Development of a Competitive Time-Resolved Fluoroimmunoassay for Paclitaxel.

INTRODUCTION: Paclitaxel (Tax) is a diterpene alkaloid isolated from Taxus species and has proved clinically effective in treating a number of malignancies. Current quantitative analytical methods for Tax such as high-performance liquid chromatography (HPLC) often involve complicated sample preparation procedures with low recovery rates. OBJECTIVE: To establish a rapid and sensitive time-resolved fluoroimmunoassay (TRFIA) for measuring Tax in Taxus materials with convenient sample preparation and a high recovery rate. METHODS: Rabbit anti-mouse IgG was coated onto a 96-well microplate, which was then incubated with standard solutions of Tax and anti-Tax monoclonal antibody 3A3. A Eu3+ -labelled conjugate of Tax and human serum albumin was used as the tracer. The luminescent system was enhanced with a solution containing 2-naphthoyltrifluoroacetone. RESULTS: The established TRFIA showed a linear response within the Tax concentration range of 3.2 to 80 ng/mL, with a limit of detection of 1.4 ng/mL. The intra- and inter-assay coefficient of variation of the assay was 9.6% and 9.7%, respectively, with an average recovery rate from spiked samples of 108.5%. Tax contents in Taxus samples were determined using both the established TRFIA system and a previously established enzyme-linked immunosorbent (ELISA), and the results of two assays were well correlated. CONCLUSION: This TRFIA system shows a high sensitivity, precision and accuracy for detection of Tax. This assay, which is convenient and less time-consuming, allows rapid analysis of Tax and provides another option for Tax measurement for quality control of Taxus materials and products. Copyright © 2017 John Wiley & Sons, Ltd.

[Preliminary study of determination of Shougong in Jinlong Capsules based on fluorescence quantitative PCR method].

A pair of species-specific primer (GZG1/GZG2) based on COⅠ sequence regions for identification of Gekko chinensis were designed. A fluorescent quantitative PCR method was established to identify and quantify G. chinensis from Jinlong Capsules Formula. A standard curve for quantitative analysis of G. chinensis was established (the standard curve equation: y=-3.012 7x+34.501, y is Ct value, x is lg N, N is the copies of COⅠ fragment from G. chinensis). Samples included G. chinensis appeared amplification, while falsify group (not included G. chinensis) and negative control did not have amplification products. The copy number of COⅠ region of G. chinensis was respectively 11.511×106, 6.416×106, 2.553×106 copies/µL in all quality goods, quality goods-adulterants 1:1, quality goods-adulterants 1:4. The results accorded with proportion of adding amount roughly. This study can provide a new strategy for quality control of Chinese patent medicine containing animal drug ingredients.

A DNA Barcode-Based RPA Assay (BAR-RPA) for Rapid Identification of the Dry Root of Ficus hirta (Wuzhimaotao).

Background: Wuzhimaotao (the dry root of Ficus hirta) is used as both medicine and food ingredient by the locals in areas around Nanling Mountains of China. Due to its very similar external morphologies with Duanchangcao (the root of Gelsemium elegans, which contains gelsemine that is extremely neurotoxic) and the associated growth of these two plants, incidents of food poisoning and even death frequently occur, resulting from the misuse of Duanchangcao as Wuzhimaotao. The aim of this study is to develop a fast, even, on-spot approach to identification of Wuzhimaotao. Methods: We used DNA barcode-based recombinase polymerase amplification (BAR-RPA) with species-specific primers targeting the internal transcribed spacer (ITS) region of the rDNA of F. hirta. BAR-RPA reaction time and temperature were optimized and the specificity and sensitivity of BAR-RPA species-specific primers were assessed. Results: This technique showed a high specificity and sensitivity to amplify the genomic DNA of F. hirta and allowed for rapid amplification (within 15 min) of the ITS region under a constant and mild temperature range of 37-42 °C without using thermocyclers. Conclusions: The BAR-RPA assay with a fast DNA extraction protocol provides a simple, energy-saving, and rapid method for identification of Wuzhimaotao in both laboratory and field settings.

[Identification of Original Species of Fish Maw by DNA Barcoding].

Objective: To identify the original species of fish maw sold in Guangzhou market by DNA barcoding technology. Methods: Mitochondrial cytochrome C subunit I (CO I) gene fragment of eleven fish maw samples were amplified and sequenced with the self-designed primers. UPGMA phylogenetic tree were constructed for clustering analysis. The species origin of each sample was identified with the identification engine provided in the Barcode of Life Data Systems (BOLD). Results: The self-designed primers were effective in fish maw CO I amplification and sequencing, with success rates both of 100%. BOLD identification and UPGMA clustering analysis indicated the fish maw samples were derived from five fish species of three families. Conclusion: DNA barcoding combined with BOLD identification system can accurately identify the species origin of commercial fish maw.

Study on mechanism of transdermal administration of eugenol for pain treatment by network pharmacology and molecular docking technology.

The objective of this study was to explore the pharmacological mechanism of transdermal administration of eugenol (EUG) for pain treatment. Firstly, network pharmacology techniques were employed to identify the potential targets responsible for the analgesic effect of EUG. Subsequently, molecular docking technology was used to validate interactions between EUG and the crystal structure of the core target protein. Finally, the impact of EUG on the expression and activation of TRPV1 receptors in HaCaT cells was evaluated through in vitro experiments, thus confirming the analysis of network pharmacology. The study suggested that the transdermal administration of EUG for pain treatment might target the TRPV1 receptor. Molecular docking revealed that EUG could spontaneously bind to the TRPV1 receptor with a high binding ability. The analysis of Western blot (WB) and intracellular Ca2+ levels demonstrated that EUG could increase the expression of TRPV1 in HaCaT cells, activating TRPV1 to induce intracellular Ca2+ influx (P < 0.05). These findings suggested that the initial application of EUG would cause a brief stimulation of TRPV1 receptors and upregulation of TRPV1 expression. Upon continued exposure, EUG would act as a TRPV1 agonist, increasing intracellular Ca2+ levels that might be associated with desensitization of pain sensations.

Molecular quantification, a new strategy for quality control of Chinese patent medicine containing animal-derived crude drug: Qi She in Jinlong capsule as an example.

Quality control for Chinese patent medicine (CPM) containing animal-derived crude drug(s) is rather difficult. The methods based on chemical composition analysis, which are commonly used in CPM consisted of plant-derived crude drugs, are often not applicable for CPM containing animal-derived crude drug, because the effective constituents of most animal-derived crude drugs remain unknown. Even if there are such methods, they are usually qualitative rather than quantitative, and the specificity is generally poor. Here we proposed a molecular quantification method for CPM containing animal-derived crude drug, based upon the hypothesis that the amount of remnant DNA fragments could reflect feeding quantity of the crude drugs and thus ensure the quality of the CPM. Take Jinlong capsule [a hepatocellular carcinoma-resisting Chinese patent medicine comprising of three fresh animal drugs, i.e. Shougong (Peking gecko, Gekko swinhonis), Qi She (sharp-snouted pitviper, Deinagkistrodon acutus), and Jinqian Baihua She (many-banded krait, Bungarus multicinctus)] as an example, we established a qPCR assay for Qi She in the capsule, which verified the feasibility of the quality control method based on molecular quantification. Species-specific primers and TaqMan probe for Qi She were designed, and the qPCR assay system was then established. The assay exhibited a good specificity; there's a good linearity between Ct values and logarithm of the target amplicon copy numbers within the range of 8.8 × 101 to 8.8 × 106 copies/µL, and the limit of detection was 88 copies/µL. The method was validated through reproducibility, stability assessment. Recovery of spiked samples was between 91.59% and 101.69%. It was verified that the copy numbers reflected the original feeding amount of an animal-derived crude drug by self-made Jinlong capsules. The assay was successfully applied in Qi She-specific amplicon determination in 20 batches of Jinlong capsule. The study was expected to provide a new strategy for quality control of CPM containing animal-derived crude drug.

Extract of Stellera Chamaejasme L. Inhibits the Progression of Hepatocellular Carcinoma by Regulating miR-134-5p and JAK1/STAT3 Pathway.

Background: Hepatocellular carcinoma (HCC) poses a growing threat to humans due to poor prognosis. Extract of stellera chamaejasme L. (ESC) is reported to inhibit metastasis of HCC. However, the underlying mechanism of ESC in regulating the progression of HCC needs to be further investigated. Methods: 3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to measure cell proliferation. Flow cytometry was employed to check cell apoptosis. Transwell assay was conducted to assess the abilities of cell migration and invasion. The protein levels of proliferating cell nuclear antigen, cleaved caspase 3 (c-caspase 3), E-cadherin, janus kinase 1 (JAK1), signal transducer and activator of transcription 3 (STAT3), and phosphorylated STAT3 were detected by Western blot. The interaction between miR-134-5p and JAK1 was predicted by starBase, which was verified by the dual-luciferase reporter assay and RNA pull-down assay. The messenger RNA levels of miR-134-5p and JAK1 were determined by quantitative real-time polymerase chain reaction. Results: The results showed that the higher concentration or the longer time treatment of ESC led to the lower survival rate of HCC cells. Besides, ESC induced apoptosis and impeded migration and invasion of HCC cells. Moreover, downregulation of miR-134-5p inverted the effects of ESC-mediated repression on HCC progression. Further studies indicated that miR-134-5p targeted the 3'-untranslated region (3'UTR) of JAK1 and reversed JAK1-mediated impacts on HCC progression. Simultaneously, ESC inactivated JAK1/STAT3 pathway by regulating the expression of miR-134-5p. Conclusion: ESC suppressed HCC progression by upregulating the expression of miR-134-5p and blocking JAK1/STAT3 pathway.