Synthesis, pharmacological evaluation and mechanistic study of scutellarin methyl ester -4'-dipeptide conjugates for the treatment of hypoxic-ischemic encephalopathy (HIE) in rat pups.

A series of novel scutellarin methyl ester-4'-dipeptide conjugates exhibiting active transport characteristics and protection against pathological damage caused by hypoxic-ischemic encephalopathy (HIE) were successfully designed and synthesized. The physiochemical properties of the obtained compounds, as well as the Caco-2 cell-based permeability and uptake into hPepT1-MDCK cells were evaluated using various analytical methods. Scutellarin methyl ester-4'-Val-homo-Leu dipeptide (5k) was determined as the optimal candidate with a high apparent permeability coefficient (Papp A to B) of 1.95 ± 0.24 × 10-6 cm/s, low ER (Papp BL to AP/Papp AP to BL) of 0.52 in Caco-2 cells, and high uptake of 25.47 µmol/mg/min in hPepT1-MDCK cells. Comprehensive mechanistic studies demonstrated that pre-treatment of PC12 cells with 5k resulted in more potent anti-oxidative activity, which was manifested by a significant decrease in the malondialdehyde (MDA) and reactive oxygen species (ROS) levels, attenuation of the H2O2-induced apoptotic cell accumulation in the sub-G1 peak, and improvement in the expression of the relevant apoptotic proteins (Bcl-2, Bax, and cleave-caspase-3). Moreover, evaluation of in vivo neuroprotective characteristics in hypoxic-ischemic rat pups revealed that 5k significantly reduced infarction and alleviated the related pathomorphological damage. The compound was also shown to ameliorate the neurological deficit at 48 h as well as to decrease the brain tissue loss at 4 weeks. Conjugate 5k was demonstrated to reduce the amyloid precursor protein (APP) and ß-site APP-converting enzyme-1 (BACE-1) expression. Pharmacokinetic characterization of 5k indicated favorable druggability and pharmacokinetic properties. The conducted docking studies revealed optimal binding of 5k to PepT1. Hydrogen bonding as well as cation-π interactions with the corresponding amino acid residues in the target active site were clearly observed. The obtained results suggest 5k as a potential candidate for anti-HIE therapy, which merits further investigation.

Xylooligosaccharides production from a sugarcane biomass mixture: Effects of commercial enzyme combinations on bagasse/straw hydrolysis pretreated using different strategies.

Xylooligosaccharides (XOS) are non-digestible food ingredients with prebiotic properties for selectively promoting the growth of probiotics, which provide many health benefits and several applications in the food and pharmaceutical industry. The objective of this study was to optimize the concentration of commercial hemicellulases for the production of XOS, with a 2-6 polymerization degree, using a mixture of sugarcane bagasse and straw pretreated with ionic liquid or diluted sulfuric acid. The concentrations of enzymes endo-1,4-xylanase (NS50030, Novozyme®) and α-L-arabinofuranosidase (GH51) (Megazyme®) were optimized using a central composite rotatable design (CCRD). The xylooligosaccharides (XOS) released by hydrolysis were analyzed via capillary electrophoresis and quantified with HPAEC-PAD. The XOS profile obtained from the hydrolisis of the pretreated sugarcane biomass mixture (MPSA) was similar to that obtained with the hydrolisis of MBX, which provided higher xylobiose (X2) concentration. Our results also demonstrated that pretreatment with an ionic liquid favored the requirement of lower enzyme concentration in enzymatic hydrolysis for having provided a biomass with lower lignin content than the pretreatment with dilute sulfuric acid. It required up to 20% less of the optimum concentration of the endo-1,4-xylanase mixture to achieve similar values to those obtained with the biomass pretreated with dilute sulfuric acid, representing a possible alternative to reduce enzymatic cost.

Demystifying a hexuronic acid ligand that recognizes Toxoplasma gondii and blocks its invasion into host cells.

Toxoplasma gondii is a ubiquitous eukaryotic pathogen responsible for toxoplasmosis in humans and animals. This parasite is an obligate intracellular pathogen and actively invades susceptible host cells, a process which is mediated by specific receptor-ligand interactions. Here, we have identified an unnatural 2,4-disulfated d-glucuronic acid (Di-S-GlcA), a hexuronic acid composed of heparin/heparan sulfate, as a potential carbohydrate ligand that can selectively bind to T. gondii parasites. More importantly, the gelatin conjugated Di-S-GlcA multivalent probe displayed strong inhibition of parasite entry into host cells. These results open perspective for the future use of Di-S-GlcA epitopes in biomedical applications against toxoplasmosis.

Total synthesis of scutellarin and apigenin 7-O-ß-d-glucuronide.

A general protocol for direct glucuronic linkages formation featuring Au(I)-catalyzed appropriately protected glucuronyl o-alkynylbenzoate-involved glycosylation reaction, as well as a concise approach for easy access of scutellarein prominent for the mild and efficient hydroxyl group installation via borylation-oxidation sequence from flavanone derivative, has been established, based on which a novel route for scutellarin derivatives preparation has been devised. The developed strategies, among which the stepwise deprotection process was also included, guarantee the high whole synthetic efficiency, and definitely will find broad application in diversity-oriented synthesis of bioactive flavonoid glycosides.

Synthesis of Scutellarein Derivatives with a Long Aliphatic Chain and Their Biological Evaluation against Human Cancer Cells.

Scutellarin is the major active flavonoid extracted from the traditional Chinese herbal medicine Erigeron breviscapus (Vant.) Hand-Mazz., which is widely used in China. Recently, accumulating evidence has highlighted the potential role of scutellarin and its main metabolite scutellarein in the treatment of cancer. To explore novel anticancer agents with high efficiency, a series of new scutellarein derivatives with a long aliphatic chain were synthesized, and the antiproliferative activities against Jurkat, HCT-116 and MDA-MB-231 cancer cell lines were assessed. Among them, compound 6a exhibited the strongest antiproliferative effects on Jurkat (IC50 = 1.80 µM), HCT-116 (IC50 = 11.50 µM) and MDA-MB-231 (IC50 = 53.91 µM). In particular, 6a even showed stronger antiproliferative effects than the positive control NaAsO2 on Jurkat and HCT-116 cell lines. The results showed that a proper long aliphatic chain enhanced the antiproliferative activity of scutellarein.

Highly Specific near-Infrared Fluorescent Probe for the Real-Time Detection of ß-Glucuronidase in Various Living Cells and Animals.

ß-Glucuronidase (GLU) is an important biomarker for primary cancers and intestinal metabolism of drugs or endogenous substances; however, an effective optical probe for near-infrared (NIR) monitoring in vivo is still lacking. Herein, we design an enzyme-activated off-on NIR fluorescent probe, HC-glu, based on a hemicyanine keleton, which is conjugated with a d-glucuronic acid residue via a glycosidic bond, for the fluorescent quantification and trapping of endogenous GLU activity in vitro and in vivo. The newly developed NIR probe exhibited prominent features including prominent selectivity, high sensitivity, and ultrahigh imaging resolution. It has been successfully used to detect and image endogenous GLU in various hepatoma carcinoma cells, tumor tissues, and tumor-bearing mouse models, for cancer diagnosis and therapy. Moreover, it could detect the in vivo activity of GLU in the intestinal tracts of animals including mice and zebrafish, where GLU performs a vital biological function and is mainly distributed. It could also evaluate real intestinal distribution and real-time variations of GLU in development and growth, all of which are very helpful to guide rational drug use in the clinic. Our results fully demonstrated that HC-glu may serve as a promising tool for evaluating the biological function and process of GLU in living systems.

Anti-Dengue, Cytotoxicity, Antifungal, and In Silico Study of the Newly Synthesized 3-O-Phospo-α-D-Glucopyranuronic Acid Compound.

The aim of the current study was to synthesize new bioactive compounds and evaluate their therapeutic relevance. The chemical structure of compound 7 (methyl 3-O-phospo-α-D-glucopyranuronic acid was elucidated by physical and advance spectral technique. Also, this compound was assessed for various in vitro biological screening. The results showed that compound 7 has promising antifungal activity against selected fungal strains. Computational study was also carried out to find antimalarial efficacy of the synthesized compounds. Compounds (2-7) were tested for cytotoxicity by MTT assay, and no considerable cytotoxicity was observed. Molecular docking study was performed to predict the binding modes of new compound (7). The docking results revealed that the compound has strong attraction towards the target protein, as characterized by good bonding networks. On the basis of the acquired results, it can be predicted that compound (7) might show good inhibitory activity against dengue envelope protein.

Production of High Commercial Value Xylooligosaccharides from Meranti Wood Sawdust Using Immobilised Xylanase.

The present study explores the utilisation of a new raw material from lignocellulose biomass, Meranti wood sawdust (MWS) for high commercial value xylooligosaccharides (XOS) production using immobilised xylanase. The xylanase was immobilised by a combination of entrapment and covalent binding techniques. The hemicellulosic xylan from MWS was extracted using a standard chlorite delignification method. The production of total and derivatives of XOS from the degradation of the hemicellulosic xylan of MWS were compared to the production from the commercial xylan from Beechwood. The utilisation of the extracted xylan from MWS yielded 0.36 mg/mL of total XOS after 60 h of hydrolysis. During the hydrolysis reaction, the immobilised xylanase released a lower degree of polymerisation (DP) of XOS, mainly X2 and X3, which were the major products of xylan degradation by xylanase enzymes. The production of XOS with a lower DP from MWS demonstrated the biotechnological potential of the MWS in the future. The XOS production retained about 70% of its initial XOS production during the second cycle. This is also the first report on the utilisation of MWS wastes in enzymatic hydrolysis using immobilised xylanase for XOS production.

Scutellarin derivatives as apoptosis inducers: Design, synthesis and biological evaluation.

To explore novel antitumor agents with high efficiency and low toxicity, a series of NO-donating scutellarin derivatives (14-17) were synthesized and the antiproliferative activities against MCF-7, HCT-116, PC-3 and HepG2 cancer cell lines were assessed. Among them, compound 14b was the strongest with IC50 values of 2.96 µM, 7.25 µM, 0.09 µM and 0.50 µM, respectively, and displayed low toxicity against normal human liver L-O2 cells with an IC50 of 47.96 µM, showing good selectivity between normal and malignant liver cells. Moreover, NO releasing ability of the derivatives has been studied. Mechanism studies of the most promising compounds 14b and 15a were carried out. The results indicated that 14b and 15a could induce apoptosis, cell cycle arrest at the S phase and led to mitochondrial dysfunction in the HepG2 and PC-3 cell lines, respectively. Furthermore, Human Apoptosis Protein Array kit assay demonstrated that 14b could induce apoptosis through down-regulating the levels of procaspase-3 and inhibiting the expression of survivin, c-IAP1, HSP27, HSP60, HSP70, HO-1/HMOX1/HSP32 and HO-2/HMOX2 in HepG2 cell line. These results guaranteed compound 14b to be a drug candidate against liver cancer for further investigation.

Exploitation of new structurally diverse d-glucuronamide-containing N-glycosyl compounds: synthesis and anticancer potential.

The synthesis and anticancer evaluation of novel N-glycosyl derivatives containing N-substituted glucuronamide moieties, as nucleoside analogs or as prospective mimetics of glycosyl phosphates or of nucleotides, is reported. These compounds comprise N-anomerically-linked nucleobases or motifs that are surrogates of a phosphate group, such as sulfonamide or phosphoramidate moieties. 1-Sulfonamido glucuronamides containing N-benzyl, N-propargyl or N-dodecyl carboxamide units were synthesized through glycosylation of methanesulfonamide with tetra-O-acetyl glucuronamides. 1-Azido glucuronamides were accessed by microwave-assisted reactions of tetra-O-acetyl glucuronamides with TMSN3 and were further converted into N-glycosylphosphoramidates by treatment with trimethyl phosphite. Potential glucuronamide-based nucleotide mimetics comprising both an anomeric sulfonamide/phosphoramidate group and a benzyltriazolylmethyl amide system at C-5, as nucleobase mimetics, were synthesized via 'click' cycloaddition of N-propargyl glucuronamide derivatives with benzyl azide. N-Dodecyl tetra-O-acetyl glucuronamides were converted into uracil and purine nucleosides via N-glycosylation of the corresponding silylated nucleobases. Biological screening revealed significant antiproliferative activities of the N-dodecyl glucuronamide-containing sulfonamide, phosphoramidate and nucleosides in K562 and MCF-7 cells. The highest effect was exhibited by the N9-linked purine nucleoside in the breast cancer cell MCF-7 with a GI50 value similar to that of clinically used 5-fluorouracil. Immunoblotting and cell cycle analysis of K562 cells treated with the most active compound as well as evaluation of the effect of this nucleoside on the activities of caspases 3 and 7 showed induction of apoptosis as the mechanism of cell death.

Co-production of functional xylooligosaccharides and fermentable sugars from corncob with effective acetic acid prehydrolysis.

A novel and green approach for the coproduction of xylooligosaccharides (XOS), in terms of a series of oligosaccharide components from xylobiose to xylohexose, and fermentable sugars was developed using the prehydrolysis of acetic acid that was fully recyclable and environmentally friendly, followed by enzymatic hydrolysis. Compared to hydrochloric acid and sulfuric acid, acetic acid hydrolysis provided the highest XOS yield of 45.91% and the highest enzymatic hydrolysis yield. More than 91% conversion of cellulose was achieved in a batch-hydrolysis using only a cellulase loading of 20FPU/g cellulose and even a high solid loading of 20% without any special strategies. The acetic acid pretreated corncob should be washed adequately before saccharification to achieve complete hydrolysis. Consequently, a mass balance analysis showed that 139.8g XOS, 328.1g glucose, 25.1g cellobiose, and 147.8g xylose were produced from 1000g oven dried raw corncob.

Chemical input reduction in the arabinoxylan and lignocellulose alkaline extraction and xylooligosaccharides production.

Lignocellulosic material breakdown by hydrolysis is an important step to open new perspectives for bioenergy and special foods production like prebiotic xylooligosaccharides. Improvement of lignocellulose and arabinoxylan alkaline extraction from sugarcane bagasse and enzymatic hydrolysis were performed. Treatments 1 (10% KOH at 70°C), 3 (5% KOH at 121°C) and ZD method (24% KOH at 35°C) showed solid lignocellulose recovery of respectively 75.2%, 74.2% and 73%. A range of 24.8-27% extracted material with high arabinoxylan content (72.1-76.3%) was obtained with these treatments. Treatment 1 and 3 exhibited great KOH reduction in the method reaction, 54.1% and 76.2%, respectively. Likewise, in treatment 3 there was a decrease in ethanol consumption (40.9%) when compared to ZD method. The extracted arabinoxylan showed susceptibility to enzymatic hydrolysis with high solid loading (7%) since Trichoderma reesei xylanases were advantageous for xylose production (54.9%), while Aspergillus fumigatus xylanases achieved better XOS production (27.1%).

Production of xylooligosaccharides by microwave-induced, organic acid-catalyzed hydrolysis of different xylan-type hemicelluloses: Optimization by response surface methodology.

A feasible approach to produce xylooligosaccharides (XOS) using organic acids as catalysts by microwave-induced hydrolysis of different hemicelluloses was developed. The effects of different acids (oxalic acid, maleic acid, citric acid and sulfuric acid), acid concentration, reaction temperature and reaction time on the hemicelluloses hydrolysis were investigated. Results demonstrated that organic acid was more beneficial to the XOS production than the conventional sulfuric acid. Higher acid concentration, higher reaction temperature and longer reaction time accelerated the further depolymerization of XOS to form monosaccharide. Response surface methodology was employed to optimize the reaction conditions (temperature and time) for the production of XOS from beechwood xylan (BX), corncob hemicelluloses (CH) and recovered hemicelluloses from the industrial waste liquor of dissolving pulp (RH), respectively. The predicted highest XOS yields were achieved to 39.31% (126.54°C-7.95min), 27.29% (120.00°C-0min), 30.32% (122.63°C-15.85min), respectively, being close to the experimental value (39.42%, 27.46% and 30.89%) from BX, CH and RH, indicating the fitted models of XOS yield were in good agreement with the experimental results.

An Efficient Chemical Synthesis of Scutellarein: An in Vivo Metabolite of Scutellarin.

Scutellarein (2), which is an important in vivo metabolite of scutellarin (1), was synthesized from 3,4,5-trimethoxyphenol (3) in high yield in four steps. This strategy relies on acetylation, aldolization, cyclization and hydrolysis reactions, respectively.

Synthesis and evaluation of vitamin D receptor-mediated activities of cholesterol and vitamin D metabolites.

A systematic study with phase 1 and phase 2 metabolites of cholesterol and vitamin D was conducted to determine whether their biological activity is mediated by the vitamin D receptor (VDR). The investigation necessitated the development of novel synthetic routes for lithocholic acid (LCA) glucuronides (Gluc). Biochemical and cell-based assays were used to demonstrate that hydroxylated LCA analogs were not able to bind VDR. This excludes VDR from mediating their biological and pharmacological activities. Among the synthesized LCA conjugates a novel VDR agonist was identified. LCA Gluc II increased the expression of CYP24A1 in DU145 cancer cells especially in the presence of the endogenous VDR ligand 1,25(OH)2D3. Furthermore, the methyl ester of LCA was identified as novel VDR antagonist. For the first time, we showed that calcitroic acid, the assumed inactive final metabolite of vitamin D, was able to activate VDR-mediated transcription to a higher magnitude than bile acid LCA. Due to a higher metabolic stability in comparison to vitamin D, a very low toxicity, and high concentration in bile and intestine, calcitroic acid is likely to be an important mediator of the protective vitamin D properties against colon cancer.

Design, synthesis and DNA interaction study of new potential DNA bis-intercalators based on glucuronic acid.

A series of novel potential DNA bis-intercalators were designed and synthesized, in which two glucuronic acids were linked by ethylenediamine, and the glucuronic acid was coupled with various chromophores, including quinoline, acridine, indole and purine, at the C-1 position. The preliminary binding properties of these compounds to calf thymus DNA (CT-DNA) have been investigated by UV-absorption and fluorescence spectroscopy. The results indicated that all the target compounds can interact with CT-DNA, and the acridine derivative, 3b, showed the highest key selection vector (KSV) value, which suggested that compound 3b binds most strongly to CT-DNA.

Improved and large-scale synthesis of different protected D-glucuronals.

Although different protected D-glucuronals are used as precursors for the preparation of many compounds, standard procedures and large-scale syntheses are still not described in the literature. In the course of the development of different protected d-glucuronyl donors we developed several versatile methods that can be used for fast and reproducible preparation of large amounts of the key intermediates. D-Glucuronolactone was converted to methyl 3,4-di-O-acetyl-D-glucuronal applying a novel one-pot protocol, which allowed for large-scale synthesis. Introduction of silyl and benzyl groups was achieved using optimized procedures. Furthermore 3,4,6-tri-O-acetyl-D-glucal was used as starting material for an improved preparation of benzyl protected D-glucuronal, which significantly accelerates and simplifies similar methods described in the literature.

Scutellarin-cyclodextrin conjugates: synthesis, characterization and anticancer activity.

A series of scutellarin-cyclodextrin conjugates (SCU-CD conjugates), in which scutellarin was covalently bound to one of the primary hydroxyl groups of ß-CD, were prepared, and their structures were determined using NMR and MS. These conjugates were further characterized by XRD and TG. The results showed that the aqueous solubility of the conjugates was much higher than that of scutellarin, and the conjugates could hardly be hydrolyzed to scutellarin in aqueous solutions. The cytotoxicity of SCU-CD conjugates on human colon cancer cell lines HT-29, SW480, Lovo and HTC116 indicated that the antitumor activities of the conjugates were better than that of scutellarin. This high antitumor activity, along with the satisfactory aqueous solubility and high stability of the conjugates, will be potentially useful for their application on human colon cancer chemotherapies.

The potential for oligosaccharide production from the hemicellulose fraction of biomasses through pretreatment processes: xylooligosaccharides (XOS), arabinooligosaccharides (AOS), and mannooligosaccharides (MOS).

Hemicellulosic oligosaccharides are sugar molecules that contain xylose, mannose, and arabinose in variable concentrations ranging from 3 to 10 molecules. These medium and long chain sugars can be classified as non-digestible carbohydrates, thus playing an important role in gastrointestinal health as prebiotics. Their physiological benefits, primarily stimulation of the proliferation of lactic acid bacteria and bifidobacteria in the colon informs their significance as high value nutraceuticals in the food and pharmaceutical industry. In addition they are well known as useful components of important pharmaceutical products. There are two main ways of producing these sugars from biomass, which include enzymatic and non-enzymatic pretreatments. Each of the two processes has advantages and disadvantages. Enzymatic processes are associated with high costs, higher concentration of monomeric sugars, and low oligosaccharide yields while thermo-chemical processes are usually associated with undesirable byproducts such as furfural and lower oligosaccharide yields. In this paper we discuss the benefits and constraints for optimization of different methods for the production of oligosaccharides from biomass.

18F-FEAnGA for PET of ß-glucuronidase activity in neuroinflammation.

UNLABELLED: Activation of microglia is a hallmark of inflammatory, infectious, and degenerative diseases of the central nervous system. Several studies have indicated that there is an increase in release of ß-glucuronidase by activated microglia into the extracellular space at the site of neuroinflammation. ß-glucuronidase is involved in the hydrolysis of glycosaminoglycans on the cell surface and the degradation of the extracellular matrix. Therefore, ß-glucuronidase might be a biomarker for ongoing neurodegeneration induced by neuroinflammation. In this study, we investigated whether the PET tracer (18)F-FEAnGA was able to detect ß-glucuronidase release during neuroinflammation in a rat model of herpes encephalitis. METHODS: Male Wistar rats were intranasally inoculated with herpes simplex virus 1 (HSV-1) or phosphate-buffered saline as a control. (11)C-(R)-PK11195 and (18)F-FEAnGA small-animal PET scans were acquired for 60 min. Logan graphical analysis was used to calculate (18)F-FEAnGA distribution volumes (DV(Logan)) in various brain areas. RESULTS: After administration of (18)F-FEAnGA, the area under the activity concentration-versus-time curve of the whole brain was 2 times higher in HSV-1-infected rats than in control rats. In addition, the DV(Logan) of (18)F-FEAnGA was most increased in the frontopolar cortex, frontal cortex, bulbus olfactorius, cerebral cortex, cerebellum, and brainstem of HSV-1-infected rats, when compared with control rats. The conversion of (18)F-FEAnGA to 4-hydroxy-3-nitrobenzyl alcohol was found to be 1.6 times higher in HSV-1-infected rats than in control rats and correlated with the DV(Logan) of (18)F-FEAnGA in the same areas of the brain. Furthermore, the DV(Logan) of (18)F-FEAnGA also correlated with ß-glucuronidase activity in the same brain regions. In addition, DV(Logan) of (18)F-FEAnGA showed a tendency to correlate with (11)C-(R)-PK11195 uptake (marker for activated microglia) in the same brain regions. CONCLUSION: Despite relatively low brain uptake, (18)F-FEAnGA was able to detect an increased release of ß-glucuronidase during neuroinflammation.