General instrumentation

Instruments used during this study for isolation and analyses of the isolated compounds can be found in the supplementary information (Appendix A ).

Plant material

The stem bark and the roots of F. sycomorus were collected at Malentouen (GPS coordinates: 5°30′20″N, 10°00′00″E, Elevation 1100 m), a locality of Foumban, West region, Cameroon, in January 2017. Mr Victor Nana, a botanist of the National Herbarium of Cameroon, Yaounde, made the identification of the plant and a specimen has been kept under the voucher number 13750/HNC.

Extraction and isolation

The air-dried and powdered stem bark (2.10 kg) and roots (2.00 kg) of F. sycomorus were separately macerated twice in methanol for 48 h and 24 h, respectively. The crude extracts of stem bark (200.55 g) and roots (34.55 g) were obtained after removing the solvent. The purification processes of the two extracts were separately conducted and led to the isolation of 24 distinct compounds as summarized in Figure 1 and Figure 2.

Figure 1

Summarized protocol for isolation of compounds from F. sycomorus bark

Figure 2

Summarized protocol for isolation of compounds from F. sycomorus root

Briefly, part of the stem bark extract (195.00 g) was submitted to flash chromatography using EtOAc, EtOAc/MeOH (70:30, v/v) and MeOH as solvents, to afford EtOAc fraction (25.15g); EtOAc/MeOH fraction (80.05 g) and MeOH fraction (85.10 g). A part of the EtOAc fraction (22.15 g) was chromatographed on silica gel column chromatography with an increasing amount of EtOAc in n-Hexane (Hex) from 5% to 100% (v/v). A total of 232 fractions (100 ml each) have been collected and combined on the basis of TLC analyses into seven sub-fractions (F1‒F7). Sub-fraction F1 [225.55 mg, 5% of EtOAc in Hex (v/v)] was purified on silica gel column chromatography, with a gradient solvent system starting by 2.5 % of EtOAc in Hex (v/v) and gave compounds 14 (8.5 mg), the mixture of 2 and 4 (20.1 mg) and 8 (12.8 mg), then, with the solvent system 5% of EtOAc in Hex (95:5, v/v) to give compound 9 (13.5 mg). The mixture of 12 and 15 (20.0 mg) precipitated from F2 [135.10 mg, Hex–EtOAc (90:10, v/v)] purified following elution with an isocratic solvent system of Hex–EtOAc (92.5:7.5, v/v). Following the same process, the sub-fraction F3 [134.50 mg, Hex–EtOAc (85:15, v/v)] gave compounds 1 (8.2 mg) and 10 (10.5 mg). The mixture of compounds 3 and 5 precipitated from sub-fraction F4 [76.55 mg, Hex–EtOAc (80:20, v/v)] while compounds 11 (9.1 mg), 21 (11.5 mg) and the mixture of 23 and 24 (21.5 mg). Were obtained from the sub-fraction F5 [126.50 mg, Hex–EtOAc (75:25, v/v)]. Furthermore, compound 22 (5.8 mg) was obtained from sub-fraction F6 [90.50 mg, Hex–EtOAc (70:30, v/v)] and compound 16 (80.7 mg) precipitated from F7 [95.55 mg, Hex– EtOAc (25:75, v/v)].

Likewise, part of the methanol crude extract of roots (30.00 g) was fractionated following the same procedure as performed with the stem bark extract. Therefore, the EtOAc fraction from the root gave six fractions (F1'‒F6'). From purification using silica gel column chromatography as described earlier, Fraction F1' [105.50 mg, Hex–EtOAc (90:10, v/v)] afforded compound 12 (7.2 mg), 13 (7.6 mg), 9 (10.1 mg) and 7 (13.8 mg). Fraction F2' [200.50 mg, Hex–EtOAc (85:15, v/v)] led to the obtention of compounds 17 (14.6 mg), 18 (8.56 mg), 19 (13.4 mg) and 20 (25.20 mg), while fraction F3' [155.50 mg, Hex–EtOAc (80:20, v/v)] led to compounds 3 (9.2 mg) and 5 (12.3 mg). Compounds 23 (15.5 mg) and 24 (25.3 mg) were obtained after elution of the fraction F4' [99.50 mg, Hex–EtOAc (75:25, v/v)] and compounds 6 and 16 were obtained from Fraction F5' [90.50 mg, Hex–EtOAc (70:30, v/v)] and the recrystallization of F6' [75.50 mg, Hex–EtOAc (40:60, v/v)], respectively.

Spectral data of isolated compounds

The ¹H and ¹³C NMR spectra (Figures 1S‒45S, Appendix A) as well as the full assignations (Tables 1S‒24S, Appendix A) of all the carbon and hydrogen atoms of the isolated compounds 1‒24 are provided in the supplementary information attached to this paper (Appendix A ).

Antimicrobial and cytotoxicity evaluations

Some compounds obtained in sufficient amounts for biological tests have been evaluated for their antibacterial, antifungal and cytotoxicity potencies. The full protocol for each test can be consulted in the supplementary material (Appendix A ). For instance, eight bacterial strains namely Salmonella typhimurium cpc, S. enteritidis cpc, S. typhi cpc, Staphylococcus aureus MR, S. aureus (ATCC25922), Klebssiella pneumonae (ATCC13883), Escherishia coli (ATCC35218), Pseudomonas aeruginosa ; seven fungal strains including Candida albicans, C. krusei, C. parasilosis, Cryptococcus neoformans, Trichophyton mentagrophytes, Microsporium audouinii, Epidermophyton flocosum as well as the VERO cell line ATCC CRT-1586 have been used during the three biological evaluations.

The antibacterial assay has been done using the broth microdilution method was used for susceptibility testing of bacteria species in 96 well-microtiter sterile plates as described by (Newton, 2002). The lowest concentration at which no visible colour change was observed was considered the Minimum Inhibitory Concentration (MIC) while the smallest concentration at which no colour change was observed was considered the Minimum Bactericidal Concentration (MBC). The tests were performed in duplicates. The ratio MBC/MIC was calculated to determine the bactericidal (MBC/MIC ≤ 4) and bacteriostatic (MBC/MIC > 4) effects (Mativandlela, Lall, & Meyer, 2006).

In antifungal activity evaluation, the MIC of each sample was determined by using broth microdilution techniques according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI, formerly National Committee for Clinical and Laboratory Standards, NCCLS) for yeasts (M27-A2). MIC values were assessed visually after the corresponding incubation period and were taken as the lowest product concentration at which there was no growth or virtually no growth, while the lowest concentration that yielded no growth after the subculturing was taken as the Minimum Fungicidal Concentration (MFC). The assay was repeated three times.

Finally, cytotoxicity activity was investigated on VERO cell line ATCC CRT-1586 using a rezasurin-based assay as previously described by Mosmann (1983). The results were expressed as a percentage of viability of the control cells and CC₅₀ values were calculated as a sigmoidal dose-response curve using GraphPad Prism 4.03 software using the nonlinear regression log (inhibitor) vs. response algorithm.

Phytochemical study

The chemical investigations of the stem bark and roots of the medicinal plant Ficus sycomorus led to the isolation and identification of twenty-four distinct compounds (Figure 3) including ten triterpenoids belonging to five different classes including one taraxastane-type triterpenoid named epi-ψ-taraxastanonol or 3-oxo-20R-hydroxytaraxastane (1) (Anjaneyulu et al., 1999), two ursane-type triterpenoids α-amyrin acetate (2) and ursolic acid (3) (Tabekoueng et al., 2020; Wouamba et al., 2020), three oleanane-type triterpenoids β-amyrin acetate (4), oleanolic acid (5) and 2-O-trans-p-coumaroyl maslinic acid (6) (Yan et al., 2014), one friedelane-type triterpenoid named canophyllol (7) (Ngouamegne et al., 2008), as well as three lupane-type triterpenoids lupeol (8), betulinic acid (9) and lupeol acetate (10) (Javed et al., 2021; Mbougnia et al., 2021). Additionally to the ten triterpenoids, we isolated one diterpenoid called ent-kauran-2β,3α,16α-triol (11) (Dongmo et al., 2019); five steroids including stigmasterol (12), stigmast-22-ene-3,6-dione (13) (Lima, Diaz, & Diaz, 2013), stigmast-7-en-3-one (14) (Wu et al., 1990), β-sitosterol (15) and β-sitosterol-3-O-β-D-glucopyranoside (16) (Mbougnia et al., 2021); four flavonoids among which three isoflavones namely alpinumisoflavone (17) (Kuete et al., 2008), derrone (18) (Chibber & Sharma, 1980) and 3'-(3-methylbut-2enyl)-biochanin A (19) (Abiy, Jacob, Mathias, & Martin, 1998), as well as one flavone named atalantoflavone (20) (Nsangou et al., 2021). Further compounds have been characterized as one benzoquinone identified as 2,6-dimethoxybenzoquinone (21) (Dongmo et al., 2019); one phenylethanol derivative named 2-(4-hydroxyphenyl)-ethyldocyoctadecanoate (22) (Acevedo et al., 2000) and two peptide derivatives viz. asperphenamate (23) and asperglaucide (24) (Popwo et al., 2019).

Figure 3

Structures of compounds 1–24 from stem bark and roots of F. sycomorus.

The results showed that the plant produces chemical constituents with a high structural diversity from terpenoid derivatives (triterpenoids and steroids) identified as the major classes of compounds to flavonoids and other phenolic compounds. The significant change observed in the core structures from one compound to another is mainly oxidation of some functional groups and it might play an important role in their potencies and the understanding of their action mechanisms (Dzouemo, Happi, Happi, Tsopgni, & Wansi, 2022; Happi et al., 2020).

Antimicrobial and cytotoxicity activities of the isolated compounds

Eleven compounds (1‒4, 10, 13, 14, 17‒19 and 22), the crude methanolic extracts of stem bark and roots as well as the EtOAc fraction from stem bark have been tested for their potency against eight bacterial strains and seven fungal strains as well as for their cytotoxicity against the VERO cell line ATCC CRT-1586.

The antibacterial assays have been carried out against bacterial strains listed in section 2.4 and ciprofloxacin was the standard drug. The results (Table 1) indicated that compound 18 showed good activity against Escherichia coli (MIC = 31.25 µg/ml) and moderate potency against Salmonella typhimurium cpc and Pseudomonas aeruginosa (MIC = 125 µg/ml, each). Compounds 10, 13 and 14 showed moderate activities on almost all the tested strains and compound 13 was the most potent against E. coli (MIC = 125 µg/ml). All the two crude extracts demonstrated moderate activities against almost all the tested strains (MIC = 250 µg/ml, MBC/MIC = 2) on S. enteritidis and E. coli. These results showed that the other compounds in the extracts might play an antagonistic effect on compound 18 (MIC = 31.25 µg/ml) which is the most active we found so far in this study.

Furthermore, the results of the antifungal activity (Table 2) showed that the methanol extract of the stem bark exhibited good activity against Candida albicans with a MIC value of 31.5 µg/ml and a fungicidal effect with an MFC/MIC ratio of 4. Almost all the isolated compounds showed weak activity against some yeast and dermatophytes with MIC values from 125 µg/ml to 500 µg/ml and fungicidal with an MFC/MIC ratio of either 2 or 4. The good activity of the methanol extract from the stem bark may justify its use in folk medicine for the treatment of infectious illnesses.

Finally, the samples were evaluated for their cytotoxic activity on VERO cell line ATCC CRT-1586. Their potencies are reported in Table 3. According to the results, all the tested extracts showed cell viability greater than 100 μg/ml, indicating that they have no risk for living cells. Compounds 18 and 19 showed the higher inhibition of cell lines with CC₅₀ values of 52.60 μg/ml and 69.10 μg/ml, respectively, indicating that they have a risk for living cells.

Chemophenetic significance of the study

In the present work, we have reported the identification of twenty-four known extractives from F. sycomorus, including ten pentacyclic triterpenoids (1‒10), one diterpenoid (11), five sterols (12–16), four flavonoids (17‒20), one benzoquinone (21), one phenyl ethanol derivative (22) and two peptide derivatives (23–24). Based on an extensive literature survey, and to the best of our knowledge, compounds 6, 11 and 22 are reported herein for the first time from Moraceae family and compound 20 from Ficus genus. However, the four compounds have been already isolated from other plants species including Hippophae rhamnoides L. (Elaeagnaceae) (Yang et al., 2007), Psiadia punctulata (DC.) (Asteraceae) (Piaz et al., 2018) and Buddleja cordata (Scrophulariaceae) (Acevedo et al., 2000), respectively.

Pentacyclic triterpenes (2–9) constituted the main class of secondary metabolites herein reported, which were already obtained from several Ficus species. Thus, compounds 2‒5 and 7 were reported from F. aripuanensis (Nascimento, Arruda, Arruda, Muler, & Yoshiok, 1999), compound 8 from F. natalensis (Mbougnia et al., 2021), F. benjamina (Simo et al., 2008), F. pseudopalma (Santiago & Mayor, 2014), F. polita (Kamga et al., 2010) and F. auriculata (El-Fishawy, Rawia, & Sherif, 2011). Moreover, compound 9 was reported from F. natalensis (Mbougnia et al., 2021), F. benjamina (Simo et al., 2008), F. polita (Kamga et al., 2010), F. auriculata Lour (El-Fishawy et al., 2011) and F. conraui (Kengap et al., 2011), while compound 10 was reported from F. racemosa (Kosankar & Aher, 2018), F. sansibarica (Awolola et., 2014), F. thonningii  (Ango et al., 2015) and F. mucuso (Bankeu et al., 2010). From this evidence, we can partially conclude that the genus Ficus is a rich source of different classes of triterpenoids including ursane-, lupane- and oleanane-type triterpenoids as the most prominent in the genus.

Steroids 12 and 14‒16 are the main constituents of many species and were reported from many Ficus species (Awolola, Chenia, Baijnath, & Koorbanally, 2018). They were also reported from another family including Cola rostrata (Malvaceae) (Dongmo et al., 2019). Flavonoids are also highly represented in Ficus genus. Compounds 17 and 18 were reported from many Ficus species e.g., F. nymphaefolia (Darbour et al., 2007) and F. auriculata (Qi et al., 2018) and compound 19 was reported from F. tikoua (Liao et al., 2015) and (Darbour et al., 2007). These isoflavones were also reported from Erythrina species (Fabaceae) e.g., E. senegalensis (Lee et al., 2009; Nkengfack et al., 2001) and E. lysistemon (Mvondo, Njamen, Fomum, Wandji, & Vollmer, 2011). Compound 20 was newly discovered from Ficus genus, it could be used to distinguish F. sycomorus from the other species. Compounds 23 and 25 were previously reported from Ficus exasperata (Popwo et al., 2019).