Decreased plasma exposure of clopidogrel active metabolite in rats after long-term treatment with clopidogrel.

Clopidogrel (Clop) is oxidized by cytochrome P450s (CYPs) to an active thiol metabolite, Clop-AM, to inhibit platelet activation and aggregation. As an irreversible inhibitor of CYP2B6 and CYP2C19, clopidogrel may inhibit its own metabolism after long-term administration. The study compared the pharmacokinetic profiles of clopidogrel and its metabolites in rats receiving a single or a 2 week administration of Clop. The mRNA and protein levels of hepatic clopidogrel-metabolizing enzymes and their enzymatic activities were analyzed to explore their contribution to any altered plasma exposure of Clop and its metabolites. The results showed that long-term treatment with clopidogrel significantly decreased the AUC(0-t) and Cmax values of Clop-AM in rats, accompanied with markedly impaired catalytic activities of Clop-metabolizing CYPs including CYP1A2, CYP2B6, CYP2C9, CYP2C19, and CYP3A4. It suggests that consecutive administration of Clop to rats decreases hepatic CYPs activities, which may, in turn, inhibit clopidogrel metabolism and then reduce Clop-AM plasma exposure. Therefore, long-term treatment with clopidogrel has the potential to reduce its anti-platelet activity and to increase the risk of drug-drug interaction.

Study on the Interaction between Deuterium Clopidogrel and Omeprazole.

INTRODUCTION: Dual antiplatelet therapy with aspirin in combination with a P2Y12 receptor antagonist is a cornerstone for treating patients with acute coronary syndrome and in percutaneous coronary intervention. However, as this combination of antiplatelet therapy increases the risk of bleeding, proton pump inhibitors (PPIs) are currently recommended to prevent gastrointestinal ulcers and bleeding. The cytochrome P450 (CYP450) isoenzyme system metabolizes both clopidogrel (CLP) and PPIs. Unfortunately, omeprazole (OM) reduce the antiplatelet activity of CLP and increases the probability of recurrence of cardiovascular events by competitively inhibiting the CYP450 isoenzyme CYP2C19. METHODS: To address these abovementioned problems, we designed and synthesized deuterium CLP (D-CL) using selective deuterium technology. Our previous research results showed that D-CL had better pharmacokinetic and pharmacodynamic properties. Thus, the HPLC-MS/MS method, cocktail method, Born method, electro-stimulated thrombus generation, and thrombus elastography were used to detect the production of thiol active metabolites (AM), CYP450 enzyme activities, platelet aggregation, time and length of thrombus formation, and the maximum clot strength after combination therapy. We investigated the pharmacokinetics and pharmacodynamics properties of D-CL combined with OM. RESULTS: As compared to CLP, D-CL was less affected when combined with OM, which was reflected in lower inhibitory effects of CYP450 enzyme activities, a greater area under the curve of AM, and better antiplatelet and antithrombotic effects. CONCLUSION: D-CL may reduce drug-drug interactions and address the clinical disadvantages of CLP.

Ultrahigh-throughput absolute quantitative analysis of linezolid in human plasma by direct analysis in real time mass spectrometry without chromatographic separation and its application to a pharmacokinetic study.

Therapeutic drug monitoring (TDM) is necessary in the clinical management of linezolid to improve its efficacy and reduce the risk of time- and dose-dependent toxicity. A novel and ultrahigh-throughput analytical method for the determination of linezolid in human plasma was developed based on direct analysis in real-time tandem mass spectrometry (DART-MS/MS) without chromatographic separation. After solid-phase extraction with Waters Oasis HLB, the linezolid and internal standard linezolid-d3 were detected by positive ion electrospray ionization followed by multiple reaction monitoring (MRM) of the transition at m/z 338.1 → 296.2 and 341.2 → 297.3, respectively. The use of DART-MS obviates the need for chromatographic separation and allowed determination of linezolid in a total run time of only 24 s per sample. The method was linear in the concentration range 0.20-25 µg mL-1 with intraday and interday precision <14.5% and accuracy ranging from -3.85% to 12.7%. The method was successfully applied to a pharmacokinetic study of linezolid in healthy male volunteers after oral administration of a 600 mg tablet. DART-MS/MS provides a rapid and sensitive method for the determination of linezolid that does not require chromatographic separation. It is eminently suitable to meet the high-throughput challenge of clinical TDM. Graphical abstract.

Development and Application of an MSALL-Based Approach for the Quantitative Analysis of Linear Polyethylene Glycols in Rat Plasma by Liquid Chromatography Triple-Quadrupole/Time-of-Flight Mass Spectrometry.

Polyethylene glycols (PEGs) are synthetic polymers composed of repeating ethylene oxide subunits. They display excellent biocompatibility and are widely used as pharmaceutical excipients. To fully understand the biological fate of PEGs requires accurate and sensitive analytical methods for their quantitation. Application of conventional liquid chromatography-tandem mass spectrometry (LC-MS/MS) is difficult because PEGs have polydisperse molecular weights (MWs) and tend to produce multicharged ions in-source resulting in innumerable precursor ions. As a result, multiple reaction monitoring (MRM) fails to scan all ion pairs so that information on the fate of unselected ions is missed. This Article addresses this problem by application of liquid chromatography-triple-quadrupole/time-of-flight mass spectrometry (LC-Q-TOF MS) based on the MSALL technique. This technique performs information-independent acquisition by allowing all PEG precursor ions to enter the collision cell (Q2). In-quadrupole collision-induced dissociation (CID) in Q2 then effectively generates several fragments from all PEGs due to the high collision energy (CE). A particular PEG product ion (m/z 133.08592) was found to be common to all linear PEGs and allowed their total quantitation in rat plasma with high sensitivity, excellent linearity and reproducibility. Assay validation showed the method was linear for all linear PEGs over the concentration range 0.05-5.0 µg/mL. The assay was successfully applied to the pharmacokinetic study in rat involving intravenous administration of linear PEG 600, PEG 4000, and PEG 20000. It is anticipated the method will have wide ranging applications and stimulate the development of assays for other pharmaceutical polymers in the future.

[Effects of isocaloric diets with different percentage energy from fat on blood glucose and lipids in rats].

OBJECTIVE: To investigate the effects of isocaloric diets with different percentage energy from fat on blood glucose and lipids in rats. METHODS: Forty male SD rats were assigned to low fat group, normal fat group, medium fat group and high fat group using randomized blocks method. Rats in four groups were fed with isocaloric diets whose percentage energy from fat were 5%, 15%, 25% and 40% respectively for 10 weeks. Body weight and body length of rats were measured every week. Blood glucose, blood lipids and insulin were determined at 0, 5th and 10 th week. The perirenal fat and epididymal fat pad were separated and weighed at the end of the 10 th week and the body fat rate was calculated. RESULTS: At the end of the 5th and 10 th week, there were no significant differences among four groups in body weight, Lee's index, body fat rate, insulin and low density lipoprotein cholesterol. At the end of the 10 th week, the level of blood glucose was higher for rats in the high fat group than those in the low fat group( P <0. 01). The level of total cholesterol, triglyceride and high density lipoprotein cholesterol were lower for rats in the high fat group than those in the low fat group( P < 0. 01). The level of blood glucose of the 10 th week was higher for rats in the high fat group than the level at the beginning( P < 0. 05). The level of total cholesterol, low density lipoprotein cholesterol and high density lipoprotein cholesterol of the 10 th week was higher for rats in the high fat group than the level at the beginning( P < 0. 01). CONCLUSION: When rats were fed with a isocaloric diet and in the condition of normal growth, diet with high proportion of energy derived from fat would not lead to overweight in rats, whereas it may change the metabolism of blood glucose and lipids.

Rapid and sensitive liquid chromatography with tandem mass spectrometry method for the simultaneous quantification of yonkenafil and its major metabolites in rat plasma.

Yonkenafil is a promising drug for treatment of male erectile dysfunction. Previous studies showed that the piperazine-N,N'-deethylation metabolite, piperazine-N-deethylation metabolite, and piperazine-N-deethylation-N,N'-deethylation metabolite were the major metabolites of yonkenafil after extensive metabolism. We developed a sensitive and selective method for the simultaneous quantification of yonkenafil and its major metabolites using high-throughput liquid chromatography with tandem mass spectrometry. Analytes and internal standard were extracted from a small quantity of plasma (50 µL) using liquid-liquid extraction with diethyl ether/dichloromethane (60:40, v/v), and the baseline separation was achieved on Zorbax SB-C18 column using ammonia/water/methanol (0.2:20:80, v/v/v) as the mobile phase. The assay was performed with an electrospray positive ionization mass spectrometry through the multiple-reaction monitoring mode within 2 min. Calibration curve of the method was linear within the range of 1.00-1000 ng/mL for all the analytes with the intra- and interday precisions of 4.0-5.2 and 4.0-5.3% for yonkenafil, 3.1-4.9 and 3.1-5.2% for the piperazine-N,N'-deethylation metabolite, 4.8-6.8 and 4.8-7.3% for the piperazine-N-deethylation metabolite, and 2.9-6.1 and 5.4-6.3% for the piperazine-N-deethylation-N,N'-deethylation metabolite, respectively. The recoveries were above 90% with low matrix effects. The validated assay was successfully applied to support a preclinical pharmacokinetic study in six rats using a single oral dose of yonkenafil (8 mg/kg).

Trantinterol, a novel ß2-adrenoceptor agonist, noncompetitively inhibits P-glycoprotein function in vitro and in vivo.

P-glycoprotein (P-gp)-mediated drug-drug interactions are important factors causing adverse effects of drugs in clinical use. The aim of this study was to determine whether trantinterol (also known as SPFF), a novel ß2-adrenoceptor agonist, was a P-gp inhibitor or substrate. The results showed that trantinterol was not a substrate of P-gp but increased rhodamine 123 (Rho 123) uptake by MDCK-MDR1 cells and decreased the efflux transport of both Rho 123 and cyclosporine A (CsA) in bidirectional transport studies across MDCK-MDR1 cell monolayers. This suggested that trantinterol was a P-gp inhibitor but not a P-gp substrate. The mechanism of inhibition was investigated in the P-gp-Glo assay system, where it was found that trantinterol inhibited P-gp ATPase activity in a dose-dependent manner. A subsequent study using the antibody binding assay with the conformation-sensitive P-gp-specific antibody UIC2 confirmed that trantinterol decreased UIC2 binding at 10 µM in contrast to the competitive inhibitor, verapamil. This suggested that trantinterol was a noncompetitive inhibitor of P-gp. Finally, a pharmacokinetic study in rat showed that trantinterol significantly increased the area under the plasma concentration-time curve (AUC) and maximum plasma concentration (Cmax) of digoxin and paclitaxel (PAC), and the Cmax of cyclosporine A (CsA). In summary, trantinterol is a potent noncompetitive P-gp inhibitor which may increase the bioavailability of other P-gp substrate drugs coadministered with it.

Determination of lansoprazole enantiomers in dog plasma by column-switching liquid chromatography with tandem mass spectrometry and its application to a preclinical pharmacokinetic study.

Lansoprazole, a selective proton pump inhibitor, has a chiral benzimidazole sulfoxide structure and is used for the treatment of gastric acid hypersecretory related diseases. To investigate its stereoselective pharmacokinetics, a column-switching liquid chromatography with tandem mass spectrometry method was developed for the determination of lansoprazole enantiomers in dog plasma using (+)-pantoprazole as an internal standard. After a simple protein precipitation procedure with acetonitrile, matrix components left behind after sample preparation were further eliminated from the sample by reversed-phase chromatography on a C18 column. The fluent was fed to a chiral column for the separation of lansoprazole enantiomers. Baseline separation of lansoprazole enantiomers was achieved on a Chiralcel OZ-RH column using acetonitrile/0.1% formic acid in water (35:65, v/v) as the mobile phase at 40°C. The linearity of the calibration curves ranged from 3 to 800 ng/mL for each enantiomer. Intra- and inter-day precisions ranged from 2.1 to 7.3% with an accuracy of ±1.7% for (+)-lansoprazole, and from 1.6 to 6.9% with an accuracy of ±3.5% for (-)-lansoprazole, respectively. The validated method was successfully applied for the stereoselective pharmacokinetic study of lansoprazole in beagle dog after intravenous infusion.

Determination of thymopentin in beagle dog blood by liquid chromatography with tandem mass spectrometry and its application to a preclinical pharmacokinetic study.

The pentapeptide thymopentin (Arg-Lys-Asp-Val-Tyr, RKDVY) corresponds to amino acids 32-36 of the 49 amino acid immunomodulatory polypeptide, thymopoietin, whose biological activity is partially reproduced. Thymopentin is widely used in the clinic and represents a promising target for drug design but bioanalytical and pharmacokinetic data are limited due to its enzymatic instability. This paper reports a rapid and sensitive method based on liquid chromatography with tandem mass spectrometry for the determination of thymopentin in beagle dog blood. To inactivate peptidases and stabilize thymopentin, acetonitrile was added to blood samples immediately after collection followed by addition of stable isotope-labeled thymopentin as internal standard and washing with dichloromethane. Chromatography was carried out on an Ascentis Express Peptide ES-C18 column using gradient elution with methanol and aqueous 0.1% formic acid at a flow rate of 0.6 mL/min. Positive electrospray ionization mass spectrometry with selected reaction monitoring achieved linearity in the range of 1.5-800 ng/mL with good accuracy/precision and minimal matrix effects. The method was successfully applied to a pharmacokinetic study in beagle dogs after intravenous administration of 0.2 mg/kg thymopentin.

[Effect of different dietary fat intake on blood lipids, body fat, adiponectin and leptin on energy balance status in rats].

OBJECTIVE: To investigate the effects of different dietary fat intake on body fat, adiponectin and leptin on energy balance status in rats. METHODS: Forty male SD rats were randomly assigned to four groups. Rats in low fat, normal fat, medium fat and high fat group were fed equal energy diets of low fat diet (5% energy from fat), normal diet (15% energy from fat), medium fat diet (25% energy from fat) and high fat diet (40% energy from fat) respectively. Blood glucose and lipids were analyzed at 0, 5 and 10 weeks. The level of serum adiponectin and leptin was tested at 0 and 10 weeks. At the end of 10 weeks, the rats were sacrificed, the perirenal and periepididymis fat were separated and weighed. The mRNA of adiponectin and leptin in fat tissues were determined by realtime PCR. RESULTS: After the 5 and 10 weeks, the levels of serum triglyceride of rats in medium fat group and high fat group were lower than those in low fat group and normal fat group. At the end of 10 weeks, the expression of adiponectin mRNA in fat tissues in medium fat group was lower than those in low fat group. There were no significant differences among four groups in body fat, blood glucose, blood cholesterol, serum adiponectin and leptin, and the expression of leptin mRNA in fat tissues. CONCLUSION: In energy balance status, different dietary fat intake had no effects on body fat, blood glucose, blood cholesterol, serum adiponectin and leptin in rats.

Evaluation of calcipotriol transdermal permeation through pig, rat and mouse skin using liquid chromatography-tandem mass spectrometry.

A rapid and sensitive liquid chromatography-tandem mass spectrometric method to evaluate the permeation and retention of calcipotriol in excised samples of pig, rat and mouse skin after application of a calcipotriol ointment has been developed and validated. After sample preparation of ointment, skin homogenate and receptor medium by liquid-liquid extraction, chromatography was performed on an Extend-C18 column using isocratic elution. Detection was by electrospray ionization in the negative ion mode using multiple-reaction monitoring of the precursor to product ion transitions of calcipotriol at m/z 411.1 → 393.5, and of lovastatin (internal standard) at m/z 403.2 → 101.2. The assay was linear in all matrices with LLOQs of 1, 0.5 and 40 ng/mL for skin homogenate, receptor medium and ointment samples respectively. In terms of the permeation profiles, it was found that calcipotriol permeated through all skins to only a limited extent over 20 h after application but was efficiently retained in all skins at a level at 20 h of between 40% (pig) and 60% (rat and mouse) of the applied dose. This indicates that calcipotriol ointment has the potential to provide sustained therapeutic benefit in the treatment of psoriasis with minimal systemic side effects.

Antagonism of androgen receptor signaling by aloe-emodin.

Over the past decades, androgen receptor (AR) signaling has been a key driver of both primary and recurrent prostate cancer. In this work, aloe-emodin was identified as a novel AR antagonist, effectively inhibiting AR signaling. Firstly, aloe-emodin can inhibit LNCaP cell growth by promoting apoptosis. Then, the results of Western blot and quantitative real-time PCR further confirmed that aloe-emodin modulated AR protein levels by promoting AR proteasomal degradation, and also inhibited the transcription of the AR downstream target genes, including PSA, KLK2, and TMPRSS2. Furthermore, the result of immunofluorescence showed that aloe-emodin prevented the nuclear translocation of AR. Molecular docking and molecular dynamics simulation suggested that aloe-emodin combined with AR to form stable complexes, which might explain that aloe-emodin prevented the translocation of AR from the cytoplasm to the nucleus by affecting the ligand binding of AR. Therefore, aloe-emodin as a novel AR antagonist may play a crucial role in promoting cancer prevention or complementing pharmacological therapies in the treatment of prostate cancer.

Identification and high-throughput quantification of baicalein and its metabolites in plasma and urine.

ETHNOPHARMACOLOGICAL RELEVANCE: Scutellaria baicalensis Georgi. contains varieties of function compounds, and it has been used as traditional drug for centuries. Baicalein is the highest amount of flavonoid found in Scutellaria baicalensis Georgi., which exerts various pharmacological activities and might be a promising drug to treat COVID-19. AIM OF THE STUDY: The present work aims to investigate the metabolism of baicalein in humans after oral administration, and study the pharmacokinetics of BA and its seven metabolites in plasma and urine. MATERIALS AND METHODS: The metabolism profiling and the identification of baicalein metabolites were performed on HPLC-Q-TOF. Then a column-switching method named MPX™-2 system was applied for the high-throughput quantificationof BA and seven metabolites. RESULTS: Seven metabolites were identified using HPLC-Q-TOF, including sulfate, glucuronide, glucoside, and methyl-conjugated metabolites. Pharmacokinetic study found that BA was extensively metabolized in vivo, and only 5.65% of the drug remained intact in the circulatory system after single dosing. Baicalein-7-O-sulfate and baicalein-6-O-glucuronide-7-O-glucuronide were the most abundant metabolites. About 7.2% of the drug was excreted through urine and mostly was metabolites. CONCLUSION: Seven conjugated metabolites were identified in our assay. A high-throughput HPLC-MS/MS method using column switch was established for quantifying BA and its metabolites. The method has good sensitivity and reproducibility, and successfully applied for the clinical pharmacokinetic study of baicalein and identified metabolites. We expect that our results will provide a metabolic and pharmacokinetic foundation for the potential application of baicalein in medicine.

Quercetin ameliorates hepatic fat accumulation in high-fat diet-induced obese mice via PPARs.

As a natural pigment in food, quercetin possesses multiple biological activities and plays a crucial role in regulating metabolic syndrome. Herein, we aim to explore the potential mechanism of quercetin to ameliorate hepatic fat accumulation. In vivo experiments showed that quercetin significantly relieved inflammation response by decreasing the serum TNF-α and IL-6 levels and also improved high-fat diet-induced hepatic steatosis without other organ injuries. Quercetin can effectively reduce lipid aggregation and down-regulate the protein expression of PCK1 in HepG2 cells induced by oleic acid and palmitic acid, indicating that inhibiting gluconeogenesis leads to hepatic fat accumulation reduction. Furthermore, molecular docking results suggested that quercetin can bind to both PPARα and PPARγ, with an even more potent binding affinity than indeglitazar, a pan-agonist of PPARs. In conclusion, quercetin may regulate gluconeogenesis to ameliorate hepatic fat accumulation via targeting PPARα/γ.

Pharmacokinetic Study of Conjugated Equine Estrogens in Healthy Chinese Postmenopausal Women Using a Parallel Two-Column LC-MS/MS Method.

BACKGROUND AND OBJECTIVE: Postmenopausal women often require estrogen supplementation to improve menopausal and postmenopausal vasomotor symptoms and maintain hormonal balance. Conjugated equine estrogens extracted from the urine of pregnant mares are commonly used to provide this estrogen replacement therapy. The complex composition of this mixture of animal sulfated metabolites makes its bioanalysis challenging such that its detailed pharmacokinetics has not been fully characterized. The purpose of this work is to reveal the pharmacokinetic behavior of conjugated equine estrogens in healthy Chinese postmenopausal women by a parallel two-column LC-MS/MS method. METHODS: An open-label study was carried out in 35 Chinese healthy postmenopausal women who received a single dose of Premarin® 0.625 mg. A high-throughput column-switching liquid chromatography-tandem mass spectrometry method was developed to determine four conjugated estrogens and two unconjugated estrogens formed by hydrolysis in vivo. The method multiplexes two high-performance liquid chromatography systems into one mass spectrometer and incorporates the positive/negative ion switching acquisition mode of mass spectrometry to significantly increase analysis efficiency. Pharmacokinetics was determined using non-compartmental methods. RESULTS: Both conjugated and unconjugated estrogens can be analyzed simultaneously in a single run with an analysis time of 13.0 minutes in the column-switching liquid chromatography-tandem mass spectrometry method as opposed to 23.0 minutes in a single-column liquid chromatography-tandem mass spectrometry system. The exposures (maximum concentration and area under the curve) of estrone and equilin in Chinese women were higher than those in the North American women. CONCLUSIONS: The fully validated assay was successfully applied to a pharmacokinetic study in healthy postmenopausal Chinese women after oral administration of a conjugated equine estrogen tablet. This study suggests that Chinese postmenopausal women achieve the same level of unconjugated estrogens in plasma at a lower dose of conjugated equine estrogens than North American women.

Evaluation of efficacy and safety after replacement of methyl hydrogen with deuterium at methyl formate of Clopidogrel.

BACKGROUND AND PURPOSE: Despite being a first-line clinical drug, thienopyridines have many unsatisfactory aspects, including the low bioavailability of clopidogrel(CLP) and the high bleeding risk of prasugrel. We synthesized deuterium clopidogrel(D-CL, patented in China) to alleviate the deficiency of CLP in clinical, such as a slow onset, a greater influence of gene polymorphism, and a high frequency of drug-drug interaction. EXPERIMENTAL APPROACH: Molecular docking was used to analyze the affinity between D-CL and the P2Y12 receptor. The levels of active metabolites of D-CL were detected using HPLC/MS-MS and the activities of main metabolic enzymes were analyzed; Subsequently, platelet aggregation function, thrombus model were used to evaluate the pharmacodynamics of D-CL. Finally, the safety of D-CL were evaluated through examination of blood routine, PT, APTT, bleeding time, serological tests, liver pathological biopsy, liver cell apoptosis and detection of apoptosis-related proteins. KEY RESULTS: The introduction of deuterium made the binding of CLP to P2Y12 receptor more stable, improved the concentration of active metabolites, and substantially reduced the inhibition of major metabolic enzymes, including CYP2B6, CYP2C9, and CYP2C19, thereby, exerting better antiplatelet effects without increasing the risk of bleeding, along with a concomitant decrease in the apoptosis of hepatocytes.

Pharmacokinetics of felbinac after intravenous administration of felbinac trometamol in rats.

Felbinac trometamol (trishydroxymethylaminomethane 4-biphenylacetate) is a new water-soluble salt of felbinac currently undergoing clinical evaluation as an intravenous (i.v.) formulation for the treatment of severe post-operative pain. This article reports the pharmacokinetics of felbinac after i.v. administration of felbinac trometamol in Sprague-Dawley rats. The maximum plasma concentration (C(0)) and area under the plasma concentration-time curve (AUC) of felbinac administered at doses of 3.36, 8.40 and 21.0 mg/kg felbinac trometamol increased linearly with dose. Felbinac was highly protein bound (~95%) at plasma concentrations up to 75 µg/ml and extensively metabolized with only small amounts being excreted unchanged in urine (0.318%), feces (0.530%) and bile (0.465%). 4'-Hydroxyfelbinac was the principal metabolite in urine, feces and bile together with felbinac glucuronide, 4'-hydroxyfelbinac glucuronide and sulfate. The majority of the administered dose was excreted in urine (63.6%) mostly as 4'-hydroxyfelbinac. Total drug in urine and feces accounted for about 72% of the dose. It would appear that felbinac trometamol has the potential to replace lipid-based NSAID formulations and progress to clinical evaluation.

Preparation, characterization, and evaluation of HP-ß-CD inclusion complex with alcohol extractives from star anise.

The active compounds in star anise alcohol extractives (SAAE) have potent bioactivity. However, their poor solubility and stability limit their applications. In this study, SAAE/hydroxypropyl-ß-cyclodextrin (HP-ß-CD) inclusion complexes were prepared as a strategy to overcome the abovementioned disadvantages. The phase solubility results indicated that the solubility of the inclusion complex was enhanced. Complexation was confirmed by complementary methods, including Fourier-transform infrared spectroscopy, nuclear magnetic resonance spectroscopy, scanning electron microscopy, and transmission electron microscopy, which proved to be extremely insightful for studying the inclusion formation phenomenon between SAAE and HP-ß-CD. Despite there being no apparent improvements in the antioxidant capacity and antimicrobial activity, the results of the stability studies presented higher thermal, volatile, and photostability after encapsulation. Further, molecular modeling was used to investigate the factors influencing complex formation and provide the most stable molecular conformation. Thus, based on the obtained results, this study strongly demonstrates the potential of the SAAE/HP-ß-CD inclusion complex in the food industry.

Elucidation of interaction between serum albumin and ginsenoside CK along with cytotoxic study.

As a main metabolite of ginsenosides, compound K (CK) has a vast array of pharmacological effects. However, due to its low polarity and insoluble in water, its oral application has been greatly limited. In this work, the interaction between serum albumin and ginsenoside CK was elucidated by multi-spectroscopic studies. The result of ultraviolet/visible absorption spectroscopy showed that the conformation of serum albumin could be changed via binding with CK. The result of fluorescence spectroscopy suggested that CK could form complex with serum albumin. CK could quench the fluorescence and the fluorescence residues of serum albumin were located in or near the binding position. Molecular docking indicated that CK bound at Sudlow's site II of serum albumin and formed hydrogen-bonding interactions with three residues. Furthermore, the flexible side chain of CK was difficult to be stabilized at the binding site, resulting in its serious perturbation during dynamics simulation. This work also performed the cytotoxic study and the result showed that serum albumin enhanced the inhibitory effect of CK on the proliferation of both Caco-2 and HCT-116 cells. To sum up, this work revealed that serum albumin might be an appropriate carrier of hydrophobic compounds, with the advantage of improving their biocompatibility.

Anti-inflammatory action of betulin and its potential as a dissociated glucocorticoid receptor modulator.

Although the medical application of betulin has been presented in previous studies, the potential mechanism of the anti-inflammatory action of betulin should be further investigated. This work aims to confirm the hypothesis that betulin has dexamethasone-like anti-inflammatory action through glucocorticoid receptor (GR)-mediated pathway. Firstly, the binding ability of betulin with GR was measured by a fluorescence polarization-based competitive binding assay, with the IC50 value of 79.18 ± 0.30 mM. Betulin could bind to GR and then induced GR nuclear translocation, but lacked GR transcriptional activity in HeLa cells. Hence, betulin exhibited the potential to be a dissociated modulator for GR, with the loss of glucocorticoid response element (GRE)-associated side effects. In addition, betulin downregulated GRE-driven protein expression of G6P involved in gluconeogenesis, namely side effect. The results of pro-inflammatory cytokines analysis showed that betulin exerted anti-inflammatory action in vitro. Both of the hydrophobic and hydrogen-bonding interactions stabilized the binding between betulin and GR during the simulation process. In conclusion, betulin might be a potential dissociated GR modulator with a reduced side effect profile yet keeping its anti-inflammatory action.