Aqueous solutions of ionic liquids (ILs) with surface active properties were used as extraction solvents, taking advantage of their impressive solvation properties, in a green microwave-assisted solid-liquid extraction method (IL-MA-SLE) for the extraction of flavonoids from passion fruit and mango leaves. The extraction method was combined with high-performance liquid chromatography and photodiode-array detection (HPLC-PDA) and optimized by response surface methodology using the Box-Behnken experimental design. Under optimum conditions, the extraction efficiency of six structurally different IL-based surfactants was evaluated. Thus, imidazolium-, guanidinium- and pyridinium-type ILs with different tailorable characteristics, such as side chain length and multicationic core, were assessed. The decylguanidinium chloride ([C10Gu+][Cl-]) IL-based surfactant was selected as key material given its superior performance and its low cytotoxicity, for the determination of flavonoids of several samples of Passiflora sp. and Mangifera sp. leaves from the Canary Islands, and using as target analytes: rutin, quercetin and apigenin. The analysis of 50 mg of plant material only required 525 µL of the low cytotoxic IL-based surfactant solution at 930 mM, 10.5 min of microwave irradiation at 30 °C and 50 W, which involves a simpler, faster, more efficient and greener method in comparison with other strategies reported in the literature for obtaining bioactive compounds profiles from plants.
- MeSH
- flavonoidy chemie izolace a purifikace MeSH
- iontové kapaliny chemie MeSH
- listy rostlin chemie MeSH
- Mangifera chemie MeSH
- mikrovlny MeSH
- Passiflora chemie MeSH
- povrchově aktivní látky chemie MeSH
- rostlinné extrakty chemie MeSH
- rozpouštědla chemie MeSH
- rutin chemie MeSH
- Publikační typ
- časopisecké články MeSH
Rutinosidases (α-l-rhamnopyranosyl-(1-6)-β-d-glucopyranosidases, EC 3.2.1.168, CAZy GH5) are diglycosidases that cleave the glycosidic bond between the disaccharide rutinose and the respective aglycone. Similar to many retaining glycosidases, rutinosidases can also transfer the rutinosyl moiety onto acceptors with a free -OH group (so-called transglycosylation). The recombinant rutinosidase from Aspergillus niger (AnRut) is selectively produced in Pichia pastoris. It can catalyze transglycosylation reactions as an unpurified preparation directly from cultivation. This enzyme exhibits catalytic activity towards two substrates; in addition to rutinosidase activity, it also exhibits β-d-glucopyranosidase activity. As a result, new compounds are formed by β-glucosylation or rutinosylation of acceptors such as alcohols or strong inorganic nucleophiles (NaN3). Transglycosylation products with aliphatic aglycones are resistant towards cleavage by rutinosidase, therefore, their side hydrolysis does not occur, allowing higher transglycosylation yields. Fourteen compounds were synthesized by glucosylation or rutinosylation of selected acceptors. The products were isolated and structurally characterized. Interactions between the transglycosylation products and the recombinant AnRut were analyzed by molecular modeling. We revealed the role of a substrate tunnel in the structure of AnRut, which explained the unusual catalytic properties of this glycosidase and its specific transglycosylation potential. AnRut is attractive for biosynthetic applications, especially for the use of inexpensive substrates (rutin and isoquercitrin).
- MeSH
- Aspergillus niger enzymologie MeSH
- disacharidy chemie metabolismus MeSH
- fungální proteiny chemie metabolismus MeSH
- glykosidhydrolasy chemie metabolismus MeSH
- glykosylace MeSH
- hydrolýza MeSH
- katalytická doména MeSH
- rekombinantní proteiny metabolismus MeSH
- rutin chemie metabolismus MeSH
- simulace molekulového dockingu MeSH
- substrátová specifita MeSH
- Publikační typ
- časopisecké články MeSH
Rutinosidases (α-l-rhamnosyl-β-d-glucosidases) catalyze the cleavage of the glycosidic bond between the aglycone and the disaccharide rutinose (α-l-rhamnopyranosyl-(1→6)-β-d-glucopyranose) of specific flavonoid glycosides such as rutin (quercetin 3-O-rutinoside). Microbial rutinosidases are part of the rutin catabolic pathway, enabling the microorganism to utilize rutin and related plant phenolic glycosides. Here, we report the first three-dimensional structure of a rutinosidase determined at 1.27-Å resolution. The rutinosidase from Aspergillus niger K2 (AnRut), a member of glycoside hydrolase family GH-5, subfamily 23, was heterologously produced in Pichia pastoris. The X-ray structure of AnRut is represented by a distorted (β/α)8 barrel fold with its closest structural homologue being an exo-β-(1,3)-glucanase from Candida albicans (CaExg). The catalytic site is located in a deep pocket with a striking structural similarity to CaExg. However, the entrance to the active site of AnRut has been found to be different from that of CaExg - a mostly unstructured section of ~ 40 residues present in CaExg is missing in AnRut, whereas an additional loop of 13 amino acids partially covers the active site of AnRut. NMR analysis of reaction products provided clear evidence for a retaining reaction mechanism of AnRut. Unexpectedly, quercetin 3-O-glucoside was found to be a better substrate than rutin, and thus, AnRut cannot be considered a typical diglycosidase. Mutational analysis of conserved active site residues in combination with in silico modeling allowed identification of essential interactions for enzyme activity and helped to reveal further details of substrate binding. The protein sequence of AnRut has been revised. DATABASES: The nucleotide sequence of the rutinosidase-encoding gene is available in the GenBank database under the accession number MN393234. Structural data are available in the PDB database under the accession number 6I1A. ENZYME: α-l-Rhamnosyl-β-d-glucosidase (EC 3.2.1.168).
- MeSH
- Aspergillus niger enzymologie MeSH
- fungální proteiny chemie genetika metabolismus MeSH
- glykosidhydrolasy chemie genetika metabolismus MeSH
- katalytická doména MeSH
- konformace proteinů MeSH
- krystalografie rentgenová MeSH
- molekulární modely MeSH
- mutace MeSH
- oxidace-redukce MeSH
- rutin chemie metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Quercetin is a flavonoid largely employed as a phytochemical remedy and a food or dietary supplement. We present here a novel biocatalytic methodology for the preparation of quercetin from plant-derived rutin, with both substrate and product being in mostly an undissolved state during biotransformation. This "solid-state" enzymatic conversion uses a crude enzyme preparation of recombinant rutinosidase from Aspergillus niger yielding quercetin, which precipitates from virtually insoluble rutin. The process is easily scalable and exhibits an extremely high space-time yield. The procedure has been shown to be robust and was successfully tested with rutin concentrations of up to 300 g/L (ca 0.5 M) at various scales. Using this procedure, pure quercetin is easily obtained by mere filtration of the reaction mixture, followed by washing and drying of the filter cake. Neither co-solvents nor toxic chemicals are used, thus the process can be considered environmentally friendly and the product of "bio-quality." Moreover, rare disaccharide rutinose is obtained from the filtrate at a preparatory scale as a valuable side product. These results demonstrate for the first time the efficiency of the "Solid-State-Catalysis" concept, which is applicable virtually for any biotransformation involving substrates and products of low water solubility.
- MeSH
- Aspergillus niger enzymologie genetika MeSH
- biokatalýza * MeSH
- disacharidy chemie metabolismus MeSH
- fungální proteiny genetika metabolismus MeSH
- glykosidhydrolasy genetika metabolismus MeSH
- Pichia genetika metabolismus MeSH
- průmyslová mikrobiologie metody MeSH
- quercetin chemie metabolismus MeSH
- rutin chemie metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
The performances of ionic liquid (1-hexyl-3-methylimidazolium-bis(trifluoromethylsulfonyl)imide, IL/CPE) and iron phthalocyanine (IP/CPE) modified carbon paste electrodes in electroanalytical determinations of rutin were evaluated and compared to the performance of unmodified carbon paste electrode (CPE). Cyclic voltammetry (CV), differential pulse voltammetry (DPV), differential pulse adsorptive stripping voltammetry (DPAdSV), and amperometry were used for rutin analysis. The best current responses of rutin were obtained at pH 4.0 for all tested techniques. IL/CPE electrode was found to perform best with DPAdSV technique, where a detection limit (LOD) as low as 5 nmol L(-1) of rutin was found. On the other hand, IP/CPE showed itself to be an optimum choice for DPV technique, where LOD of 80 nmol L(-1) was obtained. Analytical applicability of newly prepared electrodes was demonstrated on determination of rutin in the model samples and the extracts of buckwheat seeds. To find an optimum method for buckwheat seeds extraction, a boiling water extraction (BWE), Soxhlet extraction (SE), pressurized solvent extraction (PSE), and supercritical fluid extraction (SFE) were tested.
- MeSH
- elektrochemie metody MeSH
- elektrody MeSH
- Fagopyrum chemie MeSH
- molekulární struktura MeSH
- rutin chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Extensive screening for a robust producer of α-L-rhamnosidase activity from well-defined strains of filamentous fungi, including multifactorial optimization (inducers, cultivation conditions) was accomplished. Enzyme production of the optimal producer Aspergillus terreus (non-toxigenic) was scaled up to 50L. α-L-Rhamnosidase, which was fully characterized, proved to be thermo- and alkali-tolerant, thus enabling effective operation at 70°C and pH 8.0. These conditions allow for a very high substrate (rutin) load up to 100-300 g/L, thus enabling very high volumetric productivity of the reaction product quercetin-3-β-D-glucopyranoside (isoquercitrin). Here, a novel concept of "immobilised substrate" is used. Isoquercitrin is a highly effective and biocompatible antioxidant with strong anti-inflammatory activities. Rutin biotransformation was optimized and scaled up to ca 10 kg production and thus the robustness of the large-scale production was demonstrated. Isoquercitrin can be produced to a very high purity (98%) in multikilogram amounts, without any quercetin and directly applicable in nutraceuticals.
- MeSH
- alkálie farmakologie MeSH
- Aspergillus účinky léků enzymologie MeSH
- beta-glukosidasa metabolismus MeSH
- bioreaktory mikrobiologie MeSH
- biotechnologie metody MeSH
- biotransformace účinky léků MeSH
- fyziologická adaptace účinky léků MeSH
- glukosidy biosyntéza chemie MeSH
- glykosidhydrolasy metabolismus MeSH
- koncentrace vodíkových iontů účinky léků MeSH
- quercetin analogy a deriváty biosyntéza chemie MeSH
- rutin chemie metabolismus MeSH
- stabilita enzymů účinky léků MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In the food industry, in the process of creating new agricultural plant products, and in the testing of anti-cancer drugs there is often a need to assay multiple samples of low molecular weight antioxidants, plant samples and foods rich in antioxidants, with minimal additional costs and low degrees of uncertainty. With these demands in mind, we decided to study the fully automated assay of antioxidants using not only automated sample measurements but also automated processing of samples and application of reagents. The automated pipetting system epMotion 5075 and the automated spectrophotometer BS 400 were chosen for the assay purposes. Five methods were introduced for the automation: 2-diphenyl-1-picrylhydrazyl (DPPH) test, ferric reducing antioxidant power (FRAP) method, 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) based test, N,N-dimethyl-1,4-diaminobenzene (DMPD) based test and the free radicals method. Samples containing one of the four antioxidants (standard rutin, quercitrin, ferulic and gallic acid) in a range 1–1000 μg/ml were used throughout. All of the tested methods were found suitable for implementation in an automated assay. However, some of them, such as the ABTS test failed to assay all tested antioxidants. The coefficients of determination were also unequal. From the analytical point of view, FRAP methods provided the most reliable results in the automated assay; because of the capacity of the method, approximately 240 samples per hour (one sample per 15 seconds) can be assayed using the automated protocol. We were encouraged by the data received and we expect further interest in the practical performance of such automation. As a mean of testing the robustness of our method, in the next step of our study, oxidative status was assessed in model cell lines derived from prostate cancer (PC-3, PNT1A and 22RV1) that were cultured on ellipticine (0, 0.5, 1, 1.5, 2, 2.5, 5, 7.5, 10, 15 μmol/l) supplemented agar. Antioxidant activity was assessed (DPPH, ABTS, FRAP, DMPD, FR) and calculated on the phenolic antioxidant level (rutin, quercitrin, ferulic and gallic acid), and thus an estimation was formulated of the oxidative stress as a result of the impact of anti-cancer drugs. It can be demonstrated that the new method has wide applicability.
- MeSH
- antioxidancia analýza MeSH
- antitumorózní látky chemie metabolismus toxicita MeSH
- chemické techniky analytické metody přístrojové vybavení statistika a číselné údaje MeSH
- elipticiny analýza chemie metabolismus MeSH
- financování organizované MeSH
- FRAP MeSH
- kalibrace MeSH
- kyselina gallová analýza chemie MeSH
- kyseliny kumarové analýza chemie MeSH
- laboratorní automatizace metody přístrojové vybavení MeSH
- léky antitumorózní - screeningové testy metody statistika a číselné údaje MeSH
- luminiscenční měření MeSH
- nádorové buněčné linie MeSH
- oxidační stres účinky léků MeSH
- quercetin analogy a deriváty analýza chemie MeSH
- reprodukovatelnost výsledků MeSH
- rutin analýza chemie MeSH
- spektrofotometrie metody přístrojové vybavení statistika a číselné údaje MeSH
- viabilita buněk účinky léků MeSH
- volné radikály analýza MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- hodnotící studie MeSH
- statistiky MeSH
- tabulky MeSH
Preparation of coated pellets intended for rutin colon delivery, their evaluation in vitro and in vivo in experimental colitis in rats was the purpose of this study. Pellets were obtained using extrusion/spheronization and coated with three types of coatings (caffeic acid/hypromellose/alginic acid; sodium alginate/hypromellose/zinc acetate; sodium alginate/chitosan). Dissolution using buffers of pH values, β-glucosidase and times corresponding to gastrointestinal tract (GIT) was provided. Pellets coated with alginate/chitosan showed low rutin dissolution (12-14%) in upper GIT conditions and fast release (87-89%) under colon conditions; that is a good presumption of intended rutin release. After colitis induction and development, the rats were treated with pellets and rutin solution administered orally, solution also rectally. Colon/body weight ratio, myeloperoxidase activity and histological evaluation were performed. Rutin was able to promote colonic healing at the dose of 10mg/kg: colon/body weight ratio decreased and myeloperoxidase activity was significantly suppressed. Pellets coated with alginate/chitosan applied orally and rutin solution administered rectally showed the best efficacy. The combination of rutin as natural product, mucoadhesive chitosan degraded in the colon and sodium alginate as the main coating substance in the form of pellets create a promising preparation for therapy of this severe illness.
- MeSH
- algináty chemie MeSH
- antiflogistika aplikace a dávkování chemie farmakologie MeSH
- aplikace orální MeSH
- časové faktory MeSH
- chemie farmaceutická MeSH
- chitosan chemie MeSH
- farmaceutická technologie metody MeSH
- gastrointestinální látky aplikace a dávkování chemie farmakologie MeSH
- kolitida chemicky indukované farmakoterapie patologie MeSH
- kolon účinky léků patologie MeSH
- koncentrace vodíkových iontů MeSH
- krysa rodu rattus MeSH
- kyselina glukuronová chemie MeSH
- kyselina trinitrobenzensulfonová MeSH
- kyseliny hexuronové chemie MeSH
- kyseliny kávové chemie MeSH
- léky implantované MeSH
- methylcelulosa analogy a deriváty chemie MeSH
- modely nemocí na zvířatech MeSH
- octan zinečnatý chemie MeSH
- potkani Wistar MeSH
- příprava léků MeSH
- pufry MeSH
- rozpustnost MeSH
- rutin aplikace a dávkování chemie farmakologie MeSH
- stabilita léku MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Byla vypracována jednoduchá, rychlá a plně automatizovaná průtoková metoda (FIA) s chemiluminiscenčnídetekcí po oxidaci s KMnO4 v kyselém prostředí ke stanovení rutinu ve farmaceutickémpřípravku. Ke zvýšení chemiluminiscence této reakce byl využit hexametafosforečnan sodný. Kalibračníkřivka byla lineární v rozmezí 0,01–0,26 mmol.l-1 s detekčním limitem 0,005 mmol.l-1, RSDbyla 0,46 % (n=10) při počtu analýz 75.h-1 a dávkování 200 µl vzorku. Metoda byla využita kestanovení komerčně vyráběné dávkované formy rutinu: Rutin 250 tablet á 50 mg (firma Nature’sBounty, USA). FIA metoda byla statisticky porovnána s metodou podle Německého lékopisu a ukazujese, že je nejen srovnatelná ve správnosti, ale má přednosti i v jednoduchosti, rychlosti a množstvíspotřebovaných činidel.
A simple, rapid and fully automated flow injection method (FIA) with chemiluminescence detectionafter oxidation with KMnO4 in acid medium at room temperature has been developed for thedetermination of rutine in pharmaceutical formulation. Hexamethaphosphate sodium was utilizedas enhancer of chemiluminescence. The calibration curvewas linear in the range 0.01–0.26 mmol.l-1with detection limit 0.005mmol.l-1,RSD 0.46% (n=10) and a sample throughput of 75.h-1 using a 200µl sample volume. The method was used for the determination of rutine in mass-produced dosageform: Rutin 250 tablets á 50 mg, (firm Nature’s Bounty, USA). The FIA method was statisticallycompared with the official German Pharmacopoeia method and showed comparable accuracy, butwith the advantages of simplicity, speed and amounts of reagents consumed.
- MeSH
- finanční podpora výzkumu jako téma MeSH
- léčivé přípravky analýza MeSH
- lékové formy MeSH
- luminiscence MeSH
- manganistan draselný aplikace a dávkování diagnostické užití MeSH
- průtoková injekční analýza metody přístrojové vybavení MeSH
- rutin chemie izolace a purifikace terapeutické užití MeSH
- Publikační typ
- přehledy MeSH
- srovnávací studie MeSH
