Introduction

Recently, dietetics are interested in searching for functional foods with both nutritional and medicinal value and beneficial to human health1. Bee products including bee propolis, pollen, and bread are considered as potential super-foods owing to their nutritional value and myriad of health benefits1. Bee propolis is a natural gummy, and resinous substance which comprises a myriad of secondary metabolites with potential biological importance2. Honey-bees harvest resins from various plant species and when mixed with bees wax in beehives produce propolis3. Owing to its antimicrobial properties, bee propolis is formed by the honeybees during sterilization of beehives to ensure a healthy hive environment for the bee colony4. The variation in phytochemical constituents of propolis depend on several factors including it’s the bee species, geographical, and botanical source5. Secondary metabolites identified in bee propolis belonged to several phytochemical classes including flavonoids, terpenes, phenolics and their esters, sugars, hydrocarbons and mineral elements5. Triterpenes such as isomangiferolic acid, 27-hydoxymangiferonic acid, and 27-hydroxyisomangiferolic acid were identified in Tetragonula biroi propolis4. Phenolic compounds including fisetinidol and ferulic acid were isolated from Saudi propolis2. Propolis is beneficial to human health and used to treat several ailments including colds, wounds, ulcers, rheumatism, sprains, and dental caries6. Moreover, several biological activities were reported for bee propolis such as anti-inflammatory, immunomodulatory7, antimicrobial, antioxidant8, antitumor9, antiulcer10 and anti-diabetic activities11. Recently, metabolomics tools including HPLC and NMR-based are widely used for profiling of non-volatile metabolites in natural products12. The recent study aims to profiling the chemical composition of propolis samples from Nigeria using HPLC–UV–ELSD. Moreover, isolation, purification, and structure elucidation of the major compounds in Nigerian propolis was performed using different chromatographic techniques. Additionally, screening of the antitrypanosomal and cytotoxic activity was done against Trypanosoma brucei.

Results and discussion

Phytochemical profiling of red Nigerian propolis extract

The chemical profiling of red Nigerian propolis extract was carried out using many instrumental methods including high performance liquid chromatography (HPLC) coupled to different detectors such as an evaporative light scattering detector (ELSD) Figure S1A, ultraviolet detection (UV), and high-resolution mass spectrometry (HRMS) and by NMR-based tool. HPLC–UV-ELSD of the crude sample showed clearly that it contained mostly compounds with UV-absorbing activity, that could be flavonoids and phenolic compounds. Compounds without chromophores like terpenoids were detected but with low intensities (Supplementary Fig. S1A). NMR was applied to 10 mg of this extract to get more insights about the nature of the constituents (Supplementary Fig. S1B) and the LC–MS method was used for chemical profiling (Supplementary Table S1). The main constituents highlighted by 1H NMR spectrum were flavonoids and phenolics, while terpenoids and fatty acids were detected at much lesser intensity compared to flavonoids and phenolics. The crude extract was observed to contain aromatic compounds, this was shown by several signals from at the region from 6 to 8 ppm as well as phenolic hydroxyl group between 10 and 13 ppm. Considerable complexity was displayed by the LC–MS chromatogram of the crude sample, with numerous peaks that were more or less intense. As indicated in Supplementary Table S1, the extract largely consisted of flavonoids and phenolics, according to the results of LC–MS analysis and as confirmed by the 1H NMR spectra (Supplementary Fig. S1B) where there were many peaks in the aromatic region. 13 compounds were identified belonging to flavonoids, isoflavone, and phenolics (Supplementary Table S1). 6 Flavonoid peaks were annotated by comparing their MS data with the previous literature and identified as luteolinidin13, kaempferide14, malvidin15, ponciretin, hesperetin16, and liquiritigenin17. Additionally, 6 isoflavonoid compounds were identified including rosinidin18, calycosin17, medicarpin, formononetin, 7,4′-dihydroxyflavylium, and isosativan. Moreover, one phenolic compound was annotated as phloretin in addition to 15 unknown phenolic compounds.

Isolation and characterization of red Nigerian propolis isolated compounds

The ethanolic extract of red Nigerian propolis was subjected to column chromatography and elution was sequentially performed based on a gradient profile. The performance of LC–MS permitted identification of the different components and allowed combination of fractions to afford 9 fractions (Supplementary Table S2). The process of chemical analysis relies greatly on the mass of the fraction obtained following initial separation. Therefore, additional chromatographic separation could be conducted if the chemical component was available in sufficient quantity. LC–MS and HPLC–UV-ELSD analysis were conducted on fraction RN-6 revealed a rich content of compounds with varied compositions, as shown in Table 1 and Supplementary Fig. S2A,B. Based on preliminary data, the compounds were most likely isoflavonoid derivatives. Therefore, 545 mg of fraction RN-6 from CC (Supplementary Table S2) was subjected to size exclusion chromatography (SEC), yielding 38 sub-fractions (RN-6-1 to RN-6-38), which led to the acquisition of two isolated compounds from compound 1 and Compound 2.

Table 1 The most abundant components in RN-6 when analyzed by reversed phase LC–MS in negative ion mode.
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Characterization of compound 1

Compound 1 was obtained as dark orange powder isolated by using column chromatography and then SEC from the ethanolic extract of red Nigerian propolis. Compound 1 appeared as a purple spot-on TLC with Rf of 0.41 after elution with the mobile phase 20% HE in EtOAc and spraying with p-anisaldehyde-sulphuric acid reagent and exposure to heat. A molecular formula of C17H17O4- was derived from the fact that the molecular ion [M−H]− was indicated by the negative mode HRESI-MS spectrum at m/z 285.11 (Fig. 1), and the optical rotation had a value of + 5° (c = 0.1).

Fig. 1
figure 1

(A) Extracted ion chromatogram corresponding to the mass of isosativan in the negative ion mode (-ve ESI) (B) The spectrum corresponding to isosativan mass peak.

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The compound spectral data in Supplementary Figs. S3, S4 and Table 2 showed two similar sets of aromatic protons for two trisubstituted benzene rings at δ (ppm) 6.45 (d, J = 2.87), 6.50 (dd, J = 8.44, 2.48) and 7.03(d, J = 8.48) for one aromatic ring while the other aromatic ring protons appeared at 6.33, 6.42 and 7.01 ppm. Two oxymethylene protons, one methine and two methylene protons were observed at 4.37 (dd, J = 3.4, 1.8), 4.07 (d, J = 10.1), 3.53–3.55 (m), 3.01 (dd, J = 15.8, 10.3) and 2.94 (dd, J = 5.3, 1.8). The compound must have a saturated ring A of a flavonoid type of compound and the pattern is typical of a C-3 substituted isoflavan moiety. The J modulated 13C spectrum (Supplementary Fig. S5) gave 17 signals made up of six aromatic CH, two aliphatic CH2 and one CH, two methoxy and six quaternary carbons (including four phenolic ones). Analysis of it 2D spectra such as COSY (Supplementary Fig. S6), HSQC (Supplementary Fig. S7) and HMBC (Supplementary Fig. S8) correlations illustrated in Fig. 2 indicated the compound to be isosativan (Fig. 3) and the structure was confirmed by literature reports19.

Table 2 1H (400 MHz) and 13C (100 MHz) NMR data of isosativan in CDCl3.
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Fig. 2
figure 2

HSQC major correlations of isosativan, the black arrows show the correlations corresponding HSQC correlations.

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Fig. 3
figure 3

Structure of isosativan.

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Characterization of compound 2

Compound 2 was obtained as dark orange powder purified by using column chromatography and then SEC from the ethanolic extract of red Nigerian propolis. Compound 2 appeared as a purple spot-on TLC with Rf of 0.45 after elution with the mobile phase 20% HE in EtOAc and spraying with p-anisaldehyde-sulphuric acid reagent and exposure to heat. A molecular formula of C16H13O4- was derived from the fact that the deprotonated ion [M−H]− was indicated by the negative mode HRESI-MS spectrum at m/z 269.08 (Supplementary Fig. S9), and the optical rotation had a value of + 23° (c = 0.1, MeOH).

1H NMR (400 MHz) spectrum (Supplementary Fig. S10, Table 3) of the compound showed signals for two oxymethylene protons at δ (ppm) 4.25 (ddd, J = 10.9, 4.9, 0.7) and 3.64 (t, J = 10.9), one methine proton at 3.54–3.56 (m) and an oxymethine proton at 5.51 (d, J = 6.8). The proton spectrum of the compound showed two sets of aromatic ABX spin systems and that confirmed the presence of two trisubstituted benzene rings. The first set were at δH ppm 7.39 (d, J = 0.6), 6.58 (dd, J = 8.4, 2.5) and 6.44 (d, J = 2.5). The second set of the aromatic ABX protons were at 7.16 (d, J = 0.6), 6.46 (dd, J = 2.3, 0.6) and 6.48 (d, J = 2.3). From the J modulated 13C spectrum (Supplementary Fig. S11), 16 carbon signals were identified and were made up of six aromatic CH, one aliphatic CH2, two aliphatic CH, one methoxy and six quaternary carbons (including four phenolic ones). Analysis of its 2D spectra COSY (Supplementary Fig. S12), HSQC (Supplementary Fig. S13) and HMBC (Supplementary Fig. S14–15) indicated the compound to be medicarpin (Fig. 4) and the structure was confirmed by literature reports20.

Table 3 1H (400 MHz) and 13C (100 MHz) NMR data of medicarpin in CDCl3.
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Fig. 4
figure 4

Structure of medicarpin.

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In vitro antitrypanosomal activity against trypanosome (T. brucei S427 strain)

Crude, fractions and the isolated compounds (isosativan and medicarpin) isolated from red Nigerian propolis were tested against T. brucei. Pentamidine and diminazen were used as drug controls and their MIC were scored 0.0030 and 0.0313 µg/ml respectively. The results from testing the above samples against T. brucei were listed in Table 4. The results showed a varying activity against T. brucei between tested samples. Red Nigerian crude extract showed a higher activity at 6.5 µg/ml, than the fraction which has activity of 8.0 µg/ml. The obtained isolated compounds, medicarpin and isosativan, showed moderate inhibitory activity with an MIC of 7.6 µg/ml and 12.1 µg/ml respectively. Overall, crude, fractions and isolated compounds are moderately active against T. brucei S427 WT (Table 4).

Table 4 Drug sensitivity assay of red Nigerian propolis sample and its fractions on T. brucei S427 WT.
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In vitro cytotoxicity on U937 cells

Crude extract, fractions, and the isolated compounds (isosativan and medicarpin) extracted from the red Nigerian propolis sample were tested against leukemia cell line U937 cells. Pentamidine and Diminazen were used as drug controls at their IC50 values of 13.32 μg/mL and 29.58 μg/mL, respectively. The results from testing red Nigerian propolis extract and its components against leukemia cell line U937 cells were listed in Table 5. Results revealed that only RN-6 fraction showed a similar toxicity result to diminazen (as its minimum IC50 value > 26 µg/ml). Table 5 shows detailed IC50 values for the tested samples.

Table 5 Cytotoxicity assay of red Nigerian propolis sample and its fractions on U937 cells.
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Discussion

The variability in the chemical composition of propolis is significant, even in the case of propolis from the same geographical area. However, besides chemical composition, biological properties are also highly important in assessing the quality of propolis and thus in standardizing propolis samples. Propolis has been reported to consist of over 300 compounds, but not all compounds are related to biological effects. Under these circumstances, achievement of standardization of propolis samples from different geographical areas with different biological effects has not been possible so far. To link a specific chemical type of propolis to a specific biological effect, additional study is required in order to accomplish standardization of propolis types5.

Recently, ample research has been carried out on propolis derived from different geographical regions, including Europe, Asia, North America and South America, particularly Brazil21,22. In the case of Asia, comprehensive investigations23 have focused on propolis from China24, Japan25, Taiwan26, Nepal5 and Myanmar27. Given the considerable complexity of the mixture produced by the various compounds present in propolis crude extracts, it is improbable to acquire a single compound from the crude extract through with just one separation method2. Consequently, the crude extract frequently must be fractionated into several different fractions with polarities or molecular sizes that do not differ much. However, attention must be paid to the fact that detection of fractions with the compound in low concentration or detection of activity in bioassays in the case of bioassay-based isolation processes may fail if the number of fractions produced is too high, as this will result in the spreading of the compound in question across too many fractions28.

In the present study, ethanol was selected as the solvent for the extraction of different quantities of raw propolis samples from Nigeria owing to its safety for infused edibles and easily recovered and was used previously for propolis extraction4. LC–MS analysis of red Nigerian propolis extract revealed the identification of 13 compounds belonging to flavonoids, isoflavone, and phenolics. Among the identified compounds, 6 flavonoid peaks were annotated by comparing their MS data with the previous literature and identified as luteolinidin13, kaempferide14, malvidin15, ponciretin, hesperetin16, and liquiritigenin17. Additionally, 6 isoflavonoid compounds were identified including rosinidin18, calycosin17, medicarpin20, formononetin, 7,4′-dihydroxyflavylium, and isosativan20. Moreover, one phenolic compound was annotated as phloretin in addition to 15 unknown phenolic compounds.

CC and SEC were subsequently conducted for fractionation and purification of the phytochemicals of those samples. The fractionation of the ethanol extract of red Nigerian propolis, using CC followed by SEC, led to the isolation of two phenolic compounds belonging to isoflavone group identified as isosativan and medicarpin. Both compounds have been isolated from red Brazilian propolis and isosativan was previously identified in Nigerian propolis29,30 and were tested for their antibacterial and radical scavenging activity against DPPH radicals. Isosativan and medicarpin were previously reported as important antimicrobial components of red propolis, especially concerning activity against C. albicans29 and medicarpin revealed a significant antigonococcal Activity31. Such compounds belonging to pterocarpans which are well known for their antifungal activity and play a defensive role in many plants due to this activity32. Such antimicrobial and antifungal activity of isosativan and medicarpin helped in testing their effects and in comparison, with the crude extract and fraction against Trypanosoma brucei. Isolation of these compounds further supports the fact that bees target the same plants for propolis collection in the tropical regions of Brazil and Nigeria. Red propolis from Nigeria was also found to contain these compounds33 and they exhibited moderate effects against Trypanosoma brucei. The moderate activity as well has been seen upon testing activities of our Red Nigerian propolis sample against trypanosome T. brucei S427 strain, Table 4, which revealed that red Nigerian crude has a higher activity with MIC of 6.5 µg/ml, than one of fraction which was 8.0 µg/ml. Such higher activity of the crude extract may be due to its richness with several bioactive compounds including flavonoids, isofalvonoids, and phenolics. However, it showed a high level of cytotoxicity against mammalian cells, Table 5, which may explain these inhibitory effects. Moreover, the fraction RN-6 fraction revealed the higher cytotoxic effect against leukemia cell line with IC50 of 26.5 µg/ml compared to the standard diminazen with IC50 of 29.5 µg/ml. Such cytotoxic activity of the fraction is due to its richness with isoflavonoids. The abundance of isoflavonoids in Nigerian propolis, which are not found in many plant species, being limited to legumes (Leguminosae family)29, alongside isoflavans like medicarpin. In accordance with other previously reported studies, Piccinelli et al. (2005) reported that red propolis from Cuba produced mainly from the resin of Dalbergia ecastophyllum, contained the isoflavonoid medicarpin as well20. Several limitations should be considered in the future work including providing metabolite profiling using different metabolomics tools, separation and purification of other bioactive metabolites, and further biological investigation for other different biological activities including in vivo investigation should be addressed.

Conclusion

Phytochemical profiling and identification of major compounds in red Nigerian propolis followed by studying the antitrypanosomal and cytotoxic activities of the crude extract and isolated compounds was introduced herein for the first time. LC–MS profiling of red Nigerian propolis extract revealed the identification of 13 compounds belonging to flavonoids, isoflavones, and phenolics. Further chromatographic techniques including CC and SEC aid in the separation of two compounds. Two isoflavonoid compounds were purified and identified from red Nigerian propolis as isosativan and medicarpin. Red Nigerian crude extract showed a higher activity against T. brucei than that of isolated compounds with MIC of 6.5 µg/ml. Moreover, the fraction from which isosativan and medicarpin were isolated revealed a potent cytotoxic effect against human leukemia cell lines with IC50 of 26.5 µg/ml compared to the standard diminazen with IC50 of 29.5 µg/ml. Hence, the crude extract and the fraction of red Nigerian propolis were more effective than the isolated compounds which may be due to the synergistic effect of their content of phenolic compounds. The results of this study indicate the richness of red Nigerian propolis extract with phenolic compounds specially isoflavonoids with myriad health benefits which could be incorporated in pharmaceutical and dietary products as source of several nutraceuticals. Further phytochemical investigation for red Nigerian propolis is recommended in the future studies to isolate bioactive metabolites. Ultimately, screening of other biological activities is recommended.

Materials and methods

General experimental

Solvents including acetonitrile, methanol, ethyl acetate, water, formic acid (LC–MS-grade), Davisil grade 633 amorphous precipitated silica (pore size 60 A, mesh size 200–425 mm), Sephadex LH-20, p-Anisaldehyde, vanillin, sulfuric acid, and the deuterated solvents chloroform-d (CDCl3) and dimethyl sulfoxide-d6 (DMSO-d6) were obtained from Sigma-Aldrich (Gillingham, UK). Glass columns for column chromatography were obtained from Rotaflo, Fisher Sceintific, Loughborough, UK. TLC-grade silica gel (60H) and TLC silica gel 60 F254 precoated aluminum sheet and the HPLC-grade solvents ethyl acetate, methanol, acetonitrile, n-hexane, and absolute ethanol were purchased from Sigma-Aldrich (Gillingham, UK). Alamar blue® BUF 012B (AbD Serotec®, Oxford, UK), HMI-9 medium (Invitrogen, Oxford, UK), RPMI-1640 (Lonza, Verviers, Belgium), l-glutamine (Life Tech, Paisley, UK), Penicillin/Streptomycin (Life Tech, Paisley, UK), fetal bovine serum (FBS) (Sigma-Aldrich, Gillingham, UK), U937 cell cultures (obtained from ECACC, Porton Down, Salisbury, 9 of 13 UK), 96-well plates (Corning®, Sigma-Aldrich), and a plate reader (Perkin Elmer, Buckinghamshire, UK) were also obtained. For CC, silica gel 60 with a mesh size of 200–425 µm was used. HPLC–UV-ELSD was performed on an Agilent 1100 system (Agilent Technologies, Waldbronn, Germany) using a reverse-phase C18 column with water and acetonitrile as the mobile phase. The interpretation of the data was carried out using Clarity software (Data Apex).

Sample collection preparation

The propolis from the Nigerian stingless bee Tetragonula biroi Friese was collected during October 2022 from Nigeria (9.0820° N, 8.6753° E) by Mr James Fearnley, a botanical specialist, Nigeria. Raw propolis was collected in an airtight container and kept at 25 °C for 24 h. Later, propolis chunks were shipped to Saudi Arabia in ice-cold conditions, and voucher specimen were kept in Clinical Laboratory Sciences Department, College of Applied Medical Sciences, King Saud University under number RNP/22. Prior to extraction techniques, propolis was freed of impurities, such as pollen, wood, the dead remains of bees, etc., and fragmented using a mortar and pestle.

Extraction and isolation

Propolis sample (40 g) was extracted with 150 ml of ethanol three times with sonication at room temperature for 60 min. Ethanol was used for extraction as it is one of the popular solvents owing to its safety for infused edibles, compatible with any type of container, and provides consistent results while being easily recoverable4. The combined extracts were evaporated under reduced pressure at 45 °C using a rotary evaporator (SCILOGEX SCI100-Pro 5L Rotary Evaporator, Vertical Dry-Ice Condenser, Motorized Lift), and the residue was weighed. The crude extract of propolis was subjected to different chromatographic methods, including column chromatography (CC) and size-exclusion chromatography (SEC) for isolation and purification of the compounds as previously reported. The ethanolic extract of red Nigerian propolis (8.2 g) was subjected to silica column chromatography and elution was sequentially performed using a gradient profile. The total number of fractions generated was 28, and these were collected in vials with a volume of 50 ml. Fractions were collected according to chromatographic screening using TLC and with suitable solvent system. LC–MS and NMR-based profiling led to identification of the different components classes and allowed combination of fractions. The final number of fractions was nine. The column was wet packed with approximately 50 g of silica slurry, and the solvent with the lowest polarity (i.e., n-hexane) was mixed before pouring and packing in a suitably sized glass column. About 3 g of propolis extract was dissolved with ethyl acetate (5 ml) and mixed with dry silica (6 g) and let to be dried under a vacuum hood. The dried extract was then loaded on the column top and eluted with sequential performance using 200 ml of n-hexane, ethyl acetate, and methanol mixtures as follows: hexane/ethyl acetate (80:20), hexane/ethyl acetate (60:40), hexane/ethyl acetate (40:60), hexane/ethyl acetate (20:80), ethyl acetate and then ethyl acetate/methanol (80:20), ethyl acetate/methanol (60:40), ethyl acetate/methanol (40:60), and ethyl acetate/methanol (20:80). Fractions were collected according to their similarities and concentrated using rotatory evaporator, which further gathered via HPLC–UV-ELSD analysis based on similar chemical profiles. Further purification of yielded fractions from CC was carried out using SEC. Sephadex LH 20 slurry prepared by the overnight suspension of the stationary phase in 50:50 dichloromethane/methanol for nonpolar fractions and in 100% methanol for polar fractions. A glass column (2 × 100 cm) was packed with the formed sephadex LH 20 slurry and covered with cotton wool. After loading the sample, elution was performed with 100% MeOH in an isocratic manner, and fractions were collected in small vials of around 1 ml.

NMR analysis

NMR acquisition was performed by a JEOL (JNM LA400) spectrometer (400 MHz) at SIPBS and a Bruker Avance 300 (400 MHz) spectrometer with tetramethylsilane (TMS) as the internal standard. One- and two-dimensional experiments were carried out to identify the chemical structure of the compounds present in the fractions. The compounds were prepared in deuterated solvents, such as CDCl3 and DMSO-d6, based on their solubilities. Then, 500–600 µL of a suitable solvent was used for the dissolution of 10 mg of every sample, which were then poured into typical NMR tubes (5 × 178 mm) to a depth of around 4 cm.

LC–MS analysis

About 10 mg of the obtained extract or fraction was dissolved in 10 mL 100% methanol with sonication were prepared separately for chemical profiling and molecular mass determination by LC–MS. 3 µL was aliquoted and subjected to LCMS analysis using a Dionex 3000 HPLC pump/Thermo LTQ Orbitrap Velos ETD Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). A reverse-phase 5 µm C18 column (4.6 × 150 mm) (Hypersil, Thermo) was used and eluted using a gradient at a flow rate of 0.3 ml/min with 0.1% v/v formic acid in water and 0.1% v/v formic acid in acetonitrile as the two solvents (A and B) making up the mobile phase. The ESI interface in negative ionization permitted the identification of [M-H]- ion peaks. The spray voltages for the capillary and the cone were, respectively, − 4.0 kV and 35 V. The flow rates of the sheath gas and auxiliary gas were, respectively, 50 and 15 arbitrary units. The ion transfer capillary had a temperature of 275 °C, and m/z 100–1500 provided the full scan data.

Data processing and structure elucidation

The NMR spectral data were obtained using MestReNova software 8.1.2 (Mestrelab Research, A Coruña, Spain), and ChemBioDraw Ultra, Version 14 (PerkinElmer, Yokohama, Japan), was employed for illustrating figures of the structures of the isolated compounds.

Like NMR, mass spectrometry (MS) provides structural data (molecular weight and molecular formula) of the examined compounds. The sample data were acquired and processed with the Xcalibur software (Thermo Fisher Corporation, Hemel Hempstead, UK).

In vitro antitrypanosomal assay

An Alamar blue assay was carried out to evaluate the antitrypanosomal activity using a method described previously4. The test is based on viable cells metabolizing the blue resazurin dye to resorufin (a pink and fluorescent product). To perform the test, Trypanosoma brucei S427 cells at a seeding density of 2 × 105 cells and propolis samples (20 mg/ml in 100% DMSO) that were double-diluted to varying concentrations (0.19–200 µg/ml) were prepared using Hirumi’s Modified Iscove’s medium 9 (HMI-9) as a diluent. Then, 100 µl of each propolis sample was added to a 96-well plate, followed by the addition of a trypanosome suspension (100 µl) to each well and incubation for 48 h at 37 °C in 5% CO2. After incubation, resazurin dye was added and incubated for a further 24 h under the same conditions. Following incubation, the fluorescence was recorded at _Ex/EM: 544/620 nm using an FLUO star Optima (BMG Labtech, Offenburg, Germany).

Evaluation of antitumor property using cell viability assay

Antitumor evaluation was performed as previous reported work4. RPMI 1640 medium was used for the culturing and subculturing of human leukemia cell lines (U937). U937 cell cultures were obtained from ECACC, Porton Down, Salisbury, 9 of 13 UK. The medium was supplemented with penicillin and streptomycin (1% v/v), l-glutamine (1% v/v), and FCS (5% v/v), and U937 cells were cultured in desirable conditions, viz. a temperature of – 37 °C, 100% humidity, and 5% CO2. To perform the cell viability assay, 100 µl of U937 cell suspension (containing 1 × 105 cells/ml) was plated in each well and incubated for 24 h. Post incubation, cells were treated with crude and purified propolis samples prepared in varying concentrations (1.56–200 g/ml) and incubated further for 24 h. DMSO was added and served as a positive control (to kill the cells completely); the negative control was the cells with medium. Following incubation, the resazurin indicator (10% Alamar blue) was loaded into the wells and incubated for an additional 24 h. The fluorescence of the plate was read using aWallac Victor 2 microplate reader (_Ex/EM: 560/590 nm), and cell viability was calculated and expressed as mean inhibitory concentration (IC50) values.

Statistical analysis

Antitrypanosomal and cytotoxic activities were expressed as means and standard errors. A paired t-test analysis was performed to determine the significance in the mean values of the anticancer and antitrypanosomal activities between the crude and isolated compounds. P < 0.05 was considered statistically significant.