*2.1. AChE and BuChE Inhibitory Activities*

The crude alkaloid samples from the different Chilean *Rhodophiala* were tested in vitro for AChE and BuChE-inhibitory activity. The percent dry weight of the samples, *w*/*w* extraction yields, and cholinesterase inhibition are summarized in Table 1. Galanthamine was used as a control and presented AChE and BuChE inhibition with IC50 values of 0.48 ± 0.07 and 3.70 ± 0.24 μg/mL, respectively. All the alkaloid extracts tested were active against AChE. The highest AChE inhibitory potential was found in bulbs of *R. pratensis* (sample Q) followed by *R. splendens* (sample S) with IC50 values of 3.32 ± 0.26 and 3.62 ± 0.02 μg/mL, respectively. Lowest activity was measured for the aerial part of *R. pratensis* (sample N) (IC50 value: 102.27 ± 6.61 μg/mL). Nearly 50% of the samples showed some activity against BuChE, with better effect for the bulbs of *R. splendens* (sample S) (IC50 14.37 ± 1.94 μg/mL).

The bulb (I) and leaf (J) extracts of *R. montana* presented moderate activity against both enzymes with better effect of (I) against AChE and (J) towards BuChE. The differences in the chemical profiles of I and J could explain these results. However, the high number of unknown alkaloids in the extracts precludes further discussion. The bulb (Q) and leaf (R) extracts of white flowering *R. pratensis* were active towards AChE, with IC50 of 3.32 and 8.39 μg/mL, respectively but with mild to low effect against BuChE (Table 1), reducing the pharmacological interest of this species. The high AChE and BuChE inhibitory activity of *R. splendens* bulb (S) and aerial parts/leaves (T) renders this plant as the most promising species in the search for active molecules for AD therapy (Table 1).

**Table 1.** Percent dry weight, *w*/*w* extraction yields from dry starting material, percent alkaloid extract (from the crude extract), acetyl-(AChE) and butyryl- (BuChE) cholinesterase inhibitory activity of alkaloid-enriched extracts from Chilean *Rhodophiala*.


\*<sup>a</sup> after lyophilization; \*<sup>b</sup> from lyophilized material; \*<sup>c</sup> from the crude extract; \*<sup>d</sup> all IC50 were calculated using R<sup>2</sup> ≥ 0.99; \*e insufficient sample; WF: white flowers; RF: red flowers; L: with leaves; nl: no leaves; Collection place: AC: Arcos de Calán, Región del Maule; LM: Laguna del Maule; LT: Las Trancas; M: Malalcahuello, Región de la Araucanía; NC: Nevado de Chillán; SN: Sierra Nevada; VL: volcan Lonquimay.

#### *2.2. Alkaloid Identification by GC-MS*

The activity of the Chilean *Rhodophiala* towards acetylcholinesterase is a consequence of the chemical composition of the extracts. Therefore, the alkaloid composition is a key point to understand the chemical diversity of these plants as a source of potential therapies for AD. The alkaloids occurring in the different extracts were identified by comparing their GC-MS spectra and Kovats Retention Index (RI) values with those of authentic samples. Thirty-seven known alkaloids were identified in these samples (Figure 4). About 50% of them belong to three different alkaloid types, namely: lycorine, haemanthamine and crinine. The others belong to six different alkaloid types: tazettine, homolycorine, galanthamine, montanine, mesembrenone and narciclasine. Two unusual alkaloids known as ismine and galanthindole were also found. The occurrence and quantification of the alkaloids in the samples is summarized in Table 2: (**A**) (aerial parts) and (**B**) (bulbs). The number of alkaloids detected varied among extracts, ranging from 8 in the aerial parts of *R. andicola* collected in Sierra Nevada (B) to 23 in the bulb of *R. pratensis* (K). Forty structures found in these samples could not be identified, suggesting high potential of Chilean *Rhodophiala* species in the search for new alkaloids. The number of unidentified compounds ranged from 3 in aerial parts of *R. andicola* (A, B and D), *R. pratensis* (P) and *R. splendens* (T) to 12 in aerial parts of *R. montana* (J). Representative chromatograms of the samples are shown in Figures 5–9.


**Table 2.** (**A**) Identification of alkaloids occurring in the aerial parts of Chilean *Rhodophiala* species by GC-MS. Values are expressed as mg GAL/g AE; (**B**) Identificationof alkaloidsoccurringinthebulbsofChilean*Rhodophiala*speciesbyGC-MS.ValuesareexpressedasmgGAL/gAE.

*O*-methyltazettine

tazettine (**24**) epimacronine

 (**25**)

 (**23**)

 345

 331

 329

 245

 247

 261

2641.1 54.6 36.0 66.2 10.3

2685.1 - 12.5 17.0

2848.0 -

 5.1

 5.9

 T

 -

 T

 -

 -

 T

 T

 - 11.2 11.1 10.1 14.9 30.7

> 7.0

 -

 -

 -

 5.9

 9.8 24.9













BP: Base Peak; T: Traces; **B**: *R. andicola*, SN; **D**: *R. andicola*, NC; **F**: *R. andicola*, VL; **H**: *R. araucana*; **J**: *R. montana*; **L**: *R. pratensis*, RF, nl, dunes; **N**: *R. pratensis*, AP, RF, L; **P**: *R. pratensis*, RF, **R**: *R. pratensis*, WF; **T**: *R. splendens*; **A**: *R. andicola*, SN; **C**: *R. andicola*, NC; **E**: *R. andicola*, VL; **G**: *R. araucana*; **I**: *R. montana*; **K**: *R. pratensis*, RF, nl, dunes; **M**: *R. pratensis*, RF, L; **O**: *R. pratensis*, RF, nl; **Q**: *R. pratensis*, WF; **S**: *R. splendens*; - : not detected; \* proposed structure-type according to the fragmentation pattern.

parts of *R. andicola* (Volcán

Lonquimay);

 (**G**) Bulbs of *R. araucana*; (**H**) Aerial parts of *R. araucana*. IS: internal standard; U: unknown.

The highest alkaloid concentration was detected in the aerial parts of *R. andicola* (F) and in the aerial parts of *R. pratensis* (R) (311.1 and 274.1 mg GAL/g AE, respectively). Lowest content was found in the aerial parts of *R. andicola* (B) and in the aerial parts of *R. pratensis* (P) (133.2 and 138.1 mg GAL/g AE, respectively). In 70% of the samples, lycorine-, haemanthamine/crinine- and tazettine-type alkaloids were predominant. Lycorine-type alkaloids were present in all species with higher content in *R. araucana* (G) and *R. montana* bulbs (I) (79.0 and 78.6 mg GAL/g AE, respectively) and lowest values in the aerial parts of *R. andicola* (B) and (D) (8.6 and 7.5 mg GAL/g AE, respectively).

Haemanthamine/crinine-alkaloids occur in all samples except the aerial parts of *R. andicola* (B). However, the higher content was found in the bulbs of *R. andicola* from the same collection place (A) and in the aerial parts of the plant collected at Volcan Lonquimay (F). Compounds from the tazettine-type were not detected in the aerial parts and bulbs of *R. montana* (I and J). From the different Amaryllidaceae alkaloids groups, tazettine-type alkaloids were the main compounds in several samples, occurring in highest concentration in *R. andicola* (E, F) with values of 85.9 and 95.8 mg GAL/g AE of tazettine-type alkaloids in bulbs and aerial parts, respectively.

Galanthamine-type alkaloids were detected in low quantities in three species, namely *R*. *andicola*, *R. araucana* and *R. montana* (samples C, D, E, F, G, H and J) ranging between 5.1 to 19.0 mg GAL/g AE. Montanine-type alkaloids were present in all species, except *R. andicola*. The highest level of montanine-type compounds was detected in *R. pratensis* (K) (41.7 mg GAL/g AE), which presented three different alkaloids: pancratinine C, montanine and pancracine (5.3, 29.8 and 6.6 mg GAL/g AE, respectively). Mesembrenone-type was the least representative alkaloid-type. It was represented by demethylmesembrenol, detected in low quantities in three different samples of *R. pratensis* (K, M and Q) (7.2, 5.2 and 5.1 mg GAL/g AE, respectively). Narciclasine-type occurs in most samples in a range between 5.1 mg GAL/g AE in bulbs of *R. splendens* (S) to 44.5 and 32.2 mg GAL/g AE in aerial parts of *R. pratensis* with red flowers and leaves (N) and aerial parts of *R. splendens* (T), respectively. All species investigated presented ismine and/or galanthindole alkaloids, except *R. montana*. Forty structures occurring in the extracts could not be identified using the available databases. Three of the unidentified compounds were highly representative among the samples.

The compound with *m*/*z* 252 [M+ = 253] (RI 2405.0) occurs in 60% of the samples. The *m*/*z* 109 with [M<sup>+</sup> = 331] (RI 2557.5), which probably belongs to the homolycorine-type alkaloids, was detected in 40% of the samples and in high amounts in bulbs of *R. pratensis* with white flowers (46.7 mg GAL/g AE). Finally, *m*/*z* 261 with [M+ = 345] (RI 2662.6) was detected in 45% of the samples and in high quantity in aerial parts of *R. andicola* collected at Volcan Lonquimay (24.6 mg GAL/g AE).

The highest content of non-identified alkaloids was detected in the aerial parts of *R. montana* (J) and in the bulbs of *R. pratensis* (red flowers and without leaves) collected in the sand dunes at the sea shore (K) (126.9 and 92.6 mg GAL/g AE, respectively). The lowest content was detected in aerial parts of *R. pratensis* with red flowers and without leaves (P) and in aerial parts of *R. splendens* (T) (20.6 and 21.8 mg GAL/g AE, respectively).

#### *2.3. Molecular Docking*

In this study, *R. splendens* was the most active inhibitor of AChE and BuChE. Alkaloid analysis by GC-MS allowed the identification of 17 compounds in the leaf extract of *R. splendens* (T) including two unidentified constituents (Table 2). The 15 alkaloids identified in the extract were evaluated for their theoretical AChE and BuChE inhibitory potential by molecular docking (Tables 3 and 4). As expected, no alkaloid identified in sample T presented better theoretical AChE inhibitory activity than galanthamine. Molecular simulation of six alkaloids identified in sample T on the 4BDS structure theoretically showed higher enzymatic inhibition against BuChE than galanthamine by 0.80 kcal/mol.


**Table 3.** AChE and BuChE inhibitory activities of some alkaloids identified in the aerial parts of *R. splendens* (T) and the reference compound galanthamine. Values are expressed as IC50 (μg/mL).

**Table 4.** Estimated free energy binding of molecular docking between alkaloids identified in aerial parts of *R. splendens* and cholinesterases (AChE and BuChE). Values are expressed in kcal/mol.


\*<sup>a</sup> PBD code: 1DX6; \*<sup>b</sup> PBD code: 4BDS; \*<sup>c</sup> Cortes et al., 2015; \*<sup>d</sup> Cortes et al., 2017.

To gain further insight into the molecular docking results, an experiment was carried out to check the AChE and BuChE inhibitory activities of 11-hydroxyvittatine (**20**), lycorine (**9**), 8-O-demethylmaritidine (**16**), hamayne (**20b**), deacetylcantabricine (**17**) and haemanthamine (**18a**) (Table 3). The best AChE and BuChE inhibitory activities were obtained for lycorine (**9**) (IC50 101.70 ± 23.79 μg/mL) and hamayne (**20b**) (IC50 48.40 ± 1.13 μg/mL), respectively. However, their theoretical BuChE inhibition was not supported by the experimental assays. The difference in origin of the BuChE structure used in the molecular docking (human) and experimental assays (equine serum), as well as the inability of these compounds to arrive at the BuChE active site of the enzyme could help to explain the difference between theoretical and practical results.

Two important regions in the active sites of the hBuChE enzymes have been located: the first corresponding to the catalytic triad composed by the residues His438, Ser198, and Glu325 [25], while the second corresponds to a choline binding site (α-anionic site), composed principally by the residues Trp82 and Phe329 [25]. A graphical representation of molecular binding of 11-hydroxyvittatine (**20a**) and hamayne (**20b**) alkaloids with the hBuChE protein is presented in Figure 10. The alkaloid 11-hydroxyvittatine (**20a**) shows two strong interactions, hydrogen bonds, with the residues Trp82 and Trp430; however, this molecule does not present any interactions close to the catalytic triad His438, Ser198, and Glu325. On the other hand, hamayne (**20b**) shows one hydrogen bond interaction with the residue Gly115, an amino acid located close to the catalytic triad His438, Ser198, and Glu325. In the case of the interactions at the choline binding site (α-anionic site), both alkaloids show the same π–π stacking interaction with the residue Trp82. These molecular interactions suggest that the β-orientation of the hydroxyl group at C-3 in 11-hydroxyvittatine (**20a**) could theoretically increase the BuChE

inhibition on the 4BDS structure by 0.49 kcal/mol, compared to the α-orientation of the hydroxyl group at the C-3 position in hamayne (**20b**). However, in the experimental assays, hamayne (**20b**) showed BuChE inhibitory activity (48.40 ± 1.13 μg/mL). It can be hypothesized that the β-orientation of the hydroxyl group at C-3 in 11-hydroxyvittatine (**20a**) probably makes it difficult for the compound to arrive at the catalytic triad in the active site of the BuChE.

**Figure 10.** Graphical representations of the binding of (**a**) 11-hydroxivittatine (**20a**) and (**b**) hamayne (**20b**) in the gorge of the active site of *h*BuChE.

Studies on alkaloid composition associated with cholinesterase inhibition and binding-mode prediction have been reported [26,27]. A work on Argentinean Amaryllidaceae [24] reported the composition and acetylcholinesterase inhibition of four wild growing species, including *Rhodophiala mendocina*. Two *R. mendocina* samples collected in different locations presented similar activity towards AChE with IC50 values of 2.0 μg/mL but with relevant differences in the qualitative and quantitative alkaloid composition. The sample from the Provincia de San Juan showed high content of haemanthamine/crinamine (31.2%), tazettine (32.9%) and lycorine (13.3%) while the plant collected in the Provincia de Neuquen presented 6.8% haemanthamine/crinamine and 20.4% of lycorine, respectively. Galanthamine was found in both samples with 0.6 and 0.8% for the San Juan and Neuquen plants, respectively. In a report from acetylcholinesterase inhibitory alkaloids from Brazilian Amaryllidaceae [23] the bulbs of *Rhodophiala bifida* (Herb.) Traub were investigated. The activity on AChE was moderate with an IC50 value of 8.45 μg/mL, being lower than that from *R. mendocina* [24]. The alkaloid extract of *R. bifida* bulbs contained high amounts of montanine (91.94%). The alkaloid composition of the Chilean *Rhodophiala ananuca* (formerly: *Hippeastrum ananuca*) was described [28,29]. The bulbs contained phenantridine alkaloids, including hippeastidine and epi-homolycorine.

The alkaloid montanine isolated from *R. bifida* showed activity towards a panel of eight human cancer cell lines. According to [30], montanine at 2.5 μg/mL was more active than doxorubicine on the multi-drug resistant breast cell line NCLADR. Montanine also showed antimicrobial effect with MIC of 5 μg/mL against *S. aureus* ATCC 6538 and *E. coli* ATCC 24922 and 20 μg/mL against *P. aeruginosa* ATCC 27853, respectively [31]. In a screening towards *Trichomonas vaginalis*, dichloromethane and n-butanol extracts from Brazilian *Hippeastrum* species and *Rhodophiala bifida* showed activity against the protozoa [32]. The most active fractions contained the alkaloids lycorine and lycosinine.

In a study on the alkaloids of *Zephyranthes robusta* (Amaryllidaceae), the compounds isolated were evaluated as inhibitors of human cholinesterases [33]. The authors used human erythrocye AChE and serum BuChE. The compounds were tested in a range of 0.5–500 μg/mL and the inhibition was reported as IC50 values in μMolar concentration. While the activity of the reference compound galanthamine was similar in both studies, 11-hydroxyvitattine, lycorine and haemanthamine were not active on the human AChE and BuChE. 8-*O*-demethylmaritidine and hamayne were inactive on human BuChE but presented activity on erythrocyte AChE. The differences in the results can be explained by the biological source of the enzymes (human cholinesterases for [33] and electric eel AChE and horse (equine serum) BuChE in this work. For a better comparison of results, the use of enzymes from the same biological source should be recommended.

In summary, the AChE and BuChE inhibitory activity of the Chilean *Rhodophiala* species investigated led to an interesting source of inhibitors that do not contain the alkaloid galanthamine. Our results suggest that Chilean *Rhodophiala* could be a promising source of new alkaloids with effect towards cholinesterases. The difficulty in finding a species with high activity against AChE and BuChE, the similarity of the AChE and BuChE inhibitory values, the low complexity of the alkaloid profile of aerial parts of *R. splendens*, together with the absence of galanthamine-type alkaloids in this sample, prompted us to further explore the results.

## **3. Materials and Methods**

#### *3.1. Plant Material*

The samples were collected in central-southern Chile and were identified following the reference [19,34]. *Rhodophiala andicola* (Poepp.) Traub, was collected at Sierra Nevada (Región de la Araucanía, 27 January 2016), the slopes of Volcán Lonquimay (Región de la Araucanía, Provincia del Malleco, 19 December 2016) and the slopes of Nevado de Chillán (Región del Bio-Bio, 30 December 2016). *Rhodophiala araucana* (Phil.) Traub was collected at Malalcahuello, Región de la Araucanía (19 December 2016), and *Rhodophiala montana* (Phil.) Traub at the roadside to Laguna del Maule, Región del Maule (2 January 2017). Samples from *Rhodophiala pratensis* (Poepp.) Traub were collected at Arcos de Calán, Región del Maule (12 December 2016) including plants growing on sand dunes and grasslands close to the sea. Plants with red and white flowers were collected separately. According to [34], the plant with red flowers fits the description of *R. pratensis*. *Rhodophiala splendens* (Renjifo) Traub was collected at Las Trancas, Región del Bio-Bio (2 January 2016). The plants were identified by Dr. Patricio Peñailillo, Herbario de la Universidad de Talca. Voucher herbarium specimens have been deposited at the Universidad de Talca as follows: *R. andicola* (N◦ 4081); *R. araucana* (N◦ 4083); *R. montana* (N◦ 4080); *R. pratensis* (N◦ 4084); *R. pratensis* (white flower) (N◦ 8085); *R. splendens* (N◦ 4082). A map with the collection places is shown in Figure 2. Pictures of the species investigated are illustrated in Figure 3.

#### *3.2. Extraction*

The freshly collected plant material was cleaned and separated into bulbs and aerial parts, frozen and lyophilized before extraction. The dry weight percentage was determined. The lyophilized plant material was extracted with MeOH under sonication for 10 min (3×) changing the solvent each time. The plant to solvent ratio ranged from 1:10 to 1:60 and was selected according to the volume of plant material for extraction. The combined MeOH solubles were taken to dryness under reduced pressure to afford the crude extracts. The crude extracts were then acidified to pH 3 with diluted H2SO4 (2%, *v*/*v*) and the neutral material was removed with Et2O. The aqueous solutions were basified up to pH 9–10 with NH4OH (25%, *v*/*v*) and extracted with EtOAc to provide the alkaloid extracts which were used for all experiments (enzyme inhibition assays and chemical analysis by GC-MS).

#### *3.3. Acetylcholinesterase (AChE) and Butyrylcholinesterase (BuChE) Inhibitory Activity*

Cholinesterase inhibitory activities were determined according to [35] with some modifications [36]. Stock solutions with 518U of AchE from *Electrophorus electricus* (Merck, Darmstadt, Germany) and BuChE from equine serum (Merck, Darmstadt, Germany), respectively, were prepared and kept at −20 ◦C. Acetylthiocholine iodide (ATCI), *S*-butyrylthiocholine iodide (BTCI) and 5,5 -dithiobis (2-nitrobenzoic acid) (DTNB) were obtained from Merck (Darmstadt, Germany). Fifty microliters of AChE or BuChE (both enzymes used at 6.24 U) in phosphate buffer

(8 mM K2HPO4, 2.3 mM NaH2PO4, 0.15 NaCl, pH 7.5) and 50 μL of the sample dissolved in the same buffer were added to the wells. The plates were incubated for 30 min at room temperature. Then, 100 μL of the substrate solution (0.1 M Na2HPO4, 0.5 M DTNB, and 0.6 mM ATCI or 0.24 mM BTCI in Millipore water, pH 7.5) was added. After 10 min, the absorbance was read at 405 nm in a Labsystem microplate reader (Thermo Fischer, Waltham, MA, USA). Enzyme activity was calculated as percent compared to a control using buffer without any inhibitor. Galanthamine served as positive control. In a first step, samples were assessed at 10, 100 and 200 μg/mL towards both enzymes. Samples with an IC50 > 200 μg/mL were considered inactive. Samples with an IC50 < 200 μg/mL were further analyzed to determine the IC50 values. The cholinesterase inhibitory data were analyzed with the software Microsoft Office Excel 2010 (Microsoft, Redmond, WA, USA).
