Abstract

Introduction: Propolis is a resinous natural substance collected by honeybees from buds and exudates of various trees and plants; it is widely accepted that the composition of propolis depends on the phytogeographic characteristics of the site of collection. 

Glycoside of cistanche can also increase the activity of SOD in heart and liver tissues, and significantly reduce the content of lipofuscin and MDA in each tissue, effectively scavenging various reactive oxygen radicals (OH-, H₂O₂, etc.) and protecting against DNA damage caused by OH-radicals. Cistanche phenylethanoid glycosides have a strong scavenging ability of free radicals, a higher reducing ability than vitamin C, improve the activity of SOD in sperm suspension, reduce the content of MDA, and have a certain protective effect on sperm membrane function. Cistanche polysaccharides can enhance the activity of SOD and GSH-Px in erythrocytes and lung tissues of experimentally senescent mice caused by D-galactose, as well as reduce the content of MDA and collagen in lung and plasma, and increase the content of elastin, have a good scavenging effect on DPPH, prolong the time of hypoxia in senescent mice, improve the activity of SOD in serum, and delay the physiological degeneration of lung in experimentally senescent mice With cellular morphological degeneration, experiments have shown that Cistanche has the good antioxidant ability and has the potential to be a drug to prevent and treat skin aging diseases. At the same time, echinacoside in Cistanche has a significant ability to scavenge DPPH free radicals and can scavenge reactive oxygen species, prevent free radical-induced collagen degradation, and also has a good repair effect on thymine free radical anion damage.

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Objectives: This study aimed to determine the phytochemical composition of ethanolic extracts from eight propolis batches collected in different regions of Benin (north, center, and south) and Congo, Africa.

Material and methods: Characterization of propolis samples was performed by using different hyphenated chromatographic methods combined with carbon-13 nuclear magnetic resonance (13C NMR) dereplication with MixONat software. Their antioxidant or anti-advanced glycation end-product (anti-AGE) activity was then evaluated by using diphenylpicrylhydrazyl and bovine serum albumin assays, respectively. 

Results: Chromatographic analyses combined with 13C NMR dereplication showed that two samples from the center of Benin exhibited, in addition to a huge amount of pentacyclic triterpenes, methoxylated stilbenoids or phenanthrenes, responsible for the antioxidant activity of the extract for the first one. Among them, combretastatin might be cytotoxic. For the second one, the prenylated flavanones known in Macaranga-type propolis were responsible for their significant anti-AGE activity. The sample from Congo was composed of many triterpene derivatives belonging to Mangifera indica species.

Conclusion: Therefore, propolis from the center of Benin seems to be of particular interest, due to its antioxidant and anti-AGE properties. Nevertheless, as the standardization of propolis is difficult in tropical zones due to its great chemo diversity, a systematic phytochemical analysis is required before promoting the use of propolis in food and health products in Africa.

KEYWORDS 

13C NMR dereplication, methoxylated stilbenoids or phenanthrenes, pentacyclic triterpenes,  prenylated flavanones, Propolis

1. INTRODUCTION

Propolis is a natural resinous substance collected by bees from the buds and exudates of various trees and plants, mixed with beeswax and salivary enzymes.1 It is used by bees to protect the entrance from intruders, plug holes, smooth internal walls, mummify dead animals (small insects) inside the hive, and balance extreme humidity or drought conditions.2,3 Propolis has been widely used in folk medicine since ancient times due to its wide range of therapeutic properties.4 Propolis is generally composed of resin (50%), wax (30%), oils (10%),  pollen (5%), and additional phenolic compounds such as flavonoids.5,6 The chemical composition of propolis is known to be complex and varies according to plant species growing around the hive, from which bees collect exudates.7 It is accepted that several factors, such as the floristic composition of the area, the place and time of collection, the season, the type of collector, the availability and altitude, and the food activity developed during the exploitation of the propolis have an impact on the chemical composition of the propolis.8,9 There are different types of propolis, depending on the geographical area of production, botanical source, and chemical composition. The most common types of propolis are temperate, birch, tropical, Mediterranean, and Pacific.10 Propolis from temperate climatic zones, such as Europe, North America, or non-tropical regions of Asia, comes mainly from exudates of buds of Populus species (Salicaceae) and is therefore rich in flavonoids and phenolic acids and their esters; however, tropical propolis, originating from regions where neither poplar nor birch grows, is rich in prenylated derivatives of p-coumaric acids, benzophenones, or terpenoids.11 Pacific propolis, typically rich in prenylated flavanones, is another important type of propolis found in Taiwan, Japan, and the Solomon Islands, and birch propolis is found specifically in Russia.12 The study of propolis from tropical Asia has led to the discovery of Macaranga denarius L. and Mangifera indica L. as plant sources of Indonesian propolis.13 On the other hand, coniferous species of the Cupressaceae family are the main botanical source of propolis in Mediterranean regions.14 Chemical analyses revealed that propolis contains more than 300 different natural products (NPs),  including phenolic acid derivatives, coumarins, flavonoids, sesquiterpenes, diterpenes, triterpenes, steroids, lignans, or prenylated benzophenones.15 The biological effects of propolis have been described mainly about not only their antioxidant,16–18 anti-AGE (i.e., inhibition of advanced glycation end-product [AGE] formation),19 anti-inflammatories,20 and anti-tumor effects,21–23 but also their antibacterial,24–26 anti-fungal,15 anti-viral,27 and anti-parasitic28 activities. Propolis is also known to stimulate the production of antibodies, suggesting its potential use as an adjuvant in vaccines.29 The increasing use of this natural substance of diversified composition and therefore varying biological activities in the pharmaceutical, cosmetic, and food industries are generating particular interest in scientific research,  especially when it comes to defining its chemical composition. Most studies have been carried out on samples from Europe19,30–33 and Latin America, more specifically Brazil,20,23,34–36 while few studies were conducted in Africa.37–41 Information on Beninese and Congolese42 propolis remains scarce.

Thus, this study aimed to characterize major compounds from propolis samples collected in various phytogeographical zones of Benin and Congo (Africa) using 13C nuclear magnetic resonance (NMR) dereplication with MixONat software.43,44 A preliminary study using a database (DB) containing NPs previously reported from propolis together with their 13C predicted chemical shifts (δC) did not yield any usable data compared to the satisfactory results usually obtained with plant extracts.43–45 This can be explained by the chemical composition of propolis, which is highly dependent on the local flora, making it impossible to construct a  chemotaxonomic DB. Thus, to decipher major compounds from Beninese and Congolese propolis, in the present work we performed high-performance liquid chromatography coupled with ultraviolet and evaporative light scattering detection mass spectrometry (HPLC-UV-ELSD-MS) and gas chromatography coupled with mass spectrometry (GC-MS) analyses on crude propolis extracts to identify their main structural features and build corresponding DBs of NPs suitable for MixONat software.46 Then, after a coarse fractionation of the propolis samples selected according to their chemical profile, 13C NMR-based dereplication allowed the identification of major NPs without further purification. Antioxidant and anti-AGE assays were also carried out.

2. EXPERIMENTAL PROCEDURES

2.1 Chemicals

1,1-Diphenyl-2-picrylhydrazyl (DPPH), Folin–Ciocalteu reagent,  formic acid, and gallic acid, all analytical grade, were purchased from Sigma-Aldrich (St Quentin Fallavier, France). 6-Hydroxy-2,- 5,7,8-tetramethylchroman-2-carboxylic acid (Trolox®) and 50 -  caffeoylquinic acid (chlorogenic acid) were obtained from Acros Organics (Geel, Belgium).   

2.2 Propolis samples 

Eight Beninese propolis samples were collected by scraping from beehives in three phytogeographic zones (Table 1 and Figure SI-1). A  sample of Congolese propolis was collected in the artificial forest of acacia trees in the Bateke plateau in the center of the Republic of Congo (Table 1). 

2.3 Propolis Extraction

Ethanolic extracts of propolis (EEPs) were initially prepared using the following extraction protocol.19 Raw propolis was first homogenously pulverized in the presence of liquid nitrogen. For all samples, 1 g of propolis powder was macerated in 20 ml of 95% EtOH. After stirring for 2 h at room temperature, the mixture was filtered using a sintered glass disc funnel filter (16–40 μm pore size). The residue was reextracted twice using the same steps. Then, the three gathered filtrates were maintained at  18 C overnight, filtered to remove waxes,  and evaporated under reduced pressure (40 C, 10 bar) to give the EEPs.

Amounts of 8.0 g of propolis powder of BC1 (1) and BC2 (2) or 10.3 g of CG (3) were extracted again with EtOH 95% (UAE, 4 80 ml, 15 min) for further flash chromatography.

2.4 Determination of total phenolic content

The total phenolic content was determined via the Folin– Ciocalteu colorimetric method as previously described,19 and recently adapted to direct use in microplates. Briefly, 10 μl of each propolis extract in MeOH (3.5 mg/ml for BC1, 5 mg/ml for BN2, BC2, and CG, 7.5 mg/ml for BN1, BS1a, BS1b, and BS2, and 10 mg/ml for BS3) was mixed with 20 μl of distilled water and 10 μl of Folin–Ciocalteu reagent in a 96-well microplate. After 3 min, 120 μl of distilled water and 40 μl of 20% aqueous sodium carbonate were added. The absorbance was measured on a TECAN® microplate spectrophotometer (V6.5) at 760 nm after 30 min in the dark at room temperature. A  blank was prepared in the same way by using MeOH instead of the extract solution and gallic acid was used to calculate the calibration curve (0.04–0.328 mg/ml; y = 2.7241x  0.0039; r2 = 0.9982). Each sample was analyzed in triplicate. Total phenolic content was expressed in terms of gallic acid equivalents (mg) per gram of extract (mg GAE/g).

2.5 Preliminary chromatographical analyses

2.5.1 Analytical TLC

Analytical thin layer chromatography (TLC) was performed on a TLC Alugram Xtra SIL G/UV254 (Macherey-Nagel, Düren, Germany), using a mixture of cyclohexane: AcOEt as eluant. Spots in the chromatogram were visualized first under UV light (254 nm) and then by spraying with vanillin–sulfuric acid reagent (2 ml of concentrated sulfuric acid in 98 ml of a 1:99 w/v vanillin:95% ethanol solution) and heating the chromatograms to 110° C for 5 min.

2.5.2 GC-MS procedure

GC-MS analysis was performed on non-derivatized samples using a Shimadzu Gas Chromatograph GCMS-QP2010 SE (Noisiel, France)  with an ionization voltage of 70 eV (Electronic Impact). Samples were prepared in dichloromethane (DCM) at 2 mg/ml for extracts and 1 mg/ml for fractions. Separations were carried out using a Phenomenex ZB5 column (30 m * 0.250 mm internal diameter with 0.25 mm film thickness; Phenomenex, Le Pecq, France). The temperature was programmed as follows: 180° C (3 min), 180–280° C at a rate of 10 C/min, and 280° C (27 min). Helium was used as a carrier gas at a flow rate of 2.0 ml/min. Injector and detector temperatures were set at 250° C and 280° C,  respectively. Metabolites were identified by comparing their retention times (Rt), nominal mass, and/or fragmentation patterns with those of authentic samples and/or those contained in the fragmentation pattern libraries of the equipment (NIST11, NIST11s, and FFNSC2).

2.5.3 HPLC-DAD-ELSD procedure

Chromatographic analyses were carried out using a Shimadzu 2030C 3D liquid chromatograph (Noisiel, France) equipped with a diode array detector (DAD) and an ELSD (Sedere®) with a Lichrospher® column 100 RP-18 (125 mm * 4 mm i.d., 5 μm, Merck, Darmstadt, Germany)  protected with a Lichrocart® 4–4 guard cartridge (4 mm*4 mm i.d.),  using a flow rate of 1 ml/min. The mobile phase consisted of 0.1% formic acid in water (solvent A) and methanol (solvent B), and separation was performed by the following linear gradient: 25–100% B (0– 40 min), 100% (40–45 min). UV–vis spectra were recorded in the range of 190–600 nm, and chromatograms were acquired at 254 and 280 nm. ELSD was heated at 30° C, and a gain of 4 was applied to the signal. Samples were prepared at a concentration of 10 mg/ml in MeOH and centrifuged at 13,000 g for 10 min before injection (10 μl) to remove traces of suspended materials.

2.5.4 HPLC-UV-MS procedure

HPLC-UV-MS analyses were performed using a 2795 Waters separation module (Guyancourt, France) equipped with a Dual λ 2487 Waters detector. Column, mobile phases, and gradient were the same as described above for HPLC-DAD-ELSD. Chromatograms were acquired at 254 and 280 nm. The mass analyses were carried out on a Bruker (Bremen, Germany) electrospray ionization/  atmospheric pressure chemical ionization (ESI/APCI) Ion Trap Esquire 3000 + in both positive and negative modes as follows:  collision gas, He; collision energy amplitude, 1.3 V; nebulizer and drying gas, N2, 7 L/min; the pressure of nebulizer gas, 30 psi; dry temperature, 340 C; flow rate, 1.0 ml/min; solvent split ratio, 1:9; scan range, m/z 100–1,000. Samples were prepared at a concentration of 10 mg/ml in MeOH, centrifuged at 13,000 g for 10 min, and filtered through a 0.45-μm polytetrafluoroethylene (PTFE) membrane syringe filter before injection (20 μl) to remove traces of suspended materials.

2.6 EEP fractionation by flash chromatography

First, 3.8 g of BC1 (1), 3.5 g of BC2 (2), or 4.0 g of CG (3) EEPs was dissolved in a minimal volume of DCM and acetone and mixed with silica gel (silica gel: extract ratio, 2:1). For all of them, the solvent was allowed to evaporate until a fine dry powder was obtained. For  each EEP, fractionation was then performed by using a CombiFlash Teledyne ISCO apparatus (Lincoln, NE, USA) with a silica gel column (Chromabond® Flash RS 40 SiOH, 40 g, Macherey-Nagel, Hoerdt, France) at a flow rate of 25 ml/min with cyclohexane (solvent A) and  ethyl acetate (EtOAc) (solvent B) using the following gradient elution: (1) for BC1 EEP, 5% B (0–10 min), 5–20% B (10–30 min), 20–30% B (30–60 min), 30–50% B (60–85 min), and 50–100% B (85–110 min); 160 tubes of 20 ml were collected and combined into 14 fractions (BC1_F1 to BC1_F14) on the basis of their TLC chromatographic profiles (cyclohexane:EtOAc ratio, 90:10 to 50:50); (2) for BC2 EEP, 5% B (0–10 min), 5–10% B (10–20 min), 10–30% B (20–40 min), 30–40% B (40–55 min), and 40–100% B (55–70 min); 100 tubes of 20 ml were  collected and combined into 8 fractions (BC2_F1 to BC2_F8) on the  basis of their TLC chromatographic profiles (cyclohexane:EtOAc ratio, 90:10 to 60:40); (3) for CG EEP, 5% B (0–10 min), 5–10% B (10– 30 min), 10–20% B (30–50 min), 20–40% B (50–70 min), 40–60% B (70–80 min), and 60–100% B (80–90 min); 135 tubes of 20 ml were  collected and combined into 23 fractions (CG_F1 to CG_F23) on the  basis of their TLC chromatographic profiles (cyclohexane:EtOAc ratio, 80:20 to 60:40).

An additional fractionation step was performed on the BC2_F7  fraction: 200 mg was dissolved in a minimal volume of MeOH and mixed with C18-silica gel (C18-silica gel: extract ratio, 2:1). Separation was realized on a C18 column (Interchim® PF-C18HP, 4 g, Montluçon, France) at a flow rate of 15 ml/min with water (solvent A) and MeOH (solvent B) using the following gradient elution: 50–70% B (0–30 min)  and 70–100% B (30–40 min); 80 tubes of 8 ml were collected and combined into 5 fractions (BC2_F7-1 to BC2_F7-5) based on their HPLC-UV (280 nm) profiles.

2.7 1 H and 13C NMR analyses

NMR spectra (1D and 2D) of propolis fractions (7–58 mg) or sometimes NPs were recorded in deuterated chloroform (CDCl3) or deuterated methanol (CD3OD) (500 μl) on a JEOL NMR spectrometer at 400 MHz for 1 H and 100 MHz for 13C. NMR experiments (1 H NMR, 13C NMR, DEPT-135, DEPT-90, and 2D NMR) on fractions and pure NPs were performed at 298 K on a JEOL 400 MHz H spectrometer (JEOL Europe, Croissy-sur-Seine, France) equipped with an inverse 5-mm probe (ROYAL RO5). Chemical shifts (δH and δC) are expressed in ppm and J values in Hz.

For 13C NMR (100 MHz) spectra, a WALTZ-16 decoupling sequence was used with an acquisition time of 1.04 s (32,768 complex data points) and a relaxation delay of 2 s. Between 1,500 and 11,000 scans were collected to obtain a satisfactory S/N ratio. A 1 Hz exponential line broadening filter was applied to each free induction decay (FID) before the Fourier transformation. Spectra were manually phased and baseline-corrected using MestReNova software (Mestrelab Research, Santiago de Compostela, Spain) and referenced on the central resonance of the deuterated solvent at δC 77.16 ppm (CDCl3) and δC 49.00 ppm (CD3OD). For distortionless enhancement by polarization transfer (DEPT) experiments, between 512 and 5,500  scans were required and alignments with the 13C spectra were made using a given δC. A minimum intensity threshold was then used to manually collect positive 13C NMR and DEPT-90 signals and positive and negative DEPT-135 signals while avoiding potential noise artifacts.

Propolis DB1 was first built by searching for compounds described in propolis on SciFindern, 47 resulting in a DB of 1,471 NPs; δC values were predicted using Advanced Chemistry Development (ACD) NMR predictors (C, H). From such DBs containing NPs together with their δC-SDF values, the CTypeGen routine included in MixONat created a suitable DB: It read the spatial data file (SDF) and sorted chemical shifts by carbon type. The required c-type SDF was then created, i.e., c-type Propolis DB1.43,44 

Stilbenoids_Phenanthrenoids DB2 was created. A search of substances using the keywords stilbenoids and phenanthrenes in the LOTUS DB containing 276,518 NPs48 (LOTUS, 2022) allowed obtaining a Stilbenoids_Phenanthrenoids DB2 of 2,681 NPs as an SDF. Flavanones DB3 was then created: A search on SciFindern47  using the 2-phenylchroman-4-one substructure allowed to select of 56,679 molecules. They were further reduced to 2,893 referring to “Natural products occurrence” using an analysis of the substances with the filter “Reference role” proposed by SciFindern. After additional refining by a filtering step based on the expected molecular weight (MW) (i.e., 340 to 424 Da), all relevant flavanones were subsequently exported as an SDF to obtain the NPs from Flavanones DB3 (684 NPs). For each NPs of DB2 and DB3, δC-SDF values were predicted using ACD NMR predictors (C, H)  software as well as the methodology previously described by Nuzillard46 to directly obtain the c-type SDF ready for use by MixONat. The latter contains for each compound of the DB the predicted δC values organized as methyl, methylene, methine, or quaternary carbons.

Triterpenes DB4 was built from the PNMRNP3 DB49,50 imported as an SDF in ACD NMR predictors (C, H) software by searching triterpenes with an MW between 100 and 500 Da (i.e., Search data: triterpenes; Search MW: 100 to 500) to directly obtain a DB of 6,623  triterpenes in the required c-type file format.

Alk(en)yl resorcinol and phenol derivatives DB5 were built from the PNMRNP3 DB49,50 imported as an SDF in ACD NMR predictors (C, H) software by searching NPs with an m-alk(en)yl phenol scaffold and classified as phenols (i.e., 3-(hexadec-8-en-1-yl)phenol Substructure search; Search Classyfire class: phenol) to directly obtain a DB of 44 NPs in the required c-type file format.

2.8 13C NMR-based dereplication using MixONat  software

The peak list and intensity data obtained from each experimental spectrum (13C NMR, DEPT-135, and DEPT-90) were exported as a . csv file using Microsoft Excel (Microsoft 16.45) software and used as an input file in MixONat software. Such files consist of a list of δC values ordered in decreasing order associated with their intensities on the same line, separated by a comma.

Data were then processed using MixONat, which exploits any dataset that provides molecular structures in the previously described SDF (i.e., c-type DB1–5 file format). Based on this information, MixONat was used to compare the experimental δC values of the fractions to the predicted δC-SDF values from the DB and made suggestions for specific NPs potentially present in the analyzed sample. In the end, MixONat provided NP proposals with scores ranging from 0 to 1, i.e., from 0% to 100% (where 1 corresponds to a perfect match and 0 indicates no similarity for a given compound from the used DB). Acceptable values for putative identification were >0.70. Afterward,  experimental data of NPs with the best scores were compared with the literature.

2.9 Purification

2.9.1 Preparative HPLC

Purification of fractions BC1_F6 and BC1_F8 (40 mg in 2 ml of MeOH) was carried out using a Shimadzu LC-20AP preparative liquid chromatograph equipped with a UV–vis detector SPD-40 (Noisiel, France), an injection loop of 2 ml, and a fraction collector FRC-10A,  using a preparative column C18 (250 mm  21.2 mm i.d., 5 μm) (Pursuit XRs 5, Agilent, Les Ulis, France) at a flow rate of 21.24 ml/  min. The mobile phase consisted of 0.1% formic acid in water (solvent A) and methanol (solvent B) and the separation was performed using the following gradient: (1) for BC1_F6, 40% B (0–5 min), 40–70% B (5–20 min), and 70–90% (20–30 min); (2) for BC1_F8, 40–70% B (0– 15 min) and 70–90% (15-25 min). Chromatograms were acquired at 254 and 280 nm.

2.9.2 Solid phase extraction (SPE)

To obtain the most polar fraction of BC2-EPP (200 mg in 5 ml of MeOH mixed with 350 mg of C18-silica gel after evaporation), purification was carried out using a C18 solid phase extraction column (2 g/15 ml, Thermo Scientific™, Villebon-sur-Yvette, France) with water: MeOH 7:3 (10 ml) and water: MeOH 6:4 (10 ml). Tubes 4–10  were combined to form the polar fraction of BC2 (BC2_SPE) based on their HPLC-UV (330 nm) profiles.

2.10 Antioxidant and anti-AGE assays

2.10.1 Scavenging of DPPH radicals

The DPPH radical scavenging evaluation of Beninese EEPs was performed as previously described.51 Briefly, tested samples were diluted in absolute EtOH at 0.02 mg/ml from stock solutions at 1 mg/ml in dimethylsulfoxide (DMSO). Aliquots (100 μl) of these diluted solutions were placed in 96-well plates in triplicate. About 25 μl of freshly prepared DPPH solution (1 mM) was added to 75 μl of absolute EtOH using the microplate reader's injector (Infinite® 200, Tecan, France) to obtain a final volume of 200 μl per well. After 30 min in the dark at ambient temperature, the absorbance was determined at 517 nm. EtOH was used as a blank, whereas 10, 25, 50, and 75 μM solutions of Trolox (a hydrophilic α-tocopherol analog) were used for the calibration curve. A sample of chlorogenic acid ethanolic solution (0.02 mg/ml) was used as the quality control standard. Results were expressed as Trolox equivalents (micromoles of TE per g of extract).

2.10.2 Anti-AGE assay

The effects of propolis extracts and the five major isolated naringenin derivatives on AGE formation were determined as described previously.51 Briefly, bovine serum albumin (BSA) (10 mg/ml) was incubated with D-ribose (0.5 M) together with the tested compound (3 μM to 3 mM) or extract (1 μg to 1 mg) in 50 mM phosphate buffer at pH 7.4 (NaN3, 0.02%). Solutions were incubated in 96-well microtiter plates at 37° C for 24 h in a closed system before AGE fluorescence measurement. Fluorescence from the incubated sample in identical conditions without D-ribose was subtracted for each measurement. Pentosidine-like (excitation at 335 nm, emission at 385 nm) AGE fluorescence was measured by fluorometry.52 IC50 values were defined as the amount of extract (μg/ml) or positive control (μM)  required to reduce AGE formation by 50% relative to the negative control. According to the statistical validation assay,53 a single analysis is sufficient for an accurate IC50 determination.

3. RESULTS AND DISCUSSION

3.1 Chromatographic profiling

General LC profiling of the eight Beninese and the Congolese EPP  using the quasi-universal ELSD detector showed that all samples contained non-polar compounds, eluted at the end of the chromatograms (Figure 1). BC1 also presented a large amount of more polar compounds, and BC2 exhibited some medium polar compounds in smaller amounts. Non-polar compounds in CG EEP appeared to be quite different from Beninese ones.

Thus, ELSD profiling led to three different and original samples whose composition was further investigated: BC1, BC2, and CG.

Firstly, GC-MS analyses were conducted, comparing the MS fragmentation with the NIST DB, to characterize the chemical classes and,  when possible, to identify the constituents. Putative identification (based on the percentage of the match with the NIST DB) and chemical classes of compounds are presented in Table 2 (GC–total ionic current [TIC] profiling data are shown in Figure SI-2).

All Beninese EEPs presented the same profiles of non-polar compounds, labeled as letter A to F, but their amounts could vary. They were putatively identified as α- or β-amrinone (A), β-amyrin (B), lupenone (C), α-amyrin (D), α- or β-amyrin acetate (E), and lupeol acetate (F) (cf. Figure SI-3), already described as major NPs in Mexican propolis.54 Zhang et al. (2014) and Tamfu et al. (2020) also described triterpenoids as amyrin/lupeol and amyrin/lupeol acetates in various African propolis samples.41,55 Only BC1 presented additional NPs eluted between Rt 10 and 15 min suggested as methoxylated stilbenoids or phenanthrenes according to their fragmentation patterns. The Congolese EEP exhibited a different profile from the Beninese ones, with C16 and C18 acid ethyl esters as more volatile compounds associated with a good matching score of 94%, a phenol derivative at Rt 13.1 min, three resorcinol derivatives in the range of 14–17 min,  and different types of triterpene derivatives after Rt 21 min.

In addition, EPPs were analyzed by HPLC-DAD-MS to visualize non-volatile NPs (cf. Figure SI-4). The different profiling showed that BC1 EEP exhibited a large amount of medium polar compounds with chromophores absorbing at 280 nm, as well as BC2 in smaller proportions, and CG possessed a very poor UV profile at 280 nm.

3.2 Chemical composition of BC1 and BC2

To identify their major NPs using 13C NMR dereplication based on MixONat and suitable DBs, a coarse fractionation of BC1 and BC2 EEP  was then achieved by flash chromatography. Indeed, for BC1, GC-MS  analysis revealed the presence of methoxylated stilbenoids or phenanthrenes, which guided us to build the specific Stilbenoids_Phenanthrenoids DB2. For BC2, except for the more polar compounds at Rt 3.8 min, all compounds showed UV spectra of flavanones-dihydro flavonols, which suggested creating the Flavanones DB3.

As depicted in Table 3, dihydrophenanthrenes 1 and 3–5, phenanthrenes 2 and 6, and the dihydro stilbene 7 (Figure 2) were suggested in BC1 EEP by MixONat and validated by comparison with literature data (cf. supporting information). When necessary, an MW filter was used based on HPLC-MS data.

More precisely, among 9,10-dihydrophenanthrene derivatives out of 2,681 NPs in DB2, MixONat suggested 6-methoxycoelonin (2,7-dihydroxy-3,5-dimethoxy-9,10-dihydrophenanthrene; 3, rank 1, score 0.75, Figures SI-13 and SI-14) in BC1-F5 and its presence was confirmed by comparison to published NMR data. It has been yet identified from Combretaceae species56 and in the orchid Bulbophyllum vaginatum; 57 and 2,7-dihydroxy-3,4,6-trimethoxy- 9,10-dihydrophenanthrene (4, rank 1, score 0.88, Figures SI-15 and SI-16) was hypothesized in BC1_F2. Compound 4 has already been described in Senegalese propolis58 and its presence was validated  based on reported data by Pettit et al. from the African tree Combretum caffrum59 and by Lu et al. from Dioscorea nipponica Makino.60

The 13C NMR-based dereplication process also predicted 2,6-dihydroxy-3,4,7-trimethoxy-9,10-dihydrophenanthrene (5, rank 8, score 0.76, Figures SI-15 and SI-16) in BC1_F2 and 2,6,7-trihydroxy-3,4-dimethoxy-9,10-dihydrophenanthrene (1, rank 2, score 0.5, Figures SI-5 and SI-6) in BC1_F6-1 when a molecular filter at MW 288 Da was used. For 1, the score was quite low (0.5);  therefore, additional 2D NMR experiments (heteronuclear multiple quantum correlations [HMQC], heteronuclear multiple-bond connectivity [HMBC], nuclear Overhauser effect spectroscopy [NOESY],  cf. Figures SI-7–SI-10) were performed to confirm the structure; for 5,  the score was 0.76, i.e., higher than 0.70. These two NPs (1 and 5)  were first mentioned by Letcher et al. in Combretum species61,62 and 1 has already been found in Senegalese propolis.58

Among phenanthrene derivatives, MixONat hypothesized 2,6,7-trihydroxy-3,4-dimethoxyphenanthrene (2, rank 1, score 0.88, Figures SI-11 and SI-12) in BC1_F8-1, which was confirmed by comparison with its spectral data described by Letcher et al. in Combretum apiculatum. 61 2,7-Dihydroxy-3,4,6-trimethoxyphenanthrene (6, rank 5, score 0.94, Figures SI-17 and SI-18) previously isolated from Photolida Chinensis63 was suggested in BC1_F4.

Combretastatin B-2 (7), a dihydro stilbene, was suggested by MixONat in first rank (score 1.0, Figures SI-17 and SI-19) in BC1 EPP  when using an MW filter (MW 304 Da) and validated by comparison with NMR data described by Pettit et al. in Combretum caffrum. 59

Compounds 2, 6, and 7 have already been identified in Senegalese propolis.58

1 H and 13C NMR data of 1–7 are available in the supporting information.

For BC2, seven compounds were identified belonging to two structural classes of polyphenols, chromone-C-glucoside and prenylflavanone derivatives (8–14), some of which were described for the first time in propolis (Figure 3). 

The 13C NMR-based process using MixONat and DB1 to DB5 did not give relevant results suggesting original NPs. Two major products (8–9) were fully characterized as a mixture using mass spectra and 1D  and 2D NMR data (1 H, 13C, HMQC, HMBC, cf. Figure SI-20–SI-23)  and identified as florin (8) and isobiflorin (9), two chromanone-Cglucosides newly described in propolis, previously described in Pancratium biflorum64 and cloves of Eugenia caryophyllata, respectively.65

Regarding flavanone derivatives, among 688 NPs in DB3, MixONat proposed 6-prenylnaringenin (10, rank 1, score 0.95, Figures SI- 24 and SI-25) already described in Nigerian red propolis66 in BC2_F7-1. 6,8-Diprenylaromadendrin (12, rank 1, score 1.0, Figures SI-28 and SI-29) and 6,8-diprenylnaringenin (lonchocarpol A) (13, rank 10, score 0.84, Figures SI-30 and SI-31), previously found in Cameroon propolis samples,42 were validated in BC2_F7-1 and BC2_F5, respectively. 6-Geranylnaringenin (14, rank 1, score 0.84, Figures SI-32 and SI-33), previously described in propolis from the Solomon Islands,67 was hypothesized and confirmed in BC2_F7-5. Finally, 6-dimethylallyl naringenin (11) was suggested in second position by MixONat in BC2_F6 (rank 2, score 0.65, Figures SI-26 and SI- 27). The isomer in position 8 was proposed in the first position, but a comparison with literature data 68,69 showed that it is the isomer in position 6. Compound 11 was new in propolis but already described in organic extracts of Monotes africanus. 70 1 H and 13C NMR data of 8– 14 are available in the supporting information.

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