Synthesis and Preliminary Anticancer Activity Assessment of N-Glycosides of 2-Amino-1,3,4-thiadiazoles.

The addition of 2-amino-1,3,4-thiadiazole derivatives with parallel iodination of differently protected glycals has been achieved using a double molar excess of molecular iodine under mild conditions. The corresponding thiadiazole derivatives of N-glycosides were obtained in good yields and anomeric selectivity. The usage of iodine as a catalyst makes this method easy, inexpensive, and successfully useable in reactions with sugars. Thiadiazole derivatives were tested in a panel of three tumor cell lines, MCF-7, HCT116, and HeLa. These compounds initiated biological response in investigated tumor models in a different rate. The MCF-7 is resistant to the tested compounds, and the cytometry assay indicated low increase in cell numbers in the sub- G1 phase. The most sensitive are HCT-116 and HeLa cells. The thiadiazole derivatives have a pro-apoptotic effect on HCT-116 cells. In the case of the HeLa cells, an increase in the number of cells in the sub-G1- phase and the induction of apoptosis was observed.

Identification of C21 Steroidal Glycosides from Gymnema sylvestre (Retz.) and Evaluation of Their Glucose Uptake Activities.

Gymnema sylvestre (Retz.) Schult is a multi-purpose traditional medicine that has long been used for the treatment of various diseases. To discover the potential bioactive composition of G. sylvestre, a chemical investigation was thus performed. In this research, four new C21 steroidal glycosides sylvepregosides A-D (1-4) were isolated along with four known compounds, gymnepregoside H (5), deacetylkidjoladinin (6), gymnepregoside G (7) and gymnepregoside I (8), from the ethyl acetate fraction of G. sylvestre. The structures of the new compounds were established by extensive 1D and 2D nuclear magnetic resonance (NMR) spectra with mass spectroscopy data. Compounds 1-6 promoted glucose uptake by the range of 1.10- to 2.37-fold, respectively. Compound 1 showed the most potent glucose uptake, with 1.37-fold enhancement. Further study showed that compounds 1 and 5 could promote GLUT-4 fusion with the plasma membrane in L6 cells. The result attained in this study indicated that the separation and characterization of these compounds play an important role in the research and development of new anti-diabetic drugs and pharmaceutical industry.

Enzyme inhibitory assessment of the isolated constituents from Plantago holosteum Scop.

Plants of the Plantago genus are widely used in Turkish folk medicine especially for the treatment of wound, abscess, and inflammation. The aqueous extract and five phenylethanoid glycosides acteoside (1), arenarioside (2), echinacoside (3), isoacteoside (4), and leucosceptoside A (5) isolated from the aerial parts and roots of Plantago holosteum Scop. (Plantaginaceae) were tested for their possible inhibitory activity against hyaluronidase, elastase, and collagenase, related to wound pathogenesis. Even though the aqueous extract prepared from the aerial parts (36.26%) and roots (47.01%) and the isolated compounds acteoside (29.13%), echinacoside (28.73%), and isoacteoside (31.69%) exerted a notable inhibition, arenarioside and leucosceptoside A were found inactive in the hyaluronidase enzyme inhibition test. Similar results were obtained from the collagenase enzyme inhibition test. The aqueous extract prepared from the aerial parts (31.09%) and roots (35.17%), echinacoside (25.13%), and isoacteoside (23.85%) exerted a notable inhibition in this test. However, none of the extracts and isolated compounds displayed elastase enzyme inhibitory activity. The experimental data demonstrated that P. holosteum displayed a remarkable enzyme inhibitory activity against hyaluronidase and collagenase. This paper is the first report regarding the in vitro enzyme inhibitory activity of P. holosteum.

Anti-inflammatory and immune properties of the peltatoside, isolated from the leaves of Annona crassiflora Mart., in a new experimental model zebrafish.

Establishing new animal models for the study of inflammation is very important in the process of discovering new drugs, since the inflammatory event is the basis of many pathological processes. Whereas rodent models have been the primary focus of inflammation research, we defend the zebrafish (Danio rerio) test as a feasible alternative for preclinical studies. Moreover, despite all the technological development already achieved by humanity, nature can still be considered a relevant source of new medicines. In this context, the aim of this work was to evaluate the anti-inflammatory effect of a substance isolated from the medicinal plant Annona crassilfora Mart, the peltatoside, in an inflammatory model of zebrafish. It was determined: (i) total leukocyte count in the coelomate exudate; (ii) N-acetyl-ß-d-glucuronidase (NAG); (iii) myeloperoxidase (MPO); (iv) and the histology of liver, intestine and mesentery. Peltotoside (25, 50 and 100 µg) and dexamethasone (25 µg) were administered intracelomatically (i.c.) 30 min before carrageenan (i.c.). Pretreatment with peltatoside at three doses significantly inhibited leukocyte recruitment in the coelomic cavity, and inhibited NAG and MPO activity against the action of Cg, in a similar manner as dexamethasone. However, some microlesions in the evaluated organs were detected. The dose of 25 µg showed an anti-inflammatory effect with lower undesirable effects in the tissues. Our results suggest that the zebrafish test was satisfactory in performing our analyzes and that the peltotoside has a modulatory action in reducing leukocyte migration.

New γ-pyrone glycoside from Pachira glabra and assessment of its gastroprotective activity using an alcohol-induced gastric ulcer model in rats.

Pachira glabra is a medium sized tree that is famous for its delicious edible seeds and leaves. Phytochemical investigation of Pachira glabra leaf alcohol extract led to the isolation of a new γ-pyrone glycoside, 7-hydroxy maltol-3-O-ß-d-glucoside (HMGlu). In vivo assessment of the gastro-protective activity of HMGlu demonstrated superior gastro-protection at a dose of 100 mg kg-1, close to the value triggered by the standard reference omeprazole drug (evidenced by morphological and histopathological examinations). This was further confirmed by immunohistochemical staining where pretreatment with HMGlu (100 mg kg-1) markedly reduced NFκB, COX-2 and BAX positively-stained cells. Thus, it can be concluded that 7-hydroxy maltol-3-O-ß-d-glucoside can be a new pharmaceutically active agent with promising gastro-protective activity of natural origin.

Physicochemical Aspects and Sensory Profiles as Various Potential Factors for Comprehensive Quality Assessment of Nü-Er-Cha Produced from Rhamnus heterophylla Oliv.

Nü-Er-Cha, produced from the leaves of Rhamnus heterophylla Oliv., is known as an herbal tea and used in the treatment of bleeding, irregular menstruation and dysentery. A method was developed for the quality assessment of herbal tea, Nü-Er-Cha, adopting physical parameters, chemical constituents and sensory profiles as various potential factors. Their inner relationship was mined by multivariate statistical analysis tools, and the three factors were integrated by a technique for order preference by a similarity to ideal solution (TOPSIS) approach to comprehensively analyze the characters of Nü-Er-Cha. Viscosity was also introduced to the physical parameter determination besides conductivity, pH and color. Seven common peaks of eight batches of Nü-Er-Cha were marked by a high performance liquid chromatography (HPLC) fingerprint. They were further identified by HPLC mass spectrometry/mass spectrometry (HPLC-MS/MS) as hydroxybenzoic acids and flavanol glycosides. Fifty trained members participated in the sensory evaluation. Significant correlations between total sensory scores and conductivity, viscosity as well as pH were observed, a relatively innovative result for the quality assessment of herbal teas. The common peaks, belonging to hydroxybenzoic acids and flavanol glycosides, were mainly related to the color of infusions and leaves. The result of the TOPSIS analysis showed that S3 and S4 ranked as the top two in the comprehensive quality assessment. This may be related to rhamnetin triglycoside with a galactose/glucose and two rhamnoses, which had a higher peak response in S3 and S4 than that in the other samples. The present study may contribute to a better understanding of the relationship regarding physical properties, chemical composition and sensory profiles, and it may supply ideas for the comprehensive quality assessment of the herbal tea Nü-Er-Cha.

Spectroscopic Characterization and Cytotoxicity Assessment towards Human Colon Cancer Cell Lines of Acylated Cycloartane Glycosides from Astragalus boeticus L.

In several European countries, especially in Sweden, the seeds of the species Astragalus boeticus L. were widely used as coffee substitutes during the 19th century. Nonetheless, data regarding the phytochemistry and the pharmacological properties of this species are currently extremely limited. Conversely, other species belonging to the Astragalus genus have already been extensively investigated, as they were used for millennia for treating various diseases, including cancer. The current work was addressed to characterize cycloartane glycosides from A. boeticus, and to evaluate their cytotoxicity towards human colorectal cancer (CRC) cell lines. The isolation of the metabolites was performed by using different chromatographic techniques, while their chemical structures were elucidated by nuclear magnetic resonance (NMR) (1D and 2D techniques) and electrospray-ionization quadrupole time-of-flight (ESI-QTOF) mass spectrometry. The cytotoxic assessment was performed in vitro by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays in Caco-2, HT-29 and HCT-116 CRC cells. As a result, the targeted phytochemical study of A. boeticus enabled the isolation of three new cycloartane glycosides, 6-O-acetyl-3-O-(4-O-malonyl)-ß-d-xylopyranosylcycloastragenol (1), 3-O-(4-O-malonyl)-ß-d-xylopyranosylcycloastragenol (2), 6-O-acetyl-25-O-ß-d-glucopyranosyl-3-O-ß-d-xylopyranosylcycloastragenol (3) along with two known compounds, 6-O-acetyl-3-O-ß-d-xylopyranosylcycloastragenol (4) and 3-O-ß-d-xylopyranosylcycloastragenol (5). Importantly, this work demonstrated that the acetylated cycloartane glycosides 1 and 4 might preferentially inhibit cell growth in the CRC cell model resistant to epidermal growth factor receptor (EGFR) inhibitors.

Direct uptake of HCO3- in the marine angiosperm Posidonia oceanica (L.) Delile driven by a plasma membrane H+ economy.

Seagrasses access HCO3- for photosynthesis by 2 mechanisms, apoplastic carbonic anhydrase-mediated dehydration of HCO3- to CO2 and direct HCO3- uptake. Here, we have studied plasma membrane energization and the mechanism for HCO3- import in Posidonia oceanica. Classical electrophysiology and ion-selective microelectrodes were used to measure the membrane potential, cytosolic pH, and the cytosolic concentrations of Na+ and Cl- upon the addition of HCO3- . The photosynthetic response to HCO3- and to inhibitors was also measured. Results indicate that the primary pump of P. oceanica plasma membrane is a fusicoccin-sensitive H+ -ATPase. Bicarbonate depolarizes the plasma membrane voltage and transiently acidifies the cytosol, indicating that HCO3- is transported into the cells by an H+ -symport. Initial cytosolic acidification is followed by an alkalinization, suggesting an internal dehydration of HCO3- . The lack of cytosolic Na+ and Cl- responses rules out the contribution of these ions to HCO3- transport. The energetics of nH+ /HCO3- symport allows, for n = 1, an estimate of cytosolic accumulation of 0.22 mM HCO3- . Because this transporter could permit accumulation of HCO3- up to 100 times above the equilibrium concentration, it would be a significant component of a carbon-concentrating mechanism in this species.

Risk Assessment via Metabolism and Cell Growth Inhibition in a HepG2/C3A Cell Line Upon Treatment with Arpadol and its Active Component Harpagoside.

Harpagophytum procumbens (Hp) has been used as antiinflammatory and analgesic agent for the treatment of rheumatic diseases. The principal active component of Hp is harpagoside (HA). We tested the toxicity of this new therapeutic agent in a hepatic cell line (HepG2/C3A). Hp was found to be cytotoxic, and HA was found to decrease the number of cells in S phase, increase the number of cells in G2/M phase and induce apoptosis. Neither Hp nor HA was genotoxic. The expression of CDK6 and CTP3A4 was reduced by Hp, and both HA and Hp caused a significant reduction of CYP1A2 and CYP3A4 expression. It is possible that the cytotoxicity caused by HA and Hp does not involve transcriptional regulation of the cyclins and CDKs tested but is instead related to the inhibition of metabolism. This is evidenced by the results of an MTT assay and changes in the expression of genes related to drug metabolism, leading to cell death. Indeed, the cells exhibited decreased proliferation upon exposure to Hp and HA. The data show that treatment with either Hp or HA can be cytotoxic, and this should be taken into consideration when balancing the risks and benefits of treatments for rheumatic diseases. Copyright © 2016 John Wiley & Sons, Ltd.

Design, synthesis, characterization and DNA-binding studies of a triphenyltin(IV) complex of N-glycoside (GATPT), a sugar based apoptosis inducer: in vitro and in vivo assessment of induction of apoptosis by GATPT.

The novel organotin complex 1-{(2-hydroxyethyl)amino}-2-amino-1,2-dideoxy-D-glucose triphenyltin(iv) (GATPT) was synthesized by the reaction of N-glycoside ligand and triphenyltin(iv) chloride. GATPT was characterized by elemental analyses, polarimetry, IR, CD, UV and multinuclear ((1)H, (13)C, (119)Sn) 1D and 2D NMR. The interaction of GATPT with calf thymus DNA was studied by using viscometry, absorption, emission and circular dichoric spectral methods. The DNA binding results suggested the intercalative mode of binding for GATPT with DNA along with simultaneous electrostatic interaction between the Sn(iv) center and the phosphate backbone of the DNA helix. GATPT was tested for its cytotoxic properties against SY5Y, PC-12 and N2A neuronal tumor cell lines. GATPT induced significant apoptosis in the PC-12 cell line characterized by DNA fragmentation and chromosome condensation. Treatment of PC-12 cells with GATPT resulted in a dramatic up-regulation of Bax and Bak and down-regulation of the anti-apoptotic factor Bcl-2. Apoptotic induction by GATPT was shown to be mediated in a p53-dependent manner and loss of p53 impaired the release of cytochrome c from mitochondria to cytosol. Caspase-3 was found to be indispensable for the GATPT triggered apoptosis signaling pathway. Furthermore, in vivo studies using a nude mice model revealed that GATPT exhibits significant antiproliferative activity against tumor development with minimal cytotoxicity. These findings warrant further clinical investigations of GATPT as a therapeutic agent for cancer chemotherapy.

Assessment of chemoselective neoglycosylation methods using chlorambucil as a model.

To systematically assess the impact of glycosylation and the corresponding chemoselective linker upon the anticancer activity/selectivity of the drug chlorambucil, herein we report the synthesis and anticancer activities of a 63-member library of chlorambucil-based neoglycosides. A comparison of N-alkoxyamine-, N-acylhydrazine-, and N-hydroxyamine-based chemoselective glycosylation of chlorambucil revealed sugar- and linker-dependent partitioning among open- and closed-ring neoglycosides and corresponding sugar-dependent variant biological activity. Cumulatively, this study represents the first neoglycorandomization of a synthetic drug and expands our understanding of the impact of sugar structure upon product distribution/equilibria in the context of N-alkoxyamino-, N-hydroxyamino-, and N-acylhydrazine-based chemoselective glycosylation. This study also revealed several analogues with increased in vitro anticancer activity, most notably D-threoside 60 (NSC 748747), which displayed much broader tumor specificity and notably increased potency over the parent drug.

Synthesis of bivalent lactosides based on terephthalamide, N,N'-diglucosylterephthalamide, and glycophane scaffolds and assessment of their inhibitory capacity on medically relevant lectins.

Glycan recognition by lectins initiates clinically relevant processes such as toxin binding or tumor spread. Thus, the development of potent inhibitors has a medical perspective. Toward this goal, we report the synthesis of both rigid and flexible bivalent lactosides on scaffolds that include secondary and tertiary terephthalamides and N,N'-diglucosylterephthalamides. Construction of these compounds involved Schmidt-Michel glycosidation, and amide coupling or Ugi reactions of relevant glycosyl amines in key steps. A glycocluster based on a rigid glycophane was also prepared from coupling of a glucuronic acid derivative and p-xylylenediamine with subsequent ring-closing metathesis. Finally, a more flexible bivalent lactoside was produced from lactosyl azide with use of the copper-catalyzed azide-alkyne cycloaddition. Distances between lactose residues were analyzed computationally as were their orientations for the relatively rigid subset of compounds. Distinct spacing properties were revealed by varying the structure of the scaffold or by varying the location of the lactose residue on the scaffold. To relate these features to bioactivity a plant toxin and human adhesion/growth-regulatory galectins were used as sensors in three types of assay, i.e. measuring agglutination of erythrocytes, binding to glycans of a surface-immobilized glycoprotein, or binding to human cells. Methodologically, the common hemeagglutination assay was found to be considerably less sensitive than both solid-phase and cell assays. The bivalent compounds were less effective at interfering with glycoprotein binding to the plant toxin than to human lectins. Significantly, a constrained compound was identified that displayed selectivity in its inhibitory potency between galectin-3 and its proteolytically processed form. Conversely, compounds with a high degree of flexibility showed notable ability to protect human cells from plant toxin binding. The applied conjugation chemistry thus is compatible with the long-term aim to produce potent and selective lectin inhibitors.

Assessment of the antibacterial activity of phenylethanoid glycosides from Phlomis lanceolata against multiple-drug-resistant strains of Staphylococcus aureus.

Three phenylethanoid glycosides, forsythoside B (1), phlinoside C (2) and verbascoside (3), were isolated from the methanol extract of the leaves of Phlomis lanceolata, an Iranian medicinal plant, by reversed-phase preparative high-performance liquid chromatography (HPLC), and the structures of these compounds were elucidated conclusively by ultraviolet (UV), mass spectrometry (MS) and a series of 1D and 2D nuclear magnetic resonance (NMR) analyses. The antibacterial properties of 1-3 against five multi-drug-resistant (MDR) strains of Staphylococcus aureus have been assessed by the rapid and robust microtitre-plate-based serial dilution method. While compounds 1 and 3 showed considerable activities against all five strains, compound 2 was inactive at the test concentrations.

In vitro assessment of alkylglycosides as permeability enhancers.

series of alkylglycosides has been evaluated on human cell lines to determine its ability to open cellular tight junctions. Alkylglycosides were applied to cell monolayers; the resulting change in resistance was determined by transepithelial electrical resistance measurements. Change in resistance across cell monolayers is an indication of tight junction activation, whereas subsequent increase in resistance signifies monolayer recovery. Of the 13 alkylglycosides tested, 4 caused irreversible solubilization of cell membranes, 5 allowed a partial recovery of the monolayer after a relatively rapid reduction in resistance, and 4 induced a decrease in resistance with more complete cell recovery. Alkylglycosides allowing extensive cell recovery after removal may indicate tight junctions' activity dominance over membrane fluidity. Repeated application of alkylglycosides for 6 hours lowered resistance across cells, which returned to near-normal values after a recovery period of 48 hours. A model dye was transported across the cell monolayer only in the presence of an alkylglycoside, although recovery of cells was incomplete. Activity of the alkylglycosides was unrelated to either the carbon chain length or to the carbohydrate moiety. A direct correlation was established between the concentration of applied alkylglycoside and reduction in resistance over a constant time period. Dodecylmaltoside and octylglucoside were found to be optimal in decreasing resistance at low concentrations and allowing significant recovery of cells. Therefore these 2 alkylglycosides may be useful in facilitating drug transport across biological membranes.

In-vivo and in-vitro assessment of the free-radical-scavenger activity of Ginkgo flavone glycosides at high concentration.

Free radicals are involved in numerous skin diseases, especially inflammatory reactions and photosenescence. To identify possible free-radical scavenging by an original terpene-free Ginkgo biloba extract containing 33% Ginkgo flavone glycosides, mostly quercetin and kaempferol derivatives, we studied its activity by means of in-vitro and in-vivo experiments, using superoxide dismutase (SOD) as a positive control. By means of an in-vitro electron-spin resonance (ESR) assay we compared the activity of the Ginkgo extract with that of its two aglycones, quercetin and kaempferol. Quercetin and Ginkgo extract had significant antioxidant properties without pro-oxidant effect. In contrast, kaempferol, above an optimum antioxidant concentration, behaved as a pro-oxidant. The in-vivo experiments were conducted on an anti-inflammatory model. The cutaneous blood flux which reflects the skin inflammatory level was recorded by means of a laser Doppler perfusion imager. The data confirmed the free-radical-scavenging property of both Ginkgo extract and SOD. The Ginkgo extract significantly inhibited (37%) cutaneous blood flux to the same extent as SOD. These data confirmed the antioxidant property of Ginkgo extract. A complementary spin-trapping technique would enable identification of the free radicals involved. This Ginkgo extract should be useful for protection of the skin against free radicals.

Assessment of pharmacological effects on cerebral blood flow.

The effect of various drugs on hemispheric and regional cerebral blood flow (CBF) was investigated in a total of 410 patients. While a few drugs (midodrin, proxazole, vincamine, hexobendine, extract of ginkgo biloba, dextran and ouabain) were able to improve hemispheric CBF, only ephedrine combined with xanthines decreased CBF. For vincamine the dependency of the effect on certain plasma levels was established. Only ouabain of the tested cardiac glycosides effected CBF; their similar hemodynamic actions suggest here an influence of ouabain on cerebral vessels. For the evaluation of drug effects on rCBF the detection of heterogeneous responses is important. Such responses may be quantified by regression analysis. While intracerebral steal effects were observed only under certain circumstances, inverse cerebral steal phenomena may be caused by diverging actions of several drugs. If treatment is aimed at improvement of cerebral hemodynamics, only drugs with a demonstrated effect, at least on perfusion of ischemic regions, should be employed.