18 research outputs found
Quantification by LC–MS/MS of astragaloside IV and isoflavones in Astragali radix can be more accurate by using standard addition
Introduction
Astragali radix (AR), the root of Astragalus, is an important medical herb widely used in traditional Chinese medicine. Bioactive components include isoflavones and a unique class of triterpenoid saponins (named astragalosides).
Objectives
Accurate measurement of bioactive components, especially astragaloside IV, is necessary for confirming AR authenticity, quality control and future medical research.
Methodology
Liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) is a suitable technique but suffers from ion suppression effects due to sample matrix. This can be corrected by using isotopic labelled internal standards, but these are not available for many phytochemicals. We explored the use of standard addition to circumvent this issue.
Results
LC–MS/MS and liquid chromatography coupled with ultraviolet (LC-UV) detection provided linear calibration curves (R2 > 0.99). LC–MS/MS provided superior selectivity and detection limits below 10 ng/mL, which was 2–3 magnitudes lower than LC-UV detection. Precision and accuracy were overall improved by using LC–MS/MS with diluted sample extracts, resulting in an inter series coefficient of variation (CV) of 12% or less and mean recovery estimates in the 85–115% range. LC–MS/MS quantification by standard addition resulted in significantly higher concentrations of astragaloside IV measured in the samples. Concentrations calculated by standard addition were unaffected by large variation in signal response caused by matrix effects, independent of variation in slope of the standard addition curves.
Conclusion
Sample dilution was helpful but not sufficient for reducing effects of ion suppression. We have shown that LC–MS/MS quantification by standard addition can be a powerful approach for accurate measurement of phytochemicals in the absence of isotopic labelled internal standards.publishedVersio
Prediction of the anti-inflammatory mechanisms of curcumin by module-based protein interaction network analysis
AbstractCurcumin, the medically active component from Curcuma longa (Turmeric), is widely used to treat inflammatory diseases. Protein interaction network (PIN) analysis was used to predict its mechanisms of molecular action. Targets of curcumin were obtained based on ChEMBL and STITCH databases. Protein–protein interactions (PPIs) were extracted from the String database. The PIN of curcumin was constructed by Cytoscape and the function modules identified by gene ontology (GO) enrichment analysis based on molecular complex detection (MCODE). A PIN of curcumin with 482 nodes and 1688 interactions was constructed, which has scale-free, small world and modular properties. Based on analysis of these function modules, the mechanism of curcumin is proposed. Two modules were found to be intimately associated with inflammation. With function modules analysis, the anti-inflammatory effects of curcumin were related to SMAD, ERG and mediation by the TLR family. TLR9 may be a potential target of curcumin to treat inflammation
Major bioactive chemical compounds in Astragali Radix samples from different vendors vary greatly
The worldwide traditional Chinese medicine (TCM) herbs sales figures have increased considerably to 50 billion US$ (2018). Astragali Radix (AR) is amongst the most often sold TCM herbs; sales in the European Union (EU) need European Medicines Agency (EMA) approval. However, comparisons of characteristic bioactive molecules concentrations in AR from different EU vendors are lacking. This study uses liquid chromatography-tandem mass spectrometry (LC-MS/MS) with standard addition to evaluate the influence of different sample and preparation types and ammonia treatment on bioactive molecules concentrations in AR. We also compare AR samples from different EU-vendors. Astragaloside IV (AG-IV), ononin and calycosin 7-O-β-D-glucoside concentrations were higher in root powder samples when extracted with boiled water than with ultrasonication using 70% methanol. AG-IV content was by far the highest in granulates from vendor 1 (202 ± 35 μg/g) but very low in hydrophilic concentrates from vendor 1 (32 ± 7 μg/g) and granulates from vendor 4 (36 ± 3 μg/g). Ammonia-treatment significantly increased AG-IV concentrations in all samples (e.g., to 536 ± 178 μg/g in vendor 1 granulates). Comparable effects were found for most other bioactive molecules. AG-IV and other bioactive molecules concentrations differed strongly depending on sample types, extraction processes, ammonia treatment-or-not and especially between different vendors samples. Ammonia-treatment is debatable, as it is supposed to convert other astragalosides, to AG-IV. The results indicate that routine quantitative analysis of major bioactive compounds present in AR, helps in quality control of AR and to guarantee the quality of commercial products.publishedVersio
Prognostic value and reproducibility of different microscopic characteristics in the WHO grading systems for pTa and pT1 urinary bladder urothelial carcinomas
Background: European treatment guidelines for pTa and pT1 urinary bladder urothelial carcinoma depend highly on stage and WHO-grade. Both the WHO73 and the WHO04 grading systems show some intra- and interobserver variability. The current pilot study investigates which histopathological features are especially sensitive for this undesired lack of reproducibility and the influence on prognostic value. Methods: Thirty-eight cases of primary non-muscle invasive urothelial carcinomas, including thirteen cases with stage progression, were reviewed by three pathologists. Thirteen microscopic features were extracted from pathology textbooks and evaluated separately. Reproducibility was measured using Gwet’s agreement coefficients. Prognostic ability regarding progression was estimated by the area under curve (AUC) of the receiver operating characteristics (ROC) function. Results: The best reproducible features (Gwet’s agreement coefficient above 0.60) were papillary architecture, nuclear polarity, cellular maturation, nuclear enlargement and giant nuclei. Nucleoli was the strongest prognostic feature, and the only feature with an AUC above 0.70 for both grading systems, but reproducibility was not among the strongest. Nuclear polarity also had prognostic value with an AUC of 0.70 and 0.67 for the WHO73 and WHO04, respectively. The other features did not have significant prognostic value. Conclusions: The reproducibility of the histopathological features of the different WHO grading systems varied considerably. Of all the features evaluated, only nuclear polarity was both prognostic and significantly reproducible. Further validation studies are needed on these features to improve grading of urothelial carcinomas. Keywords: Papillary urothelial carcinoma, Grading, Reproducibility, PrognosispublishedVersio
The Relationship between Syntactic Structure Analysis Features, Histological Grade and High-Risk HPV DNA in Cervical Intraepithelial Neoplasia
Aim: To assess the correlation between syntactic structure analysis (SSA) features, revised dysplasia grade and the presence of high‐risk human papillomavirus DNA in cervical intraepithelial neoplasia (CIN). Materials and methods: HPV polymerase chain reaction (PCR) was assessed in 101 consecutive biopsies and consensus in CIN grade between the experts occurred in 88 cases (CIN1=16, CIN2=27, CIN3=45). SSA was performed in the diagnostic histological section of the CIN lesions in these patients and SSA features were compared with the blind review CIN grade, and presence/absence of high‐risk HPV DNA. Results: One of the SSA features (points from which the surrounding surfaces has 4 edges, PECO‐4) was significantly different between all three consensus CIN grades. Many more features revealed significant differences between CIN1 and CIN2 or between CIN2 and CIN3 cases. With stepwise discriminant analysis, the best multivariate combination of features to distinguish the different CIN grades were the Maximum MST Line Length (MML) and the Area Disorder. Crude overall classification of the consensus grades with these features was 69%. The MML and the Area Disorder is also the best combination to distinguish cases with and without high‐risk HPV DNA (77% correct classifications). Conclusions: SSA features are correlated with both CIN grade and presence of high‐risk HPV DNA, but the discrimination power is not good enough to be used as a routine method for quality control of subjective grade or as a surrogate marker for high‐risk HPV DNA presence
Influence of pre-operative oral carbohydrate loading vs. standard fasting on tumor proliferation and clinical outcome in breast cancer patients - a randomized trial
Background
Conflicting results have been reported on the influence of carbohydrates in breast cancer.
Objective
To determine the influence of pre-operative per-oral carbohydrate load on proliferation in breast tumors.
Design
Randomized controlled trial.
Setting
University hospital with primary and secondary care functions in South-West Norway.
Patients
Sixty-one patients with operable breast cancer from a population-based cohort.
Intervention
Per-oral carbohydrate load (preOp™) 18 and 2–4 h before surgery (n = 26) or standard pre-operative fasting with free consumption of tap water (n = 35).
Measurements
The primary outcome was post-operative tumor proliferation measured by the mitotic activity index (MAI). The secondary outcomes were changes in the levels of serum insulin, insulin-c-peptide, glucose, IGF-1, and IGFBP3; patients’ well-being, and clinical outcome over a median follow-up of 88 months (range 33–97 months).
Results
In the estrogen receptor (ER) positive subgroup (n = 50), high proliferation (MAI ≥ 10) occurred more often in the carbohydrate group (CH) than in the fasting group (p = 0.038). The CH group was more frequently progesterone receptor (PR) negative (p = 0.014). The CH group had a significant increase in insulin (+ 24.31 mIE/L, 95% CI 15.34 mIE/L to 33.27 mIE/L) and insulin c-peptide (+ 1.39 nM, 95% CI 1.03 nM to 1.77 nM), but reduced IGFBP3 levels (− 0.26 nM; 95% CI − 0.46 nM to − 0.051 nM) compared to the fasting group. CH-intervention ER-positive patients had poorer relapse-free survival (73%) than the fasting group (100%; p = 0.012; HR = 9.3, 95% CI, 1.1 to 77.7). In the ER-positive patients, only tumor size (p = 0.021; HR = 6.07, 95% CI 1.31 to 28.03) and the CH/fasting subgrouping (p = 0.040; HR = 9.30, 95% CI 1.11 to 77.82) had independent prognostic value. The adverse clinical outcome of carbohydrate loading occurred only in T2 patients with relapse-free survival of 100% in the fasting group vs. 33% in the CH group (p = 0.015; HR = inf). The CH group reported less pain on days 5 and 6 than the control group (p < 0.001) but otherwise exhibited no factors related to well-being.publishedVersio
Limited Prognostic Value of Cellular DNA Content to Classical and Morphometrical Parameters in Invasive Ductal Breast Cancer
MiR-18a and miR-18b are expressed in the stroma of oestrogen receptor alpha negative breast cancers
Background
Previously, we have shown that miR-18a and miR-18b gene expression strongly correlates with high proliferation, oestrogen receptor -negativity (ER−), cytokeratin 5/6 positivity and basal-like features of breast cancer.
Methods
We investigated the expression and localization of miR-18a and -18b in formalin fixed paraffin embedded (FFPE) tissue from lymph node negative breast cancers (n = 40), by chromogenic in situ hybridization (CISH). The expression level and in situ localization of miR-18a and -18b was assessed with respect to the presence of tumour infiltrating lymphocytes (TILs) and immunohistochemical markers for ER, CD4, CD8, CD20, CD68, CD138, PAX5 and actin. Furthermore, in two independent breast cancer cohorts (94 and 377 patients) the correlation between miR-18a and -18b expression and the relative quantification of 22 immune cell types obtained from the CIBERSORT tool was assessed.
Results
CISH demonstrated distinct and specific cytoplasmic staining for both miR-18a and miR-18b, particularly in the intratumoural stroma and the stroma surrounding the tumour margin. Staining by immunohistochemistry revealed some degree of overlap of miR-18a and -18b with CD68 (monocytes/macrophages), CD138 (plasma cells) and the presence of high percentages of TILs. CIBERSORT analysis showed a strong correlation between M1-macrophages and CD4+ memory activated T-cells with mir-18a and -18b.
Conclusions
Our study demonstrates that miR-18a and miR-18b expression is associated with ER- breast tumours that display a high degree of inflammation. This expression is potentially associated specifically with macrophages. These results suggest that miR-18a and miR-18b may play a role in the systemic immunological response in ER− tumourspublishedVersio