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 Trypanosoma brucei strains and displayed EC50 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 T. brucei and Leishmania mexicana, with the highest activities ≤ 15 µM. The most active compounds against T. brucei were naringenin 4',7 dimethyl ether and 4'methoxy kaempferol with activity of 15-20 µM against the three T. brucei strains. The most active compounds against L. mexicana were 4',7-dimethoxykaempferol and the coumaric acid ester mixture, with EC50 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 T. brucei strains, or the miltefosine-resistant L. mexicana strain; no clear structure activity relationship was observed for the isolated compounds. Temperate propolis yields multiple compounds with anti-kinetoplastid activity.

The Strong Anti-Kinetoplastid Properties of Bee Propolis: Composition and Identification of the Active Agents and Their Biochemical Targets.

The kinetoplastids are protozoa characterized by the presence of a distinctive organelle, called the kinetoplast, which contains a large amount of DNA (kinetoplast DNA (kDNA)) inside their single mitochondrion. Kinetoplastids of medical and veterinary importance include Trypanosoma spp. (the causative agents of human and animal African Trypanosomiasis and of Chagas disease) and Leishmania spp. (the causative agents of the various forms of leishmaniasis). These neglected diseases affect millions of people across the globe, but drug treatment is hampered by the challenges of toxicity and drug resistance, among others. Propolis (a natural product made by bees) and compounds isolated from it are now being investigated as novel treatments of kinetoplastid infections. The anti-kinetoplastid efficacy of propolis is probably a consequence of its reported activity against kinetoplastid parasites of bees. This article presents a review of the reported anti-kinetoplastid potential of propolis, highlighting its anti-kinetoplastid activity in vitro and in vivo regardless of geographical origin. The mode of action of propolis depends on the organism it is acting on and includes growth inhibition, immunomodulation, macrophage activation, perturbation of the cell membrane architecture, phospholipid disturbances, and mitochondrial targets. This gives ample scope for further investigations toward the rational development of sustainable anti-kinetoplastid drugs.

Isolation of a Novel Flavanonol and an Alkylresorcinol with Highly Potent Anti-Trypanosomal Activity from Libyan propolis.

Twelve propolis samples from different parts of Libya were investigated for their phytochemical constituents. Ethanol extracts of the samples and some purified compounds were tested against Trypanosoma brucei, Plasmodium falciparum and against two helminth species, Trichinella spiralis and Caenorhabditis elegans, showing various degrees of activity. Fourteen compounds were isolated from the propolis samples, including a novel compound Taxifolin-3-acetyl-4'-methyl ether (4), a flavanonol derivative. The crude extracts showed moderate activity against T. spiralis and C. elegans, while the purified compounds had low activity against P. falciparum. Anti-trypanosomal activity (EC50 = 0.7 µg/mL) was exhibited by a fraction containing a cardol identified as bilobol (10) and this fraction had no effect on Human Foreskin Fibroblasts (HFF), even at 2.0 mg/mL, thus demonstrating excellent selectivity. A metabolomics study was used to explore the mechanism of action of the fraction and it revealed significant disturbances in trypanosomal phospholipid metabolism, especially the formation of choline phospholipids. We conclude that a potent and highly selective new trypanocide may be present in the fraction.

New Sulphated Flavonoids from Wissadula periplocifolia (L.) C. Presl (Malvaceae).

Wissadula periplocifolia (L.) C. Presl (Malvaceae) is commonly used in Brazil to treat bee stings and as an antiseptic. The antioxidant properties of its extracts have been previously demonstrated, thus justifying a phytochemical investigation for its bioactive phenolic constituents. This has yielded five new sulphated flavonoids: 8-O-sulphate isoscutellarein (yannin) (1a); 4'-O-methyl-7-O-sulphate isoscutellarein (beltraonin) (1b); 7-O-sulphate acacetin (wissadulin) (2a); 4'-O-methyl-8-O-sulphate isoscutellarein (caicoine) (2b) and 3'-O-methyl-8-O-sulphate hypolaetin (pedroin) (3b) along with the known flavonoids 7,4'-di-O-methyl-8-O-sulphate isoscutellarein (4), acacetin, apigenin, isoscutellarein, 4'-O-methyl isoscutellarein, 7,4'-di-O-methylisoscutellarein, astragalin and tiliroside. The compounds were isolated by column chromatography and identified by NMR (¹H, (13)C, HMQC, HMBC and COSY) and LC-HRMS. A cell based assay was carried out to evaluate the preliminary cytotoxic properties of the flavonoids against UVW glioma and PC-3M prostate cancer cells as well as non-tumour cell lines. The obtained results showed that acacetin, tiliroside, a mixture of acacetin + apigenin and the sulphated flavonoids 2a + 2b exhibited inhibitory activity against at least one of the cell lines tested. Among the tested flavonoids acacetin and tiliroside showed lower IC50 values, presenting promising antitumor effects.

The isolation of antiprotozoal compounds from Libyan propolis.

Propolis is increasingly being explored as a source of biologically active compounds. Until now, there has been no study of Libyan propolis. Two samples were collected in North East Libya and tested for their activity against Trypanosoma brucei. Extracts from both samples had quite high activity. One of the samples was fractionated and yielded a number of active fractions. Three of the active fractions contained single compounds, which were found to be 13-epitorulosal, acetyl-13-epi-cupressic acid and 13-epi-cupressic acid, which have been described before in Mediterranean propolis. Two of the compounds had a minimum inhibitory concentration value of 1.56 µg/mL against T. brucei. The active fractions were also tested against macrophages infected with Leishmania donovani, and again moderate to strong activity was observed with the compounds having IC50 values in the range 5.1-21.9 µg/mL.

Phenolic constituents from Wissadula periplocifolia (L.) C. Presl. and anti-inflammatory activity of 7,4'-di-O-methylisoscutellarein.

This study reports the first phenolics from Wissadula genus (Malvaceae) and the anti-inflammatory activity of 7,4'-di-O-methylisoscutellarein. Using chromatographic methods, five phenolic compounds were isolated from aerial parts of Wissadula periplocifolia (L.) C. Presl. The compounds were identified as 4-hydroxybenzoic acid, 3-hydroxybenzoic acid, trans-cinnamic acid, tamgermanetin and 7,4'-di-O-methylisoscutellarein using spectroscopic methods. The flavone 7,4'-di-O-methylisoscutellarein showed anti-inflammatory activity by inhibiting neutrophils recruitment in a mice model of pleurisy and by decreasing significantly the production of cytokines IL-1ß and TNF-α.

Chemical and Antimicrobial Profiling of Propolis from Different Regions within Libya.

Extracts from twelve samples of propolis collected from different regions of Libya were tested for their activity against Trypanosoma brucei, Leishmania donovani, Plasmodium falciparum, Crithidia fasciculata and Mycobacterium marinum and the cytotoxicity of the extracts was tested against mammalian cells. All the extracts were active to some degree against all of the protozoa and the mycobacterium, exhibiting a range of EC50 values between 1.65 and 53.6 µg/ml. The toxicity against mammalian cell lines was only moderate; the most active extract against the protozoan species, P2, displayed an IC50 value of 53.2 µg/ml. The extracts were profiled by using liquid chromatography coupled to high resolution mass spectrometry. The data sets were extracted using m/z Mine and the accurate masses of the features extracted were searched against the Dictionary of Natural Products (DNP). A principal component analysis (PCA) model was constructed which, in combination with hierarchical cluster analysis (HCA), divided the samples into five groups. The outlying groups had different sets of dominant compounds in the extracts, which could be characterised by their elemental composition. Orthogonal partial least squares (OPLS) analysis was used to link the activity of each extract against the different micro-organisms to particular components in the extracts.

Chromatographic analysis with different detectors in the chemical characterisation and dereplication of African propolis.

Propolis or bee glue has very diverse composition and is potentially a source of biologically active compounds. Comprehensive chemical profiling was performed on 22 African propolis samples collected from the sub-Saharan region of Africa by using various hyphenated analytical techniques including Liquid Chromatography (LC)-UltraViolet Detection (UV)-Evaporative Light Scattering Detection (ELSD), LC-High Resolution Mass Spectrometry (HRMS), Gas Chromatography (GC)-MS and LC-Diode Array Detector (DAD)-HRMS/MS. The diversity of the composition of these African propolis samples could be observed by heat mapping the LC-UV and ELSD data. The characteristic chemical components were uncovered by applying Principal Component Analysis (PCA) to the LC-HRMS data and a preliminary dereplication was carried out by searching their accurate masses in the Dictionary of Natural Products (DNP). A further identification was achieved by comparing their GC-MS or LC-DAD-HRMS/MS spectra with previously published data. Generally no clear geographic delineation was observed in the classification of these African propolis samples. Triterpenoids were found as the major chemical components in more than half of the propolis samples analysed in this study and some others were classified as temperate and Eastern Mediterranean type of propolis. Based on the comparative chemical profiling and dereplication studies one uncommon propolis from southern Nigeria stood out from others by presenting prenylated isoflavonoids, which indicated that it was more like Brazilian red propolis, and more significantly a high abundance of stilbenoid compounds which could be novel in propolis.