Quickly Screening for Potential α-Glucosidase Inhibitors from Guava Leaves Tea by Bioaffinity Ultrafiltration Coupled with HPLC-ESI-TOF/MS Method.

Guava leaves tea (GLT) has a potential antihyperglycemic effect. Nevertheless, it is unclear which compound plays a key role in reducing blood sugar. In this study, GLT extract (IC50 = 19.37 ± 0.21 µg/mL) exhibited a stronger inhibitory potency against α-glucosidase than did acarbose (positive control) at IC50 = 178.52 ± 1.37 µg/mL. To rapidly identify the specific α-glucosidase inhibitor components from GLT, an approach based on bioaffinity ultrafiltration combined with high performance liquid chromatography coupled to electrospray ionization-time-of-flight-mass spectrometry (BAUF-HPLC-ESI-TOF/MS) was developed. Under the optimal bioaffinity ultrafiltration conditions, 11 corresponding potential α-glucosidase inhibitors with high affinity degrees (ADs) were screened and identified from the GLT extract. Quercetin (IC50 = 4.51 ± 0.71 µg/mL) and procyanidin B3 (IC50 = 28.67 ± 5.81 µg/mL) were determined to be primarily responsible for the antihyperglycemic effect, which further verified the established screening method. Moreover, structure-activity relationships were discussed. In conclusion, the BAUF-HPLC-ESI-TOF/MS method could be applied to determine the potential α-glucosidase inhibitors from complex natural products quickly.

High-Level Expression, Purification and Large-Scale Production of l-Methionine γ-Lyase from Idiomarina as a Novel Anti-Leukemic Drug.

l-Methionine γ-lyase (MGL), a pyridoxal 5'-phosphate-dependent enzyme, possesses anti-tumor activity. However, the low activity of MGL blocks the anti-tumor effect. This study describes an efficient production process for the recombinant MGL (rMGL) from Idiomarina constructed using the overexpression plasmid in Escherichia coli BL21 (DE3), purification, and large-scale production. The enzyme produced by the transformants accounted for 53% of the total proteins and accumulated at 1.95 mg/mL using a 500 L fermentor. The enzyme was purified to approximately 99% purity using a high-pressure mechanical homogenizer and nickel (Ni) Sepharose 6 Fast Flow (FF) chromatography. Then, the enzyme was polished by gel filtration, the endotoxins were removed using diethyl-aminoethanol (DEAE) Sepharose FF, and the final product was lyophilized with a vacuum freeze dryer at -35 °C. The specific activity of rMGL in the lyophilized powder was up to 108 U/mg. Compared to the control, the enzyme significantly inhibited cellular proliferation in a concentration-dependent manner as tested using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay and induced cellular apoptosis as analyzed by Annexin V-fluorescein isothiocyanate (FITC) with fluorescence-activated cell sorting (FACS) in leukemia cells. This paper demonstrated the cloning, overexpression, and large-scale production protocols for rMGL, which enabled rMGL to be used as a novel anti-leukemic drug.

CpG oligodeoxynucleotide protect neonatal piglets from challenge with the enterotoxigenic E. coli.

CpG motifs activates mammalian lymphocytes and macrophages to produce cytokines and polyclonal Ig. These include IFN-γ, IL-12, TNF-a, which are important in the control of bacterial infection. But thus far, the innate immunostimulatory effects of CpG ODN against pathogen have been established mainly in mouse, monkey, sheep, chicken, but not in neonatal piglets. The purpose of this study is to determine the potential protection of CpG ODN against enterotoxigenic Escherichia coli (ETEC) (with which neonatal piglets were susceptible to infection in our lab) in neonatal piglets. Here, we show intranasal (IN)-mucosal and intramuscularly (IM) systemic administration of CpG ODN could enhance innate cellular (cytokine) immunity in the sera and intestine mucosa post challenge, and thereafter the development of antigen-specific antibodies in piglets. IN and IM immunizations of neonatal piglets without antigen both reduced the ETEC excretion and alleviated diarrhoea symptoms upon challenge, and IN route had better protection effects than IM route. Protection in this study was linked to induction of a Th1 response which induced by CpG ODN. Co-delivery with Emulsigen (EM), could improve protection mediated by CpG ODN. These observations indicate that IN administration of 100 µg/kg CpG ODN with 20% EM codelivery may represent a valuable strategy for induction of innate immunity against ETEC infection in neonatal piglets.