Recent development of chemical components in propolis

Propolis is a resinous substance collected by honeybees from various plant sources. On account of its chemical composition, propolis possesses several biological and pharmacological properties. In recent years, many papers have provided information concerning its composition. This review compiles data from most studies of propolis, focusing on the chemical composition of ethanol extracts of propolis (EEP), water extracts of propolis (WEP), and volatile oils from propolis (VOP). The characteristic compounds of EEP are polyphenols including flavonoids and related phenolic acids, and flavonoids are the most abundant and effective parts. They are considered to contribute more to the antibacterial, antiviral, and antioxidant effects than the other constituents. The main flavonoids in EEP are pinocembrin, galangin, chrysin, quercetin, kaempferol, and naringenin. The constituents reported to be in WEP include phenolic acids, caffeoylquinic acid, 3-mono-O-caffeoylquinic acid, caffeic acid, flavonoids, etc. The propolis volatile compounds are benzyl alcohol, benzyl acetate, cinnamic alcohol, vanillin, eudensmol, cyclohexyl benzoate, and benzyl benzoate, which are responsible for several biological properties. As a natural mixture, propolis is widely used in medicine and cosmetics, as well as being a constituent of health foods. Since propolis has been used extensively, information on its composition is not only of interest to the academic field, but also to propolis users.     

Recent development of chemical components in propolis

Propolis is a resinous substance collected by honeybees from various plant sources. On account of its chemical composition, propolis possesses several biologi-cal and pharmacological properties. In recent years, many papers have provided information concerning its composi-tion. This review compiles data from most studies of propolis, focusing on the chemical composition of ethanol extracts of propolis (EEP), water extracts of propolis (WEP), and volatile oils from propolis (VOP). The characteristic compounds of EEP are polyphenols includ-ing flavonoids and related phenolic acids, and flavonoids are the most abundant and effective parts. They are considered to contribute more to the antibacterial, antiviral, and antioxidant effects than the other constituents. The main flavonoids in EEP are pinocembrin, galangin, chrysin, quercetin, kaempferol, and naringenin. The constituents reported to be in WEP include phenolic acids, caffeoylquinic acid, 3-mono-O-caffeoylquinie acid, caffeic acid, flavonoids, etc. The propolis volatile compounds are benzyl alcohol, benzyl acetate, cinnamic alcohol, vanillin, eudensmol, cyclohexyl benzoate, and benzyl benzoate, which are responsible for several biological properties. As a natural mixture, propolis is widely used in medicine and cosmetics, as well as being a constituent of health foods. Since propolis has been used extensively, information on its composition is not only of interest to the academic field, but also to propolis users.     

Propolis and some of its constituents down-regulate DNA synthesis and inflammatory cytokine production but induce TGF-beta1 production of human immune cells

Propolis, the resinous product collected by honey bees from plants, is used as folk medicine since ancient time. Recently, immunoregulatory and anti-inflammatory properties of propolis have been published. The detailed mechanisms of actions of propolis and its components on immune cells, however, are still unknown. Therefore, we studied the effects of different propolis extracts, of the flavonoids hesperidin and quercetin as well as of caffeic acid phenethyl ester (CAPE) on basic human immune cell functions. In detail, we measured the effects on DNA synthesis and production of different types of cytokines, namely IL-1beta, IL-12, IL-2, IL-4, IL-10 and TGF-beta1, of mitogen-activated peripheral blood mononuclear cells (PBMC) as well as of purified T lymphocytes. Our data clearly show that propolis as well as its constituents studied are capable of dose-dependently suppressing phythemagglutinin (PHA)-induced DNA synthesis of PBMC and T cells. Moreover, cytokines produced by monocytes/macrophages (IL-1beta, IL-12), by Th1 type (IL-2) as well as Th2 type (IL-4) lymphocytes were found to be also suppressed, whereas the production of TGF-beta1 by T regulatory cells was ascertained to be increased. These data convincingly demonstrate that propolis has a direct regulatory effect on basic functional properties of immune cells which may be mediated by the Erk2 MAP-kinase signal pathway. Thus, the bee product propolis can be considered as a powerful natural anti-inflammatory medicine influencing different types of immune-responses probably via immunoregulatory T cells.     

Caffeic acid derivatives in artichoke extract are metabolised to phenolic acids in vivo

The purpose of this study was to investigate the absorption and metabolism of hydroxycinnamates from artichoke extract by determining the urinary excretion of the conjugates. Ten healthy, non smoking volunteers (5 female, 5 male) were given three capsules containing artichoke extract every 4 h (0, 4, 8h) following two days of a low-polyphenol diet. One capsule contained 320 mg of artichoke extract equivalent to 34.3 ± 0.6 mg/g hydroxycinnamates (caffeic acid derivatives) and 5.6 ± 0.1 mg/g flavonoids. Polyphenols and phenolic acids present in the artichoke extract were not detected in the urine either as conjugates or aglycones. However, ferulic, isoferulic, dihydroferulic and vanillic acid were identified as major metabolites after β-glucuronidase treatment of urine. The amount excreted as well as the ratio to that of creatinine, a biomarker for the general excretion rate, increased significantly on the study day compared to the presupplementation day. Thus, the caffeic acid esters found in the artichoke extract capsule are absorbed, metabolised and excreted as methylated phenolic acids such as ferulic, isoferulic, dihydroferulic and vanillic acid.     

The in vitro effect of a lyophilized extract of wine obtained from Jacquez grapes on human chondrocytes

The present work was aimed at evaluating the in vitro effects of a lyophilized extract of wine (JW-E) obtained from Jacquez grapes (Vitis aestivalis-cinereaxVitis vinifera grapes) on the production of key molecules released in inflammatory disease utilising interleukin-1beta (IL-1beta) activated chondrocytes. The extract contains large amounts of phenolic components, in particular some flavonoids (flavan-3-ols, also known as catechins) and proanthocyanidins, as hydroxycinnamic acids and anthocyanins, that possess several biological features such as antiinflammatory and antioxidant effects and a "radical scavenger" activity too. In this study, we assayed the effect of JW-E on the production of key molecules released during chronic inflammatory events as nitric oxide (NO), prostaglandins E(2) (PGE(2)) and reactive oxygen species (ROS) in human chondrocytes culture, stimulated with proinflammatory cytokine interleukin-1beta. The JW-E proved to possess good ability against the harmfull effects of IL-1beta. Our data showed the protective effects of JW-E in cartilage alteration, that appears greater than that elicited by indomethacin, a not steroidal antiinflammatory drug (NSAID), commonly employed in joint diseases.     

Selective analysis of phenolic compounds in propolis by HPLC-MS/MS

Phenolic compounds (flavonoids and phenolic acid derivatives) are major active constituents of the resinous fraction of propolis, and also represent its allergenic principles. We have developed a chromatography electrospray ionisation tandem mass spectrometry (HPLC-ESI-MS/MS) method to characterise the polyphenolic fraction of propolis rapidly and quali-quantitatively. With precursor ion scanning, selective detection of caffeic esters was easily achieved, confirming the identification of prenyl caffeate, benzyl caffeate and phenylethyl caffeate by comparison with synthetic standards. The ionisation and fragmentation behaviour of the major propolis flavonoids was rationalised and applied to selected real samples. Taken together, the results of this study show that the introduction of precursor ion analysis leads to a significant improvement in the characterisation of the phenolic fraction of propolis, paving the way to the establishment of a better quality control for this important natural remedy. Copyright (c) 2007 John Wiley & Sons, Ltd.     

In vitro antioxidant profile of phenolic acid derivatives

Several caffeic acid esters isolated from propolis exhibit interesting antioxidant properties, but their in vivo use is compromised by hydrolysis of the ester bond in the gastrointestinal tract. Therefore, a series of caffeic acid amides were synthesized and their in vitro antioxidant profile was determined. A series of hydroxybenzoic acids, hydroxycinnamic acids, and the synthesized caffeic acid amides were tested for both their 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging and microsomal lipid peroxidation-inhibiting activity. Some of the highly active antioxidants were further tested by means of electron paramagnetic resonance for their hydroxyl radical scavenging activity. Since a promising antioxidant compound should show a lipid peroxidation-inhibiting activity at micromolar level and a low cytotoxicity, the cytotoxicity of the phenolic compounds was also studied. In all the assays used, the caffeic acid anilides and the caffeic acid dopamine amide showed an interesting antioxidant activity.     

In Vitro Antioxidant Profile of Phenolic Acid Derivatives

Several caffeic acid esters isolated from propolis exhibit interesting antioxidant properties, but their in vivo use is compromised by hydrolysis of the ester bond in the gastrointestinal tract. Therefore, a series of caffeic acid amides were synthesized and their in vitro antioxidant profile was determined. A series of hydroxybenzoic acids, hydroxycinnamic acids, and the synthesized caffeic acid amides were tested for both their 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging and microsomal lipid peroxidation-inhibiting activity. Some of the highly active antioxidants were further tested by means of electron paramagnetic resonance for their hydroxyl radical scavenging activity. Since a promising antioxidant compound should show a lipid peroxidation-inhibiting activity at micromolar level and a low cytotoxicity, the cytotoxicity of the phenolic compounds was also studied. In all the assays used, the caffeic acid anilides and the caffeic acid dopamine amide showed an interesting antioxidant activity.     

Distribution of quinic acid derivatives and other phenolic compounds in Brazilian propolis

The quinic acid derivatives (including 4-feruoyl quinic and 5-ferruoyl quinic acids characterized for first time in propolis samples) and other phenolic compounds were quantified in thirteen Brazilian propolis samples by HPLC analysis. For chemometrical analysis, the distribution of quinic acid derivatives and other phenolic compounds were considered. The results suggest that the Brazilian propolis with floral origin from Citrus sp. have the highest concentration of the quinic acid derivatives (between 11.0 to 58.4 mg/mg of the dried crude hydroalcoholic extract) and therefore would probably show a more effective hepatoprotective activity.     

Chemical properties and antioxidant and antimicrobial activities of slovenian propolis

The chemical composition as well as the antioxidant and antimicrobial activities of two EtOH extracts of propolis (PEEs) from Slovenia were determined. EtOH was used as extracting solvent at 70 and 96%, providing the extracts PEE70 and PEE96, respectively. The extraction with 70% EtOH was more efficient than that with 96% EtOH, as the PEE70 was richer in total phenolic compounds than the PEE96. The Slovenian propolis was characterized by different phenolic acids and flavonoids. The PEE96 was slightly richer in three specific compounds, i.e., caffeic acid, ferulic acid, and luteolin, while all other substances detected showed higher contents in the PEE70. The PEE70 showed a stronger reducing power and ability to scavenge free radicals and metal ions than the PEE96. Both PEEs were in the main more effective against Gram-positive bacteria than against fungi and Gram-negative bacteria like Salmonella and Escherichia coli, with the exception of Campylobacter. The PEE96 decreased the intracellular oxidation in Saccharomyces cerevisiae in a dose-dependent manner. The antimicrobial activities and antioxidant properties were related to the total phenolic contents. The two PEEs have the potential for use as natural antimicrobial and antioxidant additives in foods.     

LC-ESI-MS/MS Chemical Characterization,Antioxidant and Antidiabetic Properties of Propolis Extracted with Organic Solvents from Eastern Anatolia Region

Geographic conditions (altitude, climate, and local flora) lead to significant differences in the chemical composition of propolis. Therefore, more research is needed for propolis in different geographical regions. So, the aim of this study was to evaluate the phenolic profile, total phenolic content, antioxidant, and antidiabetic properties of Pülümür propolis from Turkey. Methanol (MeOH), chloroform (CHCl₃), and hexane extracts of propolis were analyzed. LC-ESI-MS/MS analysis of the extracts showed that the most abundant phenolic compound is caffeic acid in the MeOH extract (2943.12±11.12 μg phenolics/g extract), while on the other hand, CHCl₃ extract had the highest total phenolic content (125.75±1.02 mg GAE/g extract). Antioxidant activity was measured using ABTS and DPPH assays, whereas CHCl₃ extract (IC₅₀=6.35±0.11 and 28.84±0.10 μg/mL, respectively) and MeOH extracts (IC₅₀=5.04±0.07 and 28.80±0.09 μg/mL, respectively) showed relatively high antioxidant activity. The MeOH extract showed better antidiabetic activity than the standard compound, acarbose (IC₅₀=0.544 and 0.805 mg/mL, respectively).     

Caffeic acid derivatives in artichoke extract are metabolised to phenolic acids in vivo

The purpose of this study was to investigate the absorption and metabolism of hydroxycinnamates from artichoke extract by determining the urinary excretion of the conjugates. Ten healthy, non smoking volunteers (5 female, 5 male) were given three capsules containing artichoke extract every 4 h (0, 4, 8h) following two days of a low-polyphenol diet. One capsule contained 320 mg of artichoke extract equivalent to 34.3 ± 0.6 mg/g hydroxycinnamates (caffeic acid derivatives) and 5.6 ± 0.1 mg/g flavonoids. Polyphenols and phenolic acids present in the artichoke extract were not detected in the urine either as conjugates or aglycones. However, ferulic, isoferulic, dihydroferulic and vanillic acid were identified as major metabolites after β-glucuronidase treatment of urine. The amount excreted as well as the ratio to that of creatinine, a biomarker for the general excretion rate, increased significantly on the study day compared to the presupplementation day. Thus, the caffeic acid esters found in the artichoke extract capsule are absorbed, metabolised and excreted as methylated phenolic acids such as ferulic, isoferulic, dihydroferulic and vanillic acid.     

Antiproliferative Activity of Chemically Characterized Propolis from Turkey and Its Mechanisms of Action

The aim of this study was to evaluate the ethanolic extract of propolis originated from northern Turkey for its antiproliferative, apoptotic and cell cycle arrest promoting effects on MCF7, HGC27, A549 cancer cell lines and a healthy cell line (HUVEC) in terms of DNA content, morphological features, expression of cell cycle checkpoint proteins p21, p53, Cyclin D1 and immune checkpoint protein PD‐L1. The extract showed moderate antiproliferative activity against all tested cancer cell lines with IC₅₀ values in the range of 58.6–90.7 μg/mL in MTS assay. Further studies indicated that propolis extract exerted apoptotic effect on cancer cell lines, promoted cell cycle arrest through activation of p21 and resulted in accumulation at G0/G1 phase of cancer cells. Propolis treatment caused increased cell size, according to fluorescent imaging except for MCF7. HPTLC analysis revealed that 3‐O‐methylquercetin, chrysin, caffeic acid, CAPE, galangin and pinocembrin were the main components of the extract. The amounts of caffeic acid and CAPE in the extract were found to be 5.5 and 11.1 mg/g, respectively, by a validated HPLC method. Our study is the first one, revealing effect of propolis on PD‐L1 expression on certain cancer cell lines.     

Antimicrobial activity of Brazilian propolis extracts against rumen bacteria in vitro

The antimicrobial activity of three Brazilian propolis extracts was evaluated on bacterial strains representing major rumen functional groups. The extracts were prepared using different concentrations of propolis and alcohol, resulting in different phenolic compositions. The propolis extracts inhibited the growth of Fibrobacter succinogenes S85, Ruminococcus flavefaciens FD-1, Ruminococcus albus 7, Butyrivibrio fibrisolvens D1, Prevotella albensis M384, Peptostreptococcus sp. D1, Clostridium aminophilum F and Streptococcus bovis Pearl11, while R. albus 20, Prevotella bryantii B₁4 and Ruminobacter amylophilus H18 were resistant to all the extracts. The inhibited strains showed also different sensitivity to propolis; the hyper-ammonia-producing bacteria (C. aminophilum F and Peptostreptococcus sp. D1) being the most sensitive. Inhibition of hyper-ammonia-producing bacteria by propolis would be beneficial to the animal. The extract containing the lowest amount of phenolic compounds (LLOS C3) showed the lowest antimicrobial activity against all the bacteria. The major phenolic compounds identified in the propolis extracts (naringenin, chrysin, caffeic acid, p-coumaric acid and Artepillin C) were also evaluated on four sensitive strains. Only naringenin showed inhibitory effect against all strains, suggesting that naringenin is one of the components participating to the antibacterial activity of propolis.     

A New Type of Anatolian Propolis: Evaluation of Its Chemical Composition,Activity Profile and Botanical Origin

This study was undertaken to analyze the phenolic profiles of 19 propolis samples from Turkey by using a high‐performance thin‐layer chromatographic (HPTLC) method in order to identify their plant origins. Furthermore, their antioxidant and antimicrobial activity profiles were comparatively evaluated. For the appraisal of antioxidant potential, total phenolic (TPC) and total flavonoid contents (TFC) of propolis samples were firstly determined and then their effects on free radicals were evaluated by FRAP, ABTS^.+, CUPRAC, DPPH^. and HPTLC‐DPPH^. methods. Antimicrobial activity of propolis samples against Staphylococcus aureus (ATCC 6538), Pseudomonas aeruginosa (ATCC 15442), Escherichia coli (ATCC 11229) and Candida albicans ATCC 10231 were determined by disc diffusion and broth dilution methods. HPTLC fingerprinting analyses revealed that O‐type (botanical origin from Populus nigra L.) was the primarily available propolis type in Turkey. Moreover, 3‐O‐methylquercetin (3MQ) rich propolis was identified as a new propolis type for the first time. Principal component analysis (PCA) indicated that 3MQ‐type propolis differs from the O‐type. Antioxidant activity studies showed that O‐type of propolis possesses higher antioxidant effect than the other tested propolis types. Quercetin, caffeic acid, caffeic acid phenethyl ester (CAPE) and galangin were determined to contribute significantly to the antioxidant potential of O‐type propolis among others. Propolis extracts exerted moderate antimicrobial activity against the tested microorganisms with MIC values between the ranges of 128–512 μg/mL.     

Chemical composition of European propolis: expected and unexpected results

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.     

The effect of propolis and its components on eicosanoid production during the inflammatory response

To investigate the possible mechanism of the therapeutic action of propolis, we studied: (a) the effect of propolis, its components, caffeic acid phenethyl ester (CAPE), caffeic acid (CA), quercetin and naringenin, as well as the synthetic compounds indomethacin (IM) and nordihydroguaiaretic acid (NDGA), and a novel lipoxygenase inhibitor N,N′-dicyclohexyl-O-(3,4-dihydroxycinnamoyl)isourea (DCHCU) on eicosanoid production by mouse peritoneal macrophages in vitro; (b) the effect of IM, NDGA, CA, CAPE, DCHCU and propolis on eicosanoid production during acute inflammation in vivo; and (c) the ex vivo and in vivo effect of dietary propolis on arachidonic acid metabolism. The ethanol extract of propolis suppressed prostaglandin and leukotriene generation by murine peritoneal macrophages in vitro and during zymosan-induced acute peritoneal inflammation in vivo. Dietary propolis significantly suppressed the lipoxygenase pathway of arachidonic acid metabolism during inflammation in vivo. CAPE was the most potent modulator of the arachidonic acid cascade among the propolis components examined.     

Optimisation of an extraction procedure and chemical characterisation of Croatian propolis tinctures

Three spectrophotometric methods for the quantitative determination of different flavonoid groups and total phenolics in Croatian propolis samples were optimised and validated. The assay based on the formation of aluminium chloride complex (with galangin as a standard) was applied to the quantification of flavones and flavonols, while the 2,4-dinitrophenylhydrazine method (with pinocembrine as a reference) was used for the quantification of flavanones. Total phenolic content was measured by the Folin-Ciocalteau method using reference solution of caffeic acid:galangin:pinocembrine (1:1:1). Through analytical validation, the most suitable extraction conditions (with respect to time, temperature and concentration of extraction solvent) were determined, and final conditions for the extraction were established (80% ethanol, 1 h at the room temperature). The appropriate ratio between the mass of raw propolis and the extraction solvent volume was also established. By the application of the optimised method of extraction, 10 propolis tinctures were prepared and subjected to the analysis of general pharmacopoeial parameters, which are fundamental for the creation of quality specification (relative density, dry residue of extract, content of ethanol, methanol and 2-propanol). Additionally, the content of waxes as the main inactive constituents was determined in order to observe the level of their migration from crude propolis to the prepared tinctures.     

Phenolic Acid Composition,Antiatherogenic and Anticancer Potential of Honeys Derived from Various Regions in Greece

The phenolic acid profile of honey depends greatly on its botanical and geographical origin. In this study, we carried out a quantitative analysis of phenolic acids in the ethyl acetate extract of 12 honeys collected from various regions in Greece. Our findings indicate that protocatechuic acid, p-hydroxybenzoic acid, vanillic acid, caffeic acid and p-coumaric acid are the major phenolic acids of the honeys examined. Conifer tree honey (from pine and fir) contained significantly higher concentrations of protocatechuic and caffeic acid (mean: 6640 and 397 µg/kg honey respectively) than thyme and citrus honey (mean of protocatechuic and caffeic acid: 437.6 and 116 µg/kg honey respectively). p-Hydroxybenzoic acid was the dominant compound in thyme honeys (mean: 1252.5 µg/kg honey). We further examined the antioxidant potential (ORAC assay) of the extracts, their ability to influence viability of prostate cancer (PC-3) and breast cancer (MCF-7) cells as well as their lowering effect on TNF- α-induced adhesion molecule expression in endothelial cells (HAEC). ORAC values of Greek honeys ranged from 415 to 2129 µmol Trolox equivalent/kg honey and correlated significantly with their content in protocatechuic acid (p<0.001), p-hydroxybenzoic acid (p<0.01), vanillic acid (p<0.05), caffeic acid (p<0.01), p-coumaric acid (p<0.001) and their total phenolic content (p<0.001). Honey extracts reduced significantly the viability of PC-3 and MCF-7 cells as well as the expression of adhesion molecules in HAEC. Importantly, vanillic acid content correlated significantly with anticancer activity in PC-3 and MCF-7 cells (p<0.01, p<0.05 respectively). Protocatechuic acid, vanillic acid and total phenolic content correlated significantly with the inhibition of VCAM-1 expression (p<0.05, p<0.05 and p<0.01 respectively). In conclusion, Greek honeys are rich in phenolic acids, in particular protocatechuic and p-hydroxybenzoic acid and exhibit significant antioxidant, anticancer and antiatherogenic activities which may be attributed, at least in part, to their phenolic acid content.     

Antibacterial activity and phytochemical evidence for the plant origin of Turkish propolis from different regions

Honeybees collect propolis from practically any abundant plant source in the neighborhood of the hive, be it populus, eucalyptus, pine, sugarcane, cashew nut or orange trees. We have described that the origin plants of Turkish propolis are Populus sp., Eucalyptus sp. and Castanea sativa. In our previous study, propolis samples from Middle Anatolia displayed the typical pattern of “poplar” propolis: they contained pinobanksin, caffeic and ferulic acids and their esters. The propolis samples examined in this study were shown not to contain polar phenolics. The main components of Eucalyptus propolis were aromatic acids, mainly cinnamic acid and its esters, that are usually found in Eucalyptus species resins. The second distinct sample originated from West Anatolia. Although it contained low amounts of phenolic substances and aromatic acids, its main components were sugars and glycosides. The study revealed that there was no significant difference between propolis samples in antibacterial activity, however the yeasts were shown to be more sensitive to eucalyptus-propolis. Gram negative bacteria were susceptible to none of the samples tested.