Annotation of Alkaloids of Fusaea longifolia and Evaluation of Anti-Plasmodium Activity in vitro and in silico

Isoquinoline alkaloids, especially from the Annonaceae family, have shown biological potential against parasites. Thus, this study aimed to evaluate the potential of the alkaloid fractions of the plant Fusaea longifolia against Plasmodium falciparum and annotate the compounds present in these samples. The tentative characterization of the alkaloids from the leaves and branches of F. longifolia was performed using liquid chromatography coupled to mass spectrometry (LC-MS/MS) and molecular networks. Through manual interpretation of the MS/MS spectra, 18 alkaloids were dereplicated from F. longifolia , 17 of which were reported for the first time in this species. An unpublished putative glycosylated alkaloid was annotated by interpreting the fragmentation data profile. Regarding biological activity, the fractions studied showed high activity against P. falciparum with half-maximal inhibitory concentration (IC 50 ) of 2.42 and 1.60 µg mL -1 for branches and from the leaves, respectively, both similar to the reference standard quinine (IC 50 of 1.24 µg mL -1 ). The structures of the 17 alkaloids were subjected to in silico analysis using molecular docking against four enzymes related to anti-Plasmodium activity (wild type (dm-P f DHFR) and mutant type (qm-P f DHFR), dihydroorotate dehydrogenase (P f DHODH) and purine nucleoside phosphorylase (P f PNP)). Molecular docking revealed strong interactions, especially between oxoxylopine 17 and hydroxycassythicine N -oxide 10 , which may be potential new sources against P. falciparum .


Introduction
Plants of the Annonaceae family from the Amazon have been the target of studies aimed at elucidating their chemical composition in order to use their molecules in the fight against diseases. 1Among the molecules that have been discovered, alkaloids with an isoquinoline skeleton stand out since several biological properties are attributed to them, such as anticancer, 2 anti-inflammatory, 3 and antimalarial properties. 4On the other hand, due to the large number of species of the Annonaceae family, many species are still unstudied or understudied from the chemical and pharmacological points of view.One of these is the species Fusaea longifolia (Aubl.)Saff, which is popularly known as "envira" in Brazil and is distributed throughout the Brazilian Amazon. 5To date, only five alkaloids have been reported for Fusaea longifolia (Aubl.)Saff, one tetrahydroprotoberberine, one aporphine and three oxoaporphines. 6,7In addition to the fixed components, the composition of the essential oil from the aerial parts, which has trypanocidal activity, has been reported, in which sesquiterpenes were the majority, especially β-selinene, cis-β-guaiene and (Z)-α-bisabolene. 8alaria remains one of the most serious and potentially fatal infectious diseases in many tropical and subtropical countries, and is caused by parasites such as Plasmodium falciparum, which are transmitted to people through the bite of Anopheles mosquitoes (infected females). 9P. falciparum is considered the most dangerous species, as this protozoan is the main etiological agent involved in cases of severe malaria and, consequently, is the main cause of deaths from the disease. 10In 2021, there were 247 million new infections and 619,000 deaths worldwide.Africa accounted for 95% of all deaths and almost all cases of malaria in this region are due to infection by P. falciparum. 10 In Brazil, malaria is a major public health problem.In 2019, 157.454 cases and 1.912 hospitalizations were reported, with 83% of cases occurring in the northern region. 11In addition, P. falciparum has demonstrated resistance to existing antimalarial drugs.This generates greater suffering for those infected and is a current challenge for researchers since there is now a need to identify new antimalarial drugs. 12In this sense, the alkaloids reported in the family Annonaceae have shown promise against Plasmodium spp. 13ecause numerous proteins are essential in various metabolic processes in Plasmodium falciparum parasites, they are used as molecular targets for the evaluation of potential antimalarial agents.][16] Inhibition of these enzymes disrupts deoxyribonucleic acid (DNA) synthesis or the production of biomolecules that are necessary for parasitic multiplication, which represents a promising route for the development of new antimalarial agents. 17iven this scenario, the investigation of alkaloids that are potentially useful against the target enzymes in malaria continues to attract the interest of a number of research groups, which employ various different approaches.Among these, approaches based on mass spectrometry (MS) aiming at the rapid characterization of known molecules in extracts rich in alkaloids have been routinely applied. 18ore recently, the ability to process MS data in openaccess platforms, such as Global Natural Products Social Molecular Networking (GNPS), have aided in data interpretation and screening for unknown alkaloids. 19hus, the objective of this study was the chemical characterization of the alkaloid fractions of leaves and branches of F. longifolia by means of high-performance liquid chromatography coupled to mass spectrometry (LC-MS/MS) with the aid of molecular networks, as well as the in vitro evaluation of the alkaloid fractions against P. falciparum and cytotoxic evaluation against healthy human lung fibroblast lineage (MRC-5) cell line.In addition, the dereplicated structures in the alkaloid fractions were evaluated in silico against four different proteins that are essential for parasitic multiplication of P. falciparum.

Plant material
The leaves and branches of F. longifolia (Aubl.)Saff.were collected in February 2017 at the Museu da Amazônia (MUSA), Manaus, Amazonas, Brazil (3°00'11.4'S;59°56'22.8'W),and were identified in the Herbarium of the Instituto Nacional de Pesquisa da Amazônia (INPA), where a voucher (number No. 281627) was deposited.After collection, the botanical material was dried at room temperature (ca. 25 °C) for 72 h and then subjected to pulverization in a knife mill, which yielded 314.5 and 405.7 g of powdered leaves and branches, respectively.Subsequently, the pulverized material was subjected to extraction via maceration with n-hexane from Dinâmica (Indaiatuba, SP, Brazil) for 48 h (3 × 1 L) and then with methanol (MeOH) from Dinâmica (Indaiatuba, SP, Brazil) for 48 h (3 × 1 L), yielding 38.21 and 29.60 g of leaf and twig extract, respectively.Immediately after the macerations, aliquots of 10 g of each methanolic extract were subjected to the acid-base extraction procedure according to a previously described methodology, 20 which yielded 217.4 and 161.7 mg of alkaloid fractions of leaves and branches, respectively.This research was registered in the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (SisGen) under the code AFF9974.

LC-MS/MS analysis
The analyses were carried out on an ultra-high performance liquid chromatography (UHPLC) system (Shimadzu Nexera X2, Shimadzu, Kyoto, Japan) coupled to a quadrupole time-of-flight mass spectrometer (MicroTOF-QII; Bruker Daltonics, MA, USA) with an electrospray source, using a concentration of 1 mg mL -1 of the alkaloid fractions.The separation of 5 μL samples was performed using a Luna C18 column (150 × 4.6 mm, 2.1 µm) (Phenomenex, USA) at 50 °C, which were eluted at a flow rate of 0.35 mL min -1 , isocratically, using (15:85) MeCN/Milli-Q H 2 O (both acidified with 20 mM formic acid) from 0 to 2 min and sequential linear gradient up to 95% MeCN for 12 min.The gradient was held for 5 min, followed by a 4 min equilibration at 15% B prior to the next injection.The electrospray ionization conditions (positive mode) were set as follows: capillary potential of 4500 V, temperature of drying nitrogen gas 200 ºC at a flow rate of 9 mL min -1 , nebulizer pressure of 4 bar.Mass spectra were acquired using electrospray ionization in the positive mode over an m/z range from 50 to 1200.The QTOF instrument was operated in scan and Auto MS/MS mode, and MS/MS experiments were performed on the five most intense ions from each MS survey scan.Accurate mass data were processed using Data Analysis 4.2 software (Bruker Daltonics, Bremen, Germany). 21

Construction of molecular networks and annotation
For the comparison of the metabolite profiles of the leaves and branches, as well as alkaloid annotation, the product ion spectra resulting from the LC-MS/MS analysis of F. longifolia alkaloid fractions were analyzed and organized in molecular networks using the GNPS platform. 22Tandem mass spectra data was converted to the format .mzXMLwith MS-convert software 3.0.2113246 23and then loaded onto the GNPS platform.Parameters for molecular network generation were defined as follows: mass of precursor ions with tolerance of 0.05 Da, product ion tolerance of 0.5 Da, ions below 10 counts were removed from MS/MS spectra.Molecular networks were generated using a cosine score of 0.6.Data were visualized using the software Cytoscape 3.7.0. 24Annotation of isoquinoline alkaloids present in the samples was performed by manual interpretation of MS/MS spectra compared to the IQAMDB (IsoQuinoline and Annonaceous Metabolites Data Base) database. 20The molecular networks generated in this study are available for consultation. 25 vitro anti-Plasmodium activity Antimalarial activity against P. falciparum (FRC3 strain) was tested via flow cytometry using the traditional technique of candle burning in a desiccator providing an atmosphere rich in carbon dioxide and poor in oxygen. 26he strain was maintained in incomplete Roswell Park Memorial Institute Medium (RPMI) with 10% human serum and fed with normal human erythrocytes A+ at 37 °C.The antiparasitic test of the alkaloid fractions was performed in triplicate with 2% hematocrit and 3 to 5% parasitemia using quinine as the reference drug.The stock solutions were prepared in dimethyl sulfoxide (DMSO) from Nuclear (Diadema, SP, Brazil) (0.02-0.05% final concentration) and serially diluted in the same culture medium (concentrations from 100 to 0.01 µg mL -1 in five dilutions).The reading was performed after 72 h by quantifying the percentage of parasitemia in a flow cytometer (FAC-SCAN; Becton Dickinson, NJ, USA) with the use of ethidium bromide dye (Amresco, Solon, OH, USA).The inhibitory concentration at 50% (IC 50 ) was determined from the dose-response curve of F. longifolia fractions vs. parasitized red blood cells.The percentage of inhibition of parasite growth was determined using the formula of Lopes et al. 27 The concentration responsible for 50% inhibition of total parasitemia (IC 50 ) was calculated using GraphPad Prism 8 28 software based on a logarithmic plot of dose versus inhibition (expressed as a percentage relative to the control) using nonlinear regression analysis.

Cytotoxic activity
The cytotoxicity of the fractions was evaluated on the proliferation of non-cancerous cell lines MRC5 (human lung fibroblast), which were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, UEA).Cell viability was verified using the Alamar Blue assay as previously described 29 with minor modifications. 30Cells were cultured as recommended by ATCC guidelines, and a Mycoplasma Staining kit (Sigma-Aldrich, São Paulo, Brazil) was used to confirm that the cells were free of contamination.Doxorubicin (purity ≥ 95%, doxorubicin hydrochloride, IMA S.A.I.C. Laboratory, Buenos Aires, Argentina) was used as the positive control.The values of IC 50 with 95% confidence intervals were obtained via nonlinear regression using GraphPad Prism (Intuitive Software for Science). 28

Molecular docking
The molecular docking assay was performed according to the approach indicated by Santos et al. 31 First, the 3D structures of the alkaloids were generated and verified in relation to the protonated state at pH 7.4 and the tautomers via Marvin Sketch software. 32Then, the structures were optimized for the conformations with lower energy using the semi-empirical method PM7 using MOPAC2016 software. 33The refined structures were converted to PDBQT files via Autodock tools. 34The crystal structures of the protein targets were obtained from the Protein Data Bank (PDB).The selected structures of P. falciparum were dm-PfDHFR (PDB ID: 1J3J), PfDHODH (PDB ID: 1TV5), qm-PfDHFR (PDB ID: 1J3K) and PfPNP (PDB ID: 5ZNC).All four proteins are complexed with the reference drugs pyrimetamine, teriflunomide, WR99210 and quinine, respectively.Finally, molecular docking was performed using Autodock Vina 35 and the results were visualized in Discovery Studio. 36The validation of the docking protocol was done by means of redocking, seeking conditions of root-mean-square deviation (RMSD) < 2 Å. 34,35

LC-MS analysis and molecular networking
In order to characterize the alkaloids in the leaves and branches of F. longifolia, we performed LC-MS/MS analysis, from which 18 different chemical species were annotated (15 in the leaves and 13 in the branches) (Figure 1).All the compounds that were annotated are from the class of isoquinoline alkaloids.In particular, molecules with tetrahydroprotoberberine (two), benzylisoquinoline (seven), aporphine (five) and oxoaporphine (three) skeletons were observed.These alkaloids were identified based on the observation of m/z pairs, referring to protonated structures containing only one nitrogen atom, organization in molecular networks and were matched with the IQAMDB database. 20The annotated alkaloids stepholidine (3), reticuline (5), coclaurine (1) and N-methylcoclaurine (2) were directly indicated by the IQAMDB database, while the others were confirmed through manual interpretation of MS/MS spectra with comparison with previously published data. 18The MS/MS data, when visualized using molecular networks, allowed the observation of the grouping of the nodes according to the types of isoquinoline skeletons found in the samples.The molecular network generated (Figure 2) presented a majority cluster with seven nodes, and three smaller clusters, but with little or no significance in the chemical composition of interest of the sample.
Initially, our analysis approach was validated using the stepholidine alkaloid at m/z 328.1533 [M + H] + (3, C 19 H 21 NO 4 , -4.57ppm), which was used as a seed in this study.This pattern (3), previously isolated by our research group (Figures S3-S11, Supplementary Information (SI) section), was chosen as the seed due to its prior identification in the same species. 7Directly connected to this node, m/z 342.1707 [M + H] + was annotated as the isocorypalmine alkaloid (13, C 20 H 23 NO 4 , 0.58 ppm) (Figure S21, SI section), since they both showed a very similar fragmentation profile. 37Another group of alkaloids was identified in the same main cluster, which were of the benzylisoquinoline type (Figure 2 41 The fragmentation spectra of compounds 1, 2, 4-7, 13 and 14 showed very similar fragmentation profiles, in which the dominant losses were the exit of the southern portion of the molecule (ring C, substituted benzyl) and the northern portion (ring A, substituted isoquinoline nucleus), in which the substitution patterns of aromatic rings A and C were deduced by the masses of the fragments, as well as by characteristic losses previously described (Table 1). 18Glycosylated alkaloids are rarely found in natural sources; however, isoquinoline alkaloids with glucose portions in their structure have been reported in the literature. 43mong the substances annotated above, only stepholidine (3) is described in F. longifolia. 7Thus, the other substances identified by LC-MS/MS and via molecular networking are reported for the first time for the target species of this study and for the genus Fusaea.In addition, some of these alkaloids already have proven pharmacological properties.The alkaloid stepholidine (3), which was isolated from Annona cherimola, is a promising neuroprotector, 44 and isocorypalmine ( 13) has an insecticidal effect. 45The compounds reticuline (5) and N-methylcoclaurine (2), isolated from Peumus boldus (Monimiaceae), show promising activities of inhibition of butyrylcholinesterase, an enzyme of the cholinesterase group. 46The natural product, coclaurine (1), isolated from Annona squamosa, has cytotoxicity against colon cancer cells (HCT116), human breast cancer cells (MCF-7) and human liver cancer cells (HepG2). 2 From the analysis of the molecular network, other nodes of the main cluster that correspond to aporphine alkaloids were annotated, which were corroborated based on fragmentation patterns. 47When analyzing the MS/MS spectra, neutral losses of 17 Da (-NH 3 ) (9 and 12) and 31 Da (-NH 2 CH 3 ) (11 and 15) were observed.In addition, the fragmentation pathways for compounds 12, 15 and 10 indicated neutral losses of methanal (-CH 2 O, 30 Da) with concomitant elimination of carbon monoxide (-CO, 28 Da), which is characteristic of the presence of a methylene dioxide group in the rings A or D. 47 Compounds 9 and 11, presented pathways consistent with aporphines with adjacent methoxyl groups in the ring A, which establish competitive radical losses of 31 Da (•OCH 3 ) and 15 Da (•CH 3 ).Therefore, these ions may correspond to the alkaloids norisocorydine 9 (m/z 328.1568 [M + H] + , C 19 H 21 NO 4 , 5.78 ppm) (Figure S17, SI section), 48 corydine 11 (m/z 342.1719 [M + H] + , C 20 H 23 NO 4 , 4.09 ppm) (Figure S19, SI section), 49 S24, SI section) were attributed to fragment pathways for oxoaporphine alkaloids, as described above 53 for oxoxylopine alkaloids, 54 homomoschatoline 55 and oxobuxifoline, 56 respectively.The aporphine and oxoaporphine alkaloids mentioned above are reported for the first time in the species discussed in this study and in the genus Fusaea.

Anti-Plasmodium activity
The alkaloid fractions of F. longifolia were sent for in vitro assays against strains of P. falciparum (FRC3), with quinine as the control.The alkaloid fractions showed high anti-Plasmodium activity and exhibited an IC 50 of 2.42 µg mL -1 (branches) and 1.60 µg mL -1 (leaves), which was similar to the action of quinine, which has an IC 50 of 1.24 µg mL -1 .
Several reports in the literature contribute and strengthen these results, such as the studies by Boyom et al. 60 in which the in vitro antimalarial activity of extracts (MeOH) from the stem bark of Xylopia africana (Annonaceae) against W2 resistant strains of P. falciparum showed significant results with an IC 50 of 1.07 µg mL -1 .Extracts (MeOH) from leaves of Guatteria amplifolia (Annonaceae) have been proven to be quite active against D2 strains of P. falciparum, with an IC 50 of 1.5 µg mL -1 . 61On the other hand, the alkaloid fractions of seeds of Anonidium mannii (Annonaceae) were active with an IC 50 value of 2.4 µg mL -1 against strain W2 of P. falciparum. 60he significant activity shown in these results can be attributed to the alkaloids that are present in the fractions, and antiplasmodic activity has already been reported in the literature.In the search for new antimalarial agents, Levrier et al. 62 isolated anonaine, an alkaloid benzylisoquinoline already reported in F. longifolia, 6 from the leaves of Goniothalamus australis (Annonaceae) and revealed a significant anti-Plasmodium effect against strain 3D7 of P. falciparum, with an IC 50 of 2.7 µg mL -1 .
In this sense, the results lead to believe that the promising anti-Plasmodium activity reported in the present study is attributed to the isoquinoline alkaloid constituents present in extracts of species of the Annonaceae family.This highlights the importance of prospecting and conducting anti-Plasmodium tests with this class of substances.

Cytotoxic activity
The alkaloid fractions of the leaves and branches of F. longifolia were submitted to the cell viability test using the healthy human lung fibroblast lineage (MRC-5).The samples were considered active when they presented an IC 50 higher than 50 µg mL -1 ; therefore, the higher the IC 50 value, the better the result in this situation, with samples being less toxic to healthy cells.As such, the alkaloid fractions showed satisfactory results, with moderate cytotoxic activity, 63 and mean IC 50 values of > 50 µg mL -1 .Doxorubicin was used as the positive control and exhibited an IC 50 of 3.18 µg mL -1 .
Previous studies 64 show that extracts and alkaloids of species of the Annonaceae family demonstrate low cytotoxicity against the non-cancerous cell line MRC-5.One example of this is the recent study by Costa et al., 1 who isolated nine alkaloids (isoquinoline derivatives) from Diclinanona calycina (Annonaceae) bark and these were evaluated against non-cancerous cell lines.In the promising results, five alkaloids (thalifoline, reticuline, reticuline N β -oxide, reticuline N α -oxide and bisnorargemonine) showed low cytotoxicity against the MRC-5 cell line (> 25 µg mL -1 ).Another example is the alkaloid vincosamide, isolated from Psychotria leiocarpa (Rubiaceae), which showed moderate cytotoxicity with an IC 50 of 50 µg mL -1 , but with a high reduction in infectious diseases such as dengue.Despite vincosamide's moderate cytotoxicity, these results indicate this compound as a potential anti-dengue agent. 63Thus, the importance of finding efficient results with alkaloid fractions that are not toxic to healthy cells becomes a relevant factor for the continuity of studies with the species F. longifolia.

Molecular docking
The dereplicated compounds were subjected to molecular docking assays against four different proteins from P. falciparum.The first step of the molecular docking process was redocking, a procedure that involves the removal and repositioning of the ligand at the protein binding site, which allows the evaluation of the reproduction capacity and validity of the results obtained.The redocking results of dm-PfDHFR (PDB ID: 1J3J), PfDHODH (PDB ID: 1TV5), qm-PfDHFR (PDB ID: 1J3K) and PfPNP (PDB ID: 5ZNC), were -0.4912, -0.5461, -0.8038 and -0.9917 Å, respectively (Figure 3).These values are considered acceptable for the redocking procedure (RMSD < 2), and suggest that the model is able to reproduce the correct conformation of the ligand.These results are in agreement with virtual screening approaches of P. falciparum developed with these models. 17ext, the annotated alkaloids were similarly tested at the binding site of the target enzymes.The binding energy of the alkaloids (1-18) in relation to PfPNP (PDB ID: 5ZNC) ranged from -6.8 to -10.1 kcal mol -1 , and alkaloids 5, 1, 17 and 16 represented higher affinities when compared to quinine.Notably, the oxoaporphine alkaloid oxoxylopine 17 (-10.1 kcal mol -1 ) had a higher score than the reference drug (quinine, -8.5 kcal mol -1 , RMSD < 2).The interactions observed for alkaloid 17 showed that the oxygen of the O-methyl group established a hydrogen interaction with Ser91, while the nitrogen of the N-methyl group formed a hydrogen interaction with the residue Asp206, these being residues of the catalytic site. 65In addition, π-alkyl interactions were observed in the rings A and C with Met183, rings B and E with Val181 and rings D and E with Pro209.Whereas, π-π stacking interaction in ring D with Tyr160 and Trp212 was also shown (Figure 4 and Table S1, SI section).
The binding affinity of teriflunomide for PfDHODH (PDB ID: 1TV5) was -9.6 kcal mol -1 , while alkaloids were bound to the same protein with binding energies ranging from -5.6 to -9.8 kcal mol -1 .Alkaloids 12, 10 and 18 had higher protein binding affinities, and the aporphine alkaloid hydroxycassythicine N-oxide 10 (-9.8 kcal mol -1 , RMSD < 2) was more effective in binding-protein affinity when compared to teriflunomide (Table S2, SI section).The interactions for alkaloid 10 showed that the oxygen atom of the methylenedioxy group participated in hydrogen interaction with Cys276, and hydrogen interaction between the hydroxyl oxygen of the aromatic ring C with Ser505, and a π-alkyl interaction was observed for the methylenedioxy group with Ile263, which are considered key residues of the active site. 66he co-crystallized ligand WR99210 for the quadruple mutant qm-PfDHFR (PDB ID: 1J3K) has a binding affinity of -8.3 kcal mol -1 , and the binding energy of alkaloids in relation to the protein are between -7.1 and -9.6 kcal mol -1 .Among these, alkaloids 14, 4-6, 9-15, 17-16 have higher affinities compared to WR99210, and alkaloid 17 was superior with -9.6 kcal mol -1 , RMSD < 2 (Table S3, SI section).The interactions observed for the alkaloid oxoxylopine 17 showed that the oxygen atom of the methylenedioxy group participated in hydrogen interaction with Tyr170, while the nitrogen of the N-methyl group of ring E and the carbonyl of ring B formed hydrogen interactions with the residue Ser111, as well as the oxygen of the O-methyl group of the ring A, which participated in hydrogen interaction with Ser167.Alkaloid 17 also showed π-π stacking interaction in rings D and A with the catalytic residue Phe58 and Leu40. 15egarding dm-PfDHFR (PDB ID: 1J3J), alkaloids 4, 12, 15, 17 and 16 presented better values, between -8.0 to -9.4 kcal mol -1 .Overall, once more the alkaloid oxoxylopine 17 stood out by showing binding energy of -9.4 kcal mol -1 , RMSD < 2, while pyrimetamine was bound to the protein with binding energy of -7.9 kcal mol -1 (Table S4, SI section).The interactions observed for alkaloid 17 showed that the carbonyl of ring B formed a hydrogen interaction with Ser111, and the nitrogen of the N-methyl group of ring E formed a hydrogen interaction with the residue of the active site Asn108. 66In addition, π-alkyl interactions were observed in ring B with Leu46, and in rings C and D with Ala16.
When the docking analyses are considered, the π-π interactions can be seen dominant for the oxoaporphine alkaloid oxoxylopine 17, which favors the activity in the enzymes PfPNP, qm-PfDHFR and dh-PfDHFR.The binding site of ligands to the enzymes is strongly hydrophobic and coated by aromatic residues, thus suggesting an increase in potency via the accommodation of compounds with interactions favored by π-π stacking at the binding site.Oxoaporphines are aromatic and planar structures, which makes their structure more rigid and with limitations of degrees of freedom.These characteristics guarantee a more effective coupling; therefore, docking is favored.The literature shows that oxoaporphine alkaloids, such as liriodenine and lysicamine have potential in vitro against P. falciparum. 67Alkaloid oxoxylopine (17) showed cytotoxicity against U251e and HEPG2, with IC 50 values of 4 and 2.5 µg mL -1 , respectively. 59Oxoxylopine also has antimicrobial activity 68 and antiplatelet activity. 69he docking study provided a very useful tool for interpreting the results of the in vitro inhibitory activity of the alkaloid fractions against P. falciparum, and indicated that the dereplicated alkaloids under investigation may be able to bind effectively to the active site of target proteins are vital to the parasite.These observations, along with key interactions in the docking analyses, may be useful when planning new antimalarials.Overall, the proposed approach suggests the usefulness of alkaloids 10 and 17 as a suitable model that can be used as a prototype for the design of new therapeutic agents that are capable of disrupting the crucial functions of P. falciparum enzymes.Given this scenario, the search for new antimalarial drugs is essential and natural products have played an important role in the discovery of molecules with chemotherapeutic activity for the treatment of human diseases.

Conclusions
The combination of manual interpretation of LC-MS/MS spectra with the analysis of molecular network data allowed the dereplication of 18 isoquinoline alkaloids in F. longifolia species and, among these, an unknown glycosylated alkaloid was annotated.Among the known alkaloids, 17 are described for the first time in F. longifolia; nevertheless, NMR analysis is needed to identify the new molecules.In addition, these findings help in the tedious isolation of constituents, thus minimizing costs and optimizing the time spent in this process, and is a useful strategy to avoid the reisolation of compounds already described in the literature.
The biological tests confirmed moderate cytotoxic activity in healthy MRC-5 cells and the anti-Plasmodium potential of the species F. longifolia was observed for the branches (IC 50 of 2.42 µg mL -1 ) and the leaves (1.60 µg mL -1 ), which can be associated with synergism of bio-isoquinolinic alkaloids present in the fractions.Together with this, this study showed a good correlation of the experimental values of IC 50 with the in silico activity of molecular docking, thus providing a better understanding of the inhibitory potential of dereplicated alkaloids, especially oxoaporphine (17) and aporphine (10)  alkaloids, as new sources of protein inhibitors vital to the parasite P. falciparum.These findings deserve attention and stimulate the intensification of investigations of the full pharmacological potential of these compounds.The results obtained contribute to the knowledge of natural products of the Annonaceae family, as well as highlight F. longifolia as a source of bioactive substances.

Figure 1 .
Figure 1.Total ion chromatogram of F. longifolia from the alkaloid fraction of leaves (a) and branches (b).
The molecular formula C 24 H 31 NO 8 (4.11 ppm) was deduced from the exact mass, and this information in conjunction with the initial neutral loss of 162 Da (-hexoside, m/z 462 → m/z 300) indicated the presence of a glycosylated isoquinoline alkaloid.Then, characteristic cleavage of the amino portion was observed at 17 Da (-NH 3 ) (m/z 300 → m/z 283) with subsequent losses of 32 Da (-CH 2 OH) (m/z 283 → m/z 251) and 28 Da (-CO) (m/z 251 → m/z 223), which is characteristic of adjacent hydroxyl and methoxyl groups on ring A. Furthermore, compound 8 produced diagnostic fragment ions typical of the benzylisoquinoline skeleton at m/z 178 and 121, indicating vicinal methoxyl and hydroxyl groups in ring A and methoxyl group in ring C.

Figure 2 .
Figure 2. Annotation of the molecular network of the alkaloid-rich fraction derived from the methanolic extract of branches and leaves of F. longifolia, which shows benzylisoquinolines (blue knots), tetrahydroisoquinolines (green knots), aporphines (pink knots) and oxoaporphines (red knots).Nodes in gray, without m/z description, could not be annotated.

Figure 3 .
Figure 3. Overlapping redocked ligands (in red) with the ligand-bound conformations of the X-ray crystal structures (blue).

Figure 4 .
Figure 4. General interactions of lower energy alkaloids with amino acid residues in the binding site region of the P. falciparum protein complex.