Inhibitory activity of hibifolin on adenosine deaminase- experimental and molecular modeling study

Adenosine deaminase (ADA) is an enzyme involved in purine metabolism. ADA converts adenosine to inosine and liberates ammonia. Because of their critical role in the differentiation and maturation of cells, the regulation of ADA activity is considered as a potential therapeutic approach to prevent malignant and inflammatory disorders. In the present study, the inhibitory activity of a plant flavonoid, hibifolin on ADA is investigated using enzyme kinetic assay and isothermal titration calorimetry. The inhibitory constant of hibifolin was found to be 49.92 μM ± 3.98 and the mode of binding was reversible. Isothermal titration calorimetry showed that the compound binds ADA with binding energy of −7.21 Kcal/mol. The in silico modeling and docking studies showed that the bound ligand is stabilized by hydrogen bonds with active site residues of the enzyme. The study reveals that hibifolin can act as a potential inhibitor of ADA.     

Evaluation inhibitory activity of catechins on trypsin by capillary electrophoresis–based immobilized enzyme microreactor with chromogenic substrate

In this study, a capillary electrophoresis‐based online immobilized enzyme microreactor was developed for evaluating the inhibitory activity of green tea catechins and tea polyphenol extracts on trypsin. The immobilized trypsin activity and other kinetic parameters were evaluated by measuring the peak area of the hydrolyzate of chromogenic substrate S‐2765. The results indicated that the activity of the immobilized trypsin remained approximately 90.0% of the initial immobilized enzyme activity after 30 runs. The value of Michaelis–Menten constant (K_m) was (0.47 ± 0.08) mM, and the half‐maximal inhibitory concentration (IC₅₀) and inhibition constant (K_i) of benzamidine were measured as 3.34 and 3.00 mM, respectively. Then, the inhibitory activity of four main catechins (epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate) and three tea polyphenol extracts (green tea, white tea, and black tea) on trypsin were investigated. The results showed that four catechins and three tea polyphenol extracts had potential trypsin inhibitory activity. In addition, molecular docking results illustrated that epigallocatechin gallate, epicatechin gallate, epicatechin, and epigallocatechin were all located not only in the catalytic cavity, but also in the substrate‐binding pocket of trypsin. These results indicated that the developed method is an effective tool for evaluating inhibitory activity of catechins on trypsin.     

Immobilized capillary adenosine deaminase microreactor for inhibitor screening in natural extracts by capillary electrophoresis

A novel strategy for the preparation of in-column adenosine deaminase (ADA) microreactor and rapid screening of enzyme inhibitors in natural extracts was demonstrated. In this approach, ADA was encapsulated in anionic polyelectrolyte alginate that was immobilized on the surface of fused-silica capillary via ionic binding technique with cationic polyelectrolyte polyethylenimine (PEI). On-line enzyme inhibition study was performed by capillary electrophoresis (CE). The substrate and product were baselined separated within 75 s. The enzyme activity was determined by the quantification of peak area of the product. Enzyme inhibition can be read out directly from the reduced peak area of the product in comparison with a reference electropherogram obtained in the absence of any inhibitor. The inhibition percentage was used to evaluate relative activity of ADA microreactor. A known ADA inhibitor, erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) was employed as a model compound for the validation of the inhibitor screening method, and the screening of ADA inhibitor in 19 traditional Chinese herbal medicines was performed.     

Design and synthesis of new C-nucleosides as potential adenosine deaminase inhibitors

A number of new pyrazolo[3,4-c] and [4,3-b]pyridine C-nucleosides, which can be viewed as 4- or 6-deazaformycin analogues were synthesized and examined as potential adenosine deaminase (ADA) inhibitors. The compounds were prepared through the condensation of a suitably substituted, lithiated 2- or 4-methylpyridine with tri-O-benzyl-d-ribonolactone, followed by borohydride reduction of the resulting hemiacetals, intramolecular Mitsunobu cyclisation of the derived diols, formation of the pyrazolopyridine ring system and subsequent removal of the protecting groups. These derivatives were designed on the structural basis provided by docking simulations performed within the enzyme catalytic site, however they demonstrated weak ADA inhibitory activity. Theoretical calculations assisted in the interpretation of the obtained biological data, thus providing guidance for rational structural modifications within this molecular scaffold.     

Development of a novel electrochemical biosensor based on catalytic properties of adenosine deaminase immobilized on graphene oxide/carboxymethyl chitosan/multi-wall carbon nanotube platform

A new type of biosensor was designed based on Adenosine deaminase (ADA) immobilized on graphene oxide (GO)/carboxymethyl chitosan (CMC)/multi-wall carbon nanotube (MWCNT) platform nanostructure, fabricated and successfully applied (utilized) in Adenosine detection. Square wave voltammetry was used to study the biosensor catalytic activity. Morphological analysis of the nanostructure was performed by AFM and SEM methods. The results provided here proved that utilizing GO/CMC/MWCNT leads to effective immobilization of ADA which was confirmed by the long term stability of the biosensor during examined intervals. The immobilized ADA activity was examined and the kinetic parameters (K _m and V _max) were found to be 47.5 μM and 5.8 μM min^?1, respectively. Furthermore, benznidazole was introduced as a potent ADA inhibitor using virtual screening. Outstanding inhibition characteristics of benznidazole was observed against ADA. ADA inhibition by benznidazole was non-competitive with the inhibition constant of 0.42 μM. Such an interesting template with an easy preparation process with low cost can provide a novel matrix for developing biosensors and biocatalysts based on enzyme immobilization.     

Design and synthesis of inhibitors of adenosine and AMP deaminases

Nucleosides and nucleotides which are able to undergo covalent hydration in the aglycone ring system are potential inhibitors of the enzymes adenosine deaminase (ADA) and AMP deaminase, respectively. Calculations of the enthalpy of covalent hydration and of enzyme binding energy have been used to design new inhibitors of ADA. The ribosyl triazolotriazine 16, which was synthesized as a result of these calculations, exists predominantly as the covalent hydrate 18 in water and is a potent inhibitor of mammalian ADA (IC(50) 50 nM).     

Identification of ligands from natural products as inhibitors of glutathione S‐transferases using enzyme immobilized mesoporous magnetic beads with high‐performance liquid chromatography plus quadrupole time‐of‐flight mass spectrometry and molecular docking

Multidrug resistance is recognized as one of the main reasons leading to the failure of chemotherapy. Studies have shown that glutathione S‐transferase inhibitors could be regarded as multidrug resistance reversal agents. Herein, a method of applying enzyme immobilization, molecular docking, and high‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry was employed to screen glutathione S‐transferase inhibitors from natural products. Magnetic mesoporous silica microspheres were synthesized and modified with a poly(dopamine) layer, which has a large quantity of amino, enabling further non‐covalent binding with glutathione S‐transferase. Moreover, the immobilization conditions, namely, potential of hydrogen, catalase concentration, reaction temperature and reaction time, were optimized. In total, six potential compounds were isolated and identified from Perilla frutescens (L.) Britt leaves and green tea and molecular docking was applied to identify the binding site. Rosmarinic acid, (?)‐epigallocatechin‐3‐O‐gallate and (?)‐epicatechin‐3‐gallate showed higher binding affinity than the compounds, and their half maximal inhibitory concentration values were further determined. The results suggested that this proposed method was effective and convenient for identifying glutathione S‐transferase inhibitors from natural products.     

Antiviral-guided fractionation and isolation of phenolic compounds from Limonium densiflorum hydroalcoholic extract

A preliminary antiviral plate assay of the green solvent (hydro-ethanolic) shoot extract of Limonium densiflorum showed a potent activity against the herpes simplex virus type 1 (HSV-1). In order to isolate the active compounds, an in vitro bio-guided fractionation was undertaken by preparative chromatographic techniques. On the basis of nuclear magnetic resonance techniques, the structure of the isolated compounds was determined as gallic acid, epigallocatechin gallate, quercitrin, dihydrokaempferol, pinoresinol, N-trans-ferulolyl tyramine and (myricetin 3-O-α-rhamnopyranoside and myricetin 3-O-L-arabinofuranoside). Moreover, all isolated molecules were evaluated for their virucidality against HSV-1. Results showed that gallic acid and epigallocatechin gallate have strong activity, while pinoresinol and N-trans-ferulolyl tyramine have moderate activity. Whereas, the other molecules were inactive.     

Preparation,evaluation and characterization of quercetin-molecularly imprinted polymer for preconcentration and clean-up of catechins

Molecularly imprinted polymer (MIP) for solid extraction and preconcentration of catechins have been successfully prepared by a thermal polymerization method using quercetin as template, 4-vinylpyridine as functional monomer and ethylene glycol dimethacrylate as crosslinker. A solution mixture of acetone and acetonitrile was used as porogen. Systematic investigations of the influence of monomer, cross-linker, porogen, as well as polymerization conditions on the properties of the MIPs were carried out. The quercetin MIPs were evaluated according to their selective recognition properties for quercetin, structurally related compounds (catechin, epigallocatechin gallate and epicatechin) and a unrelated compound of similar molecular size (α-tocopherol). Good binding was observed for quercetin, catechin and epigallocatechin gallate with an optimized MIP in a solid phase extraction system. Adsorption and kinetic characteristics were evaluated for catechins which indicated that the synthesized polymer had high adsorption capacity and contained homogeneous binding sites. Chemical and morphological characterization of the MIP was investigated by FTIR, SEM and BET, which confirmed a high degree of polymerization. Finally, the MIP was successfully applied to the clean-up and preconcentration of catechins from several natural samples.     

Development and validation of a high‐throughput assay for the quantification of multiple green tea‐derived catechins in human plasma

A rapid, accurate and robust method for the determination of catechin (C), epicatechin (EC), gallocatechin (GC), epigallocatechin (EGC), catechin gallate (Cg), epicatechin gallate (ECg), gallocatechin gallate (GCg) and epigallocatechin gallate (EGCg) concentrations in human plasma has been developed. The method utilizes protein precipitation following enzyme hydrolysis, with chromatographic separation and detection using reversed‐phase liquid chromatography–tandem mass spectrometry (LC–MS/MS). Traditional issues such as lengthy chromatographic runtimes, sample and extract stability, and lack of suitable internal standards have been addressed. The method has been evaluated using a comprehensive validation procedure, confirming linearity over appropriate concentration ranges, and inter/intra‐batch precision and accuracies within suitable thresholds (precisions within 13.8% and accuracies within 12.4%). Recoveries of analytes were found to be consistent between different matrix samples, compensated for using suitable internal markers and within the performance of the instrumentation used. Similarly, chromatographic interferences have been corrected using the internal markers selected. Stability of all analytes in matrix is demonstrated over 32 days and throughout extraction conditions. This method is suitable for high‐throughput sample analysis studies.     

A rapid assay to screen adenosine deaminase inhibitors from Ligustri Lucidi Fructus against metabolism of cordycepin utilizing ultra-high-performance liquid chromatography–tandem mass spectrometry

Cordycepin has recently received increased attention owing to its extensive pharmacological activity. Adenosine deaminase (ADA) is widely distributed in mammalian blood and tissues; as a result, cordycepin is quickly metabolized upon entering into the body and converted into the inactive metabolite 3′-deoxyinosine, thus limiting its activity when administered alone. We herein present a novel ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method for screening ADA inhibitors against the metabolism of cordycepin. Cordycepin and 3′-deoxyinosine were chosen as substrate and product, respectively. A proper separation was achieved for all analytes within 3 min. 3′-Deoxyinosine was quantified in the presence or absence of potential ADA inhibitors to evaluate ADA activity. The assay can simultaneously determine substrate and product, with the endogenous substance and ADA inhibitors added not interfering in its activity. After optimizing the enzymatic incubation and UHPLC–MS/MS conditions, K_m and V_max values for ADA deamination of cordycepin were 95.18 ± 7.85 μm and 363.90 ± 12.16 μmol/min/unit, respectively. Oleanolic acid and ursolic acid from Ligustri Lucidi Fructus were chosen as ADA inhibitors with half maximal inhibitory concentration values of 21.82 ± 0.39 and 18.41 ± 0.14 μm , respectively. A non-competitive inhibition model was constructed and this assay can be used to screen other potential ADA inhibitors quickly and accurately.     

Influence of Tea Brewing Parameters on the Antioxidant Potential of Infusions and Extracts Depending on the Degree of Processing of the Leaves of Camellia sinensis

The polyphenol content of tea depends on the growing region, harvest date, the production process used, and the brewing parameters. In this study, research was undertaken that included an analysis of the influence of the brewing process parameters on the content of total polyphenols (Folin-Ciocalteu), epigallocatechin gallate (HPLC), and antioxidant activity (against DPPH radicals) of fresh tea shrub leaves grown from Taiwan and of teas obtained from them (oolong, green in bags, and green loose from the spring and autumn harvest). The antioxidant potential was determined in the methanol and aqueous extracts, as well as in infusions that were obtained by using water at 65 or 100 °C and infusing the tea for 5 or 10 min. The highest content of total polyphenols and epigallocatechin gallate was found in green tea extracts from the spring harvest. However, in the case of infusions, the highest content of these compounds was found in green tea in bags. Steaming at 100 °C for 10 min, turned out to be the most favourable condition for the extraction. Oolong tea, brewed at 100 °C for 5 min was characterised by the highest antioxidant activity against stable DPPH radicals.     

Proanthocyanidins ofPolygonum corarium. II

Eight proanthocyanidins have been isolated from the roots ofPolygonum coriarum. The structures of two dimers, two trimers, including an acylglycosylated one, a tetramer, and three oligomers have been determined. All the compounds isolated were polymers and copolymers of (-)epigallocatechin, (-)epigallocatechin gallate and (-)epicatechin with C-4β→C-8 interflavan bonds.     

Development of Adenosine Deaminase-Specific IgY Antibodies: Diagnostic and Inhibitory Application

Adenosine deaminase (ADA) is currently used as a diagnostic marker for tuberculous pleuritis. Although ADA has been suggested as a potential marker for several types of cancer, the importance of each of ADA isoforms as well as their levels and enzymatic activities in tumors need to be further investigated. Herein we developed avian immunoglobulin Y highly specific to human ADA via hens immunization with calf adenosine deaminase. The obtained antibodies were used for the development of a sensitive double-egg yolk immunoglobulin (IgY) sandwich ELISA assay with an ADA detection limit of 0.5 ng/ml and a linearity range of up to 10 ng/ml. Specific, affinity-purified IgYs were able to recognize human recombinant ADA and ADA present in human cancer cell lines. In addition, antigen-specific IgY antibodies were able to inhibit catalytic activity of calf ADA with an IC₅₀ value of 47.48 nM. We showed that generated IgY antibodies may be useful for ADA detection, thus acting as a diagnostic agent in immunoenzymatic assays.     

Ring-Open Analogues of Adenine Nucleoside. Aminoacyl Derivatives of Cyclo- and Acyclo-Nucleosides

The synthesis of acyclic analogues of ribo- and deoxyribonucleosides is described. These compounds (Table 3) are both poor substrates and poor inhibitors of adenosine deaminase. The synthesis of dinucleotides from these analogues is also described, and the activity along with the inhibitory properties of some of them are studied against deaminase enzyme. These nucleotides are resistant to degradation by phosphodiesterases. HCl impregnated on silica gel is an excellent reagent for the preparation of the chloromethyl ether precursors of acyclo-nucleosides. A general and rapid procedure is developed for the preparation and isolation of the 5′-aminoacyl derivatives of robo- and arabinonucleosides. Quinoline has a marked effect on aminoacylations without racemization. Compounds 35a , b possess remarkable antiviral effects in vitro. A procedure is also developed for the conversion of acyclo-ribonucliosides 13e , f to acyclo-deoxyribonucleosides 11e , f .     

Determination of catechins and caffeine in proposed green tea standard reference materials by liquid chromatography-particle beam/electron ionization mass spectrometry (LC-PB/EIMS)

Presented here is the quantitative analysis of green tea NIST standard reference materials (SRMs) via liquid chromatography-particle beam/electron ionization mass spectrometry (LC-PB/EIMS). Three different NIST green tea standard reference materials (SRM 3254 Camellia sinesis Leaves, SRM 3255 C. sinesis Extract and SRM 3256 Green Tea-containing Oral Dosage Form) are characterized for the content of caffeine and a series of catechin species (gallic acid, catechin, epicatechin, epigallocatechin, epicatechin gallate and epigallocatechin gallate (EGCG)). The absolute limits of detection for caffeine and the catechin species were determined to be on the nanogram level. A reversed-phase chromatographic separation of the green tea reference materials was carried out on a commercial C₁₈ column using a gradient of water (containing 0.1% TFA) and 2:1 methanol:acetonitrile (containing 0.1%TFA) at 0.9 mL min⁻¹ and an analysis time of 50 min. Quantification of caffeine and the catechin species was carried out using the standard addition and internal standard methods, with the latter providing appreciable improvements in precision and recovery.     

Camellia sinensis: Insights on its molecular mechanisms of action towards nutraceutical,anticancer potential and other therapeutic applications

The Camellia sinensis plant provides a wide diversity of black, green, oolong, yellow, brick dark, and white tea. Tea is one of the majorly used beverages across the globe, succeeds only in the water for fitness and pleasure. Generally, green tea has been preferred more as compared to other teas due to its main constituent e.g. polyphenols which contribute to various health benefits. The aim of this updated and comprehensive review is to bring together the latest data on the phytochemistry and pharmacological properties of Camellia sinensis and to highlight the therapeutic prospects of the bioactive compounds in this plant so that the full medicinal potential of Camellia sinensis can be realised. A review of published studies on this topic was performed by searching PubMed/MedLine, Scopus, Google scholar, and Web of Science databases from 1999 to 2022. The results of the analysed studies showed that the main polyphenols of tea are the four prime flavonoids catechins: epigallocatechin gallate (EGCG), epicatechin gallate (ECG), epigallocatechin (EGC), and epicatechin (EC) along with the beneficial biological properties of tea for a broad heterogeneity of disorders, including anticancer, neuroprotective, antibacterial, antiviral, antifungal, antiobesity, antidiabetes and antiglaucoma activities. Poor absorption and low bioavailability of bioactive compounds from Camellia sinensis are limiting aspects of their therapeutic use. More human clinical studies and approaching the latest nanoformulation techniques in nanoparticles to transport the target phytochemical compounds to increase therapeutic efficacy are needed in the future.     

Analysis of tea samples with a multisensor system and capillary electrophoresis

Potential of an “electronic tongue” multisensor system in identification of various tea samples and in quantitative analysis of separate tea components was studied. As a reference method for quantitative analysis of antioxidants of the polyphenolic type served micellar electrokinetic chromatography with a UV detector. The contents of epicatechin gallate, epigallocatechin gallate, epicatechin, epigallocatechin, gallic acid, gallocatechin gallate, and caffeine alkaloid in various tea samples were quantitatively estimated.