Gaps in Propolis Research: Challenges Posed to Commercialisation and the Need for an Holistic Approach

YesBoth the season and region in which propolis is collected influence its chemical composition, resulting in variations in biological activity. Significant differences in composition and concentration of certain chemical compounds in propolis make standardisation and quality control challenging. In addition, the lack of uniformity in evaluation methodology and analytical techniques, make it extremely difficult to correlate data across the climatic zones. In this report, we focus on the gaps in propolis research and the challenges they pose for commercialisation, with suggestions as to how we might address them. We hope to stimulate further research which explores the holistic nature of propolis in order to derive a propolis bioactivity standard

Temperate propolis has anti-inflammatory effects and is a potent inhibitor of nitric oxide formation in macrophages

Previous research has shown that propolis has immunomodulatory activity. Extracts from two UK propolis samples were assessed for their anti-inflammatory activities by investigating their ability to alter the production of the cytokines: tumour necrosis factor-ff (TNF-ff), interleukin-1 (IL-1), IL-6, and IL-10 from mouse bone marrow-derived macrophages co-stimulated with lipopolysaccharide (LPS). The propolis extracts suppressed the secretion of IL-1 and IL-6 with less effect on TNFff. In addition, propolis reduced the levels of nitric oxide formed by LPS-stimulated macrophages. Metabolomic profiling was carried out by liquid chromatography (LC) coupled with mass spectrometry (MS) on a ZIC-pHILIC column. LPS increased the levels of intermediates involved in nitric oxide biosynthesis; propolis lowered many of these. In addition, LPS produced an increase in itaconate and citrate, and propolis treatment increased itaconate still further while greatly reducing citrate levels. Moreover, LPS treatment increased levels of glutathione (GSH) and intermediates in its biosynthesis, while propolis treatment boosted these still further. In addition, propolis treatment greatly increased levels of uridine diphosphate (UDP)-sugar conjugates. Overall, the results showed that propolis extracts exert an anti-inflammatory effect by the inhibition of pro-inflammatory cytokines and by the metabolic reprogramming of LPS activity in macrophages

Determination of Phenolic Compounds in Various Propolis Samples Collected from an African and an Asian Region and Their Impact on Antioxidant and Antibacterial Activities

The biological activities of propolis samples are the result of many bioactive compounds present in the propolis. The aim of the present study was to determine the various chemical compounds of some selected propolis samples collected from Palestine and Morocco by the High-Performance Liquid Chromatography–Photodiode Array Detection (HPLC-PDA) method, as well as the antioxidant and antibacterial activities of this bee product. The chemical analysis of propolis samples by HPLC-PDA shows the cinnamic acid content in the Palestinian sample is higher compared to that in Moroccan propolis. The results of antioxidant activity demonstrated an important free radical scavenging activity (2,2-Diphenyl-1-picrylhydrazyl (DPPH); 2,2′-azino-bis 3-ethylbenzothiazoline-6-sulphonic acid (ABTS) and reducing power assays) with EC50 values ranging between 0.02 ± 0.001 and 0.14 ± 0.01 mg/mL. Additionally, all tested propolis samples possessed a moderate antibacterial activity against bacterial strains. Notably, Minimum Inhibitory Concentrations (MICs) values ranged from 0.31 to 2.50 mg/mL for Gram-negative bacterial strains and from 0.09 to 0.125 mg/mL for Gram-positive bacterial strains. The S2 sample from Morocco and the S4 sample from Palestine had the highest content of polyphenol level. Thus, the strong antioxidant and antibacterial properties were apparently due to the high total phenolic and flavone/flavonol contents in the samples. As a conclusion, the activities of propolis samples collected from both countries are similar, while the cinnamic acid in the Palestinian samples was more than that of the Moroccan samples

Determination of Phenolic Compounds in Various Propolis Samples Collected from an African and an Asian Region and Their Impact on Antioxidant and Antibacterial Activities

The biological activities of propolis samples are the result of many bioactive compounds present in the propolis. The aim of the present study was to determine the various chemical compounds of some selected propolis samples collected from Palestine and Morocco by the High-Performance Liquid Chromatography–Photodiode Array Detection (HPLC-PDA) method, as well as the antioxidant and antibacterial activities of this bee product. The chemical analysis of propolis samples by HPLC-PDA shows the cinnamic acid content in the Palestinian sample is higher compared to that in Moroccan propolis. The results of antioxidant activity demonstrated an important free radical scavenging activity (2,2-Diphenyl-1-picrylhydrazyl (DPPH); 2,2′-azino-bis 3-ethylbenzothiazoline-6-sulphonic acid (ABTS) and reducing power assays) with EC50 values ranging between 0.02 ± 0.001 and 0.14 ± 0.01 mg/mL. Additionally, all tested propolis samples possessed a moderate antibacterial activity against bacterial strains. Notably, Minimum Inhibitory Concentrations (MICs) values ranged from 0.31 to 2.50 mg/mL for Gram-negative bacterial strains and from 0.09 to 0.125 mg/mL for Gram-positive bacterial strains. The S2 sample from Morocco and the S4 sample from Palestine had the highest content of polyphenol level. Thus, the strong antioxidant and antibacterial properties were apparently due to the high total phenolic and flavone/flavonol contents in the samples. As a conclusion, the activities of propolis samples collected from both countries are similar, while the cinnamic acid in the Palestinian samples was more than that of the Moroccan samples

Propolis exerts an anti-inflammatory effect on PMA-differentiated THP-1 cells via inhibition of purine nucleoside phosphorylase

Previous research has shown that propolis has immunomodulatory activity. Propolis extracts from different geographic origins were assessed for their anti-inflammatory activities by investigating their ability to alter the production of tumour necrosis factor-α (TNF-α) and the cytokines interleukin-1β (IL-1β), IL-6 and IL-10 in THP-1-derived macrophage cells co-stimulated with lipopolysaccharide (LPS). All the propolis extracts suppressed the TNF-α and IL-6 LPS-stimulated levels. Similar suppression effects were detected for IL-1β, but the release of this cytokine was synergised by propolis samples from Ghana and Indonesia when compared with LPS. Overall, the Cameroonian propolis extract (P-C) was the most active and this was evaluated for its effects on the metabolic profile of unstimulated macrophages or macrophages activated by LPS. The levels of 81 polar metabolites were identified by liquid chromatography (LC) coupled with mass spectrometry (MS) on a ZIC-pHILIC column. LPS altered the energy, amino acid and nucleotide metabolism in THP-1 cells, and interpretation of the metabolic pathways showed that P-C reversed some of the effects of LPS. Overall, the results showed that propolis extracts exert an anti-inflammatory effect by inhibition of pro-inflammatory cytokines and by metabolic reprogramming of LPS activity in macrophage cells, suggesting an immunomodulatory effect

Nucleotide-binding sites can enhance N-acylation of nearby protein lysine residues.

Acyl-CoAs are reactive metabolites that can non-enzymatically S-acylate and N-acylate protein cysteine and lysine residues, respectively. N-acylation is irreversible and enhanced if a nearby cysteine residue undergoes an initial reversible S-acylation, as proximity leads to rapid S → N-transfer of the acyl moiety. We reasoned that protein-bound acyl-CoA could also facilitate S → N-transfer of acyl groups to proximal lysine residues. Furthermore, as CoA contains an ADP backbone this may extend beyond CoA-binding sites and include abundant Rossmann-fold motifs that bind the ADP moiety of NADH, NADPH, FADH and ATP. Here, we show that excess nucleotides decrease protein lysine N-acetylation in vitro. Furthermore, by generating modelled structures of proteins N-acetylated in mouse liver, we show that proximity to a nucleotide-binding site increases the risk of N-acetylation and identify where nucleotide binding could enhance N-acylation in vivo. Finally, using glutamate dehydrogenase as a case study, we observe increased in vitro lysine N-malonylation by malonyl-CoA near nucleotide-binding sites which overlaps with in vivo N-acetylation and N-succinylation. Furthermore, excess NADPH, GTP and ADP greatly diminish N-malonylation near their nucleotide-binding sites, but not at distant lysine residues. Thus, lysine N-acylation by acyl-CoAs is enhanced by nucleotide-binding sites and may contribute to higher stoichiometry protein N-acylation in vivo

Novel flavanones with anti-trypanosomal activity isolated from Zambian and Tanzanian propolis samples

A bioassay-guided phytochemical investigation of propolis samples from Tanzania and Zambia that screened for activity against Trypanosoma brucei has led to the isolation of two novel flavanones with promising antitrypanosomal activity. The compounds were characterized based on their spectral and physical data and identified as 6-(1,1-dimethylallyl) pinocembrin and 5-hydroxy-4″,4″-dimethyl-5″-methyl-5″-H-dihydrofuranol [2″,3″,6,7] flavanone. The two compounds, together with the propolis extracts and fractions, were assayed against a standard drug-sensitive strain of T. b. brucei (s427 wild-type), multi-drug resistant-resistant T. b. brucei (B48), drug-sensitive T. congolense (1L300) and a derived diminazene-resistant T. congolense strain (6C3), and for toxicity against U947 human cells and RAW 246.7 murine cells. Activity against T. b. brucei was higher than against T. congolense. Interestingly, the Tanzanian propolis extract was found to be more active than its fractions and purified compounds in these assays, with an IC of 1.20 μg/mL against T. b. brucei. The results of a cytotoxicity assay showed that the propolis extracts were less toxic than the purified compounds with mean IC values > 165.0 μg/mL

Using exomarkers to assess mitochondrial reactive species in vivo

Background: The ability to measure the concentrations of small damaging and signalling molecules such as reactive oxygen species (ROS) in vivo is essential to understanding their biological roles. While a range of methods can be applied to in vitro systems, measuring the levels and relative changes in reactive species in vivo is challenging. Scope of review: One approach towards achieving this goal is the use of exomarkers. In this, exogenous probe compounds are administered to the intact organism and are then transformed by the reactive molecules in vivo to produce a diagnostic exomarker. The exomarker and the precursor probe can be analysed ex vivo to infer the identity and amounts of the reactive species present in vivo. This is akin to the measurement of biomarkers produced by the interaction of reactive species with endogenous biomolecules. Major conclusions and general significance: Our laboratories have developed mitochondria-targeted probes that generate exomarkers that can be analysed ex vivo by mass spectrometry to assess levels of reactive species within mitochondria in vivo. We have used one of these compounds, MitoB, to infer the levels of mitochondrial hydrogen peroxide within flies and mice. Here we describe the development of MitoB and expand on this example to discuss how better probes and exomarkers can be developed. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn. Abbreviations: EPR, electron paramagnetic resonance; GFP, green fluorescent protein; 4-HNE, 4-hydroxynonenal; MitoB, 3-(dihydroxyboronyl)benzyltriphenylphosphonium bromide; MitoP, (3-hydroxybenzyl)triphenylphosphonium bromide; ROS, reactive oxygen species; SOD, superoxide dismutase; TPMP, methyltriphenylphosphonium; TPP, triphenylphosphonium catio

Activity of Compounds from Temperate Propolis against Trypanosoma brucei and Leishmania mexicana

Ethanolic extracts of samples of temperate zone propolis, four from the UK and one from Poland, were tested against three strains and displayed EC values < 20 µg/mL. The extracts were fractionated, from which 12 compounds and one two-component mixture were isolated, and characterized by NMR and high-resolution mass spectrometry, as 3-acetoxypinobanksin, tectochrysin, kaempferol, pinocembrin, 4'-methoxykaempferol, galangin, chrysin, apigenin, pinostrobin, cinnamic acid, coumaric acid, cinnamyl ester/coumaric acid benzyl ester (mixture), 4',7-dimethoxykaempferol, and naringenin 4',7-dimethyl ether. The isolated compounds were tested against drug-sensitive and drug-resistant strains of and , with the highest activities ≤ 15 µM. The most active compounds against were naringenin 4',7 dimethyl ether and 4'methoxy kaempferol with activity of 15-20 µM against the three strains. The most active compounds against were 4',7-dimethoxykaempferol and the coumaric acid ester mixture, with EC values of 12.9 ± 3.7 µM and 13.1 ± 1.0 µM. No loss of activity was found with the diamidine- and arsenical-resistant or phenanthridine-resistant strains, or the miltefosine-resistant strain; no clear structure activity relationship was observed for the isolated compounds. Temperate propolis yields multiple compounds with anti-kinetoplastid activity