Antiplasmodial and Antileishmanial Activities of a New Limonoid and Other Constituents from the Stem Bark of Khaya senegalensis

Plasmodium falciparum and Leishmania sp. resistance to antiparasitic drugs has become a major concern in malaria and leishmaniasis control. These diseases are public health problems with significant socioeconomic impacts, and mostly affect disadvantaged populations living in remote tropical areas. This challenge emphasizes the need to search for new chemical scaffolds that preferably possess novel modes of action to contribute to antimalarial and antileishmanial research programs. This study aimed to investigate the antimalarial and antileishmanial properties of a methanol extract (KS-MeOH) of the stem bark of the Cameroonian medicinal plant Khaya senegalensis and its isolated compounds. The purification of KS-MeOH led to the isolation of a new ordered limonoid derivative, 21β-hydroxybourjotinolone A (1a), together with 15 known compounds (1bc–14) using a repeated column chromatography. Compound 1a was obtained in an epimeric mixture of 21α-melianodiol (1b) and 21β-melianodiol (1c). Structural characterization of the isolated compounds was achieved with HRMS, and 1D- and 2D-NMR analyses. The extracts and compounds were screened using pre-established in vitro methods against synchronized ring stage cultures of the multidrug-resistant Dd2 and chloroquine-sensitive/sulfadoxine-resistant 3D7 strains of Plasmodium falciparum and the promastigote form of Leishmania donovani (1S(MHOM/SD/62/1S). In addition, the samples were tested for cytotoxicity against RAW 264.7 macrophages. Positive controls consisted of artemisinin and chloroquine for P. falciparum, amphotericin B for L. donovani, and podophyllotoxin for cytotoxicity against RAW 264.7 cells. The extract and fractions exhibited moderate to potent antileishmanial activity with 50% inhibitory concentrations (IC50) ranging from 5.99 ± 0.77 to 2.68 ± 0.42 μg/mL, while compounds displayed IC50 values ranging from 81.73 ± 0.12 to 6.43 ± 0.06 μg/mL. They were weakly active against the chloroquine-sensitive/sulfadoxine-resistant Pf3D7 strain but highly potent toward the multidrug-resistant PfDd2 (extracts, IC50 2.50 ± 0.12 to 4.78 ± 0.36 μg/mL; compounds IC50 2.93 ± 0.02 to 50.97 ± 0.37 μg/mL) with selectivity indices greater than 10 (SIDd2 > 10) for the extract and fractions and most of the derived compounds. Of note, the limonoid mixture [21β-hydroxylbourjotinolone A (1a) + 21α-melianodiol (1b) + 21β-melianodiol (1c)] exhibited moderate activity against P. falciparum and L. donovani. This novel antiplasmodial and antileishmanial chemical scaffold qualifies as a promising starting point for further medicinal chemistry-driven development of a dually active agent against two major infectious diseases affecting humans in Africa.


Introduction
Secondary metabolites play an important role in the identification of plants through chemotaxonomy, and exhibit a panel of bioactivities.Secondary metabolites are reported from soil, animals, fungi, bacteria, and green plants, and they are grouped into several classes and subclasses, such as terpenoids, flavonoids, and polyketides.The presence, type, and abundance of these compounds are usually related to the origin, species, and environment where the studied samples were collected.The search for secondary metabolites began when scientists examined the factors responsible for the sweetness or bitterness of fruit [1,2].Limonoids are a subclass of well-known triterpenoids in the plant kingdom, and are among the secondary metabolites responsible for the bitter taste of fruit.The occurrence of limonoids in the plant kingdom is limited to plants of the Rutales order, and they are most abundant in the Rutaceae and Meliaceae families [1,3].They are less abundant in the Simaroubaceae and Cneoraceae families [2].The structural modification of Rutaceae limonoids is usually restricted to the modification of A and B rings, while Meliaceae limonoids are the most complex limonoids of the Rutale order with a high oxidation degree [2,3].Limonoids are usually divided into two main groups, of which the first group contains a moiety of a well-known triterpenoid called an ordered limonoid, while the second group is called the degraded (modified) triterpenoid without a fixed skeleton.Both groups of limonoids are highly oxygenated with a skeleton of 4,4,8-trimethyl-17furanylsteroid or a protolimonoid as a prototypical precursor [2,4].Almost all naturally reported limonoids contain either a furan or a pyran ring linked to the D-ring at C-17, and the preferable oxygenated carbons are C23, C21, C-17, C-16, C-7, C-4, C-3, and C1 [2,[5][6][7][8].Limonoids exhibit a panel of biological activities, such as insect antifeedant, and insecticidal, antimalarial, antimicrobial, antiviral, and anticancer activities [2,3,9].
Khaya senegalensis (Desr.) A. Juss.(Meliaceae) is a plant species known as Caïlcédrat (French).K. senegalensis is a tree of approximately 25 m in height whose leaves, fruit, and stems are largely used by many African tribes in Cameroon and Nigeria for the treatment of malaria, headache, and yellow fever [9][10][11][12].Previous chemical and biological studies of different parts of K. senegalensis have revealed the occurrence of flavonoids, alkaloids, triterpenoids, steroids, and limonoids with cytotoxic, antimicrobial, hepatoprotective, antiviral, and antioxidant activities [2,9,[11][12][13].Limonoids are reported to be the most abundant and chemophenetic taxon of K. senegalensis as well as the Khaya genus [3,13].In our search for bioactive compounds from medicinal plants growing in Cameroon [11,14,15], we have undertaken a chemical investigation of the methanol extract of the stem bark of K. senegalensis.This study aimed to investigate the antimalarial and antileishmanial properties of the extract and fractions of the stem bark of the Cameroonian medicinal plant Khaya senegalensis as well as its isolated compounds.This investigation led to the isolation of 16 secondary metabolites (1abc-14).All of the isolated compounds were characterized by NMR and MS techniques, and compound 1a was characterized as an unprecedentedly ordered limonoid-type bourjotinolone.The chemophenetic aspect was also discussed.In addition, the methanolic crude extract, the EtOAc and n-hexane fractions and the isolated compounds (1abc-14) were subjected to antiplasmodial, antileishmanial, and cytotoxicity testing.

Results
A mass of 275.0 g of the MeOH extract of K. senegalensis was partitioned into n-hexane (15.6 g) and EtOAc (160.8 g) fractions.Repeated column chromatography with Sephadex as well as silica gel as the stationary phase of the EtOAc (160.0 g) and n-hexane (15.0 g) extracts led to the isolation of 16 secondary metabolites, including nine triterpenoids (1abc-7), four steroids (11)(12)(13)(14), two flavonoids (8)(9) and one ceramide (10) (Figure 1).led to the isolation of 16 secondary metabolites (1abc-14).All of the isolated compounds were characterized by NMR and MS techniques, and compound 1a was characterized as an unprecedentedly ordered limonoid-type bourjotinolone.The chemophenetic aspect was also discussed.In addition, the methanolic crude extract, the EtOAc and n-hexane fractions and the isolated compounds (1abc-14) were subjected to antiplasmodial, antileishmanial, and cytotoxicity testing.

Structure Elucidation of Compound 1
Compound 1 was isolated as a shiny white powder whose molecular formula was established as C30H48O5; HR-ESI-MS (positive) m/z 511.3394 [M + Na] + ; calcd.m/z 511.3399 (for C30H48O5Na + ), indicating seven double bond equivalents (Figure S1).The UV spectrum (Figure S2) of compound 1 showed absorption maxima at 268 and 365 nm, while its IR spectrum (Figure S3) revealed a broad absorption band ranging from 3669 to 3200 cm −1  S1).The UV spectrum (Figure S2) of compound 1 showed absorption maxima at 268 and 365 nm, while its IR spectrum (Figure S3) revealed a broad absorption band ranging from 3669 to 3200 cm −1 attributable to hydroxyl groups and other important absorption bands at 1697 and 1488 cm −1 assigned to carboxyl and olefinic functions, respectively.

Discussion
Parasitic diseases, including malaria and leishmaniasis, represent a public health challenge, particularly for poor populations living in developing countries.Although significant progress has been made through the development of control methods such as chemotherapy, the impact of these diseases remains in the countries concerned, causing enormous socioeconomic losses [41,42].This is due in particular to the drawbacks associated with approved drugs, including toxicity, duration of treatment regimens, and the spreading resistance of parasites to first-line drugs [42,43].For several decades, medicinal plants and natural products have played a key role in the discovery of lead compounds, which have been used to advance the discovery and development of drugs against infectious diseases.In fact, 20 antiparasitic agents were approved by the Food and Drug Administration between 1981 and 2019, among which nine (45%) were derived from natural products [44].Moreover, in the case of malaria, the two famous compounds that have positively revolutionized the management of this disease are quinine and artemisinin isolated from Cinchona sp.stem bark and Artemisia annua, respectively [45,46].This supports the hypothesis that remarkable discoveries can be made from medicinal plants for the identification of new scaffolds against infectious diseases, including malaria and leishmaniasis.
The goal of this study was to search for bioactive secondary metabolites from the stem bark of K. senegalensis (Meliaceae) that are endowed with antiplasmodial and antileishmanial activities.The methanol extract (KS-MeOH) of K. senegalensis showed moderate antiplasmodial activity against Pf 3D7, a chloroquine-sensitive strain, but exhibited a more promising effect against multidrug-resistant Pf Dd2 and Leishmania donovani promastigotes (Table 2, Figure 3).A similar trend was observed with the Sudanese methanolic extract from K. senegalensis stem bark against Pf 3D7 and Pf Dd2, with IC 50 values of 25 µg/mL and 150 µg/mL, respectively [47].Moreover, two other distinct studies have previously demonstrated the antiplasmodial activity of extracts from this plant species against Pf 3D7 and Pf W2; however, the bioactive compounds were not reported [48,49].With regard to its antileishmanial properties, very limited studies have reported the activity of K. senegalensis against Leishmania sp.parasites [50,51].In addition, the extract KS-MeOH was shown to be selective against Pf Dd2 (SI > 19) and L. donovani (SI > 9.28) but not against Pf 3D7 compared to the cytotoxic effect on RAW 264.7 cell lines.Climate change, geographical location, and harvesting period can modify the qualitative and quantitative content of secondary metabolites in medicinal plants and consequently their biological activities [52].This further suggests that KS-MeOH preferentially inhibits Pf Dd2 and Leishmania parasites rather than mammalian cells.Recently, plants from the Meliaceae family have been recognized as a vast source of lead candidates for the development of antiplasmodial and insecticidal drugs [53].Therefore, our phytochemical investigation of the antiprotozoal activity of Khaya senegalensis confirmed the activities previously reported [52].The subsequently afforded hexane (KS-Hex) and ethyl acetate (KS-EtOAc) fractions from the KS-MeOH extract displayed the same antileishmanial, antiplasmodial, and selectivity profiles as the crude KS-MeOH extract.It can be alluded that the potent activity of KS-MeOH crude extract and fractions KS-Hex and KS-EtOAc (Pf Dd2 IC 50 2.5-4.78µg/mL, SI 10.59-19.93;L. donovani IC 50 2.68-5.99µg/mL, SI 8.45-30.12) is consequential of synergistic interactions between limonoids and eventually other secondary metabolites that are present in the extracts.
Compounds 1b, 1c, and 10 are reported for the first time from the Khaya genus, while compound 1a is an unprecedented compound and reported for the first time from the Meliaceae family.Compound 1 (1abc), a mixture of three limonoids (hydroxybourjotinolone A, 21α-melianodiol, and 21β-melianodiol), displayed moderate activity against Pf 3D7 (IC 50 84.3µg/mL), promising activity against both Pf Dd2 (IC 50 27.38 µg/mL), high activity against Leishmania donovani (IC 50 14.31µg/mL), and high selectivity (SI > 10) for all strains.This is particularly relevant, as previous research has revealed antiplasmodial and antileishmanial activities for compound 7α-obacunyl acetate (IC 50 5.14 µg/mL) and trigilgianin (IC 50 6.044µg/mL), which are two limonoids isolated from Trichilia gilgiana (Meliaceae) [62] and Entandrophragma angolense (Meliaceae), respectively [62].Previous studies have shown that melanodiol is an anticancer [62], antioxidant, anti-inflammatory [63], and antibacterial agent [64].The presence of such compounds with different properties could therefore explain the good activity profile obtained with this mixture.The results once again highlight the extensive antiparasitic properties of secondary metabolites belonging to the limonoid class.It is also important to point out that limonoids are reported to be the most abundant and best chemophenetic markers of the Meliaceae family as well as the K. senegalensis species [1,3,8,11,65].This study therefore agrees with the classification of this plant species and improves knowledge of the classes of secondary metabolites found in K. senegalensis as well as their biological properties.

General Experimental Procedures
An alpha spectrometer (Brüker) spectrophotometer was used to record the IR spectrum in KBr.For NMR analysis, samples were first dissolved (85 µL) in their dissolving solvent before introduction into Eurisotop tubes (ref.D048T) and then analysis with Brüker Avance II+ at 500 MHz and 600 MHz equipped with a TCI Cryoprobe working under TopSpin 3.2.5.The mass spectrometry (ESI-MS, positive mode) of the samples was performed on a pneumatically assisted API (Atmospheric Pressure Ionization) mass spectrometer (Waters) type SYNAPT G2 HDMS.HRESIMS were measured with a TOF (Time-of-flight) analyzer.Silica gel (0.063-0.200 mm) was used as the stationary phase for CC (chromatographic column) for the fractionation and purification of compounds.TLC and UV (CN-6 UV, 254 nm & 365 nm) were used to check the purity of the isolated compounds.Ceric sulfate and 20% sulfuric acid were used to visualize the spotted samples on TLC after heating at approximately 90 • C. Iodine vapor was also used for visualization.

Plant Material
The stem bark of Khaya senegalensis was harvested on 15 September 2019, at Tchatibali Village (14 • 5 0 E) in the Mayo-Danay Division of the Far-North Region of Cameroon.The plant material was taken to the National Herbarium of Cameroon, where it was identified in comparison with an available voucher specimen deposited under registration number 56853/HNC.

Extraction and Isolation
The fresh stem bark of K. senegalensis was collected and allowed to dry at room temperature, then powdered (7.5 kg) and extracted for 3 days (72 h) with 22 L MeOH, leading to 500.7 g of MeOH crude extract.A total of 275.0 g of the MeOH crude extract was subjected to liquid-liquid extraction to afford 15.6 g of n-hexane (KS-Hex), 160.8 g of EtOAc (KS-EtOAc), and 10.1 g of MeOH residue.
A T-75 cm 2 cell culture flask was filled with complete Medium 199 [M199 supplemented with 10% heat-inactivated fetal bovine serum (HIFBS) and 100 IU/mL penicillin/streptomycin (Sigma, Darmstadt, Germany)] and used to grow the promastigote forms of Leishmania donovani.Every 72 h, flasks were subcultured and incubated at 28 • C.
The murine macrophage RAW 264.7 cell line was grown in DMEM (Dulbecco's modified Eagle's medium) containing 1% penicillin-streptomycin, 1% nonessential amino acids (NEA), and 10% HIFBS at 37 • C, and 5% CO 2 in a humidified environment.A reverse Etaluma fluorescence microscope was used to view the cells every day, and the growth media was replaced every 72 h.At 80% confluence, cells were isolated using 0.25% Trypsine-EDTA (Gibco, MT, USA).

Sample Preparation for Biological Assays
In 100% dimethyl sulfoxide (DMSO, Sigma Aldrich), stock solutions of crude extracts, fractions, and compounds were prepared for final concentrations of 100 mg/mL, 50 mg/mL, and 20 mg/mL, respectively.Chloroquine phosphate (CQ) (Sigma Aldrich) and artemisinin (Art) (Sigma Aldrich) were used as antiplasmodial reference medicines, and were prepared at 10 mM in sterile distilled water and 100% DMSO, respectively.For antileishmanial tests, amphotericin B (10 mM in 100% DMSO, Sigma Aldrich) was included in the experiment as a positive control.

Antiplasmodial Activity of Compounds, Fractions and Extracts
The antiplasmodial efficacy of the samples was evaluated using the SYBR Green I-based fluorescence assay, as previously reported by Smilkstein et al. [66], on a sorbitolsynchronized ring stage culture of Plasmodium falciparum (Pf Dd2 and Pf3D7 strains).The test relies on the development of intense fluorescence following SYBR green binding to the exposed DNA found in wells containing healthy cells.Ninety microliters of parasites prepared with 1% hematocrit and 2% parasitemia and 10 µL of test sample extracts, fractions, isolated chemicals, chloroquine, and artemisinin were each added in triplicate into wells of a flat-bottomed microplate.The final test concentration values for the samples varied from 50 µg/mL-0.08µg/mL and 100 µg/mL-0.16µg/mL for extract/fractions.Following a 72 h incubation at 37 • C, 100 µL of SYBR Green I solution prepared with lysis buffer (0.2 µL of 10,000 SYBR Green I per mL of lysis buffer) made of EDTA (5 mM), Tris (20 mM; pH 7.5), Triton X-100 (0.08%; v/v), and saponin (0.008%; m/v) was added to each well.Fluorescence measurements were taken using an Infinite M200 microplate reader (Tecan, MT, USA) at emission and excitation wavelengths of 538 and 485 nm, respectively.

Antileishmanial Potency of Compounds, Fractions, and Extracts
The resazurin-based assay was used to evaluate the antileishmanial activity of the extract (100 µg/mL-0.16µg/mL), major fractions (100 µg/mL-0.16µg/mL), and isolated compounds (50 µg/mL-0.08 µg/mL) against the promastigote form of Leishmania donovani [67].Briefly, 10 µL of inhibitors at various triplicate doses (as prescribed for the antiplasmodial assay) were added to 90 µL of promastigotes from a logarithmic phase culture at a density of 4.10 5 parasites/mL.The plates were incubated for 28 h at 28 • C, and 10 µL of resazurin solution (Sigma, Darmstadt, Germany) was added.Amphotericin B (Sigma, Darmstadt, Germany) (10 µg-0.016µg/mL) and 0.1% DMSO-treated wells were used as the negative and positive controls, respectively.After a further 44 h of incubation, plates were read using a Magelan Infinite M200 fluorescence plate reader (Tecan, Männedorf, Switzerland) at excitation and emission wavelengths of 530 and 590 nm, respectively.

Cytotoxicity Assay
The murine macrophages, RAW 264.7 cells, were plated into a 96-well cell culture treated flat-bottomed plate (SARSTEDT, Inc., Newton, NC 28658, USA) at a density of 10 × 10 4 cells/100 µL/well and incubated overnight to enable cell adhesion.A fresh complete medium containing serially diluted concentrations of crude extract, fractions, and compounds was used to replace the previous medium.The final assay concentration values for extracts/fractions and compounds varied from 400 µg/mL to 0.8 µg/mL and 100-0.16µg/mL, respectively.Podophyllotoxin (Sigma-Aldrich, Munich, Germany) was used as a positive control at a maximum dose of 10 µM.After 48 h of incubation, 10 µL of resazurin solution (0.15 mg/mL dissolved in PBS) was added to each well, and the plates were incubated for an additional 4 h [68].Fluorescence data were finally recorded with a Magellan Infinite M200 plate reader (Tecan, Germany) at excitation and emission wavelengths of 530 nm and 590 nm, respectively.

Data Analysis for the Performed Assays
The resulting fluorescence readings were used to calculate the growth inhibition percentages relative to the negative control for each sample and for each of the tested doses.GraphPad Prism 8.0 software was used to generate concentration-response curves from the nonlinear regression fit to determine the median inhibitory or cytotoxic concentration (IC 50 /CC 50 ).The resistance index (RI) for antiplasmodial tests was calculated as the ratio between the samples' IC 50 values for the chloroquine-sensitive strain 3D7 and the multiresistant strain Dd2 [69].Inhibitors with an RI value below 1 were considered to act preferentially on the resistant strain.Based on each inhibitor's antiparasitic efficacy (IC 50 ) and cell toxicity (CC 50 ), selectivity indices (SI) were calculated [SI = CC 50 (RAW)/IC 50 (Parasites)].Samples were classified as poorly toxic to RAW cells if their SI values were greater than 10 [70].

Conclusions
The chemical investigation undertaken on the stem bark of K. senegalensis led to the isolation of a new limonoid named 21β-hydroxybourjotinolone A (1a) in a C-21 epimeric mixture of 21α-melianodiol (1b) and 21β-melianodiol (1c), together with 13 known compounds.Compounds 2, 3-9, and 11-14 have previously been reported from K. senegalensis; while 1b, 1c, and 10 are reported for the first time from the Khaya genus, and 1a is reported for the first time from the Meliaceae family.The mixture of bourjotinolone (1a) and 21α/β-melianodiol (1b/1c) was found to possess cross activity against L. donovani and P. falciparum, and a good selectivity profile toward the parasitic strains.Other compounds displayed moderate to high antiparasitic activity and selectivity, qualifying them as interesting starting points for novel antiprotozoal agent discovery.Concurrently, it would be useful to investigate the mechanisms through which these natural products exert their antiprotozoal effects as well as the correlations between structural activity and pharmacokinetic properties.

Figure 3 .Figure 3 .
Figure 3. Column diagram summarizing the biological activities of the compounds, fractions, and extract from Khaya senegalensis.The results with the same letters are not significantly different (p < 0.05).The error bars represent the standard deviation of measurement of samples from duplicate runs.

Table 2 .
Antileishmanial and antiplasmodial activities and selectivity of compounds, fractions, and crude extract from K. senegalensis.ethyl acetate fraction of K. senegalensis extract, SI: selectivity index, RI: reactivity index, ND: not determined, NC: not calculated, P. falciparum: IC 50 : 50% inhibitory concentration in µg/mL; CC 50 : 50% cytotoxic concentration in µg/mL.The results are expressed as the mean ± standard deviation; the SI (selective index) of bioactive compounds was determined as a measure of their toxicity against RAW cell line macrophages.SI = CC 50 against macrophages/IC 50 against promastigotes.