Antileishmanial Activity of Clinanthus milagroanthus S. Leiva & Meerow (Amaryllidaceae) Collected in Peru

Leishmaniasis is a worldwide infectious parasitic disease caused by different species of protozoa of the genus Leishmania, which are transmitted to animals and humans through the bite of insects of the Psychodidae family. In the present work, the antileishmanial activity of an alkaloid extract of the bulbs of Clinanthus milagroanthus S. Leiva & Meerow (Amaryllidaceae) was evaluated in vitro, in vivo, and in silico against the parasite Leishmania braziliensis, and the chemical profile of the sample was determined by GC-MS analysis. At concentrations of 1, 10, and 100 µg·mL−1, the alkaloid extract presented inhibition percentages of 8.7%, 23.1%, and 98.8%, respectively, against L. braziliensis with a p < 0.05, and IC50 values of 18.5 ± 0.3 µg·mL−1. Furthermore, at a dose of 1.0 mg·kg−1, a greater decrease in lesion size was observed (90%) for in vivo assays, as well as a decrease in infection (96%), finding no significant differences (p > 0.05) in comparison with amphotericin B (92% and 98%, respectively). Eleven alkaloids were identified in C. milagroanthus bulbs: galanthamine, vittatine/crinine, 8-O-demethylmaritidine, anhydrolycorine, 11,12-dehydroanhydrolycorine, hippamine, lycorine, 2-hydroxyanhydrolycorine, 7-hydroxyclivonine, 2α-hydroxyhomolycorine, and 7-hydroxyclivonine isomer. A molecular model of Leishmania braziliensis trypanothione reductase (TRLb) was built using computational experiments to evaluate in silico the potential of the Amaryllidaceae alkaloid identified in C. milagroanthus toward this enzyme. The structures galanthamine, 7-hydroxyclivonine isomer, and crinine showed better estimated free energy of binding than the reference compound, amphotericin B. In conclusion, this is the first in vitro, in vivo, and in silico report about the antileishmanial potential and alkaloid profiling of the extract of C. milagroanthus bulbs, which could become an interesting source of bioactive molecules.


Introduction
One of the top ten neglected tropical diseases worldwide, leishmaniasis is endemic in 97 countries and affects more than 12 million people, with more than 350 million people at risk of developing the disease [1]. Caused by different species of protozoa from the genus Leishmania, it is transmitted to animals and humans through the bite of infected female sandflies of the Psychodidae family [2]. The incidence of leishmaniasis is directly related to poverty, but environmental and climate factors are also involved [3].

In Vivo Antileishmanial Activity
The in vivo test was performed on hamsters (Mesocricetus auratus) infected with Leishmania braziliensis, and the alkaloid extracts were evaluated at doses of 0.1 mg·kg −1 , 0.5 mg·kg −1 , 1.0 mg·kg −1 starting the treatment after three weeks of infection. Four weeks later, a decrease in the size of the cutaneous lesions in the hamsters' noses was distinguished, at the different doses tested, with values of 71%, 80% and 90% respectively. Likewise, these values when compared with amphotericin (92%), showed no significant differences at the dose of 1.0 mg·kg −1 (p > 0.05) ( Figure 3. In addition, at the first and second doses tested, a reduction in infection of 19% and 42% was observed respectively, while the reduction shown with the last dose (96%) was close to that of amphotericin B (98%), with no statistically significant differences between the latter (p > 0.05) (Figure 4). The plant family Amaryllidaceae is recognizable for its isoquinoline alkaloids, which present a wide spectrum of biological activity, including antiprotozoal [9,21]. A search of the literature revealed the antileishmanial potential of alkaloids isolated from different Amaryllidaceae species, such as Amaryllis belladonna L., Crinum x amabile Donn., Galanthus trojanus A.P. Davis & Özhatay, Narcissus angustifolius Curtis ex Haw., and Phaedranassa dubia (Kunth) J.F. Macbr. [22][23][24][25][26]. Among them, haemanthamine, O-methylnorbelladine, and stylopine, isolated from G. trojanus, showed weak activity against L. donovani, with IC 50 values of 21.9, 52.9, and 38.1 µg·mL −1 , respectively [25], while 3-O-acetylhamayne, obtained from A. belladonna, displayed moderate activity against this protozoan species, with an IC 50 value of 17.9 µg·mL −1 [23].
As shown in Table 1, lycorine (7) was the most representative alkaloid quantified in C. milagroanthus. Some authors report that this structure, isolated from the species Crinum stuhlmannii Baker, Crinum macowanii Baker, and Zephyranthes citrina Baker, is not active against L. donovani [21,[27][28][29]. As also indicated in Table 1, the second most representative alkaloid detected in C. milagroanthus was compound 11, which may have contributed to the antileishmanial activity of the bulb extract. The mechanism of action of Amaryllidaceae alkaloids against leishmaniasis is not known, although evidence suggests that the methylenedioxy group may enhance their antiprotozoal activity [30].

In Vivo Antileishmanial Activity
The in vivo test was performed on hamsters (Mesocricetus auratus) infected with Leishmania braziliensis, and the alkaloid extracts were evaluated at doses of 0.1 mg·kg −1 , 0.5 mg·kg −1 , 1.0 mg·kg −1 starting the treatment after three weeks of infection. Four weeks later, a decrease in the size of the cutaneous lesions in the hamsters' noses was distinguished, at the different doses tested, with values of 71%, 80% and 90% respectively. Likewise, these values when compared with amphotericin (92%), showed no significant differences at the dose of 1.0 mg·kg −1 (p > 0.05) (Figure 3. In addition, at the first and second doses tested, a reduction in infection of 19% and 42% was observed respectively, while the reduction shown with the last dose (96%) was close to that of amphotericin B (98%), with no statistically significant differences between the latter (p > 0.05) (Figure 4).   Currently there are no previous publications about the in vivo potential of Amaryllidaceae alkaloids against leishmaniosis. Ancistrocladiniums B, an isoquinoline alkaloid obtained from the leaves of an Ancistrocladaceae species, and a synthetically prepared isoquinoline salt were effective against intracellular Leishmania major amastigotes at submicromolar concentrations, and most likely act directly on the parasites, suggesting that  Currently there are no previous publications about the in vivo potential of Amaryllidaceae alkaloids against leishmaniosis. Ancistrocladiniums B, an isoquinoline alkaloid obtained from the leaves of an Ancistrocladaceae species, and a synthetically prepared isoquinoline salt were effective against intracellular Leishmania major amastigotes at submicromolar concentrations, and most likely act directly on the parasites, suggesting that these molecules are promising candidates as antileishmanial drugs [31]. The alkaloid extracts of the species Annona crassiflora Mart. (Annonaceae) and Cissampelos ovalifolia DC. Currently there are no previous publications about the in vivo potential of Amaryllidaceae alkaloids against leishmaniosis. Ancistrocladiniums B, an isoquinoline alkaloid obtained from the leaves of an Ancistrocladaceae species, and a synthetically prepared isoquinoline salt were effective against intracellular Leishmania major amastigotes at sub-Plants 2023, 12, 322 6 of 13 micromolar concentrations, and most likely act directly on the parasites, suggesting that these molecules are promising candidates as antileishmanial drugs [31]. The alkaloid extracts of the species Annona crassiflora Mart. (Annonaceae) and Cissampelos ovalifolia DC. (Menispermaceae) were effective against Leishmania (L.) chagasi, reducing the number of infected macrophages by approximately 90% compared with the non-treated group [32]. Two aziridine-2,3-dicarboxilates, which are inhibitors of the cathepsin L subfamily of the papain clan of cysteine proteases-enzymes of great significance as virulence factors-showed activity against L. major promastigotes [33].

Molecular Docking In Silico Analysis
The ability of Leishmania (V.) braziliensis to resist oxidative stress seems to be correlated with its survival capacity [34]. Lacking conventional redox control systems, the parasites base their defense on trypanothione reductase, an enzyme which is considered a promising target for antileishmanial therapy [35,36]. Because in vitro antileishmanial activity assays were performed using Leishmania braziliensis promastigotes, computational assays of inhibitory activity should be performed on enzymes from the same molecular target; however, no crystallized protein structures are currently available for this species. For this reason, a theoretical model of trypanothione reductase (TRLb) from Leishmania braziliensis was implemented using a molecular modeling procedure, which provides reasonable results based on the hypothesis that the tertiary structures of two proteins will be similar if their sequences are phylogenetically related [37].
The structure of Leishmania infantum trypanothione reductase (PDB: 2JK6) [38] was selected as a model due to its similarity with the TRLb sequence (>80%). The Ramachandran analysis for the modeled structure detected that more than 93% of the amino acids are in favorable regions [39,40]. Additionally, the quality of the modeling was assessed by comparing the predicted structure with the model's structure through the evaluation of overlap and root-mean-square deviation (RMSD) of atoms. The RMSD of Cα atoms for the homology model is less than 1.00 Å. The results for the best interaction between Leishmania (V.) braziliensis TR and the alkaloids are presented in Table 2. The lowest estimated free energies of binding were obtained for galanthamine (1), crinine (2b), and 7-hydroxyclivonine isomer (11) alkaloids, with values of −8.29, −8.14, and −8.24 kcal·mol −1 , respectively. These alkaloids show higher ligand-protein stability than an Amphotericin B (−7.94 kcal·mol −1 ) molecule used as a positive control. A second group of alkaloids with values relatively close to those of Amphotericin B are: hippamine (6), lycorine (7), 7-hydroxyclivonine (9), and 2α-hydroxyhomolycorine (10)  The stabilization of the galanthamine (1) alkaloid is achieved by the presence of three hydrogen bonds interactions with Glu381, Val362, and Thr374 residues, as well as four hydrophobic interactions with Cys364, Val362, Cys375, and Leu377. Those interactions in the active site are close to Ala365, an important contact reported in other work [34]. For crinine (2b), the molecular docking experiments located two hydrogen bonds interactions with Thr160 and Arg290, two ionic interactions with Asp327 and Glu35, and two hydrophobic interactions with Ala159 and Ala46. Those interactions are close to the redox-active disulfides Cys52 and Cys57 residues at the bottom of the cleft [34,36,41,42]. The stability of the 7-hydroxyclivonine isomer (11) alkaloid is achieved by three hydrogen bonds interactions with Val362 and Gly376, and three hydrophobic interactions with Cys364, Val362, and Cys375. This alkaloid is stabilized in a similar way to galanthamine (1), which explains the closeness in the binding energy values. Finally, the 2D ligand-protein diagram for amphotericin B (positive control) indicates the presence of multiple contacts within the surface of the protein due to the large size of amphotericin B; nevertheless, the molecular docking experiments showed that the stabilization is produced by four hydrogen bond interactions with Glu436, Pro435, and Lys61.

Plant Material
The bulbs of Clinanthus milagroanthus S. Leiva & Meerow were collected in 2021 in the Salpo district, La Libertad region, Peru, at an altitude of 2824 m. This plant currently has been reclassified as Paramongaia milagroantha (Leiva & Meerow) Meerow [43]. The species was authenticated by the botanist Prof. Dr. Alan Meerow (Florida University, Gainesville, FL, USA) and a specimen voucher 5795 was deposited in the Herbario Antenor Orrego (HAO), Perú.

Extraction
The alkaloid extract was obtained using 1 g of dry bulb of Clinanthus milagroanthus. The extraction process is described in a previous publication by our research group [44]. Herein, it was possible to obtain 14 mg of alkaloid extract (AE) of this species.

GC-MS Analysis
Two mg of the alkaloid extract of Clinanthus milagroanthus was dissolved in 1 mL of MeOH: HCCl 3 (1:1, v/v) and analyzed using gas chromatography coupled mass spectrometry (GC-MS). The chromatograph used was a GC-MS 6890N apparatus (Agilent Technologies, Santa Clara, CA, USA) coupled to an Agilent MSD5975 Inert XL operating in the electron ionization (EI) mode at 70 eV. A Sapiens-X5 MS column (30 m × 0.25 mm i.d., film thickness 0.25 µm) was used. 1 µL of the sample was injected using the splitless mode. Codeine was used as an internal standard in the sample injected. The chromatographic conditions are available in a previous publication [44].

Alkaloid Identification and Quantification
The results obtained using GC-MS were evaluated using the software AMDIS 2.64. The peaks were analyzed using our library database which was developed by the Natural Products Group of Barcelona University (Catalonia, Spain); the NIST 05 Database (Gaithersburg, MD, USA); and by comparison with data in the literature. Each constituent detected in the chromatogram was quantified through a calibration curve of galanthamine, using codeine as the internal standard. The amounts are shown as mg of GAL (galanthamine), which was finally related to the alkaloid extract (AE). More details can be found in a previous publication [44].

Parasite Culture
The promastigotes of the WHO reference vaccine strain of Leishmania braziliensis (MHOM/BR/75/ M2904) were grown in M199 medium containing 100 mg·L −1 L-glutamine, 100 U/mL penicillin-G, 100 µg·mL −1 streptomycin and complemented with 10% heatinactivated fetal bovine serum. Incubation of parasites was carried out at 25 • C. Promastigotes were harvested on day 4 or 5 of the culture and used to assess antileishmanial activity [45,46].

In Vitro Assay
The antileishmanial activity of the alkaloid extract against promastigotes was determined using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay [32]. Promastigotes (2 × 10 5 cells/mL) were cultured in freshly prepared M-199 medium supplemented with 10% heat-inactivated fetal bovine serum in the presence or absence of varying concentrations of the alkaloid extract (1-100 µg·mL −1 ) for 72 h at 25 • C. Amphotericin B (1-100 µg/mL) was used as the positive control, and 5% DMSO as the Plants 2023, 12, 322 9 of 13 negative control. After incubation, 5 mg/mL MTT solution was added to the promastigotes in each well and the well plate was incubated for 3 h at 25 • C. In this assay, the yellow tetrazolium MTT dye was reduced to insoluble formazan crystals (purple color) in living cells using NADH as the reducing agent. The formazan crystals formed after incubation were solubilized with acidified isopropanol and incubated at 37 • C for 30 min. The change in color from yellow to purple was read at an absorbance of 570 nm. Bioassays were performed in triplicate, and data were expressed as mean ± standard deviation. The % cell viability and IC 50 were determined from the concentration response curve generated using GraphPad Prism 6.0 software [46,47].

Infection and Treatment of Animals
The animals were separated into 5 experimental groups (n = 6 animals each). Each group was inoculated intradermally in the nose with 1 × 10 6 stationary-phase promastigotes (L. braziliensis) in a volume of 50 µL PBS. These animals had been previously anesthetized with ketamine (50 mg·mL −1 ) and xylazine (5 mg·kg −1 ) intraperitoneally. The animals remained under supervision for three weeks until the appearance of lesions, and were divided into the following groups: Group I: untreated infected; Group II: infected treated with alkaloid extract 0.1 mg·kg −1 day −1 orally; Group III: infected treated with alkaloid extract 0.5 mg·kg −1 day −1 orally; Group IV: infected treated with alkaloid extract 5.0 mg·kg −1 day −1 orally; Group V: infected treated with amphotericin B (5 mg·kg −1 day −1 intramuscularly). To assess the course of the infection, the lesion (mm 2 ) per week was measured, using a digital caliper. After four weeks of treatment, the animals were sacrificed using ketamine combined with xylazine, following the protocols of the Ethics Commission on the Use of Animals. A tissue sample sectioned from the lesion area of each animal was also used for an impression on glass slides. The slides were stained with Giemsa (Sigma), and the infectivity index was determined by multiplying the percentage of macrophages having at least one intracellular parasite by the average number of intracellular parasites per infected macrophage (at least 200 cells/animal were examined) [48][49][50].

Statistical Analysis
The results relating to the antileishmanial activity of Clinanthus milagroanthus were presented as the mean ± standard deviation (SD) of three independent experiments. The software GraphPad Prism 6.0 was used in the analysis of the data. In vitro leishmanicidal activity, indicated as IC 50 , was derived using nonlinear regression analysis. Statistically significant differences for the different groups were determined using the Student's t test, with the p-value < 0.05 being considered significant. For in vivo leishmanicidal activity, a one-way ANOVA statistical test was used to assess the importance of the differences between the various groups, followed by the Tukey test to compare the means of the different treatment groups, with a 95% confidence to be considered significant (p < 0.05).

In Silico Assay-Homology Modeling
For the implementation of the molecular docking experiments, a homology modeling of Leishmania (V.) braziliensis trypanothione reductase (GI: XP_001561849) was created using the methodology proposed in [40]. The search and recover of the template structure was obtained through the Protein Blast (http://blast.ncbi.nlm.nih.gov, accessed on 24 October 2022) and Protein Data Bank (PDB) (http://www.pdb.org, accessed on 24 October 2022) [51] web platforms. The alignment of the sequence and the template design were performed using the software MODELLER v9.10 [52]. Initially 1000 models were created, and subsequently the best model was located based on the lowest DOPE scores calculated by MODELLER [52]. The general stereochemical quality of the final model for Leishmania braziliensis was assessed using the software PROCHECK [39]. Interactive visualization and comparative analysis of molecular structures were performed in the UCSF Chimera [53].

In Silico Assay-Molecular Docking
In this approach, the software AutoDock 4.2 [54] was used through the autodocktools interface [55]. The three-dimensional structures of amphotericine B (positive control), and the alkaloids, were downloaded from pubchem web page and minimized using the UFF force field implemented in the Maestro program [56]. Calculations of the binding energy were performed based on the Lamarckian genetic algorithm [56]. The simulation grid was placed at the active site of the TRLb; specifically, it was centered around Ala 365, Cys 52 and Cys 53 residues, at the position 21.83 Å, 46.28 Å, and 3.36 Å on the x-, y-, and z-axes, respectively, with dimensions of 90 Å × 90 Å × 90 Å and spacing of 0.375 Å between the points of the grid. Analysis of the interactions was performed using the software Maestro [57].

Conclusions
The alkaloid extract of C. milagroanthus collected in Peru showed in vitro activity against L. braziliensis, with an IC 50 value of 18.5 ± 0.3 µg·mL −1 . Furthermore, in vivo assays showed a decrease in lesion size (90%) and in infection (96%) at the highest dose, 1.0 mg·kg −1 ; and in silico experiments, using a built molecular model, suggest important interactions of galanthamine, 7-hydroxyclivonine isomer, and crinine with TRLb. This is the first report on the alkaloid profiling and antileishmaniasis activity of this species, which may be an interesting source of bioactive compounds for leishmaniasis treatment.