Application of molecular imprinting for target isolation of bioactive natural compounds for the regulation of cells' response to hypoxia

Hypoxia Inducible Factor 1 (HIF-1) is a transcriptional factor highly involved in the pathology of cancer. HIF-1 inhibition retards tumor growth and enhances treatment efficiency when used in combination with chemo- or radiation therapy. The recent validation of HIF-1 as an important drug target in cancer treatment has stimulated efforts to identify and isolate natural or synthetic HIF-1 inhibitors. In the present study, quercetin, a known inhibitor of HIF-1, was imprinted in a polymer matrix in order to produce the 3D synthetic imitation of the quercetin action site in the HIF-1 signalling pathway. The Molecularly Imprinted Polymer (MIP), which contains functionalized cavities complementary in size, shape and functionality to the initial template (quercetin), was used for the selective isolation of new HIF-1 inhibitors from frankincense, a gum resin collected from the trees of the genus Boswellia that was used as anticancer remedy in Greek antiquity, traditional Chinese and Indian medicine. By the application of the frankincense acetone extract onto the lab made SPE catridge (packed with 45-90μm particles of the imprinted polymers) a number of compounds were selectively retained on the catridge with imprinting effect from 5±4 to 8±4,6 (with reference to non imprinted polymer, n=3). The mixture of compounds eluted from the catridge, efficiently inhibited the transcriptional activity of HIF-1 and decreased the protein levels of HIF-1α, the regulated subunit of HIF-1. On the contrary non-retained compouds did not have any inhibitory effect on HIF-1. Acetyl 11-ketoboswellic acid (AKBA), one of the main bioactive components of frankincense, was identified as one of the main components of the selectively retained fraction. Determination of the biological activity of AKBA, revealed that it inhibits HIF-1α induction and HIF-1 transcriptional activity. By subsequent semi-preparative fractionation of the acetone extract, AKBA was separated from the other selectively retained compounds. Interestingly, these compounds also exhibited inhibitory effect on HIF-1 transcriptional activity. Additionally the ATP analog tri-O-acetyladenosine was imprinted in order to produce the approximate synthetic imitation of the ATP binding cleft in kinases. Appropriate kinases are implicated in HIF-1α post-translational modification and their inhibition can negativily regulate HIF-1. Occupation of the ATP-binding cleft by ATP mimetic compounds is the main mechanism of kinase inhibition (ATP competitive mechanism). The production of the aproximate synthetic imitation of the ATP binding cleft could create a polymer suitable for screening and isolation of new protein kinases ATP-mimetic inhibitors from different sources. For this reason tri-O-acetyladenosine was successfully imprinted giving polymers with high affinity to the initial template (Kd as low as 1,2μΜ), sufficient concentration of binding sites N and pronounced specificity (k’imprinted >> k’ control). Overall, this study demonstrates the potential of MIP application in the screening, identification and isolation of new bioactive compounds that aim selected molecular targets, a potential that has been poorly appreciated until now.Ο επαγόμενος από την υποξία μεταγραφικός παράγοντας 1 (HIF-1) εμπλέκεται σε σημαντικό βαθμό στην παθολογία του καρκίνου. Η αναστολή του HIF-1 επιβραδύνει την ανάπτυξη του όγκου και ενισχύει την αποτελεσματικότητα της αγωγής όταν συνδυάζεται με χημειο-ραδιο θεραπεία. Η πρόσφατη καθιέρωση του HIF-1 ως σημαντικού φαρμακευτικού στόχου στη θεραπεία του καρκίνου αποτέλεσε έναυσμα σειράς προσπαθειών για την ανεύρεση και απομόνωση αναστολέων του HIF-1, φυσικής ή συνθετικής προέλευσης. Στην παρούσα μελέτη, η κερκετίνη, ένας γνωστός αναστολέας του HIF-1, αποτυπώθηκε σε πολυμερική μήτρα προκειμένου να αναπαραχθεί η 3D συνθετική απομίμηση των σημείων/θέσεων δράσης της κερκετίνης στο σηματοδοτικό μονοπάτι του HIF-1. Το μοριακά αποτυπωμένο πολυμερές (MIP), το οποίο διαθέτει λειτουργικές κοιλότητες, συμπληρωματικές σε σχήμα, μέγεθος και λειτουργικότητα προς το αρχικό υπόστρωμα (κερκετίνη), χρησιμοποιήθηκε για την επιλεκτική απομόνωση νέων αναστολέων του HIF-1 από τη ρητίνη του δέντρου Boswellia serrata (λιβάνι), ευρέως χρησιμοποιούμενο αντικαρκινικό φάρμακο στην ελληνική αρχαιότητα και την παραδοσιακή κινέζικη και ινδική ιατρική. Η εφαρμογή εκχυλίσματος της ρητίνης (σε ακετόνη) σε στήλες SPE (πληρωμένες στο εργαστήριο με κλάσμα αποτυπωμένου πολυμερούς 45-90μm) οδήγησε στην ειδική συγκράτηση ενός αριθμού ενώσεων με αποτέλεσμα αποτύπωσης από 5±4 εως 8±4,6 (με αναφορά σε μη αποτυπωμένο πολυμερές, n=3). Το μείγμα των ενώσεων αυτών (μείγμα έκλουσης) ανέστειλλε τη μεταγραφική ενεργότητα του HIF-1, με αναστολή των πρωτεϊνικών επιπέδων του HIF-1α, της ρυθμιστικής υπομονάδας του HIF-1. Αντιθέτως οι ενώσεις που δε συγκρατήθηκαν στη στήλη δεν εμφάνισαν ανασταλτική δράση στο HIF-1. Το ακετυλο-11-κετο-β-μποσβελλικό οξύ (ΑΚΒΑ), ένα από τα κύρια βιοδραστικά συστατικά του λιβανιού, ταυτοποιήθηκε ως ένα από τα κύρια συστατικά του κλάσματος που συγκρατήθηκε ειδικά. Ο προσδιορισμός της βιολογικής δραστικότητας του ΑΚΒΑ, αποκάλυψε ότι αναστέλλει την επαγωγή του HIF-1α και τη μεταγραφική δραστικότητα του HIF-1. Με επακόλουθη ημι-παρασκευαστική χρωματογραφία του μείγματος έκλουσης, το ΑΚΒΑ διαχωρίστηκε χρωματογραφικά από τα υπόλοιπα συστατικά που συγκρατήθηκαν ειδικά. Τα συστατικά αυτά βρέθηκαν επίσης να αναστέλλουν τη μεταγραφική ενεργότητα του HIF-1. Επιπρόσθετα αποτυπώθηκε το ανάλογο του ATP, τρι-Ο-ακετυλοαδενοσίνη, προκειμένου να παραχθεί η προσεγγιστική συνθετική απομίμηση του σημείου πρόσδεσης του ATP στις κινάσες. Η φωσφορυλίωση αποτελεί μία μεταμεταφραστική τροποποίηση η οποία μπορεί να επιφέρει αρνητική ρύθμιση του HIF-1, μέσω αναστολής της δράσης των κατάλληλων κινασών. Βασικό μηχανισμό αναστολής των πρωτεϊνικών κινασών αποτελεί η κατάληψη της περιοχής πρόσδεσης ATP από ενώσεις που παρουσιάζουν δυνατότητα σύνδεσης με τον ίδιο τρόπο (ATP ανταγωνιστές). Η παρασκευή της προσεγγιστικής συνθετικής απομίμησης του σημείου πρόσδεσης του ATP στοχεύει στη δημιουργία ενός πολυμερούς κατάλληλου για σκανάρισμα και απομόνωση νέων ATP μιμητικών αναστολέων πρωτεϊνικών κινασών. Η επιτυχής αποτύπωση της τρι-Ο-ακετυλοαδενοσίνης είχε ως αποτέλεσμα τη σύνθεση πολυμερών με υψηλή συγγένεια προς το αρχικό υπόστρωμα, (Kd ≥ 1,2μΜ), επαρκή αριθμό θέσεων δέσμευσης Ν και σημαντική εξειδίκευση προς το υπόστρωμα (Kd imprinted << Kd control). Συνολικά, η μελέτη αυτή καταδεικνύει το δυναμικό εφαρμογής των MIP για το σκανάρισμα, την αναγνώριση και απομόνωση νέων βιοδραστικών ενώσεων οι οποίες στοχεύουν συγκεκριμένους μοριακούς στόχους, ένα δυναμικό το οποίο ελάχιστα έχει μέχρι σήμερα αποτιμηθεί

Hydroxypropyl-β-Cyclodextrin as a Green Co-Solvent in the Aqueous Extraction of Polyphenols from Waste Orange Peels

There is, to-date, an expanding interest concerning the use of cyclodextrins as green food-grade co-solvents in the aqueous extraction of polyphenols, however, data regarding polyphenol extraction from waste orange peels (WOP) are lacking. On this ground, hydroxypropyl &beta;-cyclodextrin (HP-&beta;-CD), a highly water-soluble cyclodextrin, was used to develop a simple and straightforward methodology for the effective recovery of WOP polyphenols. Process optimization by response surface showed that maximum total polyphenol recovery (26.30 &plusmn; 1.49 mg gallic acid equivalents g&minus;1 dry mass) could be accomplished with 15 mM HP-&beta;-CD at 40 &deg;C. On the other hand, integration of ultrasonication pretreatment was found unsuitable, as it resulted in reduced polyphenol yield. Examination of solvent acidity indicated that polyphenol extraction may be enhanced at pH 4, but the difference was non-significant (p &gt; 0.05) compared to yields attained at pH 2, 3, and 5. Extraction of WOP polyphenols with HP-&beta;-CD was shown to provide significantly higher hesperidin yield compared to 60% (v/v) aqueous ethanol, which suggested selectivity of HP-&beta;-CD toward this polyphenolic metabolite

Development of a Low-Temperature and High-Performance Green Extraction Process for the Recovery of Polyphenolic Phytochemicals from Waste Potato Peels Using Hydroxypropyl β-Cyclodextrin

Potato peels (PP) are a major agri-food side-stream originating from potato processing, but to date, their green valorization as a bioresource of antioxidant polyphenols is limited to extraction processes involving mainly water/ethanol-based solvents, whereas other eco-friendly methodologies are scarce. This study aimed at developing a simple, straight-forward and green extraction methodology to effectively recover PP polyphenols, using hydroxypropyl &beta;-cyclodextrin (HP-&beta;-CD). After an initial assay to identify the optimal HP-&beta;-CD concentration that would provide increased extraction yield, optimization based on response surface methodology enabled maximization of the extraction performance, providing a total polyphenol yield of 17.27 &plusmn; 0.93 mg chlorogenic acid equivalent g&minus;1 dry mass, at 30 &deg;C. Testing of temperatures higher than 30 &deg;C and up to 80 &deg;C did not favor higher yields. The extracts obtained with HP-&beta;-CD were slightly richer in polyphenols than extracts prepared with conventional solvents, such as aqueous ethanol and methanol, displaying similar antioxidant characteristics. The major polyphenols that could be identified in the extracts were neochlorogenic, chlorogenic, caffeic and ferulic acids. The outcome of this study demonstrated that HP-&beta;-CD may be used as a highly effective green means of recovering PP polyphenols, at near-ambient temperature

Evaluation of Pulsed Electric Field Polyphenol Extraction from Vitis vinifera, Sideritis scardica and Crocus sativus

This study exploited the application of pulsed electric field (PEF) on the recovery of polyphenols from aerial parts of Sideritis scardica, tepals of Crocus sativus, and fruits of Vitis vinifera. Short pulses of 10 μs in a period of 1 ms were applied to the plant material, while different electric field intensities, 1.2 to 2.0 kV/cm were tested to optimize the procedure. The content in total polyphenols and the polyphenolic profile of the plant extracts were evaluated. Along with PEF samples, control samples were prepared for comparison. PEF treatment enhanced the recovery in total polyphenols for all the three plants examined. A significant increase was noticed in each plant tested and PEF condition applied, though lower electric field intensities up to 1.4 kV/cm proved to be more effective. Under the optimum electric field intensities, 1.4 kV/cm for V. vinifera and 1.2 kV/cm for S. scardica and C. sativus, increases of 49.15%, 35.25%, and 44.36% in total polyphenol content, respectively, were achieved. Additionally, an 85% increase of quercetin 3-rutinoside for V. vinifera, a 56% of apigenin 7-O-glucoside for S. scardica, and a 64% increase for kaempferol 3-O-glucoside for C. sativus were obtained

Development of a Green Methodology for Simultaneous Extraction of Polyphenols and Pigments from Red Winemaking Solid Wastes (Pomace) Using a Novel Glycerol-Sodium Benzoate Deep Eutectic Solvent and Ultrasonication Pretreatment

In this examination, two glycerol-based deep eutectic solvents (DESs) were tested for their efficiency in the recovery of antioxidant polyphenols and anthocyanin pigments from red grape pomace (RGP). The two DESs synthesized had sodium acetate and sodium benzoate as hydrogen bond acceptors, to test the role of the hydrogen bond acceptor polarity on the extraction performance. Furthermore, the process was enhanced by an ultrasonication pretreatment stage. After initial testing with respect to water content, ultrasonication power and liquid-to-solid ratio, the DES composed of glycerol and sodium benzoate (GL-SBz) was shown to be significantly more efficient than the one made of glycerol and sodium acetate (GL-SAc). Further optimization of the extraction with regard to time and temperature demonstrated GL-SBz to be a highly effective solvent for the production of RGP extracts rich in polyphenols including gallic acid, catechin and quercetin, and pigments including malvidin 3-O-glucoside p-coumarate and malvidin 3-O-glucoside. The extracts produced also had significantly higher antiradical activity and reducing power compared to those generated with aqueous ethanol or water. From this study, evidence emerged regarding the role of the hydrogen bond acceptor nature in the extraction efficiency of polyphenols. The process developed is proposed as a green, high-performing methodology for the production of RGP extracts with enhanced polyphenolic content and antioxidant activity

Green Valorization of Olive Leaves to Produce Polyphenol-Enriched Extracts Using an Environmentally Benign Deep Eutectic Solvent

Olive leaves (OLL) are considered to be a highly appreciated bioresource of bioactive polyphenolic phytochemicals, embracing several different structures. However, extraction processes based on deep eutectic solvents (DES) are very limited despite the wide range of techniques developed for the efficient recovery of polyphenols. This study had as objective the development of a simple, green, high-performance extraction methodology for OLL polyphenols, using a recently reported effective DES, composed of L-lactic acid and glycine. Initially, a screening was performed to select the most appropriate L-lactic/glycine molar ratio and process optimization was then carried out with response surface methodology. The optimized process variable values were DES/water (78% w/v), liquid-to-solid ratio of 36 mL g&minus;1, and stirring speed of 500 rounds per minute, and the total polyphenol yield amounted to 97.53 &plusmn; 3.54 mg gallic acid equivalents per g dry matter. Extraction with DES at 80 &deg;C did not significantly increase the total polyphenol yield, but it did enhance the total flavonoid yield and antioxidant activity. High-performance liquid chromatography analyses revealed that extraction with the DES resulted in extended oleuropein hydrolysis, to the favor of hydroxytyrosol formation. This finding might have a prospect in using properly tuned DES for polyphenol modification with improved bioactivities

Polyphenol Extraction from Humulus lupulus (Hop) Using a Neoteric Glycerol/L-Alanine Deep Eutectic Solvent: Optimisation, Kinetics and the Effect of Ultrasound-Assisted Pretreatment

The investigation presented herein had as its scope the development of an integrated process for the efficient extraction of polyphenols from hop. For this purpose, a novel, natural deep eutectic solvent (DES) was synthesised, composed of glycerol and L-alanine, and the process was optimised by deploying a response surface methodology based on a Box&ndash;Behnken design. The variables considered were the DES/water proportion, the liquid-to-solid ratio and the stirring speed. Under the optimised conditions, the yield in total polyphenols achieved was 118.97 &plusmn; 8.27 mg gallic acid equivalents per g of dry mass. Ultrasonication, incorporated into the process as a pretreatment step, was shown to significantly change the kinetic pattern of polyphenol extraction and contributed to attaining higher yields only at 80 &deg;C, whereas at lower temperatures a supressing effect was observed. Furthermore, increasing temperature was negatively correlated with the second-order extraction rates, evidencing a slow-down of the extraction rate at elevated temperatures

Saffron Processing Wastes as a Bioresource of High-Value Added Compounds: Development of a Green Extraction Process for Polyphenol Recovery Using a Natural Deep Eutectic Solvent

The current investigation was undertaken to examine saffron processing waste (SPW) as a bioresource, which could be valorized to produce extracts rich in antioxidant polyphenols, using a green, natural deep eutectic solvent (DES). Initially, there was an appraisal of the molar ratio of hydrogen bond donor/hydrogen bond acceptor in order to come up with the most efficient DES composed of L-lactic acid/glycine (5:1). The following step was the optimization of the extraction process using response surface methodology. The optimal conditions thus determined were a DES concentration of 55% (w/v), a liquid-to-solid ratio of 60 mL g&minus;1, and a stirring speed of 800 rounds per minute. Under these conditions, the extraction yield in total polyphenols achieved was 132.43 &plusmn; 10.63 mg gallic acid equivalents per g of dry mass. The temperature assay performed within a range of 23 to 80 &deg;C, suggested that extracts displayed maximum yield and antioxidant activity at 50&ndash;60 &deg;C. Liquid chromatography-mass spectrometry analysis of the SPW extract obtained under optimal conditions showed that the predominant flavonol was kaempferol 3-O-sophoroside and the major anthocyanin delphinidin 3,5-di-O-glucoside. The results indicated that SPW extraction with the DES used is a green and efficient methodology and may afford extracts rich flavonols and anthocyanins, which are considered to be powerful antioxidants

Enhancement of Polyphenols Recovery from Rosa canina, Calendula officinalis and Castanea sativa Using Pulsed Electric Field

The current study evaluates the Pulsed Electric Field (PEF) technique for the extraction of polyphenols from the plants Rosa canina, Calendulaofficinalis and Castanea sativa. These plants are traditionally used both for the preparation of therapeutic decoctions and the aromatization of beverages (alcoholic or not). Pulses of 10 μs duration were used to apply electric field intensities ranging from 1.2 to 2.0 kV cm−1. The period of the phenomenon was set to 1 ms, with a total extraction time of 20 min. The total polyphenol content as well as the identified polyphenolic compounds of the extracts were determined for monitoring and evaluation. To estimate the PEF effect, control extracts were prepared using the same process as PEF extracts but without the application of electric field. For all the three plant materials studied, the PEF technique appeared to be successful in increasing polyphenols extraction. The application of a moderate to high electric field, up to 1.4 kV cm−1, resulted in increased total and individual polyphenols recovery, reaching 63.79% and 84%, respectively, in the case of Rosa canina fruits

Optimization of Pulsed Electric Field as Standalone “Green” Extraction Procedure for the Recovery of High Value-Added Compounds from Fresh Olive Leaves

Olive leaves (OLL) are reported as a source of valuable antioxidants and as an agricultural by-product/waste. Thus, a twofold objective with multi-level cost and environmental benefits arises for a “green” standalone extraction technology. This study evaluates the OLL waste valorization through maximizing OLL extracts polyphenol concentration utilizing an emerging “green” non-thermal technology, Pulsed Electric Field (PEF). It also provides further insight into the PEF assistance span for static solid-liquid extraction of OLL by choosing and fine-tuning important PEF parameters such as the extraction chamber geometry, electric field strength, pulse duration, pulse period (and frequency), and extraction duration. The produced extracts were evaluated via comparison amongst them and against extracts obtained without the application of PEF. The Folin-Ciocalteu method, high-performance liquid chromatography, and differential scanning calorimetry were used to determine the extraction efficiency. The optimal PEF contribution on the total polyphenols extractability (38% increase with a 117% increase for specific metabolites) was presented for rectangular extraction chamber, 25% v/v ethanol:water solvent, pulse duration (tpulse) 2 μs, electric field strength (E) 0.85 kV cm−1, 100 μs period (Τ), and 15 min extraction duration (textraction), ascertaining a significant dependence of PEF assisting extraction performance to the parameters chosen