Gang Bai

Structural insight into substrate specificity of human intestinal maltase-glucoamylase

Human maltase-glucoamylase (MGAM) hydrolyzes linear alpha-1,4-linked oligosaccharide substrates, playing a crucial role in the production of glucose in the human lumen and acting as an efficient drug target for type 2 diabetes and obesity. The amino- and carboxyl-terminal portions of MGAM (MGAM-N and MGAM-C) carry out the same catalytic reaction but have different substrate specificities. In this study, we report crystal structures of MGAM-C alone at a resolution of 3.1 Å, and in complex with its inhibitor acarbose at a resolution of 2.9 Å. Structural studies, combined with biochemical analysis, revealed that a segment of 21 amino acids in the active site of MGAM-C forms additional sugar subsites (+ 2 and + 3 subsites), accounting for the preference for longer substrates of MAGM-C compared with that of MGAM-N. Moreover, we discovered that a single mutation of Trp1251 to tyrosine in MGAM-C imparts a novel catalytic ability to digest branched alpha-1,6-linked oligosaccharides. These results provide important information for understanding the substrate specificity of alphaglucosidases during the process of terminal starch digestion, and for designing more efficient drugs to control type 2 diabetes or obesity.

Bioactivity-based liquid chromatography-coupled electrospray ionization tandem ion trap/time of flight mass spectrometry for beta(2)AR agonist identification in alkaloidal extract of Alstonia scholaris

Although chromatographic fingerprinting combined with chemometrics, is a rational method for the quality control of traditional Chinese medicine (TCM), chemometrics cannot fully explore the relationship between chemical information and the efficacy of the potential activity. In the present work, a cell-based β₂ adrenergic receptor (β₂AR) agonist functional evaluation model coupled with high-performance liquid chromatography was developed to screen the potential β₂AR agonist components in the alkaloidal extract of Alstonia scholaris leaves. Using a liquid chromatography with ion trap time-of-flight mass spectrometry (LCMS-IT-TOF) system, the potential bioactive compounds in the prescription were identified and deduced based on the mass spectrometric fragmentation patterns, tandem mass spectrometry (MS/MS) data, and relevant literature. Several new β₂AR agonists of indole alkaloids were successfully found, and their activities were confirmed through an in vivo relaxant test on guinea pig tracheal muscles. The developed method is rapid and reliable compared with conventional fingerprinting and showed high sensitivity and resolution for the identification of β₂AR agonists in TCM prescriptions. This strategy clearly demonstrates that bioactivity-integrated fingerprinting is a powerful tool not only in screening and identifying potential lead compounds and in determining the therapeutic material basis of Chinese herbal prescriptions, but also in supplying suitable chemical markers for their quality control.

Identification of the major constituents in Xuebijing injection by HPLC-ESI-MS

INTRODUCTION Xuebijing injection (XBJ) is a traditional Chinese herbal prescription widely used in the treatment of sepsis. This is the first report concerning the identification of XBJ constituents. In addition, to evaluate XBJs quality, partial least square discrimination analysis (PLS-DA) was performed on chemical fingerprint data. OBJECTIVE Establish an LC-MS method to identify the components in XBJ for the purpose of quality control. METHODOLOGY Compounds were separated by HPLC using a C(18) column and gradient elution of acetonitrile-methanol (60:40, v/v) and water-acetic acid (100 : 0.5, v/v) in 80 min. HPLC equipped with diode array detector (DAD) coupled with time-of-flight (TOF) tandem mass spectrometry and HPLC electrospray ionisation (ESI) multi-stage tandem ion-trap mass spectrometry (IT-MS(n) ) method was developed to analyse XBJs major components. Both positive and negative ionisation modes were employed. RESULTS Twenty-one compounds including amino acids, phenolic acids, flavonoid glycoside, terpene glycoside and phthalide were identified or tentatively characterised. Their retention times, UV and MS spectra were compared with those of authentic compounds or literature data. The score plot of PLS-DA clearly revealed variations among samples produced in different commercial batches. CONCLUSIONS The analytical method developed is highly effective for the discrimination and quality control of XBJ.

Development, Characterization, and Evaluation of a Fusion Protein of a Novel Glucagon-Like Peptide-1 (GLP-1) Analog and Human Serum Albumin in Pichia pastoris

Glucagon-like peptide-1 (GLP-1) has considerable potential as a possible therapeutic agent for type-2 diabetes. Unfortunately, this glucoincretin is short lived due to degradation by dipeptidyl-peptidase IV and rapid clearance by renal filtration. In this study, we attempted to extend GLP-1 action through the attachment of a lysine residue at the N-terminal of GLP-1 (named KGLP-1), and to make a fusion protein with human serum albumin (HSA) in Pichia pastoris. The protein, designated KGLP-1/HSA, was purified by an immunomagnetic separation technique. High performance liquid chromatography (HPLC) showed that the purified protein had an overall purity of 92.0%, and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) confirmed the expected molecular mass of 70,297.8 Da. Additionally, the N-terminal sequence of KGLP-1/HSA was confirmed by N-terminal sequencing. The stability and biological activity of KGLP-1/HSA were then evaluated in vitro and in vivo. The findings indicated that fusion KGLP-1/HSA preserved the action of native GLP-1, and the active duration was greatly prolonged.

Identification of higenamine in Radix Aconiti Lateralis Preparata as a beta2-adrenergic receptor agonist

AbstractAim:To screen beta2-adrenergic receptor (β2-AR) agonists from Radix Aconiti Lateralis Preparata (RALP) as potential drug leads for asthma using a sensitive cell-based agonist assay.Methods:The β2-AR gene was stably expressed by Chinese hamster ovary (CHO) cells also stably expressing a cyclic adenosine monophosphate (AMP) response element-linked enhanced green fluorescent protein reporter gene. The cells were used to screen agonists from high-performance liquid chromatographic fractions of an extract of RALP. The fraction with the highest activity was selected for further compound isolation and the study of the structure-activity relationship. Its active compound was further identified by chromatography and mass spectrometry.Results:Bioactivity-directed fraction-ation of the crude extract of RALP led to the isolation and characterization of the effective compound, namely hignamine. It could dose-dependently relax the isolated guinea pig trachea strip precontraction with acetylcholine with EC50 value of (2.60±0.36) × 10−5 mol/L. Further in vivo studies also displayed that hignamine could protect experimental asthma model induced by histamine in guinea pigs to prolong the latent periods of asthma.Conclusion:Hignamine, as a β2-AR agonist existing in the extract of RALP, is the key compound contributing to the successful relief of the bronchoconstriction.

Identification of NF-κB Inhibitors in Xuebijing injection for sepsis treatment based on bioactivity-integrated UPLC-Q/TOF

ETHNOPHARMACOLOGICAL EVIDENCE Inflammation plays an important role in sepsis, and NF-κB is a key mediator of inflammation. Xuebijing (XBJ) injection is a traditional Chinese medicine injection that was widely used in the treatment of sepsis in China. However, the active constituents and mechanism responsible for its actions have not been investigated experimentally. AIM OF THE STUDY To screen the anti-inflammatory components in XBJ injection and investigate the modulation of NF-κB in the treatment of sepsis. MATERIALS AND METHODS In this study, the effect of XBJ was assessed in the cecal ligation and puncture (CLP) -induced sepsis model. Subsequently, a bioactivity-integrated ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF) assay system was established for screening potential anti-inflammatory ingredients in XBJ. Ultimately, six active ingredients were confirmed through an in vitro test. RESULTS XBJ significantly reduced the mortality rate, anal temperature and expression of TNF-α, IL-1β and IL-6 induced by CLP. Nine potential anti-inflammatory ingredients, including gallic acid, danshensu, protocatechualdehyde, hydroxysafflor yellow A, oxypaeoniflorin, paeoniflorin, safflor yellow A, senkyunolide I and benzoylpaeoniflorin, were found based on the bioactivity-integrated UPLC-Q/TOF assay system. Among these compounds, the NF-κB inhibitor activity of senkyunolide I, safflor yellow A, oxypaeoniflorin, and benzoylpaeoniflorin are first reported here. CONCLUSIONS XBJ showed significant efficacy in the treatment of sepsis induced by CLP. Multiple targets of the different components were related to these anti-inflammatory effects, which contributed to the treatment of sepsis by XBJ in a clinical setting.

Cardioprotective effects of the YiQiFuMai injection and isolated compounds on attenuating chronic heart failure via NF-κB inactivation and cytokine suppression.

ETHNOPHARMACOLOGICAL RELEVANCE The YiQiFuMai injection (YQFM) is a traditional Chinese medicine for the treatment of chronic heart failure (CHF). The present study not only evaluated the cardioprotective effect and anti-inflammatory mechanism of the YQFM injection in an experimental model of CHF but also investigated its bioactive constituents in vitro. MATERIALS AND METHODS The left anterior descending coronary artery (LAD) in rats was ligated to make an animal model of CHF. From this, electrocardiographic parameters and exterior signs of rat hearts were recorded. Additionally, the histopathology of heart tissues was examined, and parameters of inflammatory stress were measured. Experiments were performed over two months in LAD-ligation rats treated with YQFM or vehicle. Treatment with Captopril was used as a positive control, which has previously been shown to prevent CHF, and rats without LAD-ligation were used as a negative control. Furthermore, we screened and identified potential anti-inflammatory constituents by ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS) combined with NF-κB activity luciferase reporter assay systems. Further cytokine detection confirmed the anti-inflammatory effects of the potential NF-κB inhibitors from YQFM. RESULTS The administration of YQFM significantly improved cardiac function and ameliorated the activity level of inflammatory mediators (such as tumor necrosis factor-alpha, interleukin-6, and interleukin-1β) in CHF rats. Eight potential anti-inflammatory ingredients, ginsenosides Rb1, Rg1, Rf, Rh1, Rc, Rb2, Ro, and Rg3, were characterized and confirmed. Among these compounds, ginsenoside Ro was revealed as a new NF-κB inhibitor. CONCLUSION The results suggested that NF-κB inactivation and cytokine suppression might be one of the main mechanisms of YQFM that caused ameliorative effects in CHF rats, and the major constituents of ginsenosides were identified playing a key role in the treatment of CHF.

Structures of human pancreatic α-amylase in complex with acarviostatins: Implications for drug design against type II diabetes

Human pancreatic α-amylase (HPA) catalyzes the hydrolysis of α-d-(1,4) glycosidic linkages in starch and is one of the major therapeutic targets for type II diabetes. Several acarviostatins isolated from Streptomyces coelicoflavus var. nankaiensis previously showed more potent inhibition of HPA than acarbose, which has been successfully used in clinical therapy. However, the molecular mechanisms by which acarviostatins inhibit HPA remains elusive. Here we determined crystal structures of HPA in complexes with a series of acarviostatin inhibitors (I03, II03, III03, and IV03). Structural analyses showed that acarviostatin I03 undergoes a series of hydrolysis and condensation reactions in the HPA active site, similar to acarbose, while acarviostatins II03, III03, and IV03 likely undergo only hydrolysis reactions. On the basis of structural analysis combined with kinetic assays, we demonstrate that the final modified product with seven sugar rings is best suited for occupying the full active site and shows the most efficient inhibition of HPA. Our high resolution structures reported here identify first time an interaction between an inhibitor and subsite-4 of the HPA active site, which we show makes a significant contribution to the inhibitory effect. Our results provide important information for the design of new drugs for the treatment of type II diabetes or obesity.

Microfractionation bioactivity-based ultra performance liquid chromatography/quadrupole time-of-flight mass spectrometry for the identification of nuclear factor-κB inhibitors and β2 adrenergic receptor agonists in an alkaloidal extract of the folk herb Alstonia scholaris.

Traditional Chinese medicines (TCMs) are generally considered complementary or alternative remedies in most Western countries. The constituents of TCMs are hard to define, and their efficacy is difficult to appraise. Thus, the development of suitable methods for evaluating the relationship between bioactivity and the chemical makeup of complex TCM mixtures remains a great challenge. In the present work, the bioactivity-integrated fingerprints of alkaloidal leaf extracts of Alstonia scholaris, a folk medicinal herb for chronic respiratory diseases, were established by ultra performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF). This method was coupled with two dual-luciferase reporter assay systems to show nuclear factor-κB (NF-κB) inhibition and β(2) adrenergic receptor (β(2)AR) activation. Using UPLC-Q/TOF, 18 potential candidates were identified according to unique mass spectrometric fragmentation. After in vitro biological evaluation, several indole alkaloids with anti-inflammatory and anti-asthmatic properties were found, including akuammidine, (E)-alstoscholarine, and (Z)-alstoscholarine. Compared with conventional fingerprints, the microfractionation based bioactivity-integrated fingerprints that contain both chemical and bioactivity details offer a more comprehensive understanding of the chemical makeup of plant materials. This strategy clearly demonstrated that dual bioactivity-integrated fingerprinting is a powerful tool for the improved screening and identification of potential dual-target lead compounds in complex herbal medicines.

Four acarviosin-containing oligosaccharides identified from Streptomyces coelicoflavus ZG0656 are potent inhibitors of α-amylase

Four aminooligosaccharides were isolated and purified from the culture filtrate of Streptomyces coelicoflavus ZG0656. Their chemical structures were determined by electrospray ionization tandem mass spectrometry (ESI-MS/MS) and two-dimensional nuclear magnetic resonance (NMR) spectroscopy. The names acarviostatins I03, II03, III03, and IV03 were given to the oligomers due to their acarviosin core structures. Acarviostatins III03 and IV03, which contain three and four acarviosin-glucose moieties, respectively, were identified as novel compounds. The four acarviostatins were all mixed noncompetitive inhibitors of porcine pancreatic alpha-amylase (PPA). The inhibition constants (K(i)) for acarviostatins III03 and IV03 were 0.008 and 0.033muM, respectively. Acarviostatin III03 is the most effective alpha-amylase inhibitor known to date, with a K(i) value 260 times more potent than acarbose.