalized with mitochondria even at E18. This finding provides another reason to explain why embryonic chick hair cells are insensitive to aminoglycoside damage.

Preparation of Neogambogic Acid Nanoliposomes and its Pharmacokinetics in Rats.

OBJECTIVE: To prepare neogambogic acid nanoliposomes (GNA-NLC) and study its pharmacokinetics (PK) in rats. STUDY DESIGN: An experimental study. PLACE AND DURATION OF STUDY: Mudanjiang Medical University, Mudanjiang, China, from January 2016 to October 2017. METHODOLOGY: GNA-NLC was prepared by emulsion evaporation-low temperature solidification. The entrapment efficiency, average particle size, and zeta potential were investigated. Male Wistar rats were injected with 1 mg/mL gambogic acid and GNA-NLC into the caudal vein respectively, and the plasma concentration was determined by UPLC- MS/MS. The pharmacokinetic parameters of the two agents were compared. RESULTS: GNA-NLC prepared in this study were mostly spherical spheroids with an average particle size of 146.35 ±1.72 nm, polydispersity coefficient of 0.26 ±0.02, zeta potential of -28.24 ±0.13 MV, entrapment efficiency of 84.63%, and drug loading capacity of 4.23%. DSC showed that neogambogic acid nanoparticles had formed and neogambogic acid was amorphous in the matrix. The pharmacokinetics results in rats showed that GNA-NLC plasma concentration was significantly higher than that of common preparation of gambogic acid, with a half-life period of 10.14 ±0.03 hours, 4.57 times that of gambogic acid. AUC0 ~ 24h of gambogic acid in GNA-NLC lipidosome was 58.36 ±0.23 μg/h/mL, 4.83 times that of gambogic acid. CONCLUSION: GNA-NLC can be prepared successfully by emulsion evaporation-low temperature solidification. The method is simple and easy to control. The GNA-NLC has a long cycle, and high blood concentration, sustained release compared with the raw material gambogic acid.

Absorption, Metabolism, and Pharmacokinetics Profiles of Norathyriol, an Aglycone of Mangiferin, in Rats by HPLC-MS/MS.

Norathyriol, an aglycone of mangiferin, is a bioactive tetrahydroxyxanthone present in mangosteen and many medicinal plants. However, the biological fate of norathyriol in vivo remains unclear. In this study, the absorption and metabolism of norathyriol in rats were evaluated through HPLC-MS/MS. Results showed that norathyriol was well absorbed, as indicated by its absolute bioavailability of 30.4%. Besides, a total of 21 metabolites of norathyriol were identified in rats, including methylated, glucuronidated, sulfated and glycosylated conjugates, which suggested norathyriol underwent extensive phase II metabolism. Among those metabolites, 15 metabolites were also identified in hepatocytes incubated with norathyriol, indicating the presence of hepatic metabolism. Furthermore, glucuronide and sulfate conjugates, rather than their parent compound, were found to be the main forms existing in vivo after administration of norathyriol, as implicated by the great increase of exposure of norathyriol determined after hydrolysis with ß-glucuronidase and sulfatase. The information obtained from this study contributes to better understanding of the pharmacological mechanism of norathyriol.

PEGylated liposomes as delivery systems for Gambogenic acid: Characterization and in vitro/in vivo evaluation.

Gambogenic acid (GNA), which possesses diverse anti-tumor activities both in vitro and in vivo, is regarded as a potential anticancer compound. However, the excessive irritation to the blood vessel, short elimination half-life and poor aqueous solubility restricted its clinical application. In this study, Gambogenic acid-loaded PEGylated liposomes (GNA-PEG-LPs) were developed to reduce toxicity, prolong the half-life and enhance anticancer efficacy both in vitro and in vivo. The average particle size of GNA-PEG-LPs was 90.13 ± 0.16 nm and their polydispersity index (PDI) was 0.092 ± 0.003. The encapsulation efficiency, drug loading and zeta potential of GNA-PEG-LPs were 88.13 ± 1.31%, 3.72 ± 0.04%, -22.10 ± 0.20 mV, respectively. Compared to free GNA, GNA-PEG-LPs showed enhanced cytotoxicity and apoptosis induction effect against A549, SGC-7901 and HepG2 cells. Mechanistically, western blot analysis revealed that up-regulation of Bax, down-regulation of Bcl-2 and activation of caspase-3 contributed to apoptosis. In addition, the blood vessel irritation test showed that the vascular irritation of GNA could be reduced by liposomal encapsulation. The hemolysis assay revealed that good hemocompatibility of the liposomes. Furthermore, pharmacokinetic study showed that the t1/2 and the AUC of GNA-PEG-LPs were higher than GNA solution approximately 2.84-fold and 2.97-fold, respectively. In vivo antitumor efficacy demonstrated that GNA-PEG-LPs significantly inhibited the tumor growth in LLC tumor-bearing C57BL/6 mouse model. Results of Immunohistochemistry indicated that GNA-PEG-LPs significantly suppressed the expression of Bcl-2 and increased the expression of Bax and caspase-3 compared with free GNA. Collectively, PEGylated liposomes could be a potential nanocarrier to prolong half-life, reduce toxicity and enhance anticancer efficacy both in vitro and in vivo.

Focused ultrasound combined with microbubble-mediated intranasal delivery of gold nanoclusters to the brain.

Focused ultrasound combined with microbubble-mediated intranasal delivery (FUSIN) is a new brain drug delivery technique. FUSIN utilizes the nasal route for direct nose-to-brain drug administration, thereby bypassing the blood-brain barrier (BBB) and minimizing systemic exposure. It also uses FUS-induced microbubble cavitation to enhance transport of intranasally (IN) administered agents to the FUS-targeted brain location. Previous studies have provided proof-of-concept data showing the feasibility of FUSIN to deliver dextran and the brain-derived neurotrophic factor to the caudate putamen of mouse brains. The objective of this study was to evaluate the biodistribution of IN administered gold nanoclusters (AuNCs) and assess the feasibility and short-term safety of FUSIN for the delivery of AuNCs to the brainstem. Three experiments were performed. First, the whole-body biodistribution of IN administered 64Cu-alloyed AuNCs (64Cu-AuNCs) was assessed using in vivo positron emission tomography/computed tomography (PET/CT) and verified with ex vivo gamma counting. Control mice were intravenously (IV) injected with the 64Cu-AuNCs. Second, 64Cu-AuNCs and Texas red-labeled AuNCs (TR-AuNCs) were used separately to evaluate FUSIN delivery outcome in the brain. 64Cu-AuNCs or TR-AuNCs were administered to mice through the nasal route, followed by FUS sonication at the brainstem in the presence of systemically injected microbubbles. The spatial distribution of 64Cu-AuNCs and TR-AuNCs were examined by autoradiography and fluorescence microscopy of ex vivo brain slices, respectively. Third, histological analysis was performed to evaluate any potential histological damage to the nose and brain after FUSIN treatment. The experimental results revealed that IN administration induced significantly lower 64Cu-AuNCs accumulation in the blood, lungs, liver, spleen, kidney, and heart compared with IV injection. FUSIN enhanced the delivery of 64Cu-AuNCs and TR-AuNCs at the FUS-targeted brain region compared with IN delivery alone. No histological-level tissue damage was detected in the nose, trigeminal nerve, and brain. These results suggest that FUSIN is a promising technique for noninvasive, spatially targeted, and safe delivery of nanoparticles to the brain with minimal systemic exposure.

Photo- and Reduction-Responsive Polymersomes for Programmed Release of Small and Macromolecular Payloads.

We report on the preparation of photo- and reduction-responsive diblock copolymers through reversible addition-fragmentation chain transfer (RAFT) polymerization of a coumarin-based disulfide-containing monomer (i.e., CSSMA) using a poly(ethylene oxide) (PEO)-based macroRAFT agent. The resulting amphiphilic PEO- b-PCSSMA copolymers self-assembled into polymersomes with hydrophilic PEO shielding coronas and hydrophobic bilayer membranes. Upon irradiating the polymersomes with visible light (e.g., 430 nm), the coumarin moieties within the bilayer membranes were cleaved with the generation of primary amine groups, which spontaneously underwent inter/intrachain amidation reactions with the ester moieties, thereby tracelessly cross-linking and permeating the bilayer membranes. Notably, this process only gave rise to the release of small molecule payloads (e.g., doxorubicin hydrochloride, DOX) while large molecule encapsulants (e.g., Texas red-labeled dextran, TR-dextran) were retained within the cross-linked polymersomes due to the preservation of the integrity of the vesicular nanostructures. However, cross-linked polymersomes undergo further structural disintegration upon incubation with glutathione (GSH) due to the scission of disulfide linkages, resulting in the release of macromolecular payloads. Thus, dual-stimuli responsive polymersomes with tracelessly cross-linkable characteristics enable sequential release of payloads with spatiotemporal precision, which could be of promising applications in synergistic loading and programmed release of therapeutics.

Validated LC-MS/MS method for quantitation of demethylbellidifolin in rat plasma and its application to pharmacokinetic and bioavailability studies.

Demethylbellidifolin, a major xanthone compound of Swertia davidi Franch, shows many beneficial pharmacological effects including antioxidation, anti-inflammation, anti-fibrosis and cardiovascular protection effects. In this research, a rapid and sensitive LC-MS/MS method for the quantitative analysis of demethylbellidifolin in rat plasma was developed. The demethylbellidifolin and internal standard of aurantio-obtusin were extracted from 50 µL of rat plasma samples with ethyl acetate, then the dried residue was reconstituted and injected in an HPLC system with Zorbax SB-C18 analytical column (2.1 × 100 mm, 3.5 µm) and eluted with the mobile phase consisting of methanol and 0.2% formic acid aqueous solution (80:20, v/v). Quantification was performed using a TSQ Quantum Ultra mass spectrometer in negative ESI using selected reaction monitoring mode of the transitions m/z 259.1 → 215.1 for demethylbellidifolin and 329.0 → 314.2 for the IS. Excellent linearity was observed between 1.92 and 960 ng/mL with a limit of quantitation of 1.92 ng/mL. Intra- and inter-day precision (RSD) values of quality control samples were both <8.3%. This study was successfully utilized for the pharmacokinetic profiles of demethylbellidifolin in rats after oral or intravenous administration. The oral bioavailability of demethylbellidifolin was 3.6%.

[Nasal resorption of prim-O-glucosylcimifugin and 5-O-methylvisammioside in rats].

In this experiment, rat nasal mucosa absorption characteristics of prim-O-glucosylcimifugin and 5-O-methylvisammioside were studied to provide a basis for drug delivery of Toutongning nasal spray. The nasal mucosa absorption test in rats was conducted with in situ nasal perfusion method after pH 6 buffer solution was used to prepare high, medium and low concentrations of prim-O-glucosylcimifugin, 5-O-methylvisammioside mixed solution as liquid circulation in nasal cavity. Then the concentrations of the circulating liquid compositions to be measured were determined by HPLC, and the absorption rates of prim-O-glucosylcimifugin and 5-O-methylvisammioside under different pH conditions were also investigated. According to the results, the absorption rate constant was (0.588±0.041)×10⁻³, (0.547±0.023)×10⁻³, (0.592±0.063)×10⁻³ min⁻¹ for prim-O-glucosylcimifugin high, middle and low concentrations, and (0.438±0.041)×10⁻³, (0.407±0.023)×10⁻³, and (0.412±0.063)×10⁻³ min⁻¹ for 5-O-methylvisammioside high, middle and low concentrations. There was no significant difference among high, middle and low concentration groups, and the absorption under pH 6 was better than that under other pH conditions. Therefore, we can get the conclusion that the main active ingredient of Toutongning nasal sprays can be absorbed through the nasal mucosa, and it is feasible to make nasal spray; in addition, pH 6 of nasal spray is scientific and reasonable.

Long circulation nanostructured lipid carriers for gambogenic acid: formulation design, characterization, and pharmacokinetic.

1. GNA-PEG-NLC and GNA-NLC were prepared by emulsification and low-temperature solidification methods. The optimized GNA-PEG-NLC and GNA-NLC were not only found to have small mean size (146.33 ± 2.11 and 144.07 ± 1.44) nm, high Zeta potential (-25.10 ± 1.35 and -28.03 ± 0.29) mV, but also great entrapment efficiency (79.07 ± 1.11 and 84.65 ± 0.98%). TEM proved that particles were nearly spherical with smooth surface shape. Furthermore, in vitro release revealed a burst release initially, followed by a sustained profiles up to 48 h, and the cumulative drug release of GNA-PEG-NLC and GNA-NLC was 65.90 ± 2.34% and 69.25 ± 1.77%, respectively. 2. In pharmacokinetic, GNA-PEG-NLC exhibited prolonged MRT and higher AUC values compared with GNA-NLC and GNA solution. Moreover, the tissue distribution demonstrated a high uptake of GNA-PEG-NLC in stomach. 3. These results indicated that PEG-NLC is a promising delivery system for GNA, which could prolong drug circulation time in body and thus improved its bioavailability.

Pharmacokinetics of mangiferin and its metabolite-Norathyriol, Part 1: Systemic evaluation of hepatic first-pass effect in vitro and in vivo.

Mangiferin (MGF), a glucoside of xanthone existing in phytomedicines and food, is increasingly attracting attention on diabetes treatment, while the underlying mechanism leading to its low oral bioavailability is unclear. Norathyriol (NTR), an active metabolite with hypoglycemic activity and its exposure after MGF dosing remains unclear. Hence, a rapid and sensitive LC-MS/MS method was established and validated to determine MGF and NTR and applied in the PK study in rats. Correspondingly, the in vitro experiments on temperature-dependent uptake, and MGF metabolism in hepatocyte and enterobacteria samples were performed. Results revealed that hepatic first-pass effect slightly contributed to the poor bioavailability of MGF, based on the MGF exposure in portal vein plasma was nearly similar to that in systemic plasma, and the MGF accumulation in the liver was limited, so was that of NTR. Correspondingly, the in vitro study revealed the MGF uptake was mainly dependent on poor passive transport, possibly leading to its limited hepatic metabolism and accumulation. Moreover, the NTR exposure remained considerably low (Cmax < 3 ng/mL, AUCNTR /AUCMGF < 3%) in plasma after single MGF dosing, corresponding to its tiny proportion (0.1%) of MGF in MGF-incubated enterobacteria samples. However, given the low generation and elimination rates of NTR, NTR might accumulate in plasma and exert effects after repeated MGF dosing, although requires further study. This work is the first systemic study on PK profiles of MGF and NTR in vitro and in vivo, which is important for the interpretation on the poor bioavailability and pharmacodynamics of MGF. © 2016 BioFactors, 42(5):533-544, 2016.

Pharmacokinetics of mangiferin and its metabolite-norathyriol, Part 2: Influence of UGT, CYP450, P-gp, and enterobacteria and the potential interaction in Rhizoma Anemarrhenae decoction with timosaponin B2 as the major contributor.

The poor bioavailability of mangiferin (MGF) is a major obstacle on its further development. Aimed to illustrate the underlying mechanism and improve its poor exposure, the compared PK profiles of MGF and norathyriol (NTR) after different MGF preparation were performed: pure MGF, the Rhizoma Anemarrhenae (Zhi-mu) decoction, MGF, and timosaponin B2 (TB-2) combination. Furthermore, the potential contributing factors, including uridine diphosphoglucuronosyltransferase (UGT), cytochrome P450 (CYP450), P-gp, and enterobacterial were investigated by comparing the PK profiles with and without the corresponding inhibitors or in different rat models. After taking MGF, CYP450 and UGT inhibition could decrease MGF and NTR exposure; P-gp inhibition slightly enhanced (48%) MGF exposure, whereas more apparent for the improved NTR exposure (302%); enterobacterial inhibition almost completely stopped the NTR production, but no such effect was observed for MGF. Compared with the limited improvement by the abovementioned inhibition, the MGF and NTR exposure could significantly increase by 11.5- and 5.9-fold in the Zhi-mu decoction compared with the MGF treatment, probably contributed to TB-2 as an absorption enhancer because the MGF and TB-2 combination produced a similar level of improvement on the PK paremeters of MGF and NTR to the herb treatment. Likewise, most of the effects by UGT, CYP450, P-gp, and enterobacteria followed a similar variation tendency between them. Therefore, the poor bioavailability of MGF possibly mainly attributed to its poor membrane permeability, but not transporters or metabolic enzymes, and the compatibility of MGF and TB-2 could probably expand the prospective application of MGF by improving its bioavailability. © 2016 BioFactors, 42(5):545-555, 2016.

Gambogenic acid inhibits LPS-simulated inflammatory response by suppressing NF-κB and MAPK in macrophages.

Inflammation is a response of body tissues to injury and infection. Compounds that can inhibit inflammation have been shown to have potential therapeutic clinical application. Gambogenic acid (GEA) has potent antitumor and anti-inflammatory activities. Herein, the molecular mechanisms of GEA's anti-inflammatory effect were investigated in lipopolysaccharide (LPS)-stimulated macrophage cells. The results showed that pretreatment with GEA could markedly inhibit interleukin (IL)-1α, IL-1ß, tumor necrosis factor-α, IFN-ß, IL-12b, and IL-23a production in a dose-dependent manner in LPS-induced model. Furthermore, this drug significantly reduced the release of nitric oxide (NO), and impaired the protein level of inducible NO synthase and the cyclooxygenase 2. The finding also showed that the effect of GEA may be related to the suppression of the nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathway. These results indicate that GEA could suppress LPS-simulated inflammatory response partially by attenuating NO synthesis and NF-κB and MAPK activation, suggesting that it may become a potent therapeutic agent for the treatment of inflammatory diseases.

High Throughput Danio Rerio Energy Expenditure Assay.

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers).

The antidepressant-like activity of 6-methoxy-2-[4-(2-methoxyphenyl)piperazin-1-yl]-9H-xanthen-9-one involves serotonergic 5-HT(1A) and 5-HT(2A/C) receptors activation.

Xanthone derivatives have been shown to posses many biological properties. Some of them act within the central nervous system and show neuroprotective or antidepressant-like properties. Taking this into account we investigated antidepressant-like activity in mice and the possible mechanism of action of 6-methoxy-2-[4-(2-methoxyphenyl)piperazin-1-yl]-9H-xanthen-9-one (HBK-11) - a new xanthone derivative. We demonstrated that HBK-11 produced antidepressant-like effects in the forced swim test and tail suspension test, comparable to that of venlafaxine. The combined treatment with sub-effective doses of HBK-11 and fluoxetine (but not reboxetine or bupropion) significantly reduced the immobility in the forced swim test. Moreover, the antidepressant-like activity of HBK-11 in the aforementioned test was blocked by p-chlorophenylalanine, and significantly reduced by serotonergic 5HT1A receptor antagonist - WAY-1006335 and 5HT2A/C receptor antagonist - ritanserin. As none of the above treatments influenced the spontaneous locomotor activity, it can be concluded that HBK-11 mediates its activity through a serotonergic system, and its antidepressant-like effect involves 5HT1A and 5HT2A/C receptor activation. Furthermore, at antidepressant-like doses HBK-11 did not cause the mice to display locomotor deficits in rotarod or chimney tests. Considering the pharmacokinetic profile, HBK-11 demonstrated rapid absorption after i.p. administration, high clearance value, short terminal half-life, very high volume of distribution and incomplete bioavailability. The compound studied had good penetration into the brain tissue of mice. Since studied xanthone derivative seems to present interesting, untypical mechanism of antidepressant-like action i.e. 5HT2A/C receptor activation, it may have a potential in the treatment of depressive disorders, and surely requires further studies.

Simultaneous determination and pharmacokinetic study of gambogic acid and gambogenic acid in rat plasma after oral administration of Garcinia hanburyi extracts by LC-MS/MS.

Gambogic acid and gambogenic acid are two major bioactive components of Garcinia hanburyi, and play a pivotal role in biologic activity. In this study, a specific and sensitive liquid chromatography-tandem mass spectrometry was developed and validated for simultaneous determination of gambogic acid and gambogenic acid in rat plasma. Chromatographic separation was achieved on a C18 column using an isocratic elution with methanol-10 m m ammonium acetate buffer-acetic acid (90:10:0.1, v/v/v) as the mobile phase. The detection was performed on a triple-quadrupole tandem mass spectrometer equipped with electrospray positive ionization using multiple reaction monitoring modes. The transitions monitored were m/z 629.3 [M + H](+) → 573.2 for gambogic acid, m/z 631.2 [M + H](+) → 507.2 for gambogenic acid and m/z 444.2 [M + NH4 ](+) → 83.1 for IS. Linear calibration curves were obtained in the concentration range of 2.00-1000 ng/mL for gambogic acid and 0.500-250 ng/mL for gambogenic acid. The lower limits of quantification of gambogic acid and gambogenic acid in rat plasma were 2.00 and 0.500 ng/mL, respectively. The intra- and inter-day precision (RSD) values were <11.7% and accuracy (RE) was -10.6-12.4% at three QC levels for both analytes. The assay was successfully applied to evaluate pharmacokinetics behavior in rats after oral administration of Garcinia hanburyi extracts.