key: cord-0982028-lgl3iu4q
authors: Chouna, Hardo Suzy Donfack; Dize, Darline; Kagho, Donald Ulrich Kenou; Bankeu, Jean Jules Kezetas; Fongang, Yannick Stéphane Fotsing; Tali, Mariscal Brice Tchatat; Ponou, Beaudelaire Kemvoufo; Bitchagno, Gabin Thierry Mbahbou; Awantu, Angelbert Fusi; Tapondjou, Léon Azefack; Lenta, Bruno Ndjakou; Fekam, Fabrice Boyom; Sewald, Norbert; Ngouela, Silvère Augustin
title: Constituents from ripe figs of Ficus vallis-choudae Delile (Moraceae) with antiplasmodial activity
date: 2022-05-17
journal: Parasitol Res
DOI: 10.1007/s00436-022-07540-5
sha: f1ef36016bf706e79b35556ee4ff3be4743cc703
doc_id: 982028
cord_uid: lgl3iu4q

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 IC(50) 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 (IC(50) = 24.6 ± 1.5 μM) and Dd2 (IC(50) = 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 IC(50) values of 13.86 and 8.18 µg/mL, respectively. • Wighteone was the most active compound against PfDd2 (IC(50) = 11.9 ± 2.4 μM). GRAPHICAL ABSTRACT: [Image: see text] SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00436-022-07540-5.

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.

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.

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.

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.

The n-hexane fraction (FH) was subjected to column chromatography (CC) over silica gel (Merck, 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 subfractions 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). 

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.

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 (Bankeu et al. , 2017 Fongang et al. 2015) .

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.

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. 

Acetyl: 171.2 (C-1′), 21.5 (C-2′)

H-2), 2.23 (3H, s, CH 3 ); 13 C RMN (CD 3 OD): δ C (ppm) = 170.3 (COOH)

50 (1H, s, H-4), 4.42 (2H, s, CH 2 O); 13 C NMR (CD 3 OD, 150 MHz): δ C (ppm) =

C-12), 84.5 (C-3), 69.5 (C-2)

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′′)

White crystals from methanol

Synthesis of lupiwighteone via a para-Claisen-Cope rearrangement

Characterization of bioactive compounds from Ficus vallis-choudae delile (Moraceae)

Isoflvone dimers and other bioactive constituents from the figs of Ficus mucuso

Compounds from Syzygium aromaticum possessing growth inhibitory activity against oral pathogens

Compounds from the root of Pueraria peduncularis (Grah. ex Benth.) Benth. and their antitumor effects

Constituents of Peperomia vulcanica Baker & C

Piperaceae) with antiparasitic activity

Antiviral flavonoids from the root bark of Morus alba L

Prenylated flavonoids of Erythrina lysistemon grown in Egypt

Flufuran, an antifungal 3,5-disubstituted furan produced by Aspergillus flavus Link

Flavonoids and other bioactive constituents from Ficus thonningii Blume (Moraceae)

Emergence of indigenous artemisinin-resistant Plasmodium falciparum in Africa

Chemical studies on Sophora tomentosa: the isolation of a new class of isoflavonoid

A new furan derivative from an endophytic Aspergillus flavus of Cephalotaxus fortunei

A new prenyl isoflavone from Ulex jussiaei

Multitargeted activity of maslinic acid as an antimalarial natural compound

Isoflavonoids from the rhizomes of Iris hungarica and antibacterial activity of the dry rhizomes extract

Urease inhibitory isoflavonoids from different parts of Calopogonium mucunoides (Fabaceae)

Beta-amyrin and alpha-amyrin acetate isolated from the stem bark of Alstonia boonei display profound anti-inflammatory activity

Medicinal plants in Nigeria

Phytochemical and biological investigation of Erythrina variegate

Biogeographical and phylogenetic origins of African fig species (Ficus section Galoglychia)

Analysis of IR, NMR and antimicrobial activity of β-sitosterol isolated from Momordica charantia

Simple and inexpensive fluorescence-based technique for highthroughput antimalarial drug screening

Microbial glycosylation of daidzein, genistein and biochanin A: two new glucosides of biochanin A

Wighteone exhibits an antitumor effect against EGFR L858R/T790M mutation non-small cell lung cancer

A new tormentic acid derivative from Luehea divaricata Mart. (Tiliaceae)

Fruitiers sauvages d'Afrique espèces du Cameroun

Study of steroidal saponins in Dioscorea zingiberensis C.H. Wright. J Nat Products (India) 2, 123˗132. World Health Organization (2019) World malaria report

A new antioxidant compound from Capparis spinosa

Prenylated flavonoids, promising nutraceuticals with impressive biological activities

13 C NMR analysis of monodesmosidic saponins from Gomphrena macrocephala

Antiplasmodial and cytotoxic activities of flavonoids and arylbenzofuran derivatives from Morus mesozygia