Enzyme activity- and chemometrics-assisted comprehensive two-dimensional liquid chromatography coupled with ion mobility quadrupole time-of-flight mass spectrometry for the analysis of honeysuckle.

A unique and effective comprehensive two-dimensional liquid chromatography system was established and applied for the analysis of bioactive components in honeysuckle. Under the optimal conditions, Eclipse Plus C18 (2.1 × 100 mm, 3.5 µm, Agilent) and SB-C18 (4.6 × 50 mm, 1.8 µm, Agilent) columns were chosen for the first dimension (1D) and the second dimension (2D) separation. The optimal flow rates of 1D and 2D were 0.12 mL/min and 2.0 mL/min, respectively. Additionally, the proportion of organic solution was optimized to enhance orthogonality and integrated shift, and full gradient elution mode was adopted to improve chromatographic resolution. Furthermore, a total of 57 compounds were identified by molecular weight, retention time and collision cross-section value obtained from ion mobility mass spectrometry. Based on the data obtained from the principal component analysis, partial least squares discriminant analysis, and hierarchical cluster analysis, the categories of honeysuckle in different regions were significantly different. Moreover, the half maximal inhibitory concentration values of most samples were between 0.37 and 1.55 mg/mL, and most samples were potent α-glucosidase inhibitors, which is better for the evaluation of the quality of drugs from two aspects of substance content and activity.

Ecofriendly microwave-assisted reaction and extraction of bioactive compounds from hawthorn leaf.

INTRODUCTION: The main active components in hawthorn leaves possess various biological activities such as anti-inflammatory, antioxidant, and hypolipidemic effects. Therefore, it is necessary to develop an effective and reliable extraction method to extract these active compounds from hawthorn leaves. OBJECTIVE: To establish a simple, rapid, and sensitive method for extraction and determination of polyphenolic compounds from hawthorn leaves. METHODS: In this study, a microwave-assisted reaction and extraction (MARE) combined with ultra-high-performance liquid chromatography with ultraviolet detector method was established to extract and determine the polyphenolic compounds in hawthorn leaves. The solid reagent aqueous solutions were applied as extraction solvents, preventing the use of organic solvents. The target analytes were identified by quadrupole time-of-flight tandem mass spectrometry. Several experimental parameters that can significantly affect the extraction efficiency were evaluated and optimised. RESULTS: The optimal conditions were as follows: 0.1 g of sodium carbonate was used as solid reagent, the amount of sodium borate was set at 0.01 g, extraction time was 10 min, extraction temperature was set at 50°C, pH value was adjusted to 7. The validation experiments demonstrated that the method had high sensitivity with the limits of detection in the range 26.5-37.7 ng/mL. The average recoveries ranged from 80.22% to 93.27%. CONCLUSION: In this work, the proposed MARE method was successfully applied to extract and determine polyphenolic compounds in hawthorn leaf samples. Compared with other reported methods, the present method was faster, greener, and more sensitive.

[Influence of acupuncture intervention on neurologic deficits, cerebrocortical cell apoptosis and Protein kinase A expression in rats with focal cerebral ischemia].

OBJECTIVE: To observe the influence of acupuncture intervention on expression of protein kinase A (PKAP in the cerebrocortex of rats with focal cerebral ischemia-reperfusion injury(CI/RI), so as to explore its underlying neuroprotection mechanism on cerebral ischemia. METHODS: Ninety male SD rats were randomly and evenly divided into sham-operation (sham), model and acupuncture groups which were further randomized into 3, 7 and 14-days (d) subgroups (10 rats/subgroup). CI/RI model was established by occlusion of the left middle cerebral artery for 2 hours and reperfusion. Manual acupuncture stimulation was applied to "Baihui" (GV 20) and "Shuigou" (GV 26), as well as the right "Quchi" (LI 11), "Hegu" (LI 4), "Neig (PC 6), "Zusanli" (ST 36),"Sanyinjiao"(SP 6) and "Taichong" (LR 3) for 30 min, once daily for 3, 7 and 14 d respectively. Rats of the sham and model groups were restrained for 30 min each day. Neurological defects were assessed by ethologic scoring according to Bederson's neurologic assessment scales. Cellular apoptosis in the ischemic cortex was detected by flow cytometry and PKA expression determined by immunohistochemistry for calculating its PKA immunoreaction (IR)-positive cell rate, respectively. RESULTS: Compared with the sham group, the neurologic scores, cortical cellular apoptosis rates and PKA IR-positive cell rates of the model group were significantly increased at the time-points of day 3, 7 and 14 post-treatment (P<0. 05). In comparison with the model group, the neurologic scores and cortical cellular apoptosis rates of the acupuncture group at the time-points of day 3, 7 and 14 post-treatment were considerably down-regulated (P<0. 05), and the cortical PKA IR-positive cell rates of the acupuncture group were remarkably increased (P<0. 05). In addition, along with the increase of acupuncture treatment sessions, lower apoptosis rates and more PKA IR-positive cells were found, suggesting a cumulative effect. CONCLUSION: Acupuncture intervention can lower cellular apoptosis rate of the ischemic cerebrocortex and up-regulate cortical PKA expression level in CI/RI rats, which may be responsible for its effect in improving neurologic deficits.

Colocalization and identification of interaction sites between IGFBP-3 and GalNAc-T14.

GalNAc-T14 was identified as a novel IGFBP-3 binding partner in previous studies. Here, we furtherly confirmed the interaction between them by confocal microscopy, and identified the binding domain and probable interaction sites of GalNAc-T14 with IGFBP-3. The result of subcellular localization indicated that GalNAc-T14 was distributed in the cytosol, whereas IGFBP-3 existed in the cytosol and nucleolus. Confocal analyses demonstrated that IGFBP-3 and GalNAc-T14 colocalized in the cytosol. The result from yeast two hybrid assay showed that the C terminus of GalNAc-T14 (408-552aa) was essential for the interaction between GalNAc-T14 and IGFBP-3, especially Tyr(408), Pro(409), and Glu(410) of GalNAc-T14 may play key roles in the interaction with IGFBP-3. In conclusion, these studies demonstrated that IGFBP-3 and GalNAc-T14 are colocalized in MCF-7 cells and confirmed the interaction between IGFBP-3 and GalNAc-T14. This interaction may play an important role in the functional regulation of IGFBP-3.