Chemical Survey of Three Species of the Genus Rauhia Traub (Amaryllidaceae)

Plant biodiversity is an important source of compounds with medicinal properties. The alkaloid galanthamine, first isolated from Galanthus woronowii (Amaryllidaceae), is approved by the FDA for the palliative treatment of mild to moderate Alzheimer’s disease due to its acetylcholinesterase (AChE) inhibitory activity. Obtaining this active pharmaceutical ingredient, still sourced on an industrial scale from the Amaryllidaceae species, is a challenge for pharmaceutical companies due to its low natural yield and the high cost of its synthesis. The aim of this work was to determine the alkaloid profile of three different Rauhia (Amaryllidaceae) species collected in Peru, and to assess the potential application of their extracts for the treatment of Alzheimer’s disease. The alkaloids were identified by gas chromatography coupled to mass spectrometry (GC-MS), and the AChE inhibitory activity of the extracts was analyzed. Thirty compounds were quantified from the Rauhia species, the R. multiflora extract being the most interesting due to its high diversity of galanthamine-type structures. The R. multiflora extract was also the most active against AChE, with the half maximal inhibitory concentration (IC50) values of 0.17 ± 0.02 μg·mL−1 in comparison with the IC50 values of 0.53 ± 0.12 μg·mL−1 for galanthamine, used as a reference. Computational experiments were carried out on the activity of the galanthamine-type alkaloids identified in R. multiflora toward five different human AChE structures. The simulation of the molecules 3-O-acetylgalanthamine, 3-O-acetylsanguinine, narwedine, and lycoraminone on the 4EY6 crystal structure theoretically showed a higher inhibition of hAChE and different interactions with the active site compared to galanthamine. In conclusion, the results of this first alkaloid profiling of the Rauhia species indicate that R. multiflora is an important natural source of galanthamine-type structures and could be used as a model for the development of biotechnological tools necessary to advance the sustainable production of galanthamine.


Introduction
According to the World Health Organization (WHO), important medical and pharmacological discoveries are made through a greater understanding of the Earth's biodiversity [1]. Nature is a source of natural products and/or natural product structures that play a significant role in the search for new drugs [2]. Alkaloids, nitrogenated compounds metabolized mainly by plants, are of particular interest in the development of new medicines due to their structural diversity [3,4]. The plant family Amaryllidaceae, specifically the Amaryllidoideae subfamily, contains exclusive isoquinoline alkaloids known as Amaryllidaceae alkaloids, which show remarkable biological activities [5]. This subfamily contains more The aim of this work was to evaluate the diversity of alkaloids in the bulb extracts of three species of the genus Rauhia collected in Peru and to verify their potential use in Alzheimer's disease therapy. The species R. staminosa, R. decora, and R. multiflora were analyzed by gas chromatography coupled to mass spectrometry (GC-MS) and in vitro and in silico experiments were carried out to analyze their acetylcholinesterase inhibitory activity. The aim of this work was to evaluate the diversity of alkaloids in the bulb extracts of three species of the genus Rauhia collected in Peru and to verify their potential use in Alzheimer's disease therapy. The species R. staminosa, R. decora, and R. multiflora were analyzed by gas chromatography coupled to mass spectrometry (GC-MS) and in vitro and in silico experiments were carried out to analyze their acetylcholinesterase inhibitory activity.

Alkaloid Profiling
GC-MS analysis revealed 30 alkaloids in the species R. staminosa, R. decora, and R. multiflora, three of which were not identified (Table 1 and Figures S1-S3). Each al-Plants 2022, 11, 3549 4 of 13 kaloid described in Table 1 was quantified as a µg of galanthamine (GAL), which was related to the mg of the dry weight (µg GAL·100 mg −1 DW). The identified structures are presented in Figure 2. More than 650 Amaryllidaceae alkaloids are reported in the literature [17] and their structures are classified into 42 skeleton types, among which lycorine, haemanthamine, homolycorine, galanthamine, and pretazettine are among the most representative [17]. As shown in Table 1, the alkaloids identified in the Rauhia species in the present study are cataloged according to the scaffold type.
Among the species listed in Table 1, R. decora (sample B) had the highest diversity of the Amaryllidaceae alkaloid groups, with lycorine-, galanthamine-, homolycorine-, haemanthamine-, and pretazettine-type skeletons being detected. The most prevalent was the lycorine-type alkaloid, identified as 2-O-acetyl-9-O-methylpseudolycorine (12) (135.6 µg GAL·100 mg −1 DW). One unidentified structure was found in this species, which exhibited the usual fragmentation pattern of homolycorine-type alkaloids. More than 650 Amaryllidaceae alkaloids are reported in the literature [17] and their structures are classified into 42 skeleton types, among which lycorine, haemanthamine, homolycorine, galanthamine, and pretazettine are among the most representative [17]. As shown in Table 1, the alkaloids identified in the Rauhia species in the present study are cataloged according to the scaffold type.
The alkaloid galanthamine has been used for the palliative treatment of mild to moderate symptoms of Alzheimer's disease since 2001 [18,19]. Different Amaryllidaceae plants metabolize galanthamine, the species Narcissus cv Carlton, Leucojum aestivum, and Lycoris radiada being the principal sources for pharmaceutical companies [20]. Additionally, high concentrations of galanthamine have been described in an in vitro culture of Hippeastrum papilio (Amaryllidaceae), patented under the number EP2999480B1 [21].
The presence of substantial amounts of other types of Amaryllidaceae alkaloids, especially lycorine, can be a hindrance for the industrial process of the purification of galanthamine [20,22]. In the preparation of galanthamine as an active pharmaceutical ingredient, no lycorine should remain due to its cytotoxicity [23,24]. The low natural yield of galanthamine and the costly and time-consuming processes required for its industrial-scale production call for the development of efficient tools that can control its biosynthesis [25].
Among the results presented in Table 1, the species R. multiflora stands out for its high diversity of galanthamine-type alkaloids and different unquantified Amaryllidaceae alkaloid scaffolds, especially lycorine-type structures. This species may therefore be a suitable candidate for use as a model plant to elucidate the biosynthesis of galanthamine-type alkaloids. The knowledge generated would contribute to developing new biotechnological approaches for the sustainable and scaled-up production of galanthamine.
Among the results presented in Table 1, the species R. multiflora stands out for its high diversity of galanthamine-type alkaloids and different unquantified Amaryllidaceae alkaloid scaffolds, especially lycorine-type structures. This species may therefore be a suitable candidate for use as a model plant to elucidate the biosynthesis of galanthaminetype alkaloids. The knowledge generated would contribute to developing new biotechnological approaches for the sustainable and scaled-up production of galanthamine.
Many studies have described the alkaloid profiles and AChE inhibitory properties of different Amaryllidaceae species from South America, but none of the published results match the findings reported here for R. multiflora. Despite differences in the extract preparation, it is of interest to briefly review the most important results reported in the literature on this topic.
In recent studies on six species of Phaedranassa and the species Crinum x amabile Donn collected in Ecuador, Phaedranassa cuencana Minga, C. Ulloa, and Oleas was the most active against EeAChE, with IC 50 values of 0.88 ± 0.11 µg·mL −1 ; three galanthamine-type alkaloids were detected: galanthamine, sanguinine, and N-demethylgalanthamine [33][34][35]. The plant Ismene amancaes (Ker Gawl.) Herb. collected in Peru showed a low activity against EeAChE, with IC 50 values of 14.6 ± 0.6 µg·mL −1 , although high concentrations of lycoramine, a galanthamine-type alkaloid, were detected [36]. Among different species of the genera Rhodophiala, Rhodolirium, and Phycella collected in Chile, the bulb extract of Rhodophiala splendens (Renj.) Traub was the most active against EeAChE, with IC 50 values of 3.62 ± 0.02 µg·mL −1 , although no galanthamine-type alkaloid was reported in this plant [32,[37][38][39]. A study on the genera Amaryllis, Zephyranthes, and Crinum collected in Venezuela found the strongest EeAChE inhibitory activity in a C. amabile extract, with  [40]; the main alkaloids found in the extract were of the crinine/haemanthamine type, and sanguinine, a galanthamine-type alkaloid, was also detected [40]. Among nine species of the genera Hippeastrum and Rhodophiala bifida (Herb.) Traub, all collected in Brazil [41,42], the species Hippeastrum papilio (Ravenna) Van Scheepen and Hippeastrum glaucescens (Mart. ex Schult. & Schult. f.) Herb. were the most active against EeAChE, with IC 50 values from 0.33 to 0.49 µg·mL −1 , and galanthamine was the main constituent in both extracts [41]. Species from the genus Habranthus, Hieronymiella, Hippeastrum, Phycella, and Rhodophiala, all collected in Argentina, were investigated as possible sources of cholinesterase inhibitors [26,[43][44][45]. Among them, the species Habranthus jamesonii (Baker) Ravenna and Zephyranthes filifolia Herb. ex Baker and Kraenzl., collected in San Juan and Mendoza, respectively, were described as the most active against AChE, with IC 50 values of 1 ± 0.01 and 1 ± 0.08 µg·mL −1 , respectively, and found to contain galanthamine-type alkaloids among their chemical profiling [45]. Recently, the species of the genus Eucharis Planch reported herein have been revised and re-cataloged as Urceolina Rchb., as well as Habranthus Herb. and Rhodophiala C. Presl, which have both been renamed as Zephyranthes Herb. [46]. A visual representation of this information is provided in Figure 4, which lists all the reported genera according to the place of collection and using the updated generic names.

Molecular Docking
As shown in Table 2, eight galanthamine-type alkaloids identified in R. multiflora were evaluated by molecular docking. Galanthamine, sanguinine, narwedine, 3-O-acetylgalanthamine, and 3-O-acetylsanguinine have a double bond between C-1 and C-2. Sanguinine, Odemethyllycoramine, and 3-O-acetylsanguinine show a hydroxyl group at C-9, whereas the other galanthamine-type structures found in R. multiflora present a methoxy group at this position. Most of the structures detected in this species have a hydroxyl group at C-3, although alkaloids with carbonyl and acetoxy groups at this position were also found in this plant extract (Figure 2). All the computational assays were carried out on five different X-ray crystals of human acetylcholinesterase (hAChE): 4EY5, 4EY6, 4EY7 [47], 4M0E, and 4M0F [48]. In a molecular docking experiment, the ligands, ions, and water molecules are eliminated from the Protein Data Bank (PDB) file, so the topological form of the active site should be distinguished by the amino acid orientation around the co-crystalized ligand. Five X-ray PDB structures co-crystalized with different ligands were selected and deleted. Our reference protein was 4EY6, a PDB structure crystalized with galanthamine as the ligand. In this protein, the geometric distribution of a monoacid around the pocket (active site) is optimum for hosting molecules similar to galanthamine. Accordingly, the molecules 3-O-acetylgalanthamine, 3-O-acetylsanguinine, narwedine, and lycoraminone have higher binding free energy (BE) values than galanthamine (upper 8.75 kcal·mol −1 ).
On the other hand, as molecular docking treats the protein as a rigid body, each hAChE crystal has an active site with a slightly different geometry, relative to each ligand co-crystalized in the X-ray diffraction experiment. For example, in 4EY5, the co-cristallized ligand was huperzine A. The molecular docking results show the same behavior for 4EY6: the alkaloids 3-O-acetylgalanthamine, 3-O-acetylsanguinine, narwedine, and lycoraminone had the highest BE values. As with 4EY5 and 4EY6, the molecular docking experiments for 4EY7 revealed that 3-O-acetylgalanthamine, 3-O-acetylsanguinine, narwedine, and lycoraminone had the highest BE values (upper 9.83 kcal·mol −1 ). For 4EY7, the co-crystallized ligand was donepezil.
In the case of 4M0E, the original co-cristallized ligand was dihydrotanshinone I, a molecule with a more planar conformation than galanthamine. The molecular docking experiment showed that lycoraminone will form the most stable ligand-protein complex. For 4M0F, the alkaloid with the lowest BE was 3-O-acetylsanguinine; however, the values for the 3-O-acetylgalanthamine, narwedine, and lycoraminone molecules were also significantly high (upper 8.74 kcal·mol −1 ). The territrem, a large molecule in a 3D conformation, was the co-crystallized ligand on 4M0F.
To understand the arrangement of amino acids around the active site in the reference protein 4EY6, a 2D ligand-protein interaction diagram for the most energetically stable alkaloids within the active site was generated and is presented in Figure 5. The stabilization of 3-O-acetylgalanthamine (20) is produced by the presence of two hydrogen bond interactions with the residues Glu 202 and Tyr 124, one π-cation interaction with Trp 86, and three hydrophobic interactions with Tyr 133, Tyr 337, and Phe 338. For 3-O-acetylsanguinine (21), the stabilization arises from the presence of one hydrogen bond interaction with Glu 202, one π-cation interaction with Trp 86, and two hydrophobic interactions with Tyr 133 and Tyr 337. For narwedine (19), the interactions with the active site are similar to those of compound 20, with two hydrogen bond interactions with Glu 202 and Tyr 124, one π-cation interaction with Trp 86, and three hydrophobic interactions with Tyr 133, Tyr 337, and Ile 451. Finally, in lycoraminone (18), there is one hydrogen bond interaction with Tyr 124, two π-cation interactions with Trp 86, and three hydropohic interactions with Tyr 337, Phe 338, and Phe 297. In conclusion, in all the molecules depicted in Figure 5, the stabilizaton is achieved by the interactions of the NH + group with the residues Trp 86, Glu 202, Ser 203, and His 447 (the last three amino acids known as the catalytic triad) [49]. In the case of galanthamine (14), as depicted in Figure 5, the best conformation estimated by the molecular docking experiment shows the NH + group oriented in the opposite direction to the catalytic triad, and this explains the low estimated BE values. In conclusion, in all the molecules depicted in Figure 5, the stabilizaton is achieved by the interactions of the NH + group with the residues Trp 86, Glu 202, Ser 203, and His 447 (the last three amino acids known as the catalytic triad) [49]. In the case of galanthamine (14), as depicted in Figure  5, the best conformation estimated by the molecular docking experiment shows the NH + group oriented in the opposite direction to the catalytic triad, and this explains the low estimated BE values.  (21), narwedine (19), lycoraminone (18), and galanthamine (14).

Plant Material Voucher
All the Rauhia Traub species, which were collected in Peru over several years, were received as bulbs from botanical gardens and identified by Dr. Alan W. Meerow. The following species have been deposited at the Fairchild Tropical Botanic Garden (FTG) and  (21), narwedine (19), lycoraminone (18), and galanthamine (14).

Plant Material Voucher
All the Rauhia Traub species, which were collected in Peru over several years, were received as bulbs from botanical gardens and identified by Dr. Alan W. Meerow. The following species have been deposited at the Fairchild Tropical Botanic Garden (FTG) and National Arboretum (NA), both in the USA: R. multiflora (Meerow 2441, FTG), R. decora (Meerow 1160, FTG), and R. staminosa (Meerow 3530, NA).

Extraction
The bulbs of each Rauhia species were dried at 40 • C, and then milled. The extraction procedure was carried out according to [50], using fifty mg of each sample to obtain the alkaloid extracts.

GC-MS Analysis
The dried alkaloid extracts of the Rauhia species were dissolved in 100 µL of chloroform and analyzed by GC-MS. A total of 1 µL of each sample was injected in a GC-MS 6890N apparatus (Agilent Technologies, Santa Clara, CA, USA) coupled to an Agilent MSD5975 Inert XL, operating in electron ionization (EI) mode at 70 eV, and with a Sapiens-X5 MS column (30 m × 0.25 mm i.d., film thickness 0.25 µm). More information about the chromatographic conditions is available [50].

Alkaloid Identification and Quantification
The chromatograms of each Rauhia species were analyzed using AMDIS 2.64 software. The alkaloid profile of each sample was obtained using the library database of the Natural Products Group of Barcelona University (Spain), the NIST 05 Database (Gaithersburg, MD, USA), and by a comparison with the data in the literature. All the alkaloids were quantified through a calibration curve of galanthamine, using codeine as the internal standard.

Enzymatic Assay
The AChE inhibitory activity of each Rauhia species was analyzed as described by [50]. The enzyme AChE from Electrophorus electricus (Merck, Darmstadt, Germany) was used. The calibration curves of the bulb alkaloid extracts (0.05, 0.1, 0.25, 0.5, 1, and 10 µg·mL −1 ) were applied to obtain the IC 50 values for the AChE inhibition, using Prism 9 software. Galanthamine was used as a positive control.

Statistical Analysis
The results were analyzed by an ANOVA, using Prism 9 software ( Figure 3). The data are expressed as the mean ± standard deviation (SD). Significant results are marked as follows: ** p < 0.01, * p < 0.1, and ns (not significant). One-way ANOVA with Dunnett's multiple comparison test was used to compare the mean of each column with the mean of a control column (galanthamine).

Molecular Docking
The molecular docking simulations of galanthamine-type alkaloids observed in the species R. multiflora were carried out using the Autodock 4.2 program [51]. These in silico experiments require the ligand and protein structures to be correctly prepared. The tridimensional alkaloid structures were downloaded from the PubChem database and were edited using the Maestro program [52] belonging to the Schrodinger suite. In this process, hydrogen atoms were added, and the protonation states were checked for a pH of 7.0 ± 2.0. As a result, nitrogen is protonated in all the evaluated configurations.
Additionally, a set of human AChE protein structures were downloaded from the Protein Data Bank web site and were prepared using the Maestro program. The preparation consisted of deleting the water molecules, ions, and ligands included in the crystallography PDB file. Additionally, the bond orders were assigned, the hydrogen atoms were added, the missing side chains were included, and the amino acid protonation states were checked. In the molecular docking simulations, the first step corresponded to computing a set of precalculated grids of affinity potentials via AutoGrid to find suitable binding positions for a ligand on a given macromolecule. In this step, a grid box with dimensions of 60 × 60 × 60 Å and centered in the coordinates −10.30, −43.46, and 30.08 was selected. The second stage in the docking experiment involved obtaining the best orientation of a ligand at the active site of a protein, treated or selected as a rigid body, through the Lamarckian genetic algorithm (LGA) [53]. For this protocol, a population size of 5000 individuals and 50 LGA runs was selected. The best ligand-protein complexes were analyzed according to the potential intermolecular interactions such as hydrogen bonding and the cation-π, π-π stacking.

Conclusions
This is the first report about the alkaloid profile and biological potential of the genus Rauhia Traub (Amaryllidaceae). The most interesting results were obtained for the species R. multiflora, which was found to contain several galanthamine-type structures, and its extracts exerted a high in vitro inhibitory activity against AChE. The theoretical interaction of the alkaloids with five different crystallographic structures of human AChE was detailed by in silico experiments. The results indicate that R. multiflora is a promising candidate for biotechnological assays to obtain new insights into the biosynthesis of galanthaminetype alkaloids, which may contribute to the development of new methodologies for the sustainable production of galanthamine.