Tomasz Tronina

Carotenoid-induced non-photochemical quenching in the cyanobacterial chlorophyll synthase–HliC/D complex

Chl synthase (ChlG) is an important enzyme of the Chl biosynthetic pathway catalyzing attachment of phytol/geranylgeraniol tail to the chlorophyllide molecule. Here we have investigated the Flag-tagged ChlG (f.ChlG) in a complex with two different high-light inducible proteins (Hlips) HliD and HliC. The f.ChlG-Hlips complex binds a Chl a and three different carotenoids, β-carotene, zeaxanthin and myxoxanthophyll. Application of ultrafast time-resolved absorption spectroscopy performed at room and cryogenic temperatures revealed excited-state dynamics of complex-bound pigments. After excitation of Chl a in the complex, excited Chl a is efficiently quenched by a nearby carotenoid molecule via energy transfer from the Chl a Qy state to the carotenoid S1 state. The kinetic analysis of the spectroscopic data revealed that quenching occurs with a time constant of ~2ps and its efficiency is temperature independent. Even though due to its long conjugation myxoxanthophyll appears to be energetically best suited for role of Chl a quencher, based on comparative analysis and spectroscopic data we propose that β-carotene bound to Hlips acts as the quencher rather than myxoxanthophyll and zeaxanthin, which are bound at the f.ChlG and Hlips interface. The S1 state lifetime of the quencher has been determined to be 13ps at room temperature and 21ps at 77K. These results demonstrate that Hlips act as a conserved functional module that prevents photodamage of protein complexes during photosystem assembly or Chl biosynthesis.

Biotransformations of Prenylated Hop Flavonoids for Drug Discovery and Production

In this review we aim to present current knowledge on biotransformation of flavonoids from hop cones with respect to type of product, catalyst and conversion. Subsequently, a comparative analysis of biological activity of prenylated hop flavonoids and their biotransformation products has been performed in order to indicate these research efforts that have good potential for application in pharmaceutical industry. There is increasing evidence that the products of biotransformation of hop prenylflavonoids, which have been little studied until recently, can be used as drugs or drug ingredients and also as standards of human drug metabolites. They can also serve as an inspiration for the design and chemical synthesis of new derivatives with higher or different biological activity. Nevertheless, much additional work, among others on determining the mechanism of action in in vivo systems, is needed to open up the way to biomedical application of these compounds.

Transformation of xanthohumol by Aspergillus ochraceus.

Microbial transformation of xanthohumol isolated from agro‐residue (spent hops), by Aspergillus ochraceus was investigated. A new aurone, (Z)‐2″‐( 2‴ ‐hydroxyisopropyl)‐dihydrofurano[4″,5″:6,7]‐3′,4′‐dihydroxy‐4‐methoxyaurone, was obtained as a main transformation product. Three minor metabolites were identified as 2″‐( 2‴ ‐hydroxyisopropyl)‐dihydrofurano[4″,5″:3′,4′]‐2′,4‐dihydroxy‐6′‐methoxychalcone, (2S,2″S)‐2″‐( 2‴ ‐hydroxyisopropyl)‐dihydrofurano[4″,5″:7,8]‐4′‐hydroxy‐5‐methoxyflavanone and (2S,2″R)‐2″‐( 2‴ ‐hydroxyisopropyl)‐dihydrofurano[4″,5″:7,8]‐4′‐hydroxy‐5‐methoxyflavanone. Their structures were elucidated on the basis of spectroscopic evidences. The antioxidant properties of xanthohumol and its metabolites were investigated using the 2,2′‐diphenyl‐1‐picrylhydrazyl (DPPH) radical scavenging method. The major biotransformation product, was 8.6‐fold stronger antioxidant than xanthohumol and 2.3‐fold than ascorbic acid.

Microbial Glycosylation of Daidzein, Genistein and Biochanin A: Two New Glucosides of Biochanin A

Biotransformation of daidzein, genistein and biochanin A by three selected filamentous fungi was investigated. As a result of biotransformations, six glycosylation products were obtained. Fungus Beauveria bassiana converted all tested isoflavones to 4″-O-methyl-7-O-glucosyl derivatives, whereas Absidia coerulea and Absidia glauca were able to transform genistein and biochanin A to genistin and sissotrin, respectively. In the culture of Absidia coerulea, in addition to the sissotrin, the product of glucosylation at position 5 was formed. Two of the obtained compounds have not been published so far: 4″-O-methyl-7-O-glucosyl biochanin A and 5-O-glucosyl biochanin A (isosissotrin). Biotransformation products were obtained with 22%–40% isolated yield.

Biotransformation of the phytoestrogen 8-prenylnaringenin.

In order to select microorganisms capable of transforming the potent phytoestrogen 8-prenylnaringenin, preliminary screening tests on 30 fungal cultures were performed. No reports concerning successful biotransformation of 8-prenylnaringenin by this class of organisms have been known so far. Fusarium equiseti converted 8-prenylnaringenin into 2”-(2”’-hydroxyisopropyl)-dihydrofuran-[2”,3”:7,8]-5,4’-dihydroxyfl avanon in high yield.

The Influence of Glycosylation of Natural and Synthetic Prenylated Flavonoids on Binding to Human Serum Albumin and Inhibition of Cyclooxygenases COX-1 and COX-2

The synthesis of different classes of prenylated aglycones (α,β-dihydroxanthohumol (2) and (Z)-6,4’-dihydroxy-4-methoxy-7-prenylaurone (3)) was performed in one step reactions from xanthohumol (1)—major prenylated chalcone naturally occurring in hops. Obtained flavonoids (2–3) and xanthohumol (1) were used as substrates for regioselective fungal glycosylation catalyzed by two Absidia species and Beauveria bassiana. As a result six glycosides (4–9) were formed, of which four glycosides (6–9) have not been published so far. The influence of flavonoid skeleton and the presence of glucopyranose and 4-O-methylglucopyranose moiety in flavonoid molecule on binding to main protein in plasma, human serum albumin (HSA), and inhibition of cyclooxygenases COX-1 and COX-2 were investigated. Results showed that chalcone (1) had the highest binding affinity to HSA (8.624 × 104 M−1) of all tested compounds. It has also exhibited the highest inhibition of cyclooxygenases activity, and it was a two-fold stronger inhibitor than α,β-dihydrochalcone (2) and aurone (3). The presence of sugar moiety in flavonoid molecule caused the loss of HSA binding activity as well as the decrease in inhibition of cyclooxygenases activity.

Bioactivity In Vitro of Quercetin Glycoside Obtained in Beauveria bassiana Culture and Its Interaction with Liposome Membranes

Quercetin (Q) was used as substrate for regioselective glycosylation at the C-7 position catalyzed by Beauveria bassiana AM278 strain. As a result the glycoside quercetin 7-O-β-d-(4″-O-methyl)glucopyranoside (Q 7-MeGlu) was formed. The goal of the studies was to determine the anti-oxidative (liposome membrane protection against free radicals IC50Q 7-MeGlu = 5.47 and IC50Q = 4.49 µM) and anti-inflammatory (COX-1 and COX-2 enzymes activity inhibition) properties of Q 7-MeGlu as compared to Q. Every attempt was made to clarify the antioxidant activity of these molecules, which are able to interact with egg phosphatidylcholine liposomes, using a fluorometric method (by applying the probes MC540, TMA-DPH and DPH). The results indicated that Q 7-MeGlu and Q are responsible for increasing the packing order, mainly in the hydrophilic but also in hydrophobic regions of the membrane (Q > Q 7-MeGlu). These observations, confirmed by a 1H-NMR method, are key to understanding their antioxidant activity which is probably caused by the stabilizing effect on the lipid membranes. The results showed that Q 7-MeGlu and Q have ability to quench the human serum albumin (HSA) intrinsic fluorescence through a static quenching mechanism. The results of thermodynamic parameters indicated that the process of formation complexes between studied molecules and HSA was spontaneous and caused through Van der Waals interactions and hydrogen bonding.

Regioselective ortho-Hydroxylations of Flavonoids by Yeast.

Natural flavonoids, such as naringenin, hesperetin, chrysin, apigenin, luteolin, quercetin, epicatechin, and biochanin A, were subjected to microbiological transformations by Rhodotorula glutinis. Yeast was able to regioselectively C-8 hydroxylate hesperetin, luteolin, and chrysin. Naringenin was transformed to 8- and 6-hydroxyderivatives. Quercetin, epicatechin, and biochanin A did not undergo biotransformation. A metabolic pathway for the degradation of chrysin has been elucidated. The metabolism of chrysin proceeds via an initial C-8 hydroxylation to norwogonin, followed by A-ring cleavage to 4-hydroxy-6-phenyl-2H-pyran-2-one.

Synthesis and Antiproliferative Activity of Minor Hops Prenylflavonoids and New Insights on Prenyl Group Cyclization

Synthesis of minor prenylflavonoids found in hops and their non-natural derivatives were performed. The antiproliferative activity of the obtained compounds against some human cancer cell lines was investigated. Using xanthohumol isolated from spent hops as a lead compound, a series of minor hop prenylflavonoids and synthetic derivatives were obtained by isomerization, cyclisation, oxidative-cyclisation, oxidation, reduction and demethylation reactions. Three human cancer cell lines—breast (MCF-7), prostate (PC-3) and colon (HT-29)—were used in antiproliferative assays, with cisplatin as a control compound. Five minor hop prenyl flavonoids and nine non-natural derivatives of xanthohumol have been synthetized. Syntheses of xanthohumol K, its dihydro- and tetrahydro-derivatives and 1″,2″,α,β-tetrahydroxanthohumol C were described for the first time. All of the minor hops prenyl flavonoids exhibited strong to moderate antiproliferative activity in vitro. The minor hops flavonoids xanthohumol C and 1″,2″-dihydroxanthohumol K and non-natural 2,3-dehydroisoxanthohumol exhibited the activity comparable to cisplatin. Results described in the article suggest that flavonoids containing chromane- and chromene-like moieties, especially chalcones, are potent antiproliferative agents. The developed new efficient, regioselective cyclisation reaction of the xanthohumol prenyl group to 1″,2″-dihydroxantohumol K may be used in the synthesis of other compounds with the chromane moiety.

Antimicrobial Properties of Spent Hops Extracts, Flavonoids Isolated Therefrom, and Their Derivatives

Hop cones preparations possess a wide range of biological activities including antimicrobial properties. In this work, we evaluated the effect of various organic extracts obtained from spent hops, as well as six hops flavonoids and their twenty natural and synthetic derivatives on human and plant microbial pathogens. Methylene chloride, acetone, ethyl acetate, and methanol were used as extractants. Seven flavonoids, among them two natural (α,β-dihydroxanthohumol and 8-prenylnaringenin) showed significant activity against methicillin sensitive and resistant Staphylococcus aureus and Staphylococcus epidermidis strains with the lowest MIC80 value of 0.5 µg/mL. The crude ethyl acetate, acetone, and methanol extracts from the spent hops exhibited antifungal activity against Fusarium oxysporum, F. culmorum, and F. semitectum with the lowest MIC50 of 0.5 mg/mL, while the methylene chloride extract exerted antifungal activity against Botrytis cinerea with the MIC50 of 1 mg/mL. The preparation obtained after the removal of xanthohumol from the spent hops crude extracts retained up to 95% of activity. These findings suggest that various spent hops extracts may be effective agents for the control of plant pathogens of economic importance, like Botrytis cinerea and Fusarium oxysporum, while some compounds from spent hops or their derivatives may become useful for staphylococcal infections.