Simple and sensitive detection of miRNA-122 based on a micro-biosensor through square wave voltammetry.

The simple and sensitive detection of miRNA-122 in blood is crucially important for early hepatocellular carcinoma (HCC) diagnosis. In this work, a platinum microelectrode (PtµE) was prepared and electrodeposited with molybdenum disulfide (MoS2) and gold nanoparticles (AuNP), respectively, and denoted as PtµE/MoS2/Au. The prepared PtµE/MoS2/Au was used as the microsensor for the detection of miRNA-122 combined with the probe DNA as a biorecognition element which is the complementary strand of miRNA-122. The PtµE/MoS2/Au conjugated with the probe DNA modified with sulfydryl units was used as the micro-biosensor for the detection of miRNA-122. The square wave voltammetry was performed for the quantitative detection of miRNA-122 using [Fe(CN)6]4-/3- as a mediator. Under the optimized conditions, the PtµE/MoS2/Au micro-biosensor shows a linear detection toward miRNA-122 ranging from 10-11 to 10-8 M (S = 6.9 nA dec-1, R2 = 0.9997), and the detection limit is 1.6 × 10-12 M (3σ/b). The PtµE/MoS2/Au micro-biosensor demonstrates good selectivity against other types of proteins and small molecules, and has good reproducibility. Moreover, the PtµE/MoS2/Au micro-biosensor was successfully applied for the measurement of miRNA-122 in real blood samples. Herein, the proposed detection assay could be a potential tool in HCC clinical diagnostics with high sensitivity.

Using the UPLC-ESI-Q-TOF-MSE method and intestinal bacteria for metabolite identification in the nonpolysaccharide fraction from Bletilla striata.

Bletilla striata (Thunb.) Reichb. f. (Orchidaceae), also known as Bai-ji, is a traditional Chinese herb that is widely used in Asia to treat hematemesis, hemoptysis, traumatic bleeding and other similar disorders. Most studies have focused on the pharmacological activities of polysaccharide extracts from B. striata. Our previous studies found that the nonpolysaccharide fraction from B. striata extract also has a hemostatic effect; however, the active constituents responsible for this pharmacological action are unclear. Thus, the metabolic profiles of the nonpolysaccharide fraction were investigated in Sprague-Dawley rats and intestinal bacteria models using ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry. Mass data were acquired by the MSE method. Eight components including five prototypes and three metabolites were identified in rat biofluids after oral administration of the nonpolysaccharide fraction. The parent compounds underwent various metabolic processes, including hydrolysis, deglucosylation, glycosylation and sulfate conjugation. The results not only reveal the possible metabolic pathway, but also indicate the potential pharmacological components. Further mechanistic studies using nonpolysaccharide compounds of the B. striata extract are required to obtain potential candidate compounds.

A Validated HPLC-MS/MS Method for Simultaneous Determination of Militarine and Its Three Metabolites in Rat Plasma: Application to a Pharmacokinetic Study.

A rapid, reliable, and sensitive HPLC-electrospray ionization-tandem mass spectrometry (HPLC-MS/MS) method was established and validated for simultaneous determination of militarine and its three metabolites (gastrodin, α-isobutylmalic acid, and gymnoside I) in rat plasma. Plasma was acidified with formic acid, and protein was precipitated with methanol. MS/MS with ESI and multiple reaction monitoring at m/z 725.3→457.3, 457.1→127, 304.3→107.2, 189→129, and 417.1→267.1 was used for determination of militarine, gastrodin, α-isobutylmalic acid, gymnoside I, and puerarin (internal standard), respectively. Chromatographic separation was conducted using an ACE UltraCore SuperC18 (2.1 × 100 mm, 2.5 µm) column with gradient mobile phase (0.1% formic acid in water and acetonitrile). The lower limits of quantitation for militarine, gastrodin, α-isobutylmalic acid, and gymnoside I were 1.02, 2.96, 1.64, and 0.3 ng/mL, respectively. The relative standard deviations of intra- and interday measurements were less than 15%, and the method accuracy ranged from 87.4% to 112.5%. The extraction recovery was 83.52%-105.34%, and no matrix effect was observed. The three metabolites (gastrodin, α-isobutylmalic acid, and gymnoside I) were synchronously detected at 0.83 h, suggesting that militarine was rapidly transformed to gastrodin, α-isobutylmalic acid, and gymnoside I. Moreover, the area under the curve (AUC) and Cmax of militarine were significantly lower than those of gastrodin and α-isobutylmalic acid, showing that militarine was largely metabolized to gastrodin and α-isobutylmalic acid in vivo. The studies on pharmacokinetics of militarine and its three metabolites were of great use for facilitating the clinical application of militarine and were also highly meaningful for the potential development of militarine.

Panax quinquefolium saponin attenuates cardiac remodeling induced by simulated microgravity.

BACKGROUND: Cardiac atrophy and reduced cardiac distensibility have been reported following space flight. Cardiac function is correspondingly regulated in response to changes in loading conditions. Panax quinquefolium saponin (PQS) improves ventricular remodeling after acute myocardial infarction by alleviating endoplasmic reticulum stress and Ca2+overload. However, whether PQS can ameliorate cardiac atrophy following exposure to simulated microgravity remains unknown. PURPOSE: To explore the protective role of PQS in cardiac remodeling under unloading conditions and its underlying mechanisms. METHODS: Hindlimb unloading (HU) model was used to simulate unloading induced cardiac remodeling. Forty-eight male rats were randomly assigned to four groups, including control, PQS, HU and HU + PQS. At 8 weeks after the experiment, cardiac structure and function, serum levels of Creatine Kinase-MB (CK-MB), Cardiactroponin T (cTnT), ischemia modified albumin (IMA), and cardiomyocyte apoptosis were measured. Network pharmacology analysis was used to predict the targets of the six major constituents of PQS, and the signaling pathways they involved in were analyzed by bioinformatics methods. Changes in the key proteins involved in the protective effects of PQS were further confirmed by Western Blot. RESULTS: Simulated microgravity led to increases in serum levels of CK-MB, cTnT and IMA, remodeling of cardiac structure, impairment of cardiac function, and increased cardiomyocyte apoptosis as compared with control. PQS treatment significantly reduced serum levels of CK-MB, cTnT and IMA, improved the impaired cardiac structure and function, and decreased cardiomyocyte apoptosis induced by unloading. The activation of AMPK and inhibition of Erk1/2 and CaMKII/HDAC4 were demonstrated in the cardiocytes of HU rats after PQS treatment. CONCLUSION: PQS provides protection against cardiac remodeling induced by simulated microgravity, partly resulting from changes in the signaling pathways related to energy metabolism reduction, calcium overloading and cell apoptosis.

[Analysis of metabolites of quercitrin in rat intestinal flora by using UPLC-ESI-Q-TOF-MS/MS].

To investigate the metabolism of quercitrin in rat intestinal flora and possible biological pathways, laying the foundation for the metabolic mechanism of traditional Chinese medicine glycosides ingredients. UPLC-Q-TOF-MS/MS method was established to detect the quercitrin and its metabolites with 0.1% formic acid solution(A)-0.1% formic acid acetonitrile(B) as the mobile phase for gradient elution at a flow rate of 0.3 mL•min⁻¹. Electrospray negative ion mode was applied to analyze the metabolites of quercitrin in rat intestinal flora. Metabolite ToolsTM, mass defect filter(MDF) and other technologies were used to screen, analyze the metabolites and infer the chemical formula of the metabolites. The results showed that quercitrin would have degalactoside, deoxygenation and acetylation reactions, and the aglycone quercetin resulted from degalactoside would have further reactions such as hydroxylation, deoxygenation, reduction, and ring opening to achieve deoxygenation metabolite kaempferol, C2-C3 double bonds hydrogenation and reduction product taxifolin, and degalactoside product quercetin. The research results showed that quercitrin can be metabolized by rat intestinal flora, which could increase their hydrophobicity and chemical diversity.

A UPLC-MS/MS Method for Simultaneous Determination of Free and Total Forms of a Phenolic Acid and Two Flavonoids in Rat Plasma and Its Application to Comparative Pharmacokinetic Studies of Polygonum capitatum Extract in Rats.

The principal active constituents of Polygonum capitatum are phenolic acids and flavonoids, such as gallic acid, quercitrin, and quercetin. The aim of this study was to develop and validate a method to determine the three constituents and the corresponding conjugated metabolites of Polygonum capitatum in vivo and to conduct pharmacokinetic studies on the herb, a well-known Miao medicinal plant in China. Gallic acid, quercitrin, and quercetin were analysed by ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS). Protein precipitation in plasma samples was performed using methanol. For the determination of total forms of analytes, an additional process of hydrolysis was conducted using ß-glucuronidase and sulphatase. The analytes were separated on a BEH C18 column (50 mm × 2.1 mm; i.d., 1.7 µm) and quantified by multiple reaction monitoring (MRM) mode. The linear regression showed high linearity over a 729-fold dynamic range for the three analytes. The relative standard deviations of intra- and inter-day measurements were less than 9.5%, and the method was accurate to within -11.1% to 12.5%. The extraction recoveries for gallic acid, quercitrin, and quercetin were 94.3%-98.8%, 88.9%-98.8%, and 95.7%-98.5%, respectively. All samples were stable under short- and long-term storage conditions. The validated method was successfully applied to a comparative pharmacokinetic study of gallic acid, quercitrin, and quercetin in their free and total forms in rat plasma. The study revealed significantly higher exposure of the constituents in total forms for gallic acid and quercetin, while quercitrin was detected mainly in its corresponding free form in vivo. The established method was rapid and sensitive for the simultaneous quantification of free and total forms of multiple constituents of Polygonum capitatum extract in plasma.