Institute of Organic Chemistry with Centre of Phytochemistry

The newest developments in propolis pharmacological research are summarized. The problem regarding biological studies, caused by the chemical variability of propolis, is discussed. The most important trends and developments in recent propolis research are outlined: biological studies performed with chemically characterized samples, bioassay-guided studies of active principles and comparative biological studies of propolis of different origin and chemical composition. These types of studies are extremely valuable with respect to propolis standardization and practical applications in therapy. They will allow scientists to connect a particular chemical propolis type to a specific type of biological activity and formulate recommendations for practitioners.

The validation of rapid, low-cost spectrophotometric procedures for the quantification of the three main groups of bioactive substances (flavones and flavonols, flavanones and dihydroflavonols, and total phenolics) in poplar-type propolis has been performed. A spectrophotometric assay based on the formation of an aluminium chloride complex was applied for the quantification of total flavones and flavonols using galangin as standard. Because of the high amount of flavanones and dihydroflavonols in "poplar type" propolis, the introduction of a distinct procedure for their quantification was considered of special significance and the DAB9 colorimetric method was applied for the purpose. Total phenolic content was measured by the Folin-Ciocalteu procedure using a mixture of pinocembrin and galangin as a reference. The procedures were validated using a model mixture of compounds representing the poplar-type propolis composition as found in previous studies. The accuracy (recovery) varied in the range 84-109%, and the relative standard deviation was 0.5-6.2%. The developed spectrophotometric procedures were applied to six poplar type propolis samples. The results were verified independently by a HPLC procedure. The two sets of results agreed satisfactory, as proven by Student's t-test.

Propolis is a sticky material collected by bees from plants, and used in the hive as building material and defensive substance. It has been popular as a remedy in Europe since ancient times. Nowadays, propolis use in over-the-counter preparations, "bio"-cosmetics and functional foods, etc., increases. Volatile compounds are found in low concentrations in propolis, but their aroma and significant biological activity make them important for propolis characterisation. Propolis is a plant-derived product: its chemical composition depends on the local flora at the site of collection, thus it offers a significant chemical diversity. The role of propolis volatiles in identification of its plant origin is discussed. The available data about chemical composition of propolis volatiles from different geographic regions are reviewed, demonstrating significant chemical variability. The contribution of volatiles and their constituents to the biological activities of propolis is considered. Future perspectives in research on propolis volatiles are outlined, especially in studying activities other than antimicrobial.

Chalcones (1,3-diaryl-2-propen-1-ones) are open chain flavonoids that are widely biosynthesized in plants. They are important for the pigmentation of flowers and, hence, act as attractants to the pollinators. As flavonoids, chalcones also play an important role in defense against pathogens and insects. A longstanding scientific research has shown that chalcones also display other interesting biological properties such as antioxidant, cytotoxic, anticancer, antimicrobial, antiprotozoal, antiulcer, antihistaminic and anti-inflammatory activities. Some lead compounds with various pharmacological properties have been developed based on the chalcone skeleton. Clinical trials have shown that these compounds reached reasonable plasma concentrations and did not cause toxicity. For these reasons, chalcones became an object of continued interest in both academia and industry. Nowadays, several chalcones are used for treatment of viral disorders, cardiovascular diseases, parasitic infections, pain, gastritis, and stomach cancer, as well as like food additives and cosmetic formulation ingredients. However, much of the pharmacological potential of chalcones is still not utilized. The purpose of this review is to describe the recent efforts of scientists in pharmacological screening of natural and synthetic chalcones, studying the mechanisms of chalcone action and relevant structure-activity relationships. Put together, these activities aimed at synthesis of pharmacologically active chalcones and their analogs.

Ten propolis samples from Bulgaria, Italy and Switzerland were analyzed by GC-MS. As expected, most samples displayed the typical chemical pattern of "poplar" propolis: they contained pinocembrin, pinobanksin and its 3-O-acetate, chrysin, galangin, prenyl esters of caffeic and ferulic acids. Two samples differed significantly: one from the Graubünden Alpine region, Switzerland, rich in phenolic glycerides, and one from Sicily which contained only a limited number of phenolics and was rich in diterpenic acids.

BACKGROUND: Propolis is widely used in apitherapy, preparations, and food and beverage additives. Various extraction techniques were applied in the extraction of the biologically active constituents of poplar type propolis in order to compare their efficiency. The methods employed were: traditional maceration extraction, ultrasound extraction (UE), and microwave assisted extraction (MAE). RESULTS: The total amounts of extracted phenolics and flavonoids were determined, and the effectiveness of the methods compared. MAE was very rapid but led to the extraction of a large amount of non-phenolic and non-flavonoid material. UE gave the highest percentage of extracted phenolics. CONCLUSION: Compared to the maceration extraction, MAE and UE methods provided high extraction yield, requiring short timeframes and less labour. UE was shown to be the most efficient method based on yield, extraction time and selectivity.

Dodecaborate anions of the type B12X12(2-) and B12X11Y(2-) (X=H, Cl, Br, I and Y=OH, SH, NH3(+), NR3(+)) form strong (K(a) up to 10(6) L mol(-1), for B12Br12(2-)) inclusion complexes with γ-cyclodextrin (γ-CD). The micromolar affinities reached are the highest known for this native CD. The complexation exhibits highly negative enthalpies (up to -25 kcal mol(-1)) and entropies (TΔS up to -18.4 kcal mol(-1), both for B12I12(2-)), which position these guests at the bottom end of the well-known enthalpy-entropy correlation for CDs. The high driving force can be traced back to a chaotropic effect, according to which chaotropic anions have an intrinsic affinity to hydrophobic cavities in aqueous solution. In line with this argument, salting-in effects revealed dodecaborates as superchaotropic dianions.

Four samples of Brazilian propolis were investigated by GC/MS of different fractions. 32 volatile compounds, (10 of them new for propolis), as well as 12 more polar compounds (one of them new for propolis) were identified. Antibacterial activity was found in some propolis fractions.

The cyclopentenone unit is a very powerful synthon for the synthesis of a variety of bioactive target molecules. This is due to the broad diversity of chemical modifications available for the enone structural motif. In particular, chiral cyclopentenones are important precursors in the asymmetric synthesis of target chiral molecules. This Review provides an overview of reported methods for enantioselective and asymmetric syntheses of cyclopentenones, including chemical and enzymatic resolution, asymmetric synthesis via Pauson-Khand reaction, Nazarov cyclization and organocatalyzed reactions, asymmetric functionalization of the existing cyclopentenone unit, and functionalization of chiral building blocks.

In a new propolis type, red Brazilian propolis, 14 compounds were identified (six of them new for propolis), among them simple phenolics, triterepenoids, isoflavonoids, prenylated benzophenones and a naphthoquinone epoxide (isolated for the first time from a natural source). Three of the major components demonstrated significant antimicrobial activity, and two (obtained as inseparable mixture) possessed radical scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH).

Propolis is one of the most fascinating honey bee (Apis mellifera L.) products. It is a plant derived product that bees produce from resins that they collect from different plant organs and with which they mix beeswax. Propolis is a building material and a protective agent in the bee hive. It also plays an important role in honey bee social immunity, and is widely used by humans as an ingredient of nutraceuticals, over-the-counter preparations and cosmetics. Its chemical
\ncomposition varies by geographic location, climatic zone and local flora. The understanding of the chemical diversity of propolis is very important in propolis research. In this manuscript, we give an overview of the available methods for studying propolis in different aspects: propolis in the bee colony; chemical composition and plant sources of propolis; biological activity of propolis with respect to bees and humans; and approaches for standardization and quality control for the purposes of industrial application.

This study investigates the influence of extraction system on the extractability of polyphenol compounds and antioxidant activity of various medicinal plants. Oxygen radical absorbance capacity (ORAC) and total polyphenol content of 25 Bulgarian medicinal plants subjected to water or 80 % acetone extractions were investigated and compared. The type of extragent significantly influenced the efficiency of the polyphenol extraction and the antioxidant activity. In all cases ORAC results and total polyphenol content were higher for acetone extraction than for water extraction. The acetone extract of peppermint had the highest ORAC value - 2917 micromol Trolox equivalent (TE)/g dry weight (DW) and polyphenol content - 20216 mg/100 g DW. For water extraction thyme exhibited the highest ORAC antioxidant activity - 1434 micromol TE/g DW. There was a significant linear correlation between the concentration of total polyphenols and ORAC in the investigated medicinal plants. It can be concluded that the solvent used affects significantly the polyphenol content and the antioxidant activity of the extract and therefore it is recommended to use more than one extraction system for better assessment of the antioxidant activity of natural products. Several of the investigated herbs contain substantial amounts of free radical scavengers and can serve as a potential source of natural antioxidants for medicinal and commercial uses.

Reactive carbonyls, especially ,-unsaturated carbonyls produced through lipid peroxidation, damage biomolecules such as proteins and nucleotides; elimination of these carbonyls is therefore essential for maintaining cellular homeostasis. In this study, we focused on an NADPH-dependent detoxification of reactive carbonyls in plants and explored the enzyme system involved in this detoxification process. Using acrolein (CH 2 CHCHO) as a model ,-unsaturated carbonyl, we purified a predominant NADPH-dependent acrolein-reducing enzyme from cucumber leaves, and we identified the enzyme as an alkenal/one oxidoreductase (AOR) catalyzing reduction of an ,-unsaturated bond. Cloning of cDNA encoding AORs revealed that cucumber contains two distinct AORs, chloroplastic AOR and cytosolic AOR. Homologs of cucumber AORs were found among various plant species, including Arabidopsis, and we confirmed that a homolog of Arabidopsis (At1g23740) also had AOR activity. Phylogenetic analysis showed that these AORs belong to a novel class of AORs. They preferentially reduced ,-unsaturated ketones rather than ,-unsaturated aldehydes. Furthermore, we selected candidates of other classes of enzymes involved in NADPH-dependent reduction of carbonyls based on the bioinformatic information, and we found that an aldo-keto reductase (At2g37770) and aldehyde reductases (At1g54870 and At3g04000) were implicated in the reduction of an aldehyde group of saturated aldehydes and methylglyoxal as well as ,-unsaturated aldehydes in chloroplasts. These results suggest that different classes of NADPH-dependent reductases cooperatively contribute to the detoxification of reactive carbonyls.

Abstract Four labdane-type diterpenic acids and syringaldehyde were isolated and identified from Brazilian propolis. A ll the compounds exhibit antibacterial activity. The diterpenes, found for the first time in propolis, are typical for some Araucaria species and thus indicate a possible plant source of Brazilian propolis.

Yariv phenylglycosides [1,3,5-tri(p-glycosyloxyphenylazo)-2,4,6-trihydroxybenzene] are a group of chemical compounds that selectively bind to arabinogalactan proteins (AGPs), a type of plant proteoglycan. Yariv phenylglycosides are widely used as cytochemical reagents to perturb the molecular functions of AGPs as well as for the detection, quantification, purification, and staining of AGPs. However, the target structure in AGPs to which Yariv phenylglycosides bind has not been determined. Here, we identify the structural element of AGPs required for the interaction with Yariv phenylglycosides by stepwise trimming of the arabinogalactan moieties using combinations of specific glycoside hydrolases. Whereas the precipitation with Yariv phenylglycosides (Yariv reactivity) of radish (Raphanus sativus) root AGP was not reduced after enzyme treatment to remove α-l-arabinofuranosyl and β-glucuronosyl residues and β-1,6-galactan side chains, it was completely lost after degradation of the β-1,3-galactan main chains. In addition, Yariv reactivity of gum arabic, a commercial product of acacia (Acacia senegal) AGPs, increased rather than decreased during the repeated degradation of β-1,6-galactan side chains by Smith degradation. Among various oligosaccharides corresponding to partial structures of AGPs, β-1,3-galactooligosaccharides longer than β-1,3-galactoheptaose exhibited significant precipitation with Yariv in a radial diffusion assay on agar. A pull-down assay using oligosaccharides cross linked to hydrazine beads detected an interaction of β-1,3-galactooligosaccharides longer than β-1,3-galactopentaose with Yariv phenylglycoside. To the contrary, no interaction with Yariv was detected for β-1,6-galactooligosaccharides of any length. Therefore, we conclude that Yariv phenylglycosides should be considered specific binding reagents for β-1,3-galactan chains longer than five residues, and seven residues are sufficient for cross linking, leading to precipitation of the Yariv phenylglycosides.

Propolis (bee glue) is a sticky material collected by bees from plant resins, it has been proven to possess numerous beneficial pharmacological properties: antibacterial, antiviral, antioxidant, antiinflammatory, immunostimulating, hepatoprotective, cytotoxic, etc. Nowadays, propolis is a popular remedy all over the world, and is available in either pure form or combined with other natural products in over-the-counter preparations, cosmetics, and as a constituent of health foods. The interest in propolis containing commercial products is steadily growing. The extraction is a key step in making use of the bioactive constituents of propolis. The present review summarizes and discusses the available data about classical and modern methods of extraction of propolis with potential large-scale application. The published data concerning maceration, Soxhlet, ultrasound-assisted and microwave-assisted extraction, supercritical CO2 extraction, high-pressure methods, and the application of different solvents are discussed. Concerning the solvents, water – ethanol mixtures remain among the most effective, oil and natural deep eutectic solvents (NADES) have demonstrated promising potential. From the extraction processes, ultrasound-assisted extraction seems to be the optimal method, taking into account extraction time and extraction yield. Of course, more studies are necessary to find the best way to extract bioactive compounds from different types of propolis, based on chemometric optimization approaches, especially with respect to large-scale application.

Crude ethanolic extracts of propolis, a natural resin, have been directly analysed using electrospray ionization mass (ESI-MS) and tandem mass spectrometry (ESI-MS/MS) in the negative ion mode. European, North American and African samples have been analyzed, but emphasis has been given to Brazilian propolis which displays diverse and region-dependent chemical composition. ESI-MS provides characteristic fingerprint mass spectra, with propolis samples being divided into well-defined groups directly related to their geographical origins. Chemometric multivariate analysis statistically demonstrates the reliability of the ESI-MS fingerprinting method for propolis. On-line ESI-MS/MS tandem mass spectrometry of characteristic [M - H](-) ion markers provides an additional dimension of fingerprinting selectivity, while structurally characterizing the ESI-MS marker components of propolis. By comparison with standards, eight such markers have been identified: para-coumaric acid, 3-methoxy-4-hydroxycinnamaldehyde, 2,2-dimethyl-6-carboxyethenyl-2H-1-benzopyran, 3-prenyl-4-hydroxycinnamic acid, chrysin, pinocembrin, 3,5-diprenyl-4-hydroxycinnamic acid and dicaffeoylquinic acid. The negative mode ESI-MS fingerprinting method is capable of discerning distinct composition patterns to typify, to screen the sample origin and to reveal characteristic details of the more polar and acidic chemical components of propolis samples from different regions of the world.

Propolis and plant secretions from three species, most frequently mentioned as botanical sources of the bee glue in Brazil (Baccharis dracunculifolia, Araucaria angustifolia and Eucalyptus citriodora) have been investigated using GC-MS. Based on chemical evidence, B. dracunculifolia was shown to be the main propolis source in São Paulo state. The antibacterial and antifungal activities of all four materials were also tested, the most active being propolis and Baccharis leaf exudate.

The objective of this work is to analyze and identify the diterpene compounds in Mediterranean propolis samples from different Greek regions by GC-MS. The chemical composition of six propolis samples was established using previously isolated diterpenes from Cretan propolis as authentic standards for identification, based on mass spectral fragmentation of the TMS derivatives and retention index. More than 30 diterpenes, among which were new propolis constituents, were identified and characterized by means of authentic standards and interpretation of MS fragmentation as well. This is the first detailed profiling of a new type of propolis, rich in diterpenes. The chromatographic and mass- spectral characteristics of the diterpenes identified could be very useful for rapid GC-MS profiling of this propolis type and for revealing its plant sources.

Summary Polyphenols, phenolic acids, fibres and antioxidant capacity were determined in water, acetone and methanol extracts of buckwheat, rice, soybean, quinoa and 3 amaranth cultivars. Their antioxidant activities were comparatively assessed by total radical‐trapping antioxidative potential (TRAP), ferric ion‐reducing antioxidant power (FRAP), cupric‐reducing antioxidant capacity (CUPRAC) and nitric oxide (NO • ) assays, which comprised of contributions from polyphenols and phenolic acids (especially from the most abundant ferulic acid). The correlation coefficients between polyphenols and antioxidant activities of cereal and pseudocereal methanol extracts with FRAP, NO • , CUPRAC and TRAP were 0.99, 0.97, 0.96 and 0.77, respectively. The weakest correlation was with dietary fibres, an average one exhibited with tannins and marked correlation was shown with the phenolics. All the applied methods have shown that pseudocereals have higher antioxidant activity than some cereals ( rice and buckwheat ) and can be successfully replaced by cereals in case of allergy.