molecules Article Alkaloids Analysis of Habranthus cardenasianus (Amaryllidaceae), Anti-Cholinesterase Activity and Biomass Production by Propagation Strategies Daniel Zaragoza-Puchol 1,2,† , Javier E. Ortiz 1,† , Alejandro A. Orden 3 , Marianela Sanchez 4,5,6 , Jorge Palermo 4,5 , Alejandro Tapia 1,7 , Jaume Bastida 8 and Gabriela E. Feresin 1,2,7, * 1 Instituto de Biotecnología, Universidad Nacional de San Juan, Av. Libertador General San Martín 1109 (O), C.P. 5400 San Juan, Argentina; josedanielzaragoza@gmail.com (D.Z.-P.); jortiz@unsj.edu.ar (J.E.O.); atapia@unsj.edu.ar (A.T.) 2 Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Godoy Cruz 2290, C.P. 1425 Ciudad Autónoma de Buenos Aires, Argentina 3 INTEQUI CONICET, Facultad de Química Bioquímica y Farmacia, Universidad Nacional de San Luis, Almirante Brown 1455, C.P. D5700HHW San Luis, Argentina; alejandroorden@gmail.com 4 Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón, C.P. 1428 Buenos Aires, Argentina; sanchezmnela@gmail.com (M.S.); palermo@qo.fcen.uba.ar (J.P.) 5 CONICET–Universidad de Buenos Aires, Unidad de Microanálisis y Métodos Físicos en Química Orgánica (UMYMFOR), Ciudad Universitaria, C.P. 1428 Buenos Aires, Argentina 6 Centro de Investigaciones y Transferencia de Villa María (CITVM-CONICET), Universidad Nacional de Villa María,  Campus Universitario, Arturo Jauretche 1555, Villa María, C.P. 5000 Córdoba, Argentina  7 Instituto de Ciencias Básicas, Universidad Nacional de San Juan, Av. Libertador General San Martín 1109 (O), C.P. 5400 San Juan, Argentina Citation: Zaragoza-Puchol, D.; Ortiz, 8 Departament de Biologia, Sanitat i Medi Ambient, Facultat de Farmàcia i Ciències de l’Alimentació, J.E.; Orden, A.A.; Sanchez, M.; Universitat de Barcelona, Avda. Joan XXIII # 27–31, 08028 Barcelona, Spain; jaumebastida@ub.edu Palermo, J.; Tapia, A.; Bastida, J.; * Correspondence: gferesin@unsj.edu.ar; Tel.: +54-264-421-1700 (ext. 410/294); Fax: +54-264-420-0289 Feresin, G.E. Alkaloids Analysis of † These authors contributed equally to this work. Habranthus cardenasianus (Amaryllidaceae), Abstract: Plants in the Amaryllidaceae family synthesize a diversity of bioactive alkaloids. Some of Anti-Cholinesterase Activity and these plant species are not abundant and have a low natural multiplication rate. The aims of this Biomass Production by Propagation work were the alkaloids analysis of a Habranthus cardenasianus bulbs extract, the evaluation of its Strategies. Molecules 2021, 26, 192. inhibitory activity against cholinesterases, and to test several propagation strategies for biomass https://doi.org/10.3390/ molecules26010192 production. Eleven compounds were characterized by GC-MS in the alkaloid extract, which showed a relatively high proportion of tazettine. The known alkaloids tazettine, haemanthamine, and the Academic Editor: Derek J. McPhee epimer mixture haemanthidine/6-epi-haemanthidine were isolated and identified by spectroscopic Received: 8 December 2020 methods. Inhibitory cholinesterases activity was not detected. Three forms of propagation were Accepted: 28 December 2020 performed: bulb propagation from seed, cut-induced bulb division, and micropropagated bulbs. Published: 2 January 2021 Finally, different imbibition and post-collection times were evaluated in seed germination assays. The best propagation method was cut-induced bulb division with longitudinal cuts into quarters Publisher’s Note: MDPI stays neu- (T1) while the best conditions for seed germination were 0-day of post-collection and two days tral with regard to jurisdictional clai- of imbibition. The alkaloids analyses of the H. cardenasianus bulbs showed that they are a source ms in published maps and institutio- of anti-tumoral alkaloids, especially pretazettine (tazettine) and T1 is a sustainable strategy for its nal affiliations. propagation and domestication to produce bioactive alkaloids. Keywords: Amaryllidaceae; bioactive alkaloids; GC-MS; propagation methods; biomass production Copyright: © 2021 by the authors. Li- censee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and con- 1. Introduction ditions of the Creative Commons At- A large number of modern medicinal drugs are obtained from plant extracts [1]. tribution (CC BY) license (https:// The use of native plants as a source of bioactive molecules motivates studies of their creativecommons.org/licenses/by/ prospection, propagation, and domestication [2]. The Amaryllidaceae family is a large 4.0/). Molecules 2021, 26, 192. https://doi.org/10.3390/molecules26010192 https://www.mdpi.com/journal/molecules Molecules 2021, 26, 192 2 of 18 group of 1100 species divided among 75 genera, which are used for floricultural-ornamental and medicinal purposes [3]. Species belonging to Amaryllidaceae have also been used in traditional medicine by indigenous people throughout the world to cure ailments: stomach ailments, skin diseases, headaches, dizziness, wounds, chest and bladder pain, renal and liver complaints, infertility, aching joints, rheumatism, snake bites, facilitation of childbirth during labor, hysteria, and as narcotics [4]. In Argentina, the bulbs and leaves of Hippeastrum parodii Hunz. & Cocucci and Zephyranthes carinata Herb. (Amaryllidaceae) are used in traditional medicine by the Toba indigenous community for the treatment of skin disorders (warts, facial pimples, acne, and skin spots) [5]. The alkaloid galanthamine is a long-acting, selective, reversible, and competitive acetylcholinesterase (AChE) inhibitor used for the treatment of Alzheimers disease and is still obtained from plants, mainly from Leucojum aestivum (summer snowflake) and Narcissus (daffodils) in Europe, Lycoris radiata in China, and Ungernia victoris in Tadzhikistan and Uzbekistan [6,7]. Amaryllidaceae family belongs to the monocot order Asparagales and is composed of three subfamilies: Amaryllidoideae, Agapanthoideae, and Allioideae. In particular, the Amaryllidoideae subfamily is an important source of bioactive molecules [8]. Habranthus Herbert is a widely distributed Amaryllidaceae genus in America, con- sisting of 25 to 30 species [9]. These species biosynthesize alkaloids from the precursor 40 -O-methylnorbelladine, which is derived from the amino acids L-phenylalanine and L-tyrosine [10]. The nomenclature of the Amaryllidaceae skeleton types, also referred to as structural types, subgroups, ring types or series, has varied over the years [3]. In this work, the Amaryllidaceae alkaloids (AA) are classified into seventeen skeleton types according to Berkov et al. [3]. A display several types of pharmacological activities, most importantly on the central nervous system, including neuroprotection against the excitotoxicity induced by gluta- mate in primary cortical neurons, antiproliferation, cytotoxicity, and apoptosis induc- tion [11,12]. Anti-viral, anti-parasitic, anti-cancer, and anti-cholinesterasic activities have also been reported [3,11,13–15]. Amaryllidaceae species, including Habranthus cardenasianus Traub & I.S. Nelson, are not abundant, and present a low natural multiplication rate [16]. Mining and agri- cultural activities, together with urban growth, are reducing the populations of plants in this family. Thus, the extraction of bioactive metabolites from natural sources is currently not viable, and it is important to develop sustainable propagation methods for Amarylli- daceae domestication [17]. Such methods include seed propagation [18,19], cut-induced bulb division [20–22], and micropropagation [23–25]. Seed propagation is a simple and economical way to obtain seedlings [26] and allows a good multiplication rate, but it has the drawback of a high genetic drift [19]. Cut-induced bulb division consists of making incisions that induce hormonal mechanisms in the plant and promote the generation of new bulbils [20]. In-vitro culture is large scale micropropagation using tissue culture techniques [23]. Micropropagation is possible due to the totipotentiality of plant cells, which is the ability to regenerate a complete plant under the appropriate stimuli [17,24]. Micropropagation is very useful in breeding programs because it produces a uniform quality of commercial plants, with a high multiplication rate, little genetic variability, and a stable production of the metabolites of interest [23]. To the best of our knowledge, this work represents the first chemical study of H. cardenasianus bulbs, the evaluation of their inhibitory activity against cholinesterases, and the analysis of biomass production by several propagation strategies to produce bioactive alkaloids. 2. Results and Discussion 2.1. Chemical Analysis 2.1.1. Alkaloid Profile by GC-MS Analysis The alkaloid extract analysis showed eleven compounds, nine of them identified by comparison of their mass fragmentation patterns with EI-MS spectra home-made database gathered from alkaloids previously isolated and identified by different spectroscopic meth- Molecules 2021, 26, 192 3 of 18 ods (1D and 2D NMR, UV, CD, IR, HRMS) in the Natural Products Laboratory of Barcelona University. Additionally, Kovats retention index (RI) values, the NIST 05 Database, and lit- erature data were used for alkaloid identification. Besides the known AA, two compounds could not be identified. The GC-MS data are shown in Table 1. Based on the AA classifi- cation of Berkov et al. [3] the identified compounds in H. cardenasianus are galanthindole (galanthindole), haemanthamine (haemanthamine, haemanthidine/6-epi-haemanthidine), homolycorine (two m/z 109 unknown alkaloids), ismine (ismine), lycorine (anhydroly- corine, 11,12-dehydroanhydrolycorine, lycorine), narciclasine (5,6-dihydrobicolorine), and pretazettine (tazettine) type alkaloids [3]. The unknown alkaloids were tentatively identified as homolycorine-type due to their fragmentation pattern, which showed a char- acteristic m/z 109 (100) base peak. The alkaloid tazettine represented more than 50% of the total ion current (TIC). It is known that tazettine is not a naturally occurring alkaloid, but an artifact produced from the chemically labile parent alkaloid pretazettine arising from a rearrangement during alkaloid extraction [27–29]. Thus, considering the basicity and reflux conditions during the H. cardenasianus extract preparation, it would be very likely that pretazettine was totally converted to tazettine. Table 1. GC-MS data for H. cardenasianus alkaloid extract. Alkaloid [M+ ] RI 1 TIC 2 MS Galanthindole-type Galanthindole (4) 281(100) 22.8 0.86 264(13), 263(16), 262(18), 252(15), 248(8), 191(11), 132(11), 107(8) Haemanthamine-type 273(19), 272(100), 242(13), 240(14), 214(14), 212(13), 211(15), Haemanthamine (8) 301(11) 24.9 7.92 186(14), 181(25), 128(13) Haemanthidine/ 284(80), 233(77), 211(53), 201(100), 199(77), 181(71), 173(64), 317(94) 26.2 6.68 6-epi-haemanthidine (10) 3 115(79), 45(53) Homolycorine-type 330(2), 329(6), 221(4), 110(8), 109(100), 108(13), 94(2), 82(2), Unknown (3) 331(5) 22.4 0.82 81(1), 42(2) 221(7), 191(1), 163(1), 110(8), 109(100), 108(13), 94(2), 82(2), Unknown (6) 331(<1) 23.2 4.29 81(1), 42(1) Ismine-type 239(20), 238(100), 211(6), 196(9), 180(8), 168(7), 167(6), Ismine (1) 257(30) 19.3 0.701 152(5), 139(6) Lycorine-type Anhydrolycorine (5) 251(41) 23 0.97 250(100), 203(6), 192(10), 130 (15), 129(5), 118(4), 57(6), 41(5) 11,12-dehydro 249(61) 24.5 2.70 248(100), 191(10), 190(24), 189(6), 164(3), 163(7), 123(6), 95(14) anhydrolycorine (7) Lycorine (11) 287(24) 26.7 2.55 286(19), 268(24), 250(15), 227(79), 226(100), 211(7), 147(15) Narciclasine-type 5,6-Dihydrobicolorine (2) 239(44) 20.2 1.90 238(100), 181(3), 180(10), 166(3), 152(5), 139(6), 118(6), 90(7) Pretazettine-type 298(19), 248(15), 247(100), 201(16), 199(14), 181(13), 115(15), Tazettine (9) 331(26) 25.3 57.77 71(15), 70(17) 1 Kovats retention index; 2 Total ion current; 3 cannot be distinguished by GC-MS. Based on the TIC proportions, the most abundant alkaloid skeletons were the pre- tazettine, haemanthamine, and homolycorine types. The relative proportion of each alkaloid was determined as a percentage of the TIC. Thus, the major components were identified as tazettine (9) (57.77%), haemanthamine (8) (7.92%), and haemanthidine/6-epi- haemanthidine (10) (6.68%) (Table 1). The main groups of hemanthamine, pretazettine, and narciclasine alkaloids originate from a para-para’ oxidative phenolic coupling of 40 -O-methylnorbelladine [27]. Thus, taking into account the relative TIC proportions of these alkaloid types, it is likely that the metabolism of H. cardenasianus favors alkaloid biosynthesis via this oxidative pathway. On the other hand, galanthamine-type alkaloids were not detected. 238(100), 181(3), 180(10), 166(3), 5,6-Dihydrobicolorine (2) 239(44) 20.2 1.90 152(5), 139(6), 118(6), 90(7) Pretazettine-type 298(19), 248(15), 247(100), 201(16), Tazettine (9) 331(26) 25.3 57.77 Molecules 2021, 26, 192 199(14), 181(13), 115(15), 71(15), 70(17) 4 of 18 1 Kovats retention index; 2 Total ion current; 3 cannot be distinguished by GC-MS. Alkaloids are a family of compounds of great interest in cancer treatment due to their anti-proliferative Alkaloids areand anti-angiogenic a family of compounds effects. Theinterest of great structural diversity in cancer of alkaloids treatment and due to their anti-proliferative their and anti-angiogenic capacity to interact effects. The with a wide spectrum ofstructural moleculesdiversity is due toofthe alkaloids and different their amino capacity acids thattoact interact with a wide as precursors spectrum for their of molecules biosynthetic is due[30]. pathways. to the AA different amino acids are particularly di- that act as precursors for their biosynthetic pathways. [30]. AA are verse, and share norbelladine as a common biosynthetic precursor, which is obtained from particularly diverse, andcondensation the share norbelladineof the as a common aromatic aminobiosynthetic precursor, which acids L-phenylalanine is obtainedThese and L-tyrosine. from theal- condensation kaloids of the aromatic have undoubtedly amino acids L-phenylalanine revolutionized and L-tyrosine. medicine as inhibitors These alkaloids of cholinesterases, and have as undoubtedly antiviral, revolutionizedantibacterial, anti-inflammatory, medicine as inhibitors and mostofimportantly, cholinesterases, and as antiviral, antitumor agents, anti-inflammatory, serving as a reservoirantibacterial, of valuable and most importantly, anticancer drugs [31].antitumor agents, serving The pharmaceutical as a reser- potential of voir of valuable anticancer drugs [31]. The pharmaceutical potential AA has been confirmed by the registration of galanthamine, an inhibitor of AChE, as a of AA has been con- firmed drug forby the registration Alzheimer’s of galanthamine, disease [6,32]. Other AA an inhibitor of AChE, interest, are of commercial as a drugbut forare Alzheimer’s available only in low amounts in the native plants. The most abundant alkaloids isolatedamounts disease [6,32]. Other AA are of commercial interest, but are available only in low from H. in the native were cardenasianus plants. The most tazettine abundant alkaloids (9) (pretazzetine), isolated from haemanthamine H. cardenasianus (8), and were haemanthidine/6- tazettine (9) (pretazzetine), epi-haemanthidine haemanthamine (10) (Figure 1), all of (8), and haemanthidine/6-epi-haemanthidine which have been reported as bioactive com- (10) (Figure 1), pounds [3,11]. all of which have been reported as bioactive compounds [3,11]. Figure 1. GC chromatogram Figure 1. GC of H. chromatogram cardenasianus of H. extract cardenasianus showing extract showing the alkaloids thebyalkaloids identified MS. (1) identified by5,6- Ismine, (2) MS. (1) Ismine, (2) 5,6-Dihydrobicolorine, (3) Unknown, (4) Galanthindole, (5) Anhydrolycorine, Dihydrobicolorine, (3) Unknown, (4) Galanthindole, (5) Anhydrolycorine, (6) Unknown, (7) 11,12-dehydro anhydrolycorine, (6) Un- known, (7) 11,12-dehydro anhydrolycorine, (8) Haemanthamine, (9) Tazettine, (10) Haeman- (8) Haemanthamine, (9) Tazettine, (10) Haemanthidine/6-epi-haemanthidine, (11) Lycorine. thidine/6-epi-haemanthidine, (11) Lycorine. A major reason for the failure of cancer treatment is multidrug resistance (MDR), which in most cases is caused by the activity of the various ABC transporters and p- glycoproteins encoded by MDR genes, which pump anticancer drugs out of the cells [33]. The efficiency of cytotoxic agents that target cancer cells can be enhanced by the inhibition of P-gp, one of the most important ABC proteins. Some AA exhibit a combination of anti- cancer activities, including a direct cytostatic effect and P-gp-inhibition, and have been pro- posed as promising starting structures for the development of future anticancer agents [34]. Pretazettine acts on several cancer cells by affecting the p-glycoprotein ABCB1, an inter- esting and uncommon molecular mechanism [27,33]. In addition, it displays excellent antiproliferative effects on both human and mouse cell lines, indicating that pretazettine- type alkaloids are an attractive option for alkaloid-based anticancer therapy [34]. There is compelling evidence for the interaction of tazettine alkaloids with macro- molecules such as DNA, RNA, and proteins, but their potential role in other areas of molec- ular cancer chemotherapeutics such as apoptosis still needs to be investigated. A recent review by Nair & Van Staden [35] informs that eleven of the AA from eight Amaryllidaceae genera have been assayed against as many as 50 different cancer cell lines. It has been shown that pretazettine is capable of inhibiting cellular protein synthesis without affecting Molecules 2021, 26, 192 5 of 18 the synthesis of DNA or RNA in KB human oral epidermoid carcinoma, P-388, and Ehrlich ascites cells. When tested against different tumoral cell lines (the HeLa cervical adenocarci- noma cell line, mouse melanoma cells, Molt4 T-lymphoma cells, L929, and MCF7 cells), pretazettine was found to be far more potent than tazettine (9). Furthermore, pretazettine activity against Rauscher leukemia has been reported in BALB/c mice [36]. Due to its high content of pretazettine, H. cardenasianus is a promising source of this compound. According to Nair & Van Staden [35], pretazettine is one of the most active AA against Molt4 lymphoid cells, with particularly striking activity at the submicromolar level, suggesting it may have application in lymphoma chemotherapeutics. Pretazettine also possesses pronounced activity against Herpes simplex type 1 virus, which could reflect a general ability to inhibit protein synthesis during viral replication [27]. Addi- tionally, previous reports show that tazettine (9) and pretazettine are relatively safe in animal models [27,35]. Promising antiproliferative activities have been described for haemanthamine (8) and haemanthidine (10) [36], both identified in H. cardenasianus bulbs. Haemanthamine (8) displays significant in vitro cytotoxic activity against several types of cancer cell lines, including MoltT-4, HepG2, HeLa, MCF7, CEM, K562, A549, Caco-2, HT-29, A2780, SW1573, and T47-D, whereas haemanthidine (10) has been reported as a promising cytotoxic agent against A549, OE21, Hs683, and SKMEL cancer cells, and has a growth inhibitory effect on both parental and multidrug resistant L5178 mouse lymphoma cell lines [12]. Haeman- thidine is also anti-inflammatory in mice and has higher analgesic activity than aspirin or indomethacin [37]. Cedrón et al. [38] suggested that some haemanthamine derivatives are effective inhibitors of the malaria-causing parasite Plasmodium falciparum and could provide a useful starting point for the preparation of new haemanthamine-type derivatives with improved antiplasmodial activity. Additionally, Nair & van Staden [39] report better overall activities for haemanthidine (10) against various strains of P. falciparum, particularly the K1 strain (IC50 0.35 µg/mL). The alkaloids haemanthamine (8) and haemanthidine (10) were the most active compounds against P. falciparum and have served as useful scaffolds for structure-activity relationship studies, affording new insights into the antiplasmodial pharmacophore and the features supporting its efficacy. 2.1.2. Alkaloid Isolation Extensive chromatographic purification afforded the known alkaloids haemanthamine (8) (12 mg), tazettine (9) (89 mg), and the haemanthidine/6-epi-haemanthidine mixture (10) (8 mg) (Figure 2). Their structures were confirmed by NMR spectroscopy showing the presence of a mixture of both epimers for alkaloid 10. The NMR results are in agreement with literature data [27]. Likewise, for the alkaloid haemanthidine (10), previous reports indicate that in many Amaryllidaceae species this alkaloid occurs naturally as a mixture of C-6 epimers [3,27,38,40]. ecules 2020, 6 of 19 Molecules 2021, 26, 192 6 of 18 reports indicate that in many Amaryllidaceae species this alkaloid occurs naturally as a mixture of C-6 epimers [3,27,38,40]. OMe OH O H O N (8) haemanthamine (10) haemanthidine: R1 H; R2 OH (9) tazettine 6-epi-haemanthidine: R1 OH; R2 H pretazettine Figure 2. Alkaloids from Figure 2. Alkaloids from H. cardenasianus. H. cardenasianus. 2.2. AChE and Butyrylcholinesterase (BuChE) Inhibitory Activities 2.2. AChE and Butyrylcholinesterase (BuChE) Inhibitory Activities The alkaloid extract from H. cardenasianus and the isolated alkaloids were evaluated The alkaloid extract from H. cardenasianus and the isolated alkaloids were evaluated in vitro for AChE and BuChE inhibitory activities. Galanthamine (Sigma-Aldrich) was in vitro for AChE and BuChE inhibitory activities. Galanthamine (Sigma-Aldrich) was used used as a positive control, showing an IC50 value of 1 μM. The isolated alkaloids 8, 9, and as a positive control, showing an IC50 value of 1 µM. The isolated alkaloids 8, 9, and the the epimeric mixture 10 had IC50 values higher than 200 μM, and were therefore consid- epimeric mixture 10 had IC50 values higher than 200 µM, and were therefore considered ered inactive, in agreement with previous data [41], as was the alkaloid extract, which had inactive, in agreement with previous data [41], as was the alkaloid extract, which had IC50 values of 250 μg/mL and 300 μg/mL for AChE and BuChE inhibition, respectively. IC50 values of 250 µg/mL and 300 µg/mL for AChE and BuChE inhibition, respectively. Galanthamine-type alkaloids arealkaloids Galanthamine-type recognised are as the most as recognised potent cholinesterases the most inhibitors inhibitors potent cholinesterases among the AA,amongandthe their AA,non-detection by GC-MS analysis and their non-detection by GC-MS in the H. cardenasianus analysis extract in the H. cardenasianus extract supports thesupports results of the cholinesterase inhibition assays. the results of the cholinesterase inhibition assays. 2.3. Propagation 2.3. from Habranthus Propagation from cardenasianus Habranthus cardenasianus Collected material Collected of H. cardenasianus material was used for of H. cardenasianus vegetative was used forand sexual propaga- vegetative and sexual propaga- tion experiments to generate biomass. Five different propagation tion experiments to generate biomass. Five different propagation methods methods were applied:were applied: T0 (control, T0 whole bulb without cuts), T1 (bulb quarters), T2 (twin scales), (control, whole bulb without cuts), T1 (bulb quarters), T2 (twin scales), T3 (bulb basal T3 (bulb basal disc); T4 (micropropagated bulbs), and T5 disc); T4 (micropropagated (seeds). bulbs), andSeven months Seven T5 (seeds). later, the following months later,pa- the following rameters were evaluated: parameters survival were percentage, evaluated: multiplication survival percentage,rate, biomass increase, multiplication bulb increase, rate, biomass equatorial diameter and length; bulb equatorial and root diameter and diameter, length; and length, and number. root diameter, length, and number. The coefficients of variation of The coefficients were less than variation were10%, lessthe determination than coefficients coefficients 10%, the determination were were greater thangreater 0.90 andthan 0.90 and p-values of p-values the model of were the model were0.05, less than lesswhich than 0.05, whichlittle indicated indicated little variation in variation in the observations the observations and a good fit and ofathe good fit of the statistical statistical model ANOVA. model TheANOVA. obtained The obtained data demonstrate data demonstrate that the propagation that the propagation method canmethod can significantly significantly affect all theaffect all the evaluated evaluated parameters parameters (p