TiO2 nanotube immobilised 5-lipoxygenase-mediated screening and isolation of anti-inflammatory active compounds from the leaves of lonicera japonica thunb.

In this work, a highly effective separation approach mediated by 5-Lipoxygenase (5-LOX) was established for screening and isolation of anti-inflammatory ingredients from leaves of Lonicera japonica Thunb. (LLJT). Using 5-LOX immobilised on TiO2 nanotubes as a microreactor, the targeted screening was exploited by combining with HPLC-MS system. Four compounds confirmed as luteolin, luteoside, lonicerin, and isochlorogenic acid C and a fraction (M1) were screened out to be potent inhibitors of 5-LOX. Their anti-inflammatory activities were further investigated and confirmed by RAW 264.7 cells inflammation model and rat foot swelling model. Furthermore, M1 was prepared by MCI GEL CHP20P column chromatography, and further separated by Pre-HPLC. One new compound confirmed to be 5,7,3',4'-tetrahydroxyflavone-7-O-sambubioside was first isolated from LLJT. The results provide a new method for the effective separation of active components derived from natural products.HighlightsA 5-LOX mediated separation method was established for isolation of anti-inflammatory compounds.An anti-inflammatory ingredient was separated by MCI GEL CHP20P column chromatography.One new compound was first isolated from leaves of Lonicera japonica Thunb.5-LOX was immobilised on TiO2 nanotubes and exploited by combining with HPLC-MS system.The anti-inflammatory activity of screened components was evaluated. [Figure: see text].

Gramicidin-S-Inspired Cyclopeptidomimetics as Potent Membrane-Active Bactericidal Agents with Therapeutic Potential.

Antimicrobial peptides (AMPs) are promising antibacterial agents often hindered by their undesired hemolytic activity. Inspired by gramicidin S (GS), a well-known cyclodecapeptide, we synthesized a panel of antibacterial cyclopeptidomimetics using ß,γ-diamino acids (ß,γ-DiAAs). We observed that peptidomimetic CP-2 displays a bactericidal activity similar to that of GS while possessing lower side-effects. Moreover, extensive studies revealed that CP-2 likely kills bacteria through membrane disruption. Altogether, CP-2 is a promising membrane-active antibiotic with therapeutic potential.

Online coupling Fe3O4@ZIF-67@α-glucosidase biomicroreactor with high performance liquid chromatography for rapid screening of α-glucosidase inhibitors in tea and their inhibitory activity research.

In this work, α-glucosidase was immobilized on a type of nanoscaledmagnetic materials Fe3O4@ZIF-67 to construct a biomicroreactor for rapid screening α-glucosidase inhibitors. The parameters that affected the immobilization efficiency were investigated. Under optimal conditions, the amount of α-glucosidase loaded was up to 79.07 µg·mg-1. Then, the enzyme biomicroreactor was immobilized by an external magnetic field in a tube connecting HPLC and micro-injection pump at both ends to form a magnetic online screening system. The screening flow rate and eluent organic solvent ratio during the screening process were optimized. XinYang MaoJian crude tea extract was tested to verify this online screening method, and three inhibitors (catechin, epigallocatechin gallate and epicatechin gallate) were fished by this magnetic online screening system. Based on the classical pNPG method, the inhibitory activities on α-glucosidase were further verified and studied. Compared with the traditional ligand fishing methods, the screening method established in this work can integrate screening, elution and analysis. It can simply, efficiently and directly screen and identify potential α-glucosidase inhibitors from natural sources. This method was expected to provide an effective basis for accelerating the development of new hypoglycemic drugs.

Development of an on-line immobilized α-glucosidase microreactor coupled to liquid chromatography for screening of α-glucosidase inhibitors.

In order to rapidly screen α-glucosidase (α-GLU) inhibitors from Chinese herbs extract, an online screening method was developed by using enzymatic microreactors in combination with HPLC. A type of dodecahedral and porous material (ZIF-90) was synthesized at room temperature and employed as supports to construct enzymatic microreactor. The amount of α-glucosidase immobilized on ZIF-90 was 58.65 µg per mg carrier under the optimized conditions. In the online screening process, the eluent of 30 s was selected for detection. For the application of this on-line screening system, three α-glucosidase inhibitors with known structure (2,4-dimethoxy-6,7-dihydroxyphenanthrene, batatasin I, 3,5-dimethoxy-2'-hydroxyaiaryl) were selectively extracted from Dioscorea opposita Thunb. Three compounds screened from honeysuckle leaves were isochlorogenic acid B, 1,5-dicaffeoylquinic acid and isochlorogenic acid C, respectively. Two compounds including (+)-catechin and (-)-epigallocatechin gallate were screened from Xinyang Maojian tea. Three unknown ingredients were also screened out from Radix Rehmanniae Praeparata. The nanomaterials in the microreactor can be conveniently replaced. The screening flow rate and elution time can be easily adjusted and controlled by microinjection pump. Considering the specificity of enzyme binding and convenience of online screening system, this method has great potential for fast real-time fishing of α-glucosidase inhibitors from Chinese herbal medicines.

Detoxification and activating blood circulation decoction reduces restenosis involving the TLR4/NF-κB pathway after balloon injury.

Restenosis is a major problem after percutaneous coronary intervention (PCI) treatment. Inflammation is one of the major core mechanisms involved in the occurrence of restenosis, and plays an important role in intimal hyperplasia. Detoxification and activating blood circulation decoction (DABCD) is a traditional Chinese medicine that is used in the treatment and prevention of atherosclerotic and inflammatory diseases. Our previous studies demonstrated that DABCD-mediated cardioprotection involves anti-inflammatory mechanisms and could be developed as a novel drug for the treatment of vascular smooth muscle cell (VSMC) proliferation and aortic restenosis. A rat model of postoperative restenosis after PCI was generated by balloon injury to determine the protective effects and potential mechanisms of DABCD. The injured segments of aortae were collected on days 14 and 28 after the operation to observe the morphological changes in the vascular structure and measure the proportion of inflammatory factors in plasma and vascular tissues, as well as test the proliferative activity of VSMCs. The expression of related proteins, namely, Toll-like receptor (TLR) 4 and nuclear factor (NF)-κB, in the mechanistic study was clarified by western blot analysis. We tested the hypothesis that the cardioprotective effects of DABCD on aortic restenosis are associated with the inhibition of aortic intimal hyperplasia in this model. Our results showed that DABCD has protective effect on rat aortic restenosis and the anti-inflammatory mechanism of DABCD on balloon-induced restenosis in rat may be due to its ability to inhibit TLR4-mediated NF-κB signaling pathways. DABCD may be a potential therapeutic agent against restenosis.

Rapidly screening of α-glucosidase inhibitors from Dioscorea opposita Thunb. peel based on rGO@Fe3O4 nanocomposites microreactor.

Present study aimed to immobilise the α-glucosidase on suitable supports to construct enzymatic microreactors and their subsequent applicability in efficient inhibitor screening from the Chinese Yam (Dioscorea opposita Thunb.) peel. A type of lamellar and porous composites (rGO@Fe3O4) were synthesised with a facile one-step solvothermal method and employed as carriers to construct enzymatic microreactors for screening α-glucosidase ligand from the Chinese Yam peel in league with the high performance liquid chromatography and mass spectrometry (HPLC-MS). The immobilisation amount of α-glucosidase on rGO@Fe3O4 under the optimised conditions was about 40 µg α-glucosidase/mg carriers. Furthermore, the binding capacities of screened inhibitors, 2,4-dimethoxy-6,7-dihydroxyphenanthrene and batatasin I, were 35.6 and 68.2%, respectively. Hence, considering their high screening efficiency and excellent magnetic separation ability, these as-prepared nanocomposite consisting of rGO and Fe3O4 may be potential supports for the enzyme (such as α-glucosidase) immobilisation for rapid α-glucosidase inhibitors screening from the diverse nature resources.

Effects of Spica prunellae on caspase-3-associated proliferation and apoptosis in human lung cancer cells in vitro.

PURPOSE: This study aimed to explore the molecular mechanisms underlying the effects of Spica prunellae on proliferation and apoptosis of lung cancer cells in vitro. MATERIALS AND METHODS: The cell viability was determined with the CCK8 assay and the apoptosis was examined by flow cytometry. Western blotting was applied to detect the protein level of caspase-3 expression. RESULTS: It was observed that Spica prunellae could promote the apoptosis and inhibit the proliferation of A549 in vitro. The analysis of Western blotting showed that the expression level of proapoptosis protein caspase-3 was generally unchanged, whereas the level of activated caspase-3 was significantly increased. CONCLUSION: The results indicated that the growth of lung cancer cells A549 might be inhibited with Spica prunellae through activating the proapoptotic protein caspase-3 and inducing cellular apoptotic pathway.

Rapid identification of α-glucosidase inhibitors from Dioscorea opposita Thunb peel extract by enzyme functionalized Fe3O4 magnetic nanoparticles coupled with HPLC-MS/MS.

Dioscorea opposita Thunb, commonly known as "yam" that has a long dietary therapy history for diabetes, is widely consumed as a botanical dietary supplement and widely cultivated in China. In this work, a method for rapid screening of α-glucosidase inhibitors from Dioscorea opposita Thunb peel extract was developed using α-glucosidase functionalized magnetic nanoparticles (αG-MNPs) as a solid phase extraction absorbent in combination with high performance liquid chromatography-mass spectrometry (HPLC-MS). Two α-glucosidase inhibitors were selectively extracted and identified as batatasin I and 2,4-dimethoxy-6,7-dihydroxyphenanthrene. Their α-glucosidase inhibitory activities (IC50 = 2.55 mM and 0.40 mM, respectively) were significantly higher than that of acarbose (as control). Taking advantage of the specificity in enzyme binding and the convenience of magnetic separation, this method has great potential for rapid and fast screening of α-glucosidase inhibitors from complex natural resources.

Characterization of a Novel Polysaccharide-Iron(III) Complex and Its Anti-Anemia and Nonspecific Immune Regulating Activities.

OBJECTIVE: Dioscorea opposita Thunb is the famous food and traditional medicine in China and it was rich in polysaccharides. Polysaccharides of Dioscorea Opposita Thunb possess immunoregulatory activity, free radical scavenging activity and anti-diabetic activity. A novel polysaccharide- iron(III) complex (CYPIC) was synthesized by using crude polysaccharide extracted from Dioscorea opposita Thunb. The component, structure, morphology and molecular weights of CYPIC were analysed, and the anti-anemia, acute toxicity and nonspecific immune regulating activities of CYPIC were assayed. The results showed that CYPIC could increase red blood cell count (RBC), hemoglobin (Hb), hematocrit (HCT), thymus and spleen index of mice with iron deficiency anemia (IDA). Although the structure and deeper mechanisms of CYPIC should be further studied, CYPIC has the potential to be used as an iron supplement for the treatment of iron deficiency anemia. CONCLUSION: The large scale industrial production was suggested due to the simple preparation processing of CYPIC.

Preparation and Characterization of Copolymer Micelles for the Solubilization and In Vitro Release of Luteolin and Luteoloside.

Luteolin (LUT) and luteoloside (LUS) belong to flavonoids with high anticancer potential and were loaded into biodegradable diblock copolymer micelles of methoxy polyethylene glycol-polycaprolactone (mPEG5K-PCL10K), methoxy polyethylene glycol-polylactide-co-glycolide (mPEG5K-PLGA10K), and methoxy polyethylene glycol-polylactide (mPEG5K-PDLLA10K) by a self-assembly method, creating water-soluble LUT and LUS copolymer micelles, respectively. The solubilization formulations of the copolymer micelles were optimized with response surface methodology (RSM). The obtained drug micelles are torispherical under transmission electron microscope (TEM) with an average diameter of about 70 nm. The mPEG5K-PLGA10K exhibited higher loading capacity for LUS which was 4.33%, and LUT- (or LUS)-loaded mPEG5K-PCL10K exhibited a better stability and encapsulation efficiency which was 65.1 and 55.8%, respectively. The in vitro drug release study showed above 47% of LUT was released from micelles at pH 7.4 PBS; however, no more than 35% of LUT was released at pH 6.4 PBS within 24 h. Meanwhile, no more than 30% of LUS was released from micelles whether at pH 6.4 or 7.4 PBS solution within 24 h.

Low-temperature bath/high-conductivity zone/stacking micellar electrokinetic chromatography for the analysis of phenolic acids in coffee drink.

Two stacking methods in micellar electrokinetic chromatography (MEKC) were investigated in this article in an attempt to increase the amount of sample injected, as well as to focus analytes onto a small zone. One employed a "high-conductivity zone", which was inserted between the sample zone and background solution to build an unequal conductivity gradient. The other employed a "low-temperature bath". A portion of the capillary was immersed in a low-temperature bath, which served as a "pseudo-low-conductivity zone". As a result, a large volume of sample injection can be achieved. Using three phenolic acids-chlorogenic acid (CGA), caffeic acid (CA) and ferulic acid (FA) in coffee as model compounds, the limit of detection (LoD) was determined to be 0.31microg/ml (S/N=3) for CGA by means of normal MEKC under suppressed electroosmotic flow (EOF). The LoD could be improved to 2.8 x10(-2), 5.3 x10(-3) and 6.0 x10(-3) microg/ml, respectively, when normal MEKC-stacking, high-conductivity zone MEKC-stacking and the low-temperature zone MEKC-stacking methods were applied. Furthermore, the high conductivity zone and the low-temperature bath were operated simultaneously on one capillary to investigate the synergism effect. The results showed that there did exist synergism effect for CGA and CA when the two were hyphenated. The stacking efficiency was higher than that of the single one used. However, there was not synergism effect for FA.

Comparison of two sample preconcentration strategies for the sensitivity enhancement of flavonoids found in Chinese herbal medicine in micellar electrokinetic chromatography with UV detection.

Two on-column preconcentration techniques named stacking with reverse migrating micelles (SRMM) and anion selective electrokinetic injection and a water plug-sweeping with reverse migrating micelles (ASIW-sweep-RMM) were used and compared for concentration and separation of flavonoids in Chinese herbs using reverse migration micellar electrokinetic chromatography (RM-MEKC). The optimal background electrolyte (BGE) used for separation and preconcentration was a solution composed of 20mM phosphoric acid (H(3)PO(4))-100mM sodium dodecyl sulfate (SDS)-20% (v/v) acetonitrile (ACN) buffer (pH 2.0), the applied voltage was -15kV. To achieve reasonable results of the two techniques, the conditions which affected preconcentration were examined. A comparison of used techniques with normal hydrodynamic injection (5s), concerning enhancement factors and limits of detection (LODs) was presented. Under the optimum stacking conditions, about 27-37- and 45-194-fold improvement in the detection sensitivity was obtained for SRMM and ASIW-sweep-RMM, respectively, compared to usual hydrodynamic sample injection (5s). The LODs (S/N=3) for SRMM and ASIW-sweep-RMM in terms of peak height, can reach down to 1.15 x 10(-2) microg/ml for hesperetin and 2.4 x 10(-3) microg/ml for nobiletin, respectively. Finally, the amounts of the six flavonoids in extract of Fructus aurantii Immaturus were successfully determined using ASIW-sweep-RMM. The six analytes were baseline separated with sample matrix under the optimum ASIW-sweep-RMM conditions and the experimental results showed that preconcentration was well achieved after the dilution of sample solutions.

Separation and determination of psoralen and isopsoralen by microemulsion electrokinetic chromatography.

The use of microemulsion electrokinetic chromatography was proposed to separate psoralen and isopsoralen in Psoralea corylitolia L. and its preparations. After conducting a series of optimizations, baseline separation was obtained for the analytes under the optimum conditions [sodium dodecyl sulfate 1.05% (m/v), ethyl acetate 0.96% (v/v), butan-1-ol 0.24% (v/v), 25 mm borate, pH 8.5, applied voltage 17.5 kV and detection at 254 nm]. Regression equations revealed linear relationships (correlation coefficients 0.9997 for psoralen and 0.9999 for isopsoralen) between the peak area of each analyte and the concentration. The limits of detection (defined as a signal-to-noise ratio of about 3) were 0.42 microg/mL for psoralen and 0.32 microg/mL for isopsoralen, respectively. The analytes were successfully determined with recoveries ranging from 95.50 to 102.03%. The method has been successfully applied for the analysis of psoralen and isopsoralen in medical samples. Furthermore, a simple and effective extraction method, with methanol in an ultrasonic water bath for 20 min three times, was used for sample preparation.