Semi-preparative Reversed-phase Research Articles

Exploring novel antioxidant cyclic peptides in corn protein hydrolysate: Preparation, identification and molecular docking analysis

Antioxidant cyclic peptides were successfully identified from a corn protein hydrolysate. Hydrolysate by Alcalase + Flavourzyme showed the highest cyclic peptide purity (48.36 ± 1.81 %) and higher antioxidant activities compared with other hydrolysate. The success of peptide cyclization in hydrolysate was demonstrated by thermogravimetric analysis and thin-layer chromatography (TLC) analysis. Thermogravimetric analysis showed that the thermal stability of hydrolysate after cyclization was significantly increased, which was related to the formation of cyclic peptides. Peptides with molecular weight less than 1000 Da accounted for more than 80 % in hydrolysate after cyclization. After separation using gel silica chromatography and semi-preparative reverse phase high performance liquid chromatography (RP-HPLC), 22 novel antioxidant cyclic peptides were identified by ultra performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC-Q-TOF-MS) and orbitrap-tandem mass spectrometry (Orbitrap-MS/MS). Synthetic cyclic peptides with the same sequence were synthesized and characterized for their antioxidant activity. Molecular docking suggested that the free radical molecules could bind with the cyclic backbone and side chain of cyclic peptides through hydrogen bonding, hydrophobic interaction as well as electrostatic interaction. This study has important implications for the high-value utilization of corn protein and new cyclic peptides drugs or functional food development.

Purification and Identification of Peptides from Hydrilla verticillata (Linn. f.) Royle with Cytoprotective and Antioxidative Effect against H2O2-Treated HepG2 Cells.

Antioxidant peptides were purified from Hydrilla verticillata (Linn. f.) Royle (HVR) protein hydrolysate by ultrafiltration, gel filtration chromatography, and semipreparative reversed-phase HPLC and identified by UPLC-ESI-MS/MS. Therein, TCLGPK and TCLGER were selected to be synthesized, and they displayed desirable radical-scavenging activity to ABTS (99.20 ± 0.56-99.20 ± 0.43%), DPPH (97.32 ± 0.59-97.56 ± 0.97%), hydroxyl radical (54.32 ± 1.27-70.42 ± 2.01%), and superoxide anion (42.93 ± 1.46-52.62 ± 1.11%) at a concentration of 0.96 μmol/mL. They possessed a cytoprotective effect against H2O2-induced oxidative stress in HepG2 cells in a dose-dependent manner. 1.6 μmol/mL of the two peptides could perfectly protect HepG2 cells from H2O2-induced injury. The TCLGPK exhibited higher antioxidant activity and cytoprotective effect than TCLGER. Western blot and molecular docking results indicated that the two peptides achieved antioxidant ability and cytoprotective effect by combining with Kelch-like ECH-associated protein 1 (Keap1) to activate the Keap1-nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response elements signaling pathway, leading to the activity and expression of the related antioxidases in the pathway significantly up-regulating and the intracellular reactive oxygen species level, lipid peroxidation, and cell apoptosis rate significantly down-regulating.

Identification of potential bioactive compounds from Aspergillus terreus against HCV NS3 serine protease.

This study was aimed to isolate bioactive compounds from the fermentation products of Aspergillus terreus, which could inhibit NS3 protease of hepatitis C virus (HCV). The bioactive compounds were isolated by reverse-phase silica-gel column chromatography, semi-preparative reverse-phase, and Sephadex LH-20, and then their structures were elucidated through spectroscopic analysis. As a result, two small molecule compounds were isolated. Compound 1 was identified as a new benzaldehyde, (E)-2,4-dihydroxy-6-propenyl-benzaldehyde. Compound 2 was identified as pleurone, which was obtained from microorganisms for the first time. Their inhibitory activities against HCV NS3 protease (IC50) were 32.6 μM and 78.9 μM, respectively. This study provided a new option for the development of anti-HCV drugs.

A Comparison between High-Performance Countercurrent Chromatography and Fast-Centrifugal Partition Chromatography for a One-Step Isolation of Flavonoids from Peanut Hulls Supported by a Conductor like Screening Model for Real Solvents.

Peanut hulls (Arachis hypogaea, Leguminosae), which are a side stream of global peanut processing, are rich in bioactive flavonoids such as luteolin, eriodictyol, and 5,7-dihydroxychromone. This study aimed to isolate these flavonoid derivatives by liquid-liquid chromatography with as few steps as possible. To this end, luteolin, eriodictyol and 5,7-dihydroxychromone were isolated from peanut hulls using two different techniques, high-performance countercurrent chromatography (HPCCC) and fast-centrifugal partition chromatography (FCPC). The suitability of the biphasic solvent system composed of n-hexane/ethyl acetate/methanol/water (1.0/1.0/1.0/1.5; v/v/v/v) was determined by the Conductor like Screening Model for Real Solvents (COSMO-RS), which allowed the partition ratio KD-values of the three main flavonoids to be calculated. After a one-step HPCCC separation of ~1000 mg of an ethanolic peanut hull extract, 15 mg of luteolin and 8 mg of eriodictyol were isolated with purities over 96%. Furthermore, 3 mg of 5,7-dihydroxychromone could be isolated after purification by semi-preparative reversed-phase liquid chromatography (semi-prep. HPLC) in purity of over 99%. The compounds were identified by electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance spectroscopy (NMR).

Screening of trypsin inhibitors in Cotinus coggygria Scop. extract using at-line nanofractionation coupled with semi-preparative reverse-phase liquid chromatography

In this study, an at-line nanofractionation (ANF) platform was successfully fabricated in parallel with mass spectrometry and trypsin inhibitory bioactivity assessment for rapid screening of trypsin inhibitors (TIs) from natural products for the first time. After systematic optimization, the ANF platform was applied to screen and identify TIs in the extract of a traditional Chinese herb, i.e., Cotinus coggygria Scop. The semi-preparative reverse-phase liquid chromatography was used subsequently to further simplify and enrich the insufficiently separated components. After comprehensive evaluation and validation, the ANF platform successfully identified 12 compounds as potential TIs, including 8 flavonoids and 2 organic acids. Additionally, a comparison study was conducted using two other ligand fishing approaches, i.e., capillary monolithic and magnetic beads-based trypsin-immobilized enzyme microreactors, which successfully identified 8 identical flavonoids as TIs. Importantly, the molecular docking study showed the molecular interactions between enzymes and inhibitors, thus strongly supporting the experimental results. Overall, this work has fully demonstrated the feasibility of the established ANF platform for screening TIs from Cotinus coggygria Scop., and proved its great prospects for screening bioactive components from natural products.

Bacteroides fragilis derived metabolites, identified by molecular networking, decrease Salmonella virulence in mice model.

In the gut microbiota, resident bacteria prevent pathogens infection by producing specific metabolites. Among bacteria belonging to phylum Bacteroidota, we have previously shown that Bacteroides fragilis or its cell-free supernatant inhibited in vitro Salmonella Heidelberg translocation. In the present study, we have analyzed this supernatant to identify bioactive molecules after extraction and subsequent fractionation using a semi-preparative reversed-phase Liquid Chromatography High-Resolution Tandem Mass Spectrometry (LC-HRMS/MS). The results indicated that only two fractions (F3 and F4) strongly inhibited S. Heidelberg translocation in a model mimicking the intestinal epithelium. The efficiency of the bioactive fractions was evaluated in BALB/c mice, and the results showed a decrease of S. Heidelberg in Peyer's patches and spleen, associated with a decrease in inflammatory cytokines and neutrophils infiltration. The reduction of the genus Alistipes in mice receiving the fractions could be related to the anti-inflammatory effects of bioactive fractions. Furthermore, these bioactive fractions did not alter the gut microbiota diversity in mice. To further characterize the compounds present in these bioactive fractions, Liquid Chromatography High-Resolution Tandem Mass Spectrometry (LC-HRMS/MS) data were analyzed through molecular networking, highlighting cholic acid (CA) and deoxycholic acid. In vitro, CA had inhibitory activity against the translocation of S. Heidelberg by significantly decreasing the expression of Salmonella virulence genes such as sipA. The bioactive fractions also significantly downregulated the flagellar gene fliC, suggesting the involvement of other active molecules. This study showed the interest to characterize better the metabolites produced by B. fragilis to make them means of fighting pathogenic bacteria by targeting their virulence factor without modifying the gut microbiota.

Purification and Characterization of a Novel Calcium-Binding Heptapeptide from the Hydrolysate of Tilapia Bone with Its Osteogenic Activity.

In this study, a calcium-binding peptide was obtained by hydrolyzing tilapia bone and its osteogenic activity was evaluated. Animal protease was selected from nine enzymes, and its hydrolysate was purified through preparative and semi-preparative reverse phase high-performance liquid chromatography. The purified peptide was identified as DGPSGPK (656.32 Da) and its calcium-binding capacity reached 111.98 µg/mg. The peptide calcium chelate (DGPSGPK-Ca) was obtained, and its structure was characterized through Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), and mass spectrometry (MS). The results of XRD and SEM showed that DGPSGPK-Ca was formed as a new compound. The carboxyl and amino groups of Lys and Asp residues may be the chelating sites of DGPSGPK according to the FTIR and MS results. The molecular simulation showed the carbonyl groups of Asp, Pro, Ser, and Lys residues involved in the binding of calcium. The interaction of DGPSGPK and different integrins was evaluated by molecular docking simulation, and the main forces involved were electrostatic interaction forces, hydrogen bonding and hydrophobic interactions. Furthermore, DGPSGPK could inhibit the differentiation of osteoclast and promote the proliferation, differentiation and mineralization of osteoblasts.

Isolation of N-Ethyl-2-pyrrolidinone-Substituted Flavanols from White Tea Using Centrifugal Countercurrent Chromatography Off-Line ESI-MS Profiling and Semi-Preparative Liquid Chromatography

N-Ethyl-2-pyrrolidinone-substituted flavanols (EPSF) are marker compounds for long-term stored white teas. However, due to their low contents and diasteromeric configuration, EPSF compounds are challenging to isolate. In this study, two representative epimeric EPSF compounds, 5′′′R- and 5′′′S-epigallocatechin gallate-8-C N-ethyl-2-pyrrolidinone (R-EGCG-cThea and S-EGCG-cThea), were isolated from white tea using centrifugal partition chromatography (CPC). Two different biphasic solvent systems composed of 1. N-hexane-ethyl acetate-methanol-water (1:5:1:5, v/v/v/v) and 2. N-hexane-ethyl acetate-acetonitrile-water (0.7:3.0:1.3:5.0, v/v/v/v) were used for independent pre-fractionation experiments; 500 mg in each separation of white tea ethyl acetate partition were fractionated. The suitability of the two solvent systems was pre-evaluated by electrospray mass-spectrometry (ESI-MS/MS) analysis for metabolite distribution and compared to the results of the CPC experimental data using specific metabolite partition ratio KD values, selectivity factors α, and resolution factors RS. After size-exclusion and semi-preparative reversed-phase liquid chromatography, 6.4 mg of R-EGCG-cThea and 2.9 mg of S-EGCG-cThea were recovered with purities over 95%. Further bioactivity evaluation showed that R- and S-EGCG-cThea possessed in vitro inhibition effects on α-glucosidase with IC50 of 70.3 and 161.7 μM, respectively.

Purification and antibacterial properties of a novel bacteriocin against Escherichia coli from Bacillus subtilis isolated from blueberry ferments

The novel bacteriocin JS17 was obtained from Bacillus subtilis JSX-5, isolated from blueberry (Vaccinium uliginosum) ferments. JS17 was purified by ÄKTA Purifier, semi-preparative reversed-phase high performance liquid chromatography, and nano liquid chromatography coupled with tandem mass spectrometry, in succession. Its molecular mass was 652.37 Da and its amino acid sequence was identified as L-F-R-A-F. A homology BLAST search confirmed JS17 as a novel bacteriocin. JS17 was found to have an extensive antibacterial activity spectrum against both Gram-positive and Gram-negative bacteria. JS17 showed heat tolerance, and it was highly stable over pH 2–10 and was sensitive to proteinase K and pepsin. This indicates that JS17 exhibits protein properties and has application potential under high temperature and acid-base conditions. The minimum inhibitory concentration and minimum bactericide concentration of JS17 against Escherichia coli were 10.56 μg/mL and 22.03 μg/mL, respectively, which is lower than that of most previously described bacteriocins. Furthermore, scanning electron microscopy showed that JS17 bactericidally destroyed the cell membrane integrity of E. coli, resulting in cell dissolution and impaired membrane permeability. This study first purified B. subtilis bacteriocin from fruit ferments, suggesting the potential for using JS17 as a food bio-preservative.

Purification and identification of novel antioxidant peptides from watermelon seed protein hydrolysates and their cytoprotective effects on H2O2-induced oxidative stress

The aim of this study was to purify and identify antioxidant peptides from watermelon seed protein hydrolysates (WSPHs-I: Mw < 1 kDa) and further evaluate their cytoprotective effects against H2O2-induced oxidative stress in HepG2 cells. After purification by Sephadex G-15 and semi-preparative reversed-phase high performance liquid chromatography (RP-HPLC), five peptides, RDPEER (P1), KELEEK (P2), DAAGRLQE (P3), LDDDGRL (P4), and GFAGDDAPRA (P5) were sequenced by LC-MS/MS and synthesized with solid-phase synthesis method. These peptides showed desirable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging capacity (IC50: 0.216 ± 0.01–0.435 ± 0.03), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging capacity (IC50: 0.54 ± 0.02–1.23 ± 0.03), and oxygen radical absorbance capacity (ORAC) (82.36 ± 1.2–130.67 ± 2.2 μM TE/mg). Among them, peptide P1 exhibited the strongest antioxidant capacity. Moreover, the results suggested that peptide P1 may protect HepG2 cells from H2O2-induced oxidative damage by significantly inhibiting reactive oxygen species (ROS), [Ca2+]i, malondialdehyde (MDA) levels and increasing antioxidative enzyme activities.

Valorization of Yak (Bos grunniens) Bones as Sources of Functional Ingredients

Yak (Bos grunniens) bone, is an underutilized biowaste that is generated as a major byproduct from yak slaughtering process. More commonly, yak bones are either made into primary processing products like whole bone powder or simply disposed, which may not only cause resources waste but also produce pressure on the sensitive and frail environment in cold plateau areas. The aim of this study was to valorize yak bone biowastes to functional ingredients while simultaneously reduce the adverse impact of yak bone biowastes discharge on plateau fragile ecological environment. Sequential purification of yak bone collagen hydrolysates was achieved by ultrafiltration, size exclusion chromatography (SEC), semi-preparative reverse phase high-performance liquid chromatography (RP-HPLC) and HPLC coupled to electrospray ionization tandem mass spectrometry (ESI–MS/MS). The bioactivity of the peptides on MC3T3-E1 osteoblast proliferation was evaluated in vitro by MTT method. Furthermore, the most potent peptide was selected and its underlying molecular mechanism was investigated through molecular docking simulation using the software of Discovery Studio 2018. 35 novel peptides were identified by mass spectrometry analysis, of which 9 were chemically synthesized and evaluated for the osteoblast proliferation-promoting activity. At a concentration of 0.05 mg/mL, peptide GPAGPPGPIGNV displayed the highest osteoblast proliferation-promoting activity with an increase of 42.7% in cell growth. Molecular docking study revealed that the main interactions of GPAGPPGPIGNV with epidermal growth factor receptor were the van der waals and hydrogen bonding. These results suggest that yak bones-derived peptides could be potentially used as osteogenic ingredients in functional foods. This work provides a feasible strategy for valorization of yak bone biowastes and sustainable development of animal husbandry industry in Qinghai-Tibetan Plateau. This work provides a feasible strategy for valorization of yak bone biowastes and sustainable development of animal husbandry industry in Qinghai-Tibetan Plateau.

Fully automated radiosynthesis and quality control of estrogen receptor targeting radiopharmaceutical 16α-[18F]fluoroestradiol ([18F]FES) for human breast cancer imaging

16α-[18F]Fluoroestradiol ([18F]FES) is the most successful estrogen receptor (ER) targeting radiopharmaceutical to date. [18F]FES has been extensively used for positron emission tomography (PET) to assess the ER expression in breast cancer and to monitor the response of breast cancer to antiestrogen therapy. To address local investigator needs for [18F]FES-PET, we sought to adapt established literature methods to our in-house multi-purpose 18F-radiosynthesis module for [18F]FES production. Here we describe facile fully automated radiosynthesis and quality control (QC) of [18F]FES using our home-built automated multi-purpose 18F-radiosynthesis module. [18F]FES was produced via two-step-one-pot synthesis using cyclic sulfate precursor, and purified by semi-preparative reversed-phase (RP) high performance liquid chromatography (HPLC) with a C18 column followed by solid-phase extraction (SPE) with a C18 Plus Sep-Pak cartridge trap/release formulation. The overall synthesis time was 75–80 min, and the radiochemical yield was 30–35% decay corrected to end of bombardment (EOB), based on H[18F]F. The radiochemical purity was >99%, and the molar activity (Am) was 182–470 GBq/μmol at EOB. The [18F]FES dose meets all QC criteria for clinical use, and is suitable for clinical PET study of breast cancer.

Total Synthesis of the Natural Products Ulmoside A and (2R,3R)-Taxifolin-6-C-β-d-glucopyranoside

An efficient first total synthesis of highly polar ulmoside A and (2R,3R)-taxifolin-6-C-β-d-glucopyranoside, useful for the prevention of metabolic disorders, has been described. Key elements of the synthesis include a Sc(OTf)3-catalyzed regio- and stereoselective C-glycosidation on taxifolin in 35% yield with d-glucose and chiral semipreparative reverse-phase high-performance liquid chromatography (HPLC) for the separation of both taxifolins and the diastereomeric mixture of taxifolin-6-C-β-d-glucopyranosides. Correlation of the analytical data of synthetic ulmoside A and its diastereomer with a natural ulmoside A sample confirmed the assigned absolute stereochemistry of the natural products.

Chemical profiling of secondary metabolites from Himatanthus drasticus (Mart.) Plumel latex with inhibitory action against the enzymes α-amylase and α-glucosidase: In vitro and in silico assays

Ethnopharmacological relevanceHimatanthus drasticus is an important medicinal plant whose latex is traditionally used in Northeast Brazil to treat various diseases, including diabetes. The use of α-amylase and α-glucosidase inhibitors can be an effective strategy to modulate levels of postprandial hyperglycemia via control of starch metabolism. Aims of the studyThis study aimed to verify if H. drasticus latex has inhibitory activity against enzymes linked to type 2 diabetes, besides chemically characterizing the metabolites responsible for such activities. In addition, in silico analysis was performed to support the traditional claim of possible antidiabetic activity of this latex. Materials and methodsLatex from H. drasticus stems was sequentially partitioned with n-hexane (FHDH), CHCl3 (FHDC) and EtOH (FHDHA). Wash extraction of the FHDHA fraction was performed to obtain the other extract fractions. The FHDHA was submitted to chromatography in a SPE C18 cartridge using gradient elution with MeOH/H2O to produce five fractions: FHDHA1, FHDHA2, FHDHA3, FHDHA4 and FHDHA5. The FHDHA1 was subjected to semi-preparative reverse phase HPLC. Lineweaver-Burk plots were used to investigate the kinetic parameters of α-amylase and α-glucosidase inhibitory activity. The interactions between plumieride and porcine pancreatic α-amylase and α-glucosidase were analyzed through an in silico molecular docking study. ResultsPhytochemical identification of compounds present in the FHDHA fraction of H. drasticus latex was possible by 1H, 13C NMR analysis and mass spectrometry, and the results were compared with the literature. The identified compounds were α-ethyl glucoside, protocatechuic acid, 3-O-caffeoylquinic acid, 15-demethylplumieride acid, 5-O-caffeoylquinic acid, caffeic acid, vanillic acid, plumieride, and catechin. The inhibition results of the fractions tested against α-amylase and α-glucosidase showed inhibitory activities dependent on the increase of fractions and compound concentrations. The IC50 results obtained from FHDHA, FHDHA1 and plumieride fractions against α-amylase were 36.46, 72.61, 33.87 μg/mL respectively. The IC50 of plumieride was the closest to that of acarbose (22.52 μg/mL), a result similar to that obtained for α-glucosidase. The type of inhibition was competitive for both enzymes. ConclusionsThere was strong inhibition of α-amylase and α-glucosidase by FHDHA, FHDHA1 and plumieride, suggesting that these enzymes slow glucose absorption.

Novel Antioxidant Peptides Purified from Mulberry (Morus atropurpurea Roxb.) Leaf Protein Hydrolysates with Hemolysis Inhibition Ability and Cellular Antioxidant Activity.

Neutrase-hydrolysates hydrolyzed from mulberry leaf proteins were separated by ion exchange chromatography, gel filtration chromatography, and semipreparative reverse-phase HPLC. Purified fractions were analyzed for their radical scavenging activity, hemolysis inhibition ability, and cellular antioxidant activity (CAA). Three new antioxidant peptides, P1 (SVL, 317 Da), P2 (EAVQ, 445 Da), and P3 (RDY, 452 Da), were obtained from the most active HPLC fraction (R1) and identified using UPLC-QTOF-MS. These three peptides were then synthesized, and their antioxidant activities were analyzed. P1 and P2 had no ability to inhibit hemolysis of erythrocytes but did show antioxidant activity on HepG2 cells. P3 showed the highest hemolysis inhibition ability (92%) and CAA value (2204 μM QE/100 g peptide). The Tyr residues at the C-terminal region play an important role in the antioxidant activity in P3. Thus, the natural peptide R1 and synthesized P3 could be used as antioxidants and might be promising components of functional foods.

Composition and antioxidant activity of anthocyanins from Aronia melanocarpa cultivated in Haicheng, Liaoning, China

Aronia melanocarpa fruits are high in phenolic substances-mainly anthocyanins. The anthocyanins, as a water-soluble plant pigments with strong antioxidant activity, have been used in food and medical industries. The fresh weight, total soluble solids, total anthocyanins and total phenols of Aronia melanocarpa were determined. Anthocyanins were purified using ion exchange resin coupled with chromatography, and identified using HPLC and mass spectrometry. Then, the antioxidant activities of anthocyanins were determined using different kit methods, and the potential application of purified anthocyanins was tested in lard. The results showed the anthocyanins content increased from 280 to 930 mg/g after being purified using semi-preparative reverse phase HPLC. After anthocyanins were purified using absorbent resin, a total of 7 different anthocyanins, including cyanidin-3,5-dihexoside, the dimer of cyanidin-hexoside, cyanidin-3-O-(galactoside, glucoside, arabinoside, xyloside) and delphinidin-3-O-rutinoside, were identified using HPLC-MS, cyanidin-3,5-dihexoside and a dimer of cyanidin-hexoside were first found in A. melanocarpa. In addition, cyanidin-3-O-(galactoside, glucoside, arabinoside, xyloside) were identified using HPLC from anthocyanins purified using absorbent resin coupled with semi-preparative reverse phase HPLC. Furthermore, the antioxidant tests showed that the antioxidant capacity of anthocyanins could be significantly improved by increasing the purity of anthocyanins, and purified anthocyanins were a good antioxidant for lard. A. melanocarpa cultivated in Haicheng is high in anthocyanins and cyanidin-3-O-(galactoside, glucoside, arabinoside, xyloside) were the main anthocyanins. Purified anthocyanins could be separated and may have further applications in food.

Bioactive Peptides from Germinated Soybean with Anti-Diabetic Potential by Inhibition of Dipeptidyl Peptidase-IV, α-Amylase, and α-Glucosidase Enzymes

Functional foods containing peptides offer the possibility to modulate the absorption of sugars and insulin levels to prevent diabetes. This study investigates the potential of germinated soybean peptides to modulate postprandial glycaemic response through inhibition of dipeptidyl peptidase IV (DPP-IV), salivary α-amylase, and intestinal α-glucosidases. A protein isolate from soybean sprouts was digested by pepsin and pancreatin. Protein digest and peptide fractions obtained by ultrafiltration (<5, 5–10 and >10 kDa) and subsequent semipreparative reverse phase liquid chromatography (F1, F2, F3, and F4) were screened for in vitro inhibition of DPP-IV, α-amylase, maltase, and sucrase activities. Protein digest inhibited DPP-IV (IC50 = 1.49 mg/mL), α-amylase (IC50 = 1.70 mg/mL), maltase, and sucrase activities of α-glucosidases (IC50 = 3.73 and 2.90 mg/mL, respectively). Peptides of 5–10 and >10 kDa were more effective at inhibiting DPP-IV (IC50 = 0.91 and 1.18 mg/mL, respectively), while peptides of 5–10 and <5 kDa showed a higher potency to inhibit α-amylase and α-glucosidases. Peptides in F1, F2, and F3 were mainly fragments from β-conglycinin, glycinin, and P34 thiol protease. The analysis of structural features of peptides in F1–F3 allowed the tentative identification of potential antidiabetic peptides. Germinated soybean protein showed a promising potential to be used as a nutraceutical or functional ingredient for diabetes prevention.

Solid Phase Synthesis of Marine Cyclopeptide Phakellistatin 13

The synthesis of the naturally occurring marine cyclopeptide phakellistatin 13 has been achieved by solid phase peptide synthesis (SPPS). A general method was described to synthesize the cyclic peptide by a two-step solid-phase/solution synthesis strategy. The linear peptide was assembled by standard Fmoc chemistry on solid-phase and subsequently cyclized in solution phase to yield fully protected cyclic heptapeptide. After removing the protected groups, phakellistatin 13 was obtained and purified by semi-preparative reverse phase high-performance RP-HPLC, and its structure was confirmed by NMR and HR-ESI-MS.

Bioassay-guided purification of Lippia citriodora polyphenols with AMPK modulatory activity

Recently, the relationship between nutrients and health is becoming since natural dietary products as polyphenols are being considered by their potential for the management of several diseases. We aimed to investigate the capacity of Lippia citriodora compounds to modulate AMP-activated protein kinase activity (AMPK) on a hypertrophic adipocyte model. HPLC semi-preparative purification method and reverse phase high performance liquid chromatography coupled to time-of-flight mass detection with electrospray ionization (RP-HPLC-ESI-TOF/MS) were used to obtain de compounds from L. citriodora extract. AMPK activity was measured on the hypertrophic 3T3-L1 adipocyte model by immunofluorescence microscopy. Four compounds of 29 total compounds have been tentatively identified in L. citriodora for the first time by HPLC-ESI-TOF-MS. Phenylpropanoids (verbascoside), iridoids (gardoside) and flavonoids (luteolin-7-diglucoronide) were the best candidates to account for activating AMPK capacity. The combination of specific polyphenols from L. citriodora, which showed strong activating AMPK capacity, could be an alternative in the management of obesity-associated diseases.

Hypaphorine, an Indole Alkaloid Isolated from Caragana korshinskii Kom., Inhibites 3T3-L1 Adipocyte Differentiation and Improves Insulin Sensitivity in Vitro.

Obesity, a major health problem worldwide, is a complex multifactorial chronic disease that increases the risk for insulin resistance, type 2 diabetes, coronary heart disease, and hypertension. In this study, we assessed methods to isolate hypaphorine, a potent drug candidate for obesity and insulin resistance. Semi-preparative reversed-phase liquid chromatography (semi-preparative RPLC) was established as a method to separate three compounds, adenosine, l-tryptophan, and hypaphorine, from the crude extracts of Caragana korshinskii Kom. Due to its specific chemical structure, the effect of hypaphorine on differentiation and dexamethasone (DXM) induced insulin resistance of 3T3-L1 cells was investigated. The structures of the three compounds were confirmed by UV, 1 H-NMR, and 13 C-NMR analysis and compared with published data. The activity results indicated that hypaphorine prevented the differentiation of 3T3-L1 preadipocytes into adipocytes by down-regulating hormone-stimulated protein expression of peroxisome proliferator activated receptor γ (PPARγ) and CCAAT/enhancer binding protein (C/EBPα), and their downstream targets, sterol regulatory element binding protein 1 c (SREBP1c) and fatty acid synthase (FAS). Hypaphorine also alleviated DXM-induced insulin resistance in differentiated 3T3-L1 adipocytes via increasing the phosphorylation level of Akt2, a key protein in the insulin signaling pathway. Taken together, we suggest that the method can be applied to large-scale extraction and large-quantity preparation of hypaphorine for treatment of obesity and insulin resistance.