Constituents from ripe figs of Ficus vallis-choudae Delile (Moraceae) with antiplasmodial activity

Abstract Ripe figs, barks, and wood of Ficus vallis-choudae are used in traditional medicine against several conditions including nausea and malaria. However, its use is still to be scientifically documented and validated. Hence, the aim of the present work was to evaluate the antiplasmodial activity of the dichloromethane-methanol (DCM-MeOH (1:1)) crude extract, their hexane, dichloromethane, ethyl acetate, and methanoli fractions, as well as the isolated chemical constituents. The chemical study of the DCM-MeOH (1:1) crude extract of F. vallis-choudae figs led to the isolation of fifteen (15) known compounds identified based on their spectroscopic data [one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR), mass spectrometry] and by comparison of these data with those reported in the literature. Some of the isolated compounds were assessed in vitro for their antiplasmodial activity against Plasmodium falciparum chloroquine-sensitive 3D7 (Pf3D7) and multidrug-resistant Dd2 strains. The dichloromethane fraction exhibited very good antiplasmodial activity against both strains with IC50 values of 13.86 μg/mL and 8.18 μg/mL, respectively. Among the tested compounds, wighteone (2) was the most active against P. falciparum 3D7 (IC50 = 24.6 ± 1.5 μM) and Dd2 (IC50 = 11.9 ± 2.4 μM) strains. The obtained results could justify the traditional uses of F. vallis-choudae against malaria. Wighteone appears to be the most active ingredient. However, further consideration of this compound as starting point for antimalarial drug discovery will depend upon its selectivity of action towards Plasmodium parasites. Highlights • 15 (fifteen) compounds were isolated from the dichloromethane-methanol extract of Ficus vallis-choudae. • Their structures were determined on the basis of their spectroscopic data. • 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 compound against PfDd2 (IC50 = 11.9 ± 2.4 μM). Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00436-022-07540-5.


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 vallischoudae 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 vallischoudae. 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.

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 F 254 ), 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 CH 2 Cl 2 -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.

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 nonlinear regression and dose-response curves were drawn to determine the inhibitory concentration that reduces parasite viability by 50% (IC 50 ).
The degree of resistance was determined by comparison of the antiplasmodial IC 50 of inhibitors on the chloroquinesensitive and multi-resistant strains of P. falciparum using the following formula: RI = IC 50 multidrug-resistant strain/IC 50 chloroquinesensitive strain.

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 IC 50 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 (IC 50 = 11.9 ± 2.4 μM) and Pf3D7 (IC 50 = 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.

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 IC 50 values of 13.86 and 8.18 µg/mL, respectively. Wighteone was the most active against PfDd2 (IC 50 = 11.9 ± 2.4 µM) and Pf3D7 (IC 50 = 24.6 ± 1.5 µM). Wighteone and maslinic acid might be suggested as the backbone of the antiplasmodial potency of Ficus vallischoudae. However, their safety towards normal mammalian cells should be demonstrated.