Bio-guided isolation of anti-leishmanial natural products from Diospyros gracilescens L. (Ebenaceae)

Background Plants represent an intricate and innovative source for the discovery of novel therapeutic remedies for the management of infectious diseases. The current study aimed at discovering new inhibitors of Leishmania spp., using anti-leishmanial activity-guided investigation approach of extracts from Diospyros gracilescens Gürke (1911) (Ebenaceae), targeting the extracellular (promastigotes) and intracellular (amastigotes) forms of Leishmania donovani. Methods The plant extracts were prepared by maceration using H20: EtOH (30:70, v/v) and further fractionated using a bio-guided approach. Different concentrations of D. gracilescens extracts, fractions and isolated compounds were tested in triplicate against L. donovani promastigotes and amastigotes in vitro. The antileishmanial potency and cytotoxicity on RAW 264.7 cells were determined using the resazurin colorimetric assay. The time kill kinetic profile of the most active sample was also investigated. The structures of all compounds were elucidated on the basis of extensive spectroscopic analyses, including 1D and 2D NMR, and HR-ESI-MS and by comparison of their data with those reported in the literature. Results The hydroethanolic crude extract of D. gracilescens trunk showed the most potent antileishmanial activity (IC50 = 5.84 μg/mL). Further fractionation of this extract led to four (4) fractions of which, the hexane fraction showed the most potent activity (IC50 = 0.79 μg/mL), and seven (07) compounds that exhibited moderate potency (IC50 = 13.69–241.71 μM) against L. donovani. Compound 1-deoxyinositol (7) inhibited the promastigote and amastigote forms of L. donovani with IC50 values of 241.71 μM and 120 μM respectively and also showed the highest selectivity against L. donovani promastigotes (SI > 5.04). To the best of our knowledge, the antileishmanial activity of this compound is being reported here for the first time. The promising hexane fraction showed significant inhibition of parasites growth at different concentrations, but with no evidence of cidal effect over an exposure period of 120 h. Conclusions The results obtained indicated that the hydroethanolic extract from the D. gracilescens trunk and the derived hexane fraction have very potent inhibitory effect on cultivated promastigotes and amastigotes of L. donovani parasite. The isolated compounds showed a lesser extent of potency and selectivity. However, further structure-activity-relationship studies of 1-deoxyinositol could lead to more potent and selective hit derivatives of interest for detailed drug discovery program against visceral leishmaniasis. Supplementary Information The online version contains supplementary material available at 10.1186/s12906-021-03279-1.


Background
Leishmaniasis is a severe, widespread zoonotic and parasitic disease caused by an intracellular flagellate protozoan of the genus Leishmania. The disease is generally transmitted between man and animals during a blood meal by the phlebotome female sandfly. About 20 different Leishmania species including L. donovani have been discovered to be pathogenic to human. The clinical features of the disease include a wide range of manifestations, including skin ulcers at the site of the infection or dissemination in visceral organs followed by anemia, leucopenia, fever and weakness [1,2]. The World Health Organization estimates that 1.3 million new cases of leishmaniasis occur every year with 20,000 to 30,000 deaths annually [3]. Therefore, a great concern has been expressed by the WHO, as leishmaniases are considered as neglected tropical diseases [4]. Visceral leishmaniasis (VL), caused by L. donovani is the most dangerous form of the disease that can be lethal in human when untreated. It is considered as a serious public health problem worldwide, and especially in Africa where its significant morbidity and mortality require more effective chemotherapy [5]. Current available chemotherapy includes the first line treatment drugs such as pentavalent antimonials, meglumine antimoniate (glucantime) and sodium stibogluconate (pentostam) and second line drugs such as amphotericin B, pentamidine, paromomycin and miltefosine [6,7]. However, these drugs are limited by factors such as emergence of drug resistance, especially with the pentavalent antimonials and challenges of toxicity, short half-life and high cost of drugs, as well as failure of patient to comply with treatment [8,9]. Due to the limitations of current chemotherapeutic regimes and in the absence of effective and sustainable vaccines, there is a persistent need for alternative and readily available sources for treatment of leishmaniasis. In this respect, natural products offer good sources for new drug discovery [10].
This paper describes the in vitro antileishmanial activity of natural products from D. gracilescens, a plant of Ebenaceae family. It is a forest tree widely distributed in West and Centre regions of Cameroon. Furthermore, there is no mention of use of D. gracilescens in traditional medicine in Cameroon. However, related species such as D. bipindensis (Gürke), D. conocarpa (Gürke & K. Schum.) and D. malabarica ((Descr.) Kostel.) are widely used by Baka Pygmies for the treatment of malaria, sleeping sickness and respiratory disorders [11]. Globally, Diospyros spp. are known above all, as fishing poisons, especially in South East Asia and in the Philippines. They are also widely-used medications in traditional African medicine, mainly against leprosy. The roots are used as purgative in the Central African Republic, against pneumonia in Zimbabwe and schistosomiasis in Malawi [12,13]. The first chemical study of D. gracilescens led to the isolation of few compounds such as: lupeol, betulin, betulinic acid, isodiospyrin (II) and sitosterol [14]. Of note, this is the first report of antileishmanial guided isolation of the chemical constituents of D. gracilescens.

Methods
Phytochemical investigation of D. gracilescens Collection of plant material and preparation of the crude extracts The plant materials (root, trunk, stem bark and leaf) of D. gracilescens, were collected in March 2017 at Eloumdem mountain (GPS coordinates: Latitude 3°49′00″N, Longitude 11°25′60″E), in the Centre Region of Cameroon and were identified by comparison with a voucher specimen (No. 2016 / SRFK) from the National Herbarium of Cameroon by Mr. Victor Nana, a botanist.
The collected plant materials were dried under shelter at room temperature and further ground to obtain the powders. The tinctures were prepared by maceration of 4000 g of each powder in aqueous ethanol 70% (15 L, 48 h × 3). The resulting macerates were filtered using Whatman filter paper No. 2 and the filtrates concentrated on a Büchi rotary evaporator (Büchi Labortechnik AG -Flawil, Switzerland) under reduced pressure at 45-55°C and further lyophilized using a freeze-dryer Alpha 2-4 LD plus (Christ, Germany) to yield the crude extracts. Each extract was kept dried in tightly stoppered bottles at 4°C until it was used for the biological screenings.

Liquid-liquid partition of the trunk crude extract
The crude extract of the trunk (150 g) which showed the best leishmanicidal activity was fractionated by liquidliquid partition as shown in Fig. 1, according to the procedure described by Xie et al. [15]. Briefly, 140 g of the crude extract was suspended in water and then extracted with n-hexane, dichloromethane, ethyl acetate and n-butanol successively (Fig. 1). Each fraction was evaporated under reduced pressure at 45-55°C and then the aqueous fraction was lyophilized. Five residues were obtained and respectively named fraction A from n-hexane [6.5 g, and fraction E for the remaining aqueous residue (45.5 g, 32.50% yield). Each of the afforded fractions was submitted to antileishmanial screening and the promising ones (fractions A, B and D) having IC 50 values below 2 μg/mL were selected and submitted to chromatographic separation.

HPLC-DAD-ESI-MS analysis of extracts from D. gracilescens
Sample preparation Each extract was dissolved in HPLC grade methanol at a concentration of 0.5 mg/mL, then filtrated through a syringe-filter-membrane. Each aliquot obtained (5 μL) was injected into the UPLC-DAD-HRESI/MS Dionex Ultimate 3000 HPLC (Germany) apparatus used to perform the analyses.
HPLC-MS conditions High resolution mass spectra were obtained with an OTOF Spectrometer (Bruker, Germany) equipped with a HRESI source and a UV-vis absorbance detector. The spectrometer was operated in positive mode (mass range: 100-1500, with a scan rate of 1.00 Hz) with automatic gain control to provide highaccuracy mass measurements with 2 ppm deviation using Na Formate as calibrant. Mass spectra were simultaneously acquired using electrospray ionization in the positive ionization mode. The following parameters were used for experiments: spray voltage of 4.5 kV, capillary temperature of 200°C. Nitrogen was used as sheath gas (10 l/min). The spectrometer was attached to an Ultimate 3000 (Thermo Fisher, USA) HPLC system consisting of LC-pump, UV traces were measured at 215, 218, 254, 280 and 330 nm and UV spectra-Diode Array Detector-(DAD) was recorded between 190 and 600 nm, auto sampler (injection volume 5 μl) and column oven (35°C). The separations were performed using a Synergi MAX-RP 100A (50 × 2 mm, 2.5 μ particle size) with a H 2 O (+ 0.1% HCOOH) (A)/acetonitrile (+ 0.1% HCOOH) (B) gradient (flow rate 500 μL/min). Samples were analyzed using a gradient program as follows: 95% A isocratic for 1.5 min, linear gradient to 100% B over 6 min, after 100% B isocratic for 2 min, the system returned to its initial condition (90% A) within 1 min, and was equilibrated for 1 min.
Identification of peaks Identification of all constituents was performed by UPLC-DAD-HRESI/MS analysis and by comparing the UV, MS spectra and MS/MS fragmentation of the selected peaks in the sample chromatogram with those of data reported the literature of SciFinder database.

Screening of extracts for biological activity
Parasite culture and maintenance The cryopreserved promastigote form of L. donovani (1S (MHOM/SD/62/1S) was obtained from Bei Resources (https://www.beiresources.org/) and is routinely cultured at the Antimicrobial and Biocontrol Agents Unit, University of Yaoundé I, in Medium 199 (Sigma, Darmstadt, Germany) supplemented with 10% Heat-Inactivated fetal Bovine Serum (HIFBS) (Sigma, Darmstadt, Germany) and 100 IU/mL penicillin and 100 μg/mL streptomycin. The culture was maintained in 75 Cm 2 cell culture flask at 28°C and checked for growth daily and sub-cultured everyday 72 h [21].

Determination of the antileishmanial activity of plant extracts and fractions
Inhibitory assay against L. donovani promastigotes The antileishmanial activity of D. gracilescens crude extracts, derived fractions and compounds against cultured L. donovani promastigotes was evaluated using the resazurin colorimetric assay as described by Siqueira-Neto et al. [22]. The stock solutions were prepared by dissolving each sample in 100% dimethyl sulfoxide (DMSO) and subsequently diluted serially in non-supplemented culture medium. To assess the antileishmanial activity, 4 × 10 5 promastigotes/mL/well were seeded in a 96 well microtiter plate and treated with 5-fold diluted concentrations of D. gracilescens extracts (0.16, 0.8, 4, 20 and 100 μg/mL) for 72 h at 28°C. The viability rate of promastigotes positively correlated with the amount of pink resorufin that was produced through the reduction of blue resazurin by the dehydrogenase enzymes in the inner mitochondrial membrane of the living parasites. Briefly, promastigotes from a logarithmic phase culture (4 × 10 5 cells/mL; 90 μL) were seeded in 96-well microtiter plates and were treated with 10 μl of inhibitors at different triplicate concentrations ranging 100 μg/mL-0.16 μg/mL for extracts and fractions and 50 μg/mL-0.08 μg/mL for compounds. The final concentration of DMSO in each well was not higher than 1%. Plates were incubated for 28 h at 28°C, followed by the addition of 1 mg/mL resazurin (Sigma, Darmstadt, Germany

Data analysis
All the activity data represent mean ± standard deviation (SD) from three independent experiments. Microsoft Excel Software was used to calculate the percentage of inhibition. The IC 50 and CC 50 values were determined using GraphPad Prism 5.0 Software with data fitted by non-linear regression.

Statistical analysis
Data were expressed as mean ± SEM (standard error of mean). Statistical analysis was performed by one-way ANOVA (analysis of variance) followed by the Bonferroni post-test using GraphPad 7 software. Difference was considered as significant at p < 0.05.

Phytochemical analysis data
The crude extract, the hexane and dichloromethane soluble fractions of the trunk of D. gracilescens were analyzed by UPLC coupled to both diode array and mass spectrometry detectors. The latter was used with an electrospray ionization (ESI) source in positive ion mode. A representative base peak chromatogram and all ions MS (Fig. 1) indicating that the used UPLC conditions allowed a good separation of a large percentage of compounds. The compounds were recognizable from their characteristic UV spectra, which were identified based on the UPLC-DAD-HRESI-MS data and subsequent confirmation by comparison with literature data.
The chromatographic profile and spectroscopic data are presented in Table 1 and Fig. 2 below.

Biological activities data
Antileishmanial activity of plant samples The inhibitory potential of D. gracilescens extracts against L. donovani promastigotes was measured by direct counting of live promastigotes after parasite exposure to various concentrations of extract. The IC 50 values of the different crude extracts are shown in Table 2 below. The results shown in Table 2 indicate that only the extract from the trunk of D. grascilisens exerted antileishmanial activity with an IC 50 value of 5.84 μg/mL. The other extracts were non-active up to 100 μg/mL. The trunk extract was therefore progressed for bio-guided investigation.

Bio-guided fractionation of the trunk extract of D. grascilisens
Fractionation of the trunk extract was performed by liquid-liquid partition to afford four (04) main fractions that were tested for activity against the promastigotes and amastigotes forms of L. donovani (Table 3).
Further fractionation of fraction A led to the isolation of lupeol (1) and a mixture of sterols (4). Fraction B led to betulin (2) and betulinic acid (3) and fraction D led to β-sitosterol glucoside (6) and 1-deoxyinositol (7) as shown in Fig. 3 below. The structures of these compounds were elucidated on the basis of spectroscopic analyses, including 1D and 2D NMR, and HR-ESI-MS and by comparison of their data with those reported in the literature (see supplementary information). These compounds were also tested for activity against the promastigote and amastigote forms of L. donovani. The results achieved are presented in Table 4.

Kinetics of parasite killing as a relation to time and inhibitor concentration
The following graphs below (Fig. 4) show the time kill kinetic of the most active (Hexane) fraction (A), compared to the positive control, amphotericin B (B).
The ability of the hexane fraction to fast-kill L. donovani promastigotes was assessed at different concentrations points (0.5x IC 50 , IC 50 , 2x IC 50 and 4x IC 50 ) relative to untreated parasites culture over a period of 120 h. The results showed that treatment with increasing concentrations of the hexane fraction and amphotericin B resulted in a significant reduction in promastigote replication after 24 h (Fig. 4). In the meantime, an exponential growth was observed in untreated parasite culture. At all tested concentrations of hexane fraction and amphotericin B, a regular reduction of the viability of treated parasite cultures was observed up to 120 h, with however no evidence of cidal effect.

Discussion
The investigation of extracts from D. grascilesens for antileishmanial activity identified the hydroethanolic trunk extract as a promising starting point for bioguided study. This is the first report describing the antileishmanial activity of extracts from D. grascilesens. However, extracts from other Diospyros spp. have been previously investigated in this direction. Of note, Lenta et al. [25] demonstrated the capacity of the dichloromethane-methanol (1:1) extract of D. canaliculata (De Wild.) to inhibit the growth of axenic amastigotes of L. donovani. On another hand, Dhar et al. [26] reported that ethanolic extracts of D. montana (Roxb) and D. peregrina ((Gaertn.) Gürke) possess antiprotozoal activity against Entamoeba histolytica, antiviral activity against Ranikhet disease virus and hypoglycemic activities in albino rats. Rocío et al. [27] also demonstrated the in vitro antimycobacterial potency of the stem bark extract from D. anisandra (S.F. Blake) against a resistant strain of M. tuberculosis. In other studies, Hazra et al. [28] demonstrated the anti-tumour activity of bark extract from D. ferrea ((Willd.) Bakh). Asolkar et al. [29] showed the antibacterial activity of leaf and seed extracts from D. montana and Satish and Sunil [30] demonstrated the anti-diabetic and antioxidant potential of the ethanolic bark extract of D. malabarica.
Based on the criteria set for antileishmanial activity of plant extracts by Camacho et al. [31], the promising hydroethanolic trunk extract of D. grascilesens was further fractionated yielding the hexanic fraction as the more active and selective against the extracellular and the intracellular forms of L. donovani parasite. Further fractionation of this fraction led to six (6) compounds  identified as lupeol, betulin, betulinic acid, mixture of sterols, β-sitosterol glucoside and 1-deoxyinositol. Among these compounds, betulinic acid showed the most potent activity and selectivity against both promastigote and amastigote forms of L. donovani. There are few studies in the literature reporting the activity of betulin and betulinic acid and derivatives against Leishmania parasites. Indeed, similarly to our findings, Sousa et al. [32] have reported moderate antileishmanial activity (23-55 μM) of semisynthetic lupane triterpenoids, betulin and betulinic acid when tested alone, and their synergistic effects with miltefosine, an alkylphosphocholine drug with demonstrated activity against various parasite species (including Leishmania infantum parasites and amoeba) and cancer cells as well as some pathogenic bacteria and fungi [33]. Alakurtti et al. [34] also determined the activity of heterocyclic betulin derivatives on L. donovani amastigotes and the in vitro activity of betulin and betulinic acid derivatives against L. donovani amastigotes and promastigotes of L. amazonensis. Dominguez et al. [35] also reported the activity of betulinic acid acetate and betulinic acid methyl ester against promastigotes of L. amazonensis. Betulinic acid has been already reported in the literature to possess a wide range of biological and medicinal properties, including anti-human immunodeficiency virus (HIV), antibacterial, antimalarial, antiinflammatory, anthelmintic, antinociceptive, anti-herpes simplex viruses-1 (HSV-1), immune-modulatory, antiangiogenic, and anticancer activities [36,37]. The activity of betulinic acid and its derivatives against the erythrocytic stage of the chloroquine-sensitive 3D7 Plasmodium falciparum strain was previously reported, as well as moderate antileishmanial activity on different Leishmania spp. [34,38,39]. Cassio et al. [40] also showed that the semi-synthetic derivatives of betulinic acid were able to prevent the parasite development and invasion into host cells, that are crucial events for Trypanosoma cruzi infection establishment, with potency similar to benznidazole.
In this study, lupeol was found to be active against promastigotes and moderately active against amastigotes of L. donovani, corroborating the previous findings. Indeed, the antileishmanial activity of lupeol against both promastigotes and amastigotes of L. donovani has been demonstrated in the literature [41]. Other previous studies indicated that lupeol isolated from aerial parts of Vernonia scorpioides displayed a weak antileishmanial activity [42,43]. Also, studies have highlighted the activity of lupeol from the latex of E. resinifera and E. officinarum against promastigote of L. infantum [44]. Other studies attempting to establish the mechanism of action of lupeol were conducted by Ramos et al. [45] and showed that this compound mediates an increased cytoplasmic membrane depolarization which may promote enhanced cell membrane damage. They also suggested that the leishmanicidal activity could lead to disruption of the cytoplasmic membrane of L. donovani promastigotes as evidenced by DISC3 mediated fluorometric analysis. Whereas lupeol might mediate reduction in intracellular parasitic load was found to be executed through the induction of pro-inflammatory cytokine response and generation of Nitrite Oxide (NO) in L. donovani infected macrophages [41]. Betulinic acid and lupeol that showed the most potent activity in this study belong to the class of terpenoids. In fact, a number of terpenes are reputed to possess antileishmanial activity. Different authors suggested that the antileishmanial activity of these compounds could be related to the inhibition of proteins and nucleic acids synthesis or of a membrane-associated calcium-dependent ATPase pump [43,46]. Indeed, previous studies have suggested that lipophilic compounds, such as triterpenes, act by a peculiar mechanism. These compounds can pass easily through the cytoplasmic membranes, affecting structures of their different layers of polysaccharides, fatty acids, and phospholipids, thus making them permeable [47]. Once they cross the membrane, the coagulation of cytoplasm can occur. These events are able to promote the interruption of specific metabolic pathways of lipids and proteins [48], interference in cell division [49,50], or stimulate the depolarization of the mitochondrial membranes, which can lead the cell to trigger necrosis or apoptosis mechanisms [51].
Among the isolated compounds, 1-deoxyinositol exerted a promising activity against both promastigote and amastigote forms of L. donovani. This compound was previously detected by GC-MS in a moderately active (with IC 50 value of 126.4 μg/mL) methanolic extract from the aerial part of Scutellaria havanensis against L.  amazonensis promastigotes [52]. The antileishmanial activity of 1-deoxyinositol adds to the novelty of this work given that, to the extent of our knowledge, no previous report has been published on the antileishmanial activity of this compound. More interestingly, 1-deoxyinositol showed the highest selectivity against L. donovani promastigotes (SI > 5.04) as well as acceptable preference for amastigotes (SI > 10. 15).
Overall, this study has indicated that the hexane fraction was 12 to 49-fold and 1.8 to 2.4-fold more active than the derived lupeol and mixture of sterols against the promastigote and amastigote forms of L. donovani respectively. Also, selectivity indexes greater than 152 and 11 for promastigotes and amastigotes respectively were obtained compared to the derived components. The activity profile of the hexane fraction portents a very probable synergistic interaction between its nonpolar components to increase activity and selectivity (safety profile). The implication of these findings is of high significance in the use of D. grascilesens plant in traditional medicine to treat neglected tropical diseases (NTDs).

Conclusion
This study reports the first detailed investigation aiming at determining the antileishmanial activity of natural products from D. gracilescens using a bio-guided approach. The hydroethanolic extract of the trunk showed promising profile, (IC 50 = 5.84 μg/mL) and its bio-guided fractionation led to the most potent hexane fraction (IC 50 = 0.79 μg/mL). Further fractionation of this fraction led to six compounds that also exhibited antileishmanial potency. The promising hexane fraction and derived active compounds represent potential raw materials for detail-oriented drug discovery against visceral leishmaniasis that exacts a very heavy toll to poor patients in remote endemic settings in Africa and elsewhere. Among the isolated compounds, 1-deoxyinositol has shown acceptable profile for further structureactivity-relationship studies in drug discovery program to unveil hits or leads adhering to the criteria defined earlier against visceral leishmaniasis. Of particular note is the activity profile of the hexane fraction that exerted the greatest potency and selectivity. It is a promising candidate for the development of a phytodrug against leishmaniasis.