Introduction

Malaria is an endemic disease in most tropical regions of Africa, Asia, and Latin America and its incidence have been increasing these recent times due to drug resistance and the emergence of the Covid-19 pandemic. An estimated 228 million cases of malaria occurred worldwide in 2018 with the largest burden of morbidity (93%) in Africa (WHO 2019). Chemotherapy remains a key component of malaria control and elimination strategies. Artemisinin-based combination therapy was the most commonly used drug-based therapy between 2010 and 2018 in African public health sectors (WHO 2019), but the high cost and the low availability in rural areas is still an important health problem. The emergence of artemisinin-resistant Plasmodium falciparum strains in Africa turned the situation worse (Huayun et al. 2017). Hence, understanding and controlling the spread of antimalarial resistance, particularly to artemisinin and its partner drugs remains a top priority. Therefore, alternative antimalarial drugs need to be developed, and medicinal plants are known as promising sources of potential antimalarial compounds.

The genus Ficus (Moraceae) is one of the largest genera of angiosperms with more than 800 species of trees, shrubs, hemiepiphytes, climbers, and creepers in the tropics and subtropics worldwide. It is an important genetic resource due to its high economic and nutritional values and also an important part of the biodiversity in the rainforest ecosystem. Ficus species are also a good source of food for fruit-eating animals in tropical areas (Rønsted et al. 2007). Ficus vallis-choudae Dellile (synonyms Ficus schweinfurthii Miquel) is a shrub or small tree up to 8 m tall. It is distributed in tropical Africa from Senegal to Cameroon, from Sudan to Ethiopia and Malawi (Vivien and Faure 1996). Fruits, bark, and wood are the most used parts of Ficus vallis-choudae against several conditions including nausea and malaria (Oliver 1960). Several phytochemical and pharmacological surveys were already done worldwide on different Ficus species. However, only a few research studies were conducted on Ficus vallis-choudae. Our research group has previously reported the isolation of one ceramide, steroids, and triterpenoids from the methanolic extract of the figs of this plant as well as the α-glucosidase inhibitory activity and DPPH radical scavenging potency (Bankeu et al. 2017). However, to provide scientific validation to the traditional use of this plant to treat malaria, in-depth investigation of stem bark and other parts of Ficus vallis-choudae for antiplasmodial was required. In the present article, we report the antiplasmodial activity of the dichloromethane-methanol extract F. vallis-choudae figs, its hexane, dichloromethane, ethyl acetate, and methanolic fractions, as well as the isolated chemical constituents.

Materials and methods

General experimental procedures

The used equipments were those reported by Chouna et al (2021). Column chromatography (CC) was performed on silica gel (230–400 mesh). Fractions were monitored by thin-layer chromatography (TLC) using Merck precoated silica gel sheets (60 F254), and the identification of spots on the TLC plate was carried out by spraying with sulfuric acid reagent solution and heating the plate to about 80 °C.

Plant material

The figs of Ficus vallis-choudae Dellile were collected in May 2019 at Egona II (Center region of Cameroon). Mr. NANA Victor, botanist at the National Herbarium, Yaoundé, by comparison with the existing voucher specimen No 5115/HNC4, identified the plant material.

Extraction and isolation

The plant material (1.5 kg) was air-dried, pulverized, and extracted three times with 4 L of CH2Cl2-MeOH (1:1) mixture at room temperature (24℃) (each time for 24 h). The solvent was evaporated under reduced pressure to afford 119.1 g of crude extract. A part of the extract (118.1 g) was partitioned with n-hexane, dichloromethane (DCM), ethyl acetate, and methanol to afford fractions of 72.9, 6.5, 11.1, and 25.6 g, respectively.

The n-hexane fraction (FH) was subjected to column chromatography (CC) over silica gel (Merck, 230–400 mesh) eluting with the mixtures of n-hexane–EtOAc (1:0 → 0:1) and EtOAc/MeOH (1:0 → 0:1) with increasing polarities. Fractions of 300 mL each were collected and combined according to their TLC profiles to afford five major sub-fractions labeled FH1–FH5. Sub-fraction FH1 (10.5 g) was submitted to CC over silica gel using n-hexane–EtOAc (1:0 → 4:6) as solvent systems to yield 7 (50.3 mg) and 8 (55.2 mg). Compound 14 (40 mg) crystallized in sub-fraction FH2 (9.7 g) and was filtered off by simple filtration. Sub-fraction FH3 (13.9 g) was submitted to purified CC over silica gel eluted with n-hexane–EtOAc (1:0 → 2:8) to afford compounds 5 (9.2 mg), 4 (8.7 mg), and 11 (25.1 mg). The purification of sub-fraction FH4 (22.8 g) over silica gel CC using n-hexane–EtOAc (1:0 → 2:8) as elution solvents yielded compounds 2 (20.5 mg), 3 (5.4 mg), and 1 (6.9 mg). The sub-fraction FH5 (15.4 g) was subjected to CC with n-hexane–EtOAc (6:4 → 0:1) and EtOAc/MeOH (1:0 → 85:15) over silica gel to afford compounds 6 (5 mg) and 15 (30 mg).

Likewise, the DCM fraction (6.5 g) was subjected to the CC over silica gel (Merck, 230–400 mesh) eluting with n-hexane–EtOAc (1:0 → 0:1) and EtOAc/MeOH (1:0 → 0:1) systems of increasing polarities. Fractions of 300 mL each were collected and combined according to their TLC profiles to afford four major sub-fractions labeled (FD1–FD4). The sub-fraction FD1 (1.43 g) was further subjected to CC over silica gel and eluted with n-hexane–EtOAc (1:0 → 0:1) to afford compound 9 (5.4 mg). Further CC of the sub-fraction FD2 (3.21 g) with n-hexane–EtOAc (8:2 → 0:1) afforded compounds 10 (20.8 mg) and 12 (6.2 mg). The sub-fraction FD3 (1.12 g) was subjected to CC over silica gel with n-hexane–EtOAc (6:4 → 0:1) to yield compound 13 (15.7 mg).

Physical and spectroscopic data of the isolated compounds

5,7,4′-trihydroxyisoflavone (Genistein) (1): Yellow powder from n-hexane–EtOAc (1:1), mp 289–291 °C [Literature (Lit.) 290–292 °C (Mykhailenko et al. 2017)], 1H NMR (CD3OD, 600 MHz): δH (ppm) = 8.08 (1H, s, H-2), 7.40 (2H, d, J = 8.9 Hz, H-2′/6′), 6.87 (2H, d, J = 8.9 Hz, H-3′/5′), 6.37 (1H, d, J = 2.2 Hz, H-8), 6.25 (1H, d, J = 2.2 Hz, H-6); 13C NMR (CD3OD, 150 MHz): δC (ppm) = 182.6 (C-4), 166.0 (C-7), 163.9 (C-5), 159.8 (C-9), 158.8 (C-4′), 154.8 (C-2), 131.4 (C-2′/6′), 123.8 (C-3), 123.3 (C-1′), 116.2 (C-3′/5′), 106.3 (C-10), 100.1 (C-6), 94.8 (C-8).

Wighteone (Erythrinin B) (2): Yellow powder from n-hexane–EtOAc (4:6), mp 218–220 °C [Lit. 219–220 °C (Kinoshita et al. 1990)], 1H NMR (CD3OD, 600 MHz): δH (ppm) = 13.03 (1H, s, HO-5), 7.98 (1H, s, H-2), 7.28 (2H, d, J = 8.5 Hz, H-2'/6'), 6.74 (1H, d, J = 8.5 Hz, H-3′/5′), 6.31 (1H, s, H-8), 5.13 (1H, m, H-2′′), 3.21 (2H, d, J = 7.3 Hz, H-1′′), 1.67 (3H, d, J = 1.5 Hz, 5′′-CH3), 1.56 (3H, d, J = 1.5 Hz, 4′′-CH3); 13C NMR (CD3OD, 150 MHz): δC 182.0 (C-4), 163.4 (C-5), 160.4 (C-7), 158.8 (C-4′), 157.3 (C-9), 154.6 (C-2), 132.2 (C-3′′), 131.5 (C-2′/6′), 124.3 (C-3), 123.5 (C-2′′), 123.4 (C-1′), 116.3 (C-3′/5′), 112.7 (C-6), 106.1 (C-10), 94.0 (C-8), 26.2 (C-4′′), 22.4 (C-1′′), 18.2 (C-5′′).

Lupiwighteone (3): Yellow powder from n-hexane–EtOAc (4:6), mp 133–135 °C [Lit. 133–134 °C (Al-Maharik and Botting 2003)], 1H NMR (CD3OD, 600 MHz): δH (ppm) = 8.16 (1H, s, H-2), 7.40 (2H, d, J = 8.9 Hz, H-2′/6′), 6.87 (2H, d, J = 0.8.9.Hz, H-3′/5′), 6.30 (1H, s, H-6), 5.23 (1H, m, H-2′′), 3.44 (2H, d, J = 7.1 Hz, H-1′′), 1.88 (3H, d, s,H-5′′), 1.70 (3H, d, s, H-4′′); 13C NMR (CD3OD, 150 MHz): δC (ppm) = 182.6 (C-4), 163.3(C-7), 161.4 (C-5), 158.8 (C-4′), 156.9 (C-9), 154.8 (C-2), 132.4 (C-3′′), 131.4 (C-2′/6′), 124.4 (C-1′), 123.4 (C-3), 123.4 (C-2′′), 116.2 (C-3′/5′); 107.9 (C-8), 106.3 (C-10), 99.6 (C-6), 25.9 (C-4′′), 22.3 (C-1′′), 17.9 (C-5′′).

Derrone (4): Yellow powder from n-hexane–EtOAc (3:7), mp 178–179 °C [Lit. 179–181 °C (Máximo et al. 2002)], 1H NMR (CDCl3, 600 MHz): δH (ppm) = 12.89 (1H, s, HO-5), 7.88 (1H, s, H-2), 7.38 (2H, d, J = 8 Hz, H-2′/6′), 6.88 (1H, d, J = 8 Hz, H-3′/5′), 6.68 (1H, J = 10.0 Hz, H-4′′), 6.33 (1H, s, H-6), 5.60 (1H, d, J = 10.0 Hz, H-3′′), 5.15 (1H, s, HO-4′), 1.48 (6H, s, CH3); 13C NMR (CDCl3, 150 MHz): δC (ppm) = 181.2 (C-4), 162.4 (C-5), 159.7 (C-7), 156.1 (C-4'), 152.4 (C-9), 130.5 (C-2′/6′), 127.6 (C-3′′), 123.8 (C-3), 123.1 (C-1′), 115.8 (C-3′/5′), 114.7 (C-4′′), 106.2 (C-10), 100.5 (C-6), 101.3 (C-8), 78.3 (C-2′′), 28.4 (C-5′′/6′′).

Alpinumisoflavone (5): Yellow powder from n-hexane–EtOAc (7:3), mp 212–214 °C [Lit. 213–214 °C (Ndemangou et al. 2013)], 1H NMR (CDCl3, 600 MHz): δH (ppm) = 13.18 (1H, s, HO-5), 7.82 (1H, s, H-2), 7.39 (2H, d, J = 8 Hz, H-2′/6′), 6.89 (1H, d, J = 8 Hz, H-3'/5'), 6.73 (1H, J = 10.0 Hz, H-4′′), 6.33 (1H, s, H-8), 5.62 (1H, d, J = 10.0 Hz, H-3′′), 5.15 (1H, s, HO-4′), 1.47 (6H, s, CH3); 13C NMR (CDCl3, 150 MHz): δC (ppm) = 181.1 (C-4), 159.7 (C-7), 157.5 (C-9), 157.0 (C-5), 156.0 (C-4′), 130.5 (C-2′/6′), 128.4 (C-4′′), 123.7 (C-3), 123.3 (C-1′), 115.7 (C-3′/5′), 115.6 (C-3′′), 106.3 (C-10), 105.8 (C-6), 96.0 (C-8), 78.2 (C-2′′), 28.5 (C-5′′/6′′).

Oleanolic acid-28-O-β-D-glucopyranosyl ester (6): white amorphous powder from n-hexane–EtOAc (3:7), 13C NMR (CDCl3, 150 MHz): Aglycone: δC (ppm) = 174.5 (C-28), 140.5 (C-13), 122.3 (C-12), 79.7 (C-3), 56.3 (C-5), 50.3 (C-9), 48.0 (C-9), 47.2 (C-19), 46.0 (C-18), 42.5 (C-17/14), 39.0 (C-8), 38.6 (C-4), 38.5 (C-1), 36.9 (C-10), 34.4 (C-21), 34.1 (C-22), 31.9 (C-7), 31.8 (C-29), 29.9 (C-20), 29.1 (C-23), 27.4 (C-2/15), 23.9 (C-27), 22.8 (C-11/16), 21.0 (C-6), 19.8 (C-30), 19.5 (C-26), 14.3 (C-25), 11.8 (C-24), Glucose: δC (ppm) = 94.0 (C-1′), 76.5 (C-3′), 76.4 (C-5′), 72.2 (C-2′), 69.6 (C-4′), 61.3 (C-6′).

β-palmitate (7): white amorphous powder from n-hexane–EtOAc (98:2), 13C NMR (CDCl3, 150 MHz): δC (ppm) = 145.5 (C-13), 121.8 (C-12), 80.7 (C-3), 55.4 (C-5), 47.7 (C-9), 47.4 (C-18), 46.9 (C-19), 41.9 (C-14), 40.0 (C-8), 38.4 (C-1), 37.9 (C-4), 37.8 (C-22), 37.3 (C-10), 34.8 (C-21), 33.5 (C-29), 32.7 (C-17 and C-20), 32.6 (C-7), 28.5 (C-28), 28.2 (C-23), 27.1 (C-2), 26.3 (C-15 and C-16), 26.1 (C-27), 23.8 (C-11 and C-30), 18.4 (C-6), 17.0 (C-26),16.8 (C-24), 15.7 (C-25); Palmitoyl: 173.9 (C-1′), 31.2 (C-2′), 29.3–29.9 (C-4′-14′), 25.3 (C-3′), 22.8 (C-15′), 14.3 (C-16′).

β-Amiryn acetate (8): white amorphous powder from n-hexane–EtOAc (97:3), 13C NMR (CDCl3, 150 MHz): δC (ppm) = 145.4 (C-13), 121.8 (C-12), 81.1 (C-3), 55.4 (C-5), 47.7 (C-9), 47.4 (C-18), 46.9 (C-19), 41.9 (C-14), 38.4 (C-1), 38.4 (C-4/8), 37.3 (C-22), 37.3 (C-10), 34.8 (C-21), 33.5 (C-29), 32.6 (C-7), 32.7 (C-20), 31.2 (C-17), 28.2 (C-24/28), 27.1 (C-2/16), 26.3 (C-15), 26.1 (C-27), 23.7 (C-30), 23.6 (C-11), 18.4 (C-6), 16.9 (C-26), 16.7 (C-25), 15.7 (C-23); Acetyl: 171.2 (C-1′), 21.5 (C-2′).

Cappariside (9): white amorphous powder from n-hexane–EtOAc (7:3), 1H NMR (CD3OD, 600 MHz): δH (ppm) = 7.87 (1H, s, H-2), 2.23 (3H, s, CH3); 13C RMN (CD3OD): δC (ppm) = 170.3 (COOH), 151.8 (C-4), 145.8 (C-3), 142.8 (C-5), 140.4 (C-2), 14.8 (CH3).

5-Hydroxymethylfuran-3-carboxylic acid (10): white powder from n-hexane–EtOAc (1:1), mp 144–146 °C [Lit. 145–147 °C (Ma et al. 2015)], 1H NMR (CD3OD, 600 MHz): δH (ppm) = 7.96 (1H, s, H-2), 6.50 (1H, s, H-4), 4.42 (2H, s, CH2O); 13C NMR (CD3OD, 150 MHz): δC (ppm) = 176.9 (COOH), 170.4 (C-5), 147.4 (C-3), 141.0 (C-2), 110.7 (C-4), 61.2 (CH2OH).

Oleanolic acid (11): White crystals from methanol, mp 304–306 °C [Lit. 303.306 °C (Cai and Wu 1996)].

Maslinic acid (12): 13C NMR (CD3OD, 150 MHz): white amorphous powder from n-hexane–EtOAc (1:1), δC (ppm) = 180.9 (C-28), 144.8 (C-13), 123.4 (C-12), 84.5 (C-3), 69.5 (C-2), 56.7 (C-5), 48.9 (C-9), 48.1 (C-1), 47.7 (C-17), 47.3 (C-19), 42.9 (C-14), 42.8 (C-18), 40.5 (C-4), 39.3 (C-8/10), 34.6 (C-21), 33.8 (C-22), 33.6 (C-29), 33.9 (C-7), 31.6 (C-20), 29.3 (C-23), 28.8 (C-15), 24.1 (C-11), 24.0 (C-16), 23.7 (C-27/30), 19.6 (C-6), 17.8 (C-26), 17.4 (C-25), 17.1 (C-24).

Erythrinin C (13): Yellow cristals from n-hexane–EtOAc (7:3), 1H NMR (CDCl3, 150 MHz): Yellow amorphous powder, 1H NMR (DMSO-d6, 400 MHz): δH (ppm) = 13.18 (1H, s, 5-OH), 9.64 (1H, s, 4′-OH), 8.10 (1H, s, H-2), 7.38 (2H, d, J = 8.5 Hz, H-2′/6′), 6.84 (2H, d, J = 8.5 Hz, H-3′/5′), 6.43 (1H, s, H-8), 4.81 (2H, m, H-2′′), 3.10 (2H, d, J = 8.5 Hz, H-3′′), 1.31 (3H, s, H-6′′), 1.26 (3H, s, H-5′′); 13C NMR (DMSO-d6, 100 MHz): δC (ppm) = 182.3 (C-4), 168.1 (C-7), 159.5 (C-5), 158.7 (C-9), 157.9 (C-4′), 93.1 (C-2′′), 154.9 (C-2), 131.4 (C-2′/6′), 124.8 (C-3), 123.1 (C-1′), 116.1 (C-3′/5′), 110.8 (C-6), 107.1 (C-10), 89.9 (C-8), 72.2 (C-4′′), 27.4 (C-3′′), 25.3 (C-6′′), 23.5 (C-5′′).

β-Sitosterol (14): White crystals from methanol, mp: 134–135 °C [Lit. 136–137 °C (Sen et al. 2012)].

β-Sitosterol-3-O-β-D-glucopyranoside (15): White crystals from methanol, mp: 290‒292 °C [Lit. 290‒291 °C (Wang et al. 2009)].

Antiplasmodial assay

Chloroquine-sensitive (Pf3D7) and multidrug-resistant (PfDd2) strains of P. falciparum were continuously cultured as reported by Chouna et al. (2021). The previously SYBR green I-based fluorescence method described by Smilkstein et al. (2004) was used to evaluate the antiplasmodial activity. The growth inhibition percentage for each test sample was determined using Microsoft Excel and data analysis was performed with GraphPad Prism 5.0 fitting by non-linear regression and dose–response curves were drawn to determine the inhibitory concentration that reduces parasite viability by 50% (IC50).

The degree of resistance was determined by comparison of the antiplasmodial IC50 of inhibitors on the chloroquine-sensitive and multi-resistant strains of P. falciparum using the following formula:

RI = IC50 multidrug-resistant strain/IC50 chloroquine-sensitive strain.

Results and discussion

Phytochemical analysis

The dichloromethane-methanol (1:1) crude extract of the figs of Ficus vallis-choudae was repeatedly subjected to silica gel column chromatography (CC) to yield fifteen compounds identified as 5,7,4′-trihydroxyisoflavone (1) (Sordon et al. 2017); wighteone (2) (Kinoshita et al. 1990); lupiwighteone (3) (Al-Maharik and Botting 2003); derrone (4) (Máximo et al. 2002); alpinumisoflavone (5) (Rahman et al. 2007; El-Masry et al. 2002); oleanolic acid-28-O-β-D-glucopyranosyl ester (6) (Young et al. 1997); β-amyrin palmitate (7) (Bankeu et al. 2017); β-amyrin acetate (8) (Du et al. 2003; Okoye et al. 2014); cappariside (9) (Yang et al. 2010); 5-hydroxymethylfuran-3-carboxylic acid (10) (Evidente et al. 2009); maslinic acid (12) (Tanaka et al. 2003); erythrinin C (13) (Chen et al. 2019). The remaining compounds were identified by co-TLC with authentic samples and melting points measurement as oleanolic acid (11) (Cai and Wu 1996), β-sitosterol (14) (Sen et al. 2012), and 3-O-β-D-glucopyranoside of β-sitosterol (15) (Wang et al. 2009) (Fig. 1). The isolation of isoflavonoids, triterpenes, and steroids is in agreement with the previous results obtained from Ficus species (Bankeu et al. 2011, 2017; Fongang et al. 2015).

Fig. 1
figure 1

Sructures of compounds 1–15

Full size image

Antiplasmodial activity

The crude extract, fractions, and isolated compounds were assessed in vitro for their antiplasmodial activity against chloroquine-sensitive (Pf3D7) and multidrug-resistant (PfDd2) strains of P. falciparum. Out of the tested fractions, the dichloromethane fraction (FVFD) exhibited good antiplasmodial activities on the Pf3D7 and PfDd2 with IC50 values of 13.86 and 8.18 µg/mL, respectively (Table 1). Four compounds (9, 10, 12, 13) were isolated through its repeated purification. The multi-targeted activity of maslinic acid (12) as an antimalarial natural compound was previously demonstrated (Moneriz et al. 2011), and might justify the activity observed for FVFD. However, the low amount of 12 isolated in this work did not allow the evaluation of its antiplasmodial activity against both P. falciparum strains. Compound 2 (wighteone) was the most active against PfDd2 (IC50 = 11.9 ± 2.4 μM) and Pf3D7 (IC50 = 24.6 ± 1.5 μM). However, a recent report indicated that wighteone inhibits cell proliferation, suppressed EGFR signalling pathway, caused cell cycle redistribution and induced cell apoptosis, and could provide a novel potential therapeutic strategy for NSCLC patients with T790M mutation (Sun et al. 2021). For further consideration of this compound (2) as starting point for antimalarial drug discovery, cytotoxicity assessment using normal mammalian cells is required. The main structural difference between 1 and 2 is the presence of a prenyl group at C-6 of 2, thus suggesting that the prenyl moiety may contribute to the high activity of 2. This result corroborates previous findings as prenyl moiety including isoflavonoids have been reported to considerably increase various biological activities such as antiplamodial, antifungal, and anticancer potencies of secondary metabolites (Zelefack et al. 2012; Yang et al. 2015). These results could support the traditional use of Ficus vallis-choudae for the treatment of malaria. Wighteone, maslinic acid, and genistein may be eventually qualified as antiplasmodial active principles of this medicinal plant.

Table 1 Antiplasmodial activity of compounds and fractions from the dichloromethane-methanolic extract of F. vallis-choudae
Full size table

Conclusion

Fifteen (15) known compounds including six isoflavonoids, five triterpenoids, two steroids, and two furan derivatives were isolated from the dichloromethane-methanol (1:1) crude extract of Ficus vallis-choudae figs. This is the first report of the isolation of these compounds (excepted 7, 11, and 14) from Ficus vallis-choudae. The dichloromethane fraction showed promising activities on the Pf3D7 and PfDd2 strains with IC50 values of 13.86 and 8.18 µg/mL, respectively. Wighteone was the most active against PfDd2 (IC50 = 11.9 ± 2.4 µM) and Pf3D7 (IC50 = 24.6 ± 1.5 µM). Wighteone and maslinic acid might be suggested as the backbone of the antiplasmodial potency of Ficus vallis-choudae. However, their safety towards normal mammalian cells should be demonstrated.