Chemical constituents of two Cameroonian medicinal plants: Sida rhombifolia L. and Sida acuta Burm. f. (Malvaceae) and their antiplasmodial activity

Abstract An extensive phytochemical investigation of the EtOH/H2O (7:3) extracts of Sida rhombifolia L. and Sida acuta Burm. f., yielded a previously undescribed ceramide named rhombifoliamide (1) and a xylitol dimer (2), naturally isolated here for the first time, as well as the thirteen known compounds viz, oleanolic acid (3), β-amyrin glucoside (4), ursolic acid (5), β-sitosterol glucoside (6), tiliroside (7), 1,6-dihydroxyxanthone (8), a mixture of stigmasterol (9) and β-sitosterol (10), cryptolepine (11), 20-Hydroxyecdysone (12), (E)-suberenol (13), thamnosmonin (14) and xanthyletin (15). Their structures were elucidated by the analyses of their spectroscopic and spectrometric data (1 D and 2 D NMR, and HRESI-MS) and by comparison with the previously reported data. The crude extracts, fractions, and some isolated compounds were tested against chloroquine-sensitive (3D7) and chloroquine-resistant (Dd2) Plasmodium falciparum strains. All the tested samples demonstrated moderate and/or significant activities against 3D7 (IC50 values: 0.18―20.11 µg/mL) and Dd2 (IC50 values: 0.74―63.09 µg/mL).


Introduction
Malaria is a parasitic disease caused by a protozoan of the genus Plasmodium asexually replicating inside the human body and transmitted from mosquitos to humans during a blood meal. Despite the continuous evolution of drug search, malaria is the deadliest and most dangerous parasitic infection in Sub-Saharan Africa, making the search for new antimalarial drugs an imperative of the overall health goal and probably one of the greatest public health challenges facing humanity (Gontijo et al. 2019). Despite extensive control efforts, the incidence of the disease is not decreasing and constitutes a major public health issue, principally in developing countries. According to the latest World malaria report, released on 30 November 2020, around 229 million malaria cases were reported compared to 228 million cases in 2018 where children under 5 years of age are the most vulnerable group affected by malaria; in 2019 and accounting for 67% (274 000) of all malaria deaths worldwide (WHO. 2020). Responsible for 99.7% of malaria cases in 2017, Plasmodium falciparum is the most prevalent malaria parasite in Sub-Saharan Africa. Until the development of an effective vaccine, chemotherapy remains a major frontline strategy for the control and future elimination of malaria. The current chemotherapy against malaria relies on Artemisinin Combination Therapy (WHO 2020). Plants of the genus Sida (Malvaceae) are widely used in indigenous communities for their nutritive values and for treating various ailments such as gonorrhoea, piles, rheumatism, gastrointestinal infections, varicella, variola, and malaria (Gupta et al. 2009). Sida genus is one of the most diverse in the Malvaceae family, with about 200 species distributed worldwide (Brandao et al. 2017). Phytochemical and pharmacological studies performed on some species have led to the identification of antibacterial lipid compounds from S. cordata Burm. f. and S. acuta Burm. f. (Adindu and Oguzie 2017), alkaloids with anti-inflammatory and antiparasitic potential from S. rhombifolia L. and S. cordifolia L. (Chaves et al. 2017;da Rosa et al. 2018). Polyphenols, triterpenes, and steroids have also been identified (da Rosa et al. 2015;Mah et al. 2017;Kumar et al. 2019). Currently, there are various empiric formulations based on Sida species (e.g., S. acuta Burm. f., S. cordifolia L. and S. rhombifolia L.) for the treatment of neurological and rheumatic problems, and which also act as antimalarial drugs (Rodrigues and Oliveira 2020) but little is known about their chemical composition. In our continuous search for bioactive compounds from Cameroonian medicinal plants (Fotso et al. 2017, Mbougnia et al. 2020, we have carried out the chemical and biological study of two Cameroonian medicinal plants: S. rhombifolia L. and S. acuta Burm. f. The choice of these plants was motivated by the fact that they are traditionally used to treat malaria, but no antiplasmodial constituent has been described from them to date. We herein report the isolation and structure elucidation of a new ceramide named rhombifoliamide (1) and a xylitol dimer (2), naturally isolated for the first time, together with thirteen known compounds as well as their antiplasmodial activity.

Results and discussion
2.1. Isolation and structure elucidation S. rhombifolia whole plant was extracted using the mixture of EtOH/H 2 O (7:3, v/v). It is worth noting that this solvent system was chosen firstly because the hydroethanolic extract displayed the best antiplasmodial activity after micro-extraction compared to the DCM-MeOH extract and secondly because of the use of white wine in traditional medicine for plant maceration. The resulting crude extract was subjected to repeated silica gel and Sephadex LH-20 column chromatography (CC) to afford a previously undescribed ceramide named rhombifoliamide (1, 10.4 mg), together with eight known compounds viz, oleanolic acid (3, 6.2 mg) , b-amyrin glucoside (4, 7.8 mg) (Alam et al. 2012), ursolic acid (5, 4.3 mg) ), b-sitosterol glucoside (6, 9.7 mg) , tiliroside (7, 8.1 mg) (Danielly et al. 2007), 1,6-dihydroxyxanthone (8, 6.4 mg) (Lien Do et al. 2020), mixture of stigmasterol (9) and b-sitosterol (10) ) and 20-hydroxyecdysone (12, 2.2 mg) (da Rosa et al. 2018). Similarly, S. acuta whole plant was extracted using the same solvent mixture. The crude extract obtained was subsequently purified using the above-mentioned chromatographic techniques to yield a xylitol dimer (2, 9.3 mg), naturally isolated for the first time, together with five known secondary metabolites namely: cryptolepine (11, 3.9 mg) (Banzouzi et al. 2004 (Figure 1). The structures of the known compounds were identified by comparison of their spectroscopic and spectrometric data with those reported in the literature.
Compound 1 was obtained as a white powder. Its molecular formula C 43 H 85 NO 5 was established from its HRESI-MS spectrum ( Figure S1), showing the pseudo-molecular ion peak [M þ H] þ at m/z 696.6506 (C 43 H 86 NO 5 þ ; calcd. 696.6501), indicating two degrees of unsaturation. Its IR spectrum ( Figure S2) showed characteristic absorption bands for free OH groups (3329-3215 cm À1 ) and an amide group (1620 cm À1 ) (Yue et al. 2001;Wonkam et al. 2020). The structure of 1 was fully assigned after careful analyses of its 1 H, 13 C, 1 H-1 H COSY, HMQC, HMBC, tandem MS spectra and methanolysis reaction ( Figure S3-S12, Table 1). Indeed, the 1 H NMR spectrum of 1 ( Figure S4) in conjunction with 13 C-NMR DEPT 135 spectra and HSQC ( Figure S5-S7) displayed a set of signals characteristic of a ceramide as described by Simo et al. 2008. This was confirmed by the signals of the carbonyl of an amide at d C 175.4 and the signal of a nitrogen-attached sp 3 carbon at d C 51.5. Specifically, the NC-H proton appeared at d H/C 4.03(1H, m)/51.5 while the broad signal centered at d H 1.18 was attributed to the methylene protons of the aliphatic long chain; a distorted triplet at d H 0.79 (6H, t, 6.9) characterized the two terminal methyl groups. In addition, the spectrum displayed two diastereotopic protons of an oxymethylene at d H/C 3.72 (1H, dd, 4.6, 11.5, H-1a)/ 60.9 and 3.66 (1H, dd, 4.6, 11.4, H-1b)/60.9 as well as three oxymethine protons at d H / d C 3.46/75.3 (C-3), 3.45/72.1 (C-4) and 3.95/71.8 (C-2 0 ) respectively. Correlations between these protons were observed on the 1 H-1 H COSY spectrum ( Figure S8). In addition, the presence of a signal at d  by the methanolysis using 0.9 N, HCl/MeOH, at 70 C for 20 H to yield the fatty acid methyl ester (1a) and the sphingosine (1 b) (Simo et al. 2008 ( Figure S3). Specifically, the peak at m/z 216.2 [M þ H] þ corresponding to molecular formula C 18 H 38 NO 3 þ was attributable to the long chain base (1 b) and implying one degree of unsaturation. Furthermore, this molecular formula of sphingosine suggested that the olefinic moiety is located in the long chain base (LCB). In the HMBC spectrum of compound 1 ( Figure  S9), 2 J correlations were observed between the olefinic proton at d H 5.32 and carbon C-12 (d C 32.5); H-15 at d H 1.21 with C-16 carbon (d C 31.8). Finally, H-17 at d H 1.20 correlates in 3 J with C-18 carbon corresponding to the terminal methyl. All of these correlations ( Figure S11) made it possible to locate the double bond at D 10 on the long basic chain. This information was confirmed by the ESI-MS/MS spectrum ( Figure S12a and S12b) on which the ions peaks [M þ H-C 9 H 17 ] þ at m/z 571.5 and [M þ H-C 7 H 15 ] þ at m/z 619.5 corresponding to the allylic cleavages of the double bond, respectively for C 9 -C 10 and C 11 -C 12 were observed ( Figure S10). The trans configuration of the C ¼ C bond was evident from the chemical shifts of the allylic C-atoms at d C 32.5 and 32.0, which should have been less than 29.0 ppm if the configuration was cis (Simo et al. 2008). In addition, the absolute configurations at C(2), C(3), C(4), and C(2 0 ) were determined as (S), (S), (R), and (R) according to biogenetic consideration and previously reported data (Ishii et al. 2006, Wonkam et al. 2020. Therefore, the structure of 1 was unambiguously determined as (2S,2 0 R,3S,4R,10E)-N-[2 0 -hydroxypentacosanoyl]-2-amino-octadec-10-ene-1,3,4-triol, to which the trivial name rhombifoliamide was given.

Antiplasmodial and cytotoxicity activities
Crude extracts, fractions and isolated compounds were screened for their antiplasmodial and cytotoxicity activities using SyBr Green-Based assay and resazurin-based assay respectively. Results showed that, extracts and fractions exhibited moderate to strong antiplasmodial activities against 3D7 (IC 50 values: 0.18-20.11 mg/mL) and Dd2 (IC 50 values: 0.74-63.09 mg/mL) Plasmodium falciparum strains. Interestingly, two compounds, oleanolic acid (3) and cryptolepine (11) isolated from the EtOAc-soluble fraction of S. rhombifolia and S. acuta displayed strong antiplasmodial activity with IC 50 of (3.56 ± 0.62 and 2.02 ± 0.27) for 3; (0.18 ± 0.01and 0.74 ± 0.09) for 11 respectively against 3D7 and Dd2 P. falciparum strains (Table S1). b-amyrin glucoside (4) and tiliroside (7) showed moderate activity only against the multidrug resistant (Dd2) and no activity against sensitive strain of P. falciparum. Except for cryptolepine (11), all extracts, fractions and compounds with activity against asexual P. falciparum parasites exhibited no cytotoxicity against Raw cells (selectivity indices (SI) > 10. To the best of our knowledge, this study provides the first report of antiplasmodial activity of isolated oleanolic acid against chloroquine-sensitive (3D7) and chloroquine-resistant (Dd2) P. falciparum strains. However, previous studies showed that the strong antiplasmodial activity of dichloromethane twig extract of keetia leucantha is attributed to the presence of eight triterpenic esters and the major antiplasmodial triterpenic acids, ursolic and oleanolic acids identified by HPLC-UV methods (Beaufay et al. 2017). In addition, cryptolepine previously showed varied interaction with the 4-aminoquinolines, amodiaquine, and chloroquine. The combination of cryptolepine with amodiaquine showed a synergistic effect in vitro (mean RFIC ¼ 0.235 ± 0.15), whereas an additive effect (mean RFIC ¼ 1.342 ± 0.34) have been seen with chloroquine (Forkuo et al. 2016). Additionally, cryptolepine has already been reported to show high inhibitory activity against the late-stage gametocytes (IC 50 ¼ 1965 nM) (Forkuo et al. 2016) of P. falciparum (NF54). Summing-up, these studies report the good potential of cryptolepine as a promising antimalarial hit for both malaria treatment and transmission-blocking therapy (Forkuo et al. 2016). Based on the pronounced antiplasmodial activity of this compound, further chemical studies such as structural-activity relationship and/or medicinal chemistry are needed to obtain a lead compound that responds to pharmacokinetics and pharmacodynamics properties for antimalarial drugs. All experiments were performed in triplicate and the main results obtained are recorded in  Table S1.

Supplementary material
Experimental section, NMR, and MS data of compounds 1 are available alongside Figures S1-S12.