In vitro Antiplasmodial Activities of Alkaloids Isolated from Roots of Worsleya procera ( Lem . ) Traub ( Amaryllidaceae )

A combined phytochemical, crystallographic and biological study of Worsleya procera roots was performed. Fifteen alkaloids were identified by gas chromatography mass spectrometry (GC-MS) and seven of them were isolated. The structures of the alkaloids were elucidated by spectroscopic methods, and a detailed crystallographic study of tazettine was carried out. The isolated alkaloids and the obtained extracts were tested in vitro against Plasmodium falciparum (3D7 and K1 strains) and human hepatocarcinoma cells (HepG2) to assess their antiplasmodial and cytotoxic effects, respectively. One of the isolated alkaloid derivatives, lycorine, exhibited antiplasmodial activity against both sensitive (3D7) and resistant (K1) parasite strains in the low micromolar range (half-maximal sample inhibitory concentration (IC50) values of 2.5 and 3.1 μM, respectively) and displayed a low cytotoxicity profile, with a selectivity index greater than 100. Our findings indicate that lycorine is a hit for antimalarial drug discovery.


Introduction
Malaria is one of the most relevant infectious diseases throughout the world.For decades, global efforts towards malaria eradication reflected in a yearly decrease in cases and mortality rates.Since 2016, however, the rate of malaria infections is rising due to resistance in both the parasite and the mosquito vectors to antimalarial drugs and insecticides, respectively. 1Artemisinin-based combination therapies (ACTs), employing artemisinin and its derivatives (dihydroartemisinin, artemether and artesunate), remain as the standard treatment against falciparum malaria.However, the emergence and spread of resistance are constant threats, as cases of parasite resistance to ACTs have been reported as early as 2008. 2 Hence, the efficiency of drugs in use has been declining, despite investment from organizations such as the Bill and Melinda Gates Foundation and national governments.Unquestionably, there is a demand for new and effective drugs for malaria treatment.
Natural products play an important role in the discovery process of new drugs. 3][10][11][12][13][14][15][16][17][18][19][20] Comprising more than 1600 species worldwide, the Amaryllidaceae family is distinguished by the production of isoquinolinic alkaloids, a privileged scaffold responsible for most of the biological activities observed in these plants.This kind of alkaloids is specifically found in the Amaryllidoideae subfamily species.In Brazil, according to Rio de Janeiro's Botanical Garden, 21 142 species of Amaryllidaceae have been described, of which 64 are endemic and some are endangered, as is the case of Worsleya procera (Lem.)][25][26] This species occurs exclusively in the State of Rio de Janeiro (Brazil) and no phytochemical study has been reported for this species so far.
In this work, we performed a combined strategy including phytochemical, crystallographic and biological studies to better characterize the isolated alkaloids from Worsleya procera.We investigated the roots' extracts for their chemical composition, identified fifteen alkaloids by gas chromatography mass spectrometry (GC-MS), and isolated seven derivatives.Next, we conducted crystallographic studies and solved the molecular structure of tazettine at high resolution.All isolated alkaloids and the obtained extracts were tested in vitro for their antiplasmodial potency against Plasmodium falciparum, as well as for their cytotoxic effect in human hepatoma cells (HepG2).The most potent alkaloid derivative, lycorine, is a low micromolar inhibitor of both sensitive and resistant strains of P. falciparum and has considerable safety profile against human cells.

Equipment
The GC-MS spectra were obtained on a GC-17A Shimadzu CG-MS QP 5000 operating in the EI mode at 70 eV using a DB5 MS column (30 m × 0.25 mm × 0.25 µm).The temperature program was as it follows: 100-180 °C at 15 °C min -1 , 1 min hold at 180 °C, then 180-300 °C at 5 °C min -1 and 10 min hold at 300 °C.The injector temperature was 280 °C.The flow rate of carrier gas (helium) was 0.8 mL min -1 and the split ratio was 1:20.
The alkaloids were identified by comparing their GC-MS spectra and Kovats retention indices (RI) with our library database.8][29] Mass spectra were deconvoluted using AMDIS 2.64 software (NIST) (WA, USA) and RIs recorded using a standard n-hydrocarbon calibration mixture (C9-C36).The proportion of individual components in the alkaloid fractions is expressed as a percentage of total alkaloid content.GC-MS peak areas are dependent on the concentration of the injected alkaloid, as well as the intensity of its mass spectral fragmentation.Although the data given in Table 1 are not representative of a validated alkaloid quantification method, they can be used for a relative comparison purpose.
Nuclear magnetic ressonance (NMR) spectra were recovered in a Varian 400 MHz instrument using CDCl 3 (CD 3 OD for compound 11) as a solvent and tetramethylsilane (TMS) as the internal standard.Chemical shifts were reported in d units (ppm) and coupling constants (J) in Hz.

Extraction and isolation of alkaloids
Fresh roots (6.7 Kg) of Worsleya procera were crushed and exhaustively extracted with MeOH (100% v/v) at room temperature for 48 h, and the combined macerate was filtered and evaporated to dryness under reduced pressure.The crude extract of roots (146.2 g) was acidified to pH 2 with diluted sulphuric acid (H 2 SO 4 ) and extracted with diethyl ether (Et 2 O) (3 × 150 mL) to remove neutral material.The aqueous solution was basified with 25% ammonia (NH 3 ) up to pH 10 and extracted with n-hexane (18 × 250 mL) to give extract A (2.9368 g).Another extraction using ethyl acetate (EtOAc) (18 × 350 mL) gave extract B (6.9987 g), and the last extraction using EtOAc and methanol (MeOH) (3:1, 3 × 200 mL) gave extract C (7.7210 g).
The crystal structure of tazettine (8) was solved by direct methods using the SHELXS 38 program and refined by full-matrix least-squares method against the intensity data using SHELXL 38 program.Anisotropic displacement parameters (ADP) were applied for all non-hydrogen atoms.Hydrogen atoms were observed by Fourier maps, and placed at geometrically calculated positions and refined using an appropriate riding model regard to the parent atom with U iso (H) = 1.2U eq (C), displacement parameters, while the hydroxyl and methyl group were set using U iso (H) = 1.5U eq (O, C methyl ).The solving and refinement were made with aid of the Olex2 39 program.The structure representations were done by ORTEP-3 40 and MERCURY 41 programs.The crystallographic information and the structure data files for tazettine are deposited at Cambridge Crystallographic Data Centre (Supplementary Information section).

P. falciparum in vitro culture and in vitro SYBR green assay
The inhibitory activity of the seven isolated alkaloids and three extracts was evaluated against P. falciparum blood parasites of the 3D7 (chloroquine sensitive) and K1 (multidrug-resistant to the antimalarial drugs chloroquine, pyrimethamine and cycloguanil) strains.An aliquot of the 3D7 or K1 strain was obtained from the Malaria Research and Reference Reagent Resource Center (MR4-BEI Resources) and cultured as previously described, 42 using complete RPMI medium (RPMI 1640 medium supplemented with 25 mM hepes (pH 7.4), 21 mM sodium bicarbonate, 11 mM D-glucose, 3.67 mM hypoxanthine, 40 µg mL -1 of penicillin-streptomycin and 0.5% (m/v) Albumax ® ).The compounds were previously solubilized in 0.05% dimethyl sulfoxide (DMSO) (v/v), distributed in duplicate in 96-well plates, using two-fold serial dilutions in complete RPMI medium to assay a range of concentrations (10-0.152µM or 10-0.152µg mL -1 ).Synchronous ring-stage cultures of the parasite were added to the plate to a final parasitemia of 0.5% and hematocrit of 2%.Previous ring-stage synchronization was performed by the sorbitol method. 43ach 96-well plate included positive and negative control wells, consisting of infected red blood cells without test compounds and non-infected red blood cells, respectively.Artesunate was used in each experiment as an antimalarial positive control.All compounds were tested in duplicate in two independent experiments.The effects of the samples on parasite growth were measured through the SYBR ® green assay. 44Briefly, the medium was removed, followed by addition of 1X PBS (phosphate buffered saline) and incubation for 30 min with lysis buffer solution [TRIS, ultra-pure 20 mM, pH 7,5; EDTA 5 mM ultrapure; Saponin 0.008% m/v; Triton X-100 0.08% v/v; water Type I] and SYBR ® green I DNA stain (in a 1:20000 dilution).Fluorescence was aquired on a fluorimeter (SpectraMax 340 PC 384) at 485/535 nm.The fluorescence of each well was used to calculate the normalized viability in comparison to the positive (100% viability) and negative (0% viability) control wells.The half-maximal sample inhibitory concentration (IC 50 ) was estimated by curve fitting using software from the OriginLab Corporation (USA) and comparing to the parasite growth in the drug-free medium.The reported antiplasmodial activities (Tables 2 and 3) are the mean values of two independent experiments.The assessed IC 50 values are based on the mass of the isolated compounds or extracts dissolved in DMSO.

Cytotoxicity tests using immortalized liver cells by MTT assay
The cytotoxicity of the all samples was evaluated in a human hepatoma cell line (HepG2, obtained from American Type Culture Collection (ATCC; Manassas, VA, USA)) using cells cultured in 75 cm 2 sterile flasks containing RPMI-1640 medium (supplemented with 10% heat-inactivated fetal bovine serum and 40 mg L -1 gentamicin) under a 5% CO 2 humidified atmosphere at 37 °C.When confluent, the cell monolayer was washed with culture medium, trypsinized, distributed in a flat-bottomed 96-well plate (5 × 10 4 cells per well) and incubated for 24 h at 37 °C for cell adherence (method adapted from Denizot and Lang). 45The samples (20 µL) at various concentrations (400-0.5 µM or 400-0.5 µg mL -1 in two-fold serial dilutions) were placed in the 96-well plates, incubated with the cultured cells for 24 h under a 5% CO 2 atmosphere at 37 °C, and then the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (5 mg mL -1 ; 20 µL per well for 3 h) was used to evaluate the mitochondrial viability.Each plate included positive control wells without addition of test compounds.The supernatants were carefully removed and 100 µL of DMSO were added to each well and mixed to solubilize the formazan crystals.The optical density was determined at 570 nm.The cell viability was expressed as the percentage of the control absorbance in the untreated cells after subtracting the appropriate background.The reported cytotoxic potency (Table 3) is the mean value of two independent experiments.
The GC-MS approach applied to enriched-alkaloidal fractions from Worsleya procera (Table 1) allowed us to distinguish 18 alkaloids from the complex mixture of metabolites, been that 15 of them were identified by comparison of their GC-MS spectra and Kovats retention indices with our in-home library database (Figure 1).The alkaloids pretazettine (expressed as tazettine) and albomaculine were the main components from the n-Hex extract, while galanthine and again pretazettine were the major alkaloids from the EtOAc extract.The observed percentages in both extracts were approximately 15 and 20% of TIC (total ion current).The labile alkaloid pretazettine undergoes a fast rearrangement to tazettine after acid-base extraction and under GC-MS conditions, and then it is detected as tazettine. 29Lycorine and galanthindole showed a remarkable percentage at the EtOAc extract (13.2 and 12.8%, respectively), although it showed no detection and less than 1% of TIC from n-Hex fraction, respectively.Homolycorine, which is biogenetically related with the lycorine skeleton, displayed 8.9 and 4.8% of TIC from n-Hex and EtOAc fractions, respectively.The alkaloids 3-epi-macronine showed 6.1% of TIC from n-Hex extract, although it was detected only as traces from EtOAc fraction.
Concerning the minor components, the considered catabolic product from haemanthamine-type skeleton ismine was detected from the n-Hex extract at percentages of 3.2 and 5.8% of TIC, respectively.The related homolycorine-type alkaloid O-methyllycorenine was only detected from n-Hex extract, but at low amount (4.0%).The alkaloids belonging to the homolycorine series displaying a typical EI-MS fragmentation pattern which is featured by a retro-Diels-Alder rearrangement generated two fragments: one, the most representative, illustrates the pyrrolidine ring (along with the substituents in position 2), and the other one (a less abundant fragment) encompasses the aromatic lactone or hemilactone moiety. 46Furthermore, a noteworthy feature is the low abundance of the molecular ion peak in all alkaloids with a double bound Δ. 3,4 In this attempt, some alkaloids were detected belonging to the homolycorine-type skeleton, even though they were not identified using only GC-MS techniques.Besides, an ungwedine derivative was detected by GC-MS, but just as traces.Further efforts must be applied in terms of collection of plant material, acid-base extraction and purification steps for isolation of these minor components and complete structure characterization using other spectroscopic techniques.

X-ray analysis
During the work with the isolation of Worleya procera chemical compounds, tazettine (8) crystallized spontaneously.This led us to an in-depth study on the crystallographic structure of this compound.
A single-crystal X-ray diffraction experiment of tazettine (8) was carried out to determine the absolute configuration of the molecule.In this sense, we initially carried out a low temperature single crystal X-ray diffraction experiment using Cu radiation.The crystallographic structure of the compound 8 is already known.However, structure was reported with error on absolute-configuration in 1996 47 because no accurate experiment was carried out.Then in 1998, 48 a more rigorous experiment at low temperature was reported, giving, therefore, a more accurate structure.In this work, the structural determination was conducted at 178 K, facilitating the scattering and allowing a more reliable absolute structure determination.Indeed, Linden et al. 48refined their structure based on the comparison with the derivative N-methyltazettine iodide, 49 once no meaningful anomalous scattering was observed for tazettine compound using Mo radiation.
Therefore, here we performed a meticulous experiment with Cu radiation at lower temperature (100 K) to avoid the hitch of the non-anomalous scattering observation in tazettine.This strategy allowed us to obtain well-defined Friedel pairs suitable to solve the absolute configuration with a coherent Flack parameter. 50,51It is important to mention that for a meaningful absolute structure characterization, the Flack parameter should be close to 0 (with significant errors also) when the correct absolute configuration over each chiral center is given correctly.
Regarding the molecular conformation, an S-configuration was observed in each chiral center during the refinement.In this way, the structure reported herein matches very well with the one reported by Linden et al., 48 with a very low root mean square deviation value (RMS 0.00681).As discussed by Linden et al., 48 all the structural parameters are in a normal range of expected values.The small differences between both structures are mainly due to the differences in the data collection temperature.The hydroxyl group (C6a−O6a) exhibits a bond length of 1.400(3) Å, which is a value lower than the expected for hydroxyl groups linked to a sp 3 -carbon atom. 52Asymmetrical C−N bonds were also observed over the N-atom which adopts a sp 3 -pyramidalization, as pointed out Linden et al. 48Meanwhile, the bond vector of the methoxy group (O3−C14) showed an anti-orientation regarding the hydroxyl group, which was settled by the torsional angles C2−C3−O3−C14 and C4−C3−O3−C14 (−171.75°and 66.57°, respectively).
Both the cyclohexene and the pyran rings exhibited distorted half-chair conformations.The cyclohexene ring was twisted over the C4−C4a bond, while the pyran ring was twisted over the C6a−O7 bond.The pyrrolidine ring also showed a half-chair conformation twisted on C6a−C13b bond.Lastly, the 1,3-benzodioxole ring showed an almost planar conformation, except for the position of the C11 atom, which was slightly deviated (0.20 Å) from the mean plane of the remaining atoms.An ORTEP type view model of tazettine asymmetric unit showing the atom-labelling and an overlay of the tazettine structure described here with one reported structure by Linden et al. 48red) can be seen in Figures 2a and 2b, respectively.
Tazettine crystallized in the non-centro symmetric orthorhombic space group P2 1 2 1 2 1 .The hydroxyl group acted as a donor center of strong OH•••O hydrogen-bonds with the methoxy groups of adjacent molecules along the crystallographic b axes, giving raise to one-dimensional infinite chains with C(8) graph set. 53We observed weak C−H•••N interactions between one C−H fragment of benzodioxole group and the sp 3 -pyramidal N atoms and could contribute to the crystal packing.Additionally, π•••π interactions between the 1,3-benzodioxole fragments helped in the stacking of the tazettine molecules along the crystallographic a axe.A stereographic view from the crystal packing and the main intermolecular interactions of tazettine crystal packing can be seen in the Figures 3  and 4, respectively.

Biological activity
Many compounds found in Amaryllidaceae plants have antiplasmodial potential, 10 and therefore, we assessed the biological relevance of the Worsleya procera root extracts by investigating the antiplasmodial potency of the samples.Thus, a total of 10 samples was tested against P. falciparum (3D7 strain, sensitive to chloroquine), including three extracts (A-C) and seven isolated compounds.The hexane (extract A) and ethyl acetate (extract B) fractions were active against the parasite, showing IC 50 values of 1.3 and 0.8 µg mL -1 , respectively (Table 2), whereas the EtOAc:MeOH fraction (extract C) was inactive under the assay condition.Next, we assessed the parasite growth inhibitory activity of the seven major isolated alkaloids to identify which extract components were related to the antiplasmodial activity (Table 3).We set a threshold concentration value of 10 µM to identify the active compounds.Based on that, lycorine was the most potent P. falciparum inhibitor, with inhibitory activity in the low  3).This result is in agreement with previous data. 10Furthermore, lycorine was tested against the P. falciparum K1 strain (resistant to chloroquine, cycloguanil and pyrimethamine) to evaluate potential cross-resistance of the compound.As indicated in Table 3, the IC 50 values against both sensitive and resistant strains were similar, thereby suggesting that lycorine's mechanism of action is distinct from those related to the K1 strain resistance.
Aiming to verify whether the antiplasmodial activities were related to cytotoxic effect, we evaluated the cytotoxicity of the active samples against HepG2 cells (hepatic human cells).A selectivity index (SI) above 10 generally indicates that the sample is well-tolerated by the cell.Interestingly, lycorine (EC 50 > 400 µM) (median effective dose (EC 50 )), as well as the ethyl acetate and hexane extracts (both with EC 50 > 400 µg mL -1 ) were not cytotoxic under the experimental conditions, showing high selectivity indexes (SI values > 129 > 300 and > 500, respectively) (Tables 2 and 3).
The Worsleya procera (Lem.)Traub is yet largely unexplored in terms of chemical diversity.In this work, we showed that the roots of this plant contain compounds identified in other members of its family, such as Lycoris radiata.The Amaryllidaceae family of plants is of particular pharmacological interest in the field of natural products for the characteristic production of isoquinolinic alkaloids, a series of compounds with confirmed antitumoral, antimalarial and anti-inflammatory activity, among others. 54Other compounds obtained from this family are the cripowellins, isolated from plants of the Crinum genus, which have presented antiplasmodial potency in the nanomolar range, 55 providing further evidence to the potential of these plants as a source of compounds with promising antiplasmodial activity.

Figure 2 .
Figure 2. (a) ORTEP type view model of tazettine asymmetric unit showing the atom-labelling Thermal displacement ellipsoids are drawn at 50% probability level.Hydrogens atoms are drawn with arbitrary radii; (b) overlay of the tazettine structure (blue) described here with one reported structure by Linden et al. 48(red).Hydrogen atoms were omitted for the sake of clarity.The CSD ref. code RENRIL01 was assigned for the Linden et al. 48structure.

Figure 3 .
Figure 3. Stereographic view from the crystal packing along the (a) a axis and (b) 1836655 axes; hydrogens atoms were omitted for the sake of clarity.

Figure 4 .
Figure 4. Main intermolecular interactions of tazettine crystal packing: (a) unidimensional hydrogen-bond chain along the crystallographic b axe; (b) weak interaction of C−H•••N type, and (c) π-stacking of the 1,3-benzodioxole groups along the crystallographic a axe.
Worsleya procera were collected in Petropolis city (State of Rio de Janeiro, Brazil) in July 2014.The plant was collected in an area of environmental protection (22°32'38.7"S43°10'11.8"W);therefore, an authorization for sampling was issued by the proper regulatory agencies (Sistema de Autorização e Informação em Biodiversidade (SISBIO) No. 41717).The sample was authenticated by Dr Julie H. A. Dutilh from University of Campinas (Unicamp, Campinas city, Brazil).A specimen voucher (No. 35307) was deposited in the Herbarium VIES of the Department of Botany of the Federal University of Espírito Santo (Vitória city, Brazil).