3.3 Chemical composition of CG

For CG, the EEP from Congo, GC-MS was not effective enough to directly identify all the different constituents but allowed us to characterize several chemical classes, including fatty esters, phenol and resorcinol derivatives, and triterpenoids. This structural information prompted us to use 13C NMR-based dereplication using customized DBs, i.e., DB4 and DB5. Thus, flash chromatography was performed on CG EEP and fractions were gathered using TLC with sulfuric vanillin revelation (cf. Figure SI-24). 13C NMR dereplication with MixONat software was performed on the different fractions using DB4 and DB5, followed by a comparison with literature for validation. Moreover, GC-MS data allowed us to determine the MW of major NPs, which were used with MixONat software to improve structural hypotheses (Table 4, Figure 4).

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As expected, fatty esters such as palmitic acid ethyl ester (15) and oleic acid ethyl ester (16), the most volatile compounds of CG, were confirmed in CG_F2 by 1 H NMR as hypothesized by GC-MS. For CG_F3, 13C NMR dereplication using MixONat software with triterpenes DB4 gave more than 50 compounds with a match score higher than 0.90. 13C NMR spectra of CG_F3 revealed a mixture of several triterpene derivatives (with MW at 424 for all proposed by GC-MS)  including one major and two minor ones. After validation of 13C NMR  data in the literature, the major compound was identified as cycloartenone71,72 (17, rank 1, score 0.90, MW filter at 424 Da, Figures SI-35  and SI-36) and the two minor ones as lupenone73,74 (18, rank 4, score 0.87) and β-amyrenone75 (19, rank 31, score 0.83, Figures SI-35 and SI-36). Similarly, in fraction CG_F8, we found a mixture of corresponding alcohols such as cycloartenol76 (21, rank 7, score 0.90, Figures SI-39 and SI-40) as major NP and lupeol77 (22) and β-amyrin78 (23) as minor ones (rank 1, score 0.97 and rank 34, score 0.83, MW filter at 426 Da; Figures SI-41 and SI-42). The isomer of 23, α-amyrin (24),  was also identified mainly based on 13C NMR data (comparison to literature data by Seo et al.78 and GC-MS (MW 426) data. All these ketone and alcohol triterpene derivatives were already known in propolis.37,38,42,54,79 For CG_F5, as GC-MS analysis showed a major compound at 13.1 min with a phenol derivative profile (Table 1), 13C NMR dereplication with the alk(en)yl resorcinol_phenol DB5 rightly identified m-heptadecenylphenol80 (20, rank 1, score 0.87 (Figures SI- 37 and SI-38) already described in Cameroonian propolis.37 In fraction CG_F10, Triterpenes DB4 accurately highlighted dipterocarpol81 (25,  rank 1, score 0.93, MW 442, Figures SI-43 and SI-44) previously described in propolis from Thailand82 and Alk(en)yl resorcinol_phenol DB5 suggested 6-heptadecenylsalicylic acid (26, rank 1, score 0.92, MW 374, Figures SI-43 and SI-45). Regarding these two NPs,  their MW did not match with those determined by GC-MS. This could be explained by the high temperature used in the GC-MS  method: Compound 25 might lose a molecule of water through dehydration (m/z 424 [M  18]) while 26 might lose a molecule of carbon dioxide through decarboxylation (m/z 330 [M-44]). For the following fraction CG_F12, the dereplication process using Triterpenes DB4 suggested a lot of compounds with high scores. Using an MW filter at 442 Da, dipterocarp (25) already found in CG_F10 (rank 20, score 0.90, Figures SI-46 and SI-47) and 24-methylene cyclobutane-3β,26-diol83 (27, rank 3, score 0.97, MW filter at 456 Da, Figures SI-46 and SI-48) were identified. In fraction CG_F15, dammarenediol II (28) was rightly hypothesized by MixONat and Triterpenes DB4 using an MW filter at 444 Da (rank 1, score 1, Figures SI-49 and SI-50; GC-MS: m/z 426 [M-18]) and confirmed by 13C NMR data.84 Using the same approach, this latter fraction was shown to contain two other triterpenoids, i.e., mangiferin acid (29, rank 10, score 0.87, MW filter at 454 Da, Figures SI-49  and SI-51) and embryonic acid (30, rank 1, score 0.94, MW filter at 468 Da, Figures SI-49 and SI-52) already found in propolis from Nigeria40 and Brazil.85 These carboxylic acids were not detected by GC-MS because of their lack of volatility (intermolecular hydrogen bond due to carboxylic acid function). Likewise, in CG_F18, two other triterpenoid acids were identified, i.e., embolic acid (34, rank 1, score 0.87, MW filter at 470 Da, Figures SI-55 and SI-56) and mangerolic acid (35, rank 9, score 0.80, MW filter at 456 Da, Figures SI-55 and SI-57) also described by Silva et al. in Brazil.85 In the penultimate fraction CG_F16, 13C NMR dereplication  with Alk(en)yl resorcinol_phenol DB5 accurately suggested a resorcinolic lipid, i.e., heptadecenylresorcinol86 (31, rank 1, score 0.91, Figures SI-53 and SI-54) associated to two other resorcinol derivatives identified as pentadecenylresorcinol87 (32, MW 318) and pentadecylresorcinol88 (33, MW 320). All were previously isolated in Cameroonian37 and Mexican propolis.89 13C NMR data of 17–35  are available in the supporting information.

For this Congolese propolis sample, most triterpenoids as well as resorcinolic lipids might originate from M. indica. 83,89,90

3.4 Antioxidant and anti-AGE assessment

Evaluation of the antioxidant and anti-AGE activities of BC1, BC2,  and CG EEPs revealed that only BC1 EEP showed a good antioxidant activity (1,172 ± 97 μmol TE/g), twice higher than that of ethanolic rosemary extract (E392). This activity was about its high total phenolic content (297.0 ± 15.6 mg GAE/g) comparable to those of poplar-type EEPs mostly found in Europe, North America, or China.19 However, the chemical analysis of BC1 EEP revealed phenanthrenes and stilbenoid polyphenols, including combretastatin B-2 (7), a dihydro stilbene. Such derivatives have already been described by Inui et al. in 202158 in Senegalese propolis showing significant anti-inflammatory activity, with compounds 4, 6, and 7 being the most active. The dihydrophenanthrene 6-methoxycoelonin (3) has also been described as displaying a cytotoxic effect against five human cancer cell lines (786-0, MCF-7, Hep2, UACC-62, and NCI/ADR-RES) with remarkable activity against UACC-62 cells (IC50 2.59 μM).91 The phenanthrene 6 was also known for its moderate cytotoxic activity against KB, MCF-7, and K562 cells and its potent inhibitory activity on CDK1/cyclin B (IC50 0.07 μM).92 Dihydrophenanthrenes from Combretum species such as combretastatin were also previously described as cell growth inhibitors59: Four types of combretastatin can be distinguished, i.e., stilbene-type (combretastatin A), dihydro stilbene-type (combretastatin B), phenanthrene-type (combretastatin C), and cyclic macrolactone-type combretastatin (combretastatin D). Among them, combretastatin A (A-4, but also A-1 and A-2)  exhibit the highest anti-tumor activity.93,94 Consequently, it seems best to use such antioxidant propolis containing phenanthrene, dihydrophenanthrene, and combretastatin derivatives with great care. It confirms the importance of the preliminary determination of the chemical composition of propolis before its use in food and health products.

Among all propolis extracts used in this study, only BC2 EEP  exhibited a moderate anti-AGE activity (IC50 0.70 mg/ml) compared to S. japonicum ethanolic extract, well known for its high anti-AGE  activity19 (cf. Table SI.1). As expected, all purified flavanone derivatives showed a good activity (IC50 0.20–0.26 mM; Table SI-1) close to the reference value (Quercetin IC50 0.20 mM) except for 13 (IC50 0.60 mM). Higher anti-AGE activity was previously reported for a French EEP (IC50 0.05 mg/ml) with much lower levels of triterpene derivatives and high amounts of pinobanksine derivatives.19

In conclusion, in the present work, GC-MS or HPLC-DAD-MS  was first used to identify different classes of NPs and determine their MWs. Then 13C NMR-based dereplication using MixONat software with custom DBs allowed us to unambiguously characterize major NPs from fractions. In addition to major triterpenoids, BC1, a propolis sample originating from the center of Benin, exhibited an original composition with high levels of dihydrophenanthrene, phenanthrene,  and bis benzyl derivatives, some of which were identified for the first time in propolis. Some of these antioxidant polyphenols are probably cytotoxic and remind us of the need for systematic analysis of propolis before use in food and health products, especially for the lesser-known propolis from tropical areas. Among the other extracts belonging to Macaranga-type propolis, BC2, collected in the same area, contained prenyl and geranyl flavanones with anti-AGE activities. Propolis from Congo contained NPs most likely associated with the botanical source M. indica: cyclobutane-type triterpenoids and resorcinolic lipids. Further investigation will be performed to associate the phytochemical composition of Beninese and Congolese propolis samples with the local flora and to evaluate their anti-fungal or anti-bacterial properties.

ACKNOWLEDGMENT 

The authors thank Dr. Ingrid Freuze from the “Plateau Astral” at the Faculty of Sciences in Angers for LC-MS analyses.

ACKNOWLEDGMENTS OF FINANCIAL SUPPORT 

The authors thank the International Foundation of Science (IFS) (Grant I-3-E-5720-2) and the Committee for Scientific and Technological Cooperation (COMSTECH) for their financial support.

DATA AVAILABILITY STATEMENT 

All data marked SI are included in the supplementary information section.

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SUPPORTING INFORMATION

Additional supporting information can be found online in the Supporting Information section at the end of this article.

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