The Anti-Cholinesterase Potential of Fifteen Different Species of Narcissus L. (Amaryllidaceae) Collected in Spain

Narcissus L. is a renowned plant genus with a notable center of diversity and is primarily located in the Mediterranean region. These plants are widely recognized for their ornamental value, owing to the beauty of their flowers; nonetheless, they also hold pharmacological importance. In Europe, pharmaceutical companies usually use the bulbs of Narcissus pseudonarcissus cv. Carlton to extract galanthamine, which is one of the few medications approved by the FDA for the palliative treatment of mild-to-moderate symptoms of Alzheimer’s disease. The purpose of this study was to evaluate the potential of these plants in Alzheimer’s disease. The alkaloid extract from the leaves of different species of Narcissus was obtained by an acid-base extraction work-up -procedure. The biological potential of the samples was carried out by evaluating their ability to inhibit the enzymes acetyl- and butyrylcholinesterase (AChE and BuChE, respectively). The species N. jacetanus exhibited the best inhibition values against AChE, with IC50 values of 0.75 ± 0.03 µg·mL−1, while N. jonquilla was the most active against BuChE, with IC50 values of 11.72 ± 1.15 µg·mL−1.


Introduction
Narcissus L. is a well-known plant genus that belongs to the Amaryllidaceae family, specifically within the Amaryllidoideae subfamily [1].These plants, commonly referred to as daffodils, are highly favored in gardens and serve as a significant commercial crop [2].This genus encompasses around 100 wild species, primarily concentrated in southwestern Europe, with a significant center of diversity in the Iberian Peninsula-where 90% of all species are present, especially in Spain, and extend throughout North Africa [2][3][4].
The native habitats of the Narcissus species exhibit remarkable diversity, encompassing a broad spectrum of landscapes that span from lowland to mountainous regions.This genus includes a rich tapestry of environments, including verdant grasslands, rugged scrublands, serene woodlands, meandering riverbanks, and secluded rocky crevices [2].The presentation of blooms exhibited by the majority of these species is a characteristic Life 2024, 14, 536 2 of 15 feature observed during late winter and spring.However, there are a limited number of species that deviate from this pattern and blossom during the autumnal season [5,6].
The hybridization of some of these species has led to the development of commercial Narcissus cultivars, which, in most instances, are larger and more robust than their wild ancestors [2,7].This genus stands out as the main choice among commercial bulb planters, showcasing remarkable importance in various horticultural and commercial contexts [8] in Western Europe, being cultivated since the 16th century in the Netherlands.Currently, the United Kingdom, the Netherlands, and the United States are the main producing countries of Narcissus bulbs.Until the late nineties, Narcissus bulb production was oriented only towards ornamental use, but since 1999, the purpose of extracting galanthamine from these bulbs started to play an important and growing role [9].Therefore, although the cultivation of Narcissus for ornamental purposes has had a long tradition, leading to extensive knowledge of its growing and breeding, as well as the production of large stocks of raw materials, only a few of the numerous cultivars developed could be cultivated in significant amounts and quality for chemical extraction [2].This question has been successfully solved by in vitro cultivation [10].
The medicinal properties of the Narcissus species have been documented in the literature for many years.Hippocrates of Kos (460-370 BCE), the father of modern medicine, advocated the use of oil from the Narcissus species to alleviate symptoms that today would be recognized as cancer [11].Dioscorides, in the 1st century, mentions some of their medicinal properties due to the presence of alkaloids, as well as the sedative and narcotic effects of their aroma [12].In recent decades, the genus Narcissus has provided a range of useful or potentially valuable compounds, of which galanthamine has been extensively studied.Galanthamine hydrobromide is one of the few medicines available used for the palliative treatment of symptoms derived from Alzheimer's disease, owing to its ability to inhibit the enzyme acetylcholinesterase.This alkaloid-specifically Amaryllidaceae alkaloid-was first isolated in 1952 from the species Galanthus woronowii and received the FDA's approval in 2001 [13,14].Pharmaceutical companies concentrated their search for galanthamine on the Amaryllidoideae subfamily, as it is the exclusive natural source of this metabolite [2,15].For Narcissus cultivars, different studies have been carried out on variations in galanthamine content in bulbs, depending on their geographical origin or changes due to the addition of fungicides [16,17].Torras-Claveria and co-workers [18] investigated the galanthamine content and bioactivity of more than one hundred Narcissus cultivars, highlighting those with higher galanthamine content and higher acetylcholinesterase inhibitory activity.In Central and Western Europe, the extraction of galanthamine is mainly derived from Narcissus pseudonarcissus cv.Carlton.Meanwhile, in Eastern Europe, Leucojum aestivum, known as snowflakes, serves as a key source, and it has been used in the past, although its collection for this purpose is currently not possible, as this species is in decline.In China, the red-tubed lily, Lycoris radiata, contributes to galanthamine production as well [9].
Amaryllidaceae alkaloids are unique structures originating from the Amaryllidoideae subfamily, covering a diverse group of compounds that exhibit interesting biological properties.Currently, there are more than 650 structures of Amaryllidaceae alkaloids reported in the literature [19].While significant progress has been made in recent years, the comprehensive exploration of the diversity of structures, reactions, and genes within the Amaryllidaceae family remains ongoing.All of these metabolites originate from the aromatic amino acids L-phenylalanine (L-Phe) and L-tyrosine (L-Tyr), which, through distinct enzymatic reactions, ultimately lead to the formation of norbelladine [20,21].The enzyme norbelladine 4 -O-methyltransferase catalyzes the production of 4 -O-methylnorbelladine, which is considered the main common precursor to Amaryllidaceae alkaloids [21].The oxidative phenolic coupling of 4 -O-methylnorbelladine leads to the three main skeleton types that form the bases for the extensive structural diversity of Amaryllidaceae alkaloids.The ortho-para' coupling of 4 -O-methylnorbelladine results in the formation of the lycorine-and homolycorine-type skeletons, para-para' originates the crinine-, haemanthamine-, tazettine-, narciclasine-and montanine-type structures, and para-ortho' gives the galanthamine-type skeleton [22].Since the initial isolation of the alkaloid lycorine from Narcissus pseudonarcissus in 1877, significant advancements have been achieved in the study of Amaryllidaceae plants.Nevertheless, they continue to represent a relatively underexplored source of phytochemicals [3].Generally, within each plant, a variety of related alkaloids is present, consisting of a few dominant metabolites and multiple minor compounds with varying substituent positions [3].
The historical bond between Amaryllidaceae and medicine stands as a testament to the enduring allure and profound significance of these remarkable compounds within the realm of human health and well-being.In the present day, continued research into Amaryllidaceae alkaloids promises to unveil further insights into their potential applications and therapeutic benefits.In this way, the aim of this study was to describe the potential different wild species of Narcissus L. collected in Spain against Alzheimer's disease through cholinesterase inhibition assays.

Plant Material
Fifteen different species of Narcissus L. were provided from the Iberian Narcissus Collection of the Torretes Biological Research Station-Botanical Garden of the UA, in Alicante, Spain.All the samples were collected during the flowering season, April 2023.The origins of the species are different localities of the Iberian Peninsula, as described in Table 1 and Figure 1.All the species were authenticated by botanists Dr. Segundo Ríos and Dr. Vanessa Martínez-Francés.Most wild daffodils are distributed in small, scattered populations, in very specific and fragile microhabitats such as wetlands, rocky areas, and deciduous forests [2,[23][24][25][26].These populations with very small numbers (less than 1000 individuals) have a small surface area (often less than 1 ha), making them extremely vulnerable to changes due to natural (climate change, predation, etc.) or anthropogenic causes (land use, grazing, collecting, etc.), which endanger their survival [27,28].The ecology of many species from the Pseudonarcissus DC. section, as N. asturiensis, N, jacetanus, N. minor, and N. vasconicus are perennial grasslands of external fringe woodlands.Other species, such as N. bujei and N. genesii-lopezii, are present in perennial mesophytic grasslands, and N. confusus is present in the secondary mesoforests of deciduous oaks (Table 1).The ecology of many species from the Pseudonarcissus DC. section, as N. asturiensis, N, jacetanus, N. minor, and N. vasconicus are perennial grasslands of external fringe woodlands.Other species, such as N. bujei and N. genesii-lopezii, are present in perennial mesophytic grasslands, and N. confusus is present in the secondary mesoforests of deciduous oaks (Table 1).
More ecological diversity is observed in the Nevadensis Zonn.section.N. nevadensis grows in the secondary mesoforests of deciduous oaks and the perennial grasslands of external fringe woodlands.The existing populations of N. alcaracensis develop on lacustrine and helophyte communities dominated by Carex hispida, and N. yepesii is present in meadows and chionophilous grassland vegetation (Table 1).
N. jonquilla, from section Jonquillae DC., grows in lacustrine and riverine helophyte graminoid communities, while N. assoanus grows in perennial succulent grassland in rocky, open communities.The representative of the Bulbocodium section, N. hedraeanthus, is present in meadows and chionophilous grassland vegetation.From the Ganymedes section, N. pallidulus grows in perennial mesophytic grasslands and bare sandy soils, and N. tazetta of section Tazettae DC. has been collected from gardens where it was cultivated (Table 1).Although ornamental domestication of the genus Narcissus by the United Kingdom and Holland began in the 16th century [7,24], today there is still continuous extraction of wild bulbs in Spain, Portugal, and North Africa to satisfy the global private collecting market, to obtain new disease-resistant genes in commercial bulbs, and to satisfy the demand for galanthamine by pharmaceutical companies.Despite the scarce data on the populations and conservation status of most of the species, the information collected for some of them has allowed for their inclusion in the IUCN Red List, considered a critical indicator of the health of biodiversity.Two species of section Nevadensis Zonn.Analyzed in this work, N. alcaracensis and N. nevadensis, and another of section Pseudonarcissus DC., N. bujei, have been assessed as Endangered (EN) in the IUCN list [30][31][32].Another species of this first mentioned section, N. yepesii, has been evaluated and classified as Vulnerable (VU) [33].
N. asturiensis and N. hedraeanthus are listed under Least Concern (LC), with no other major conservation measures recommended [34,35].N. pallidulus, although not mentioned, is included in N. triandrus, which is assessed as Least Concern (LC), considering its wide distribution, with large and stable populations; it is unlikely that existing threats More ecological diversity is observed in the Nevadensis Zonn.section.N. nevadensis grows in the secondary mesoforests of deciduous oaks and the perennial grasslands of external fringe woodlands.The existing populations of N. alcaracensis develop on lacustrine and helophyte communities dominated by Carex hispida, and N. yepesii is present in meadows and chionophilous grassland vegetation (Table 1).
N. jonquilla, from section Jonquillae DC., grows in lacustrine and riverine helophyte graminoid communities, while N. assoanus grows in perennial succulent grassland in rocky, open communities.The representative of the Bulbocodium section, N. hedraeanthus, is present in meadows and chionophilous grassland vegetation.From the Ganymedes section, N. pallidulus grows in perennial mesophytic grasslands and bare sandy soils, and N. tazetta of section Tazettae DC. has been collected from gardens where it was cultivated (Table 1).Although ornamental domestication of the genus Narcissus by the United Kingdom and Holland began in the 16th century [7,24], today there is still continuous extraction of wild bulbs in Spain, Portugal, and North Africa to satisfy the global private collecting market, to obtain new disease-resistant genes in commercial bulbs, and to satisfy the demand for galanthamine by pharmaceutical companies.Despite the scarce data on the populations and conservation status of most of the species, the information collected for some of them has allowed for their inclusion in the IUCN Red List, considered a critical indicator of the health of biodiversity.Two species of section Nevadensis Zonn.Analyzed in this work, N. alcaracensis and N. nevadensis, and another of section Pseudonarcissus DC., N. bujei, have been assessed as Endangered (EN) in the IUCN list [30][31][32].Another species of this first mentioned section, N. yepesii, has been evaluated and classified as Vulnerable (VU) [33].
N. asturiensis and N. hedraeanthus are listed under Least Concern (LC), with no other major conservation measures recommended [34,35].N. pallidulus, although not mentioned, is included in N. triandrus, which is assessed as Least Concern (LC), considering its wide distribution, with large and stable populations; it is unlikely that existing threats will cause them to seriously decrease in the near future [36].However, it should be added that the different species of Section Ganymedes (Salisb.)Schultes f. studied in [37] have not been taken into consideration in the preparation of this manuscript, and different problems are present regarding each of them, both with respect to population size and natural and anthropogenic issues, requiring, therefore, a new review.
Life 2024, 14, 536 5 of 15 Only the leaves were collected for biological assays.The use of leaves instead of bulbs for biological activity studies ensures the maintenance of the Iberian Daffodil Collection, allowing for its reproduction.Moreover, depending on the phenological stage of the plant, the alkaloid content and, therefore, the biological activity, may vary from bulb tissue to leaf tissue, and sometimes leaves may contain more alkaloids and be more active than bulbs [18,38].

Alkaloid Extracts Preparation
For the evaluation of the bioactivity, purified alkaloid extracts were performed.The species were meticulously processed as follows: First, they were cut into pieces and dried at a controlled temperature of 40 • C. Afterward, the dried plant material was finely powdered using a rotary blade mill, specifically a stainless-steel grinder (Taurus, Oliana, Spain).To extract the desired compounds, 1 g of the resulting powder was subjected to a maceration process with methanol at 25 • for three days.During this period, the solvent was replaced each day using 3 × 50 mL aliquots and submitted to an ultrasonic bath (20 min, 4 daily intervals).Following methanolic extraction, the mixture was meticulously strained, and the solvent was carefully evaporated under reduced pressure, leaving behind crude extracts.These crude extracts were subsequently acidified using 30 mL of a 2% (v/v) sulfuric acid solution, lowering the pH to 2. After acidification, an ethyl acetate treatment (using 3 × 50 mL) was employed to eliminate neutral materials.The next step involved adjusting the pH of the remaining aqueous solution to a range of 9-10, achieved by the addition of a 25% (v/v) ammonium hydroxide solution.The volume of the ammonium hydroxide solution employed was the quantity required to achieve the aforementioned pH.Finally, the alkaloids were extracted with ethyl acetate (using 3 × 50 mL).After evaporation of the solvent, the dried alkaloid extract (AE) was obtained.

Cholinesterase Inhibitory Activity
Levels of ACh and BuCh decrease in patients with Alzheimer's disease.Inhibition of AChE and BuChE has been shown to maintain ACh and BuCh levels in the brain, reducing disease progression [13,39].The activity of Narcissus extracts inhibiting these enzymes can be assessed with colorimetric AChE and BuCHE inhibition assays, based on the formation of thiobenzoate anion (yellow) following the reaction of thiocholine and 2,2 -dinitro-5 -dinitro-5 -dithiobenzoic acid when the enzymes are active.

Statistical Evaluation
The inhibition of the cholinesterase activity of the Narcissus species was assessed using three separate assays.The PRISM software was used to analyze the results.The data are presented as the average ± standard deviation (SD).The significance of the results is represented versus the control (Gal), and it is indicated as follows: **** p < 0.0001, *** p < 0.001, ** p < 0.01, and ns (not significant).A one-way ANOVA test was performed following Dunnet's multiple comparison test, comparing the differences with respect to the outcome of galanthamine with both AChE and BuChE.

Results and Discussion
The alkaloid extracts from all the Narcissus species collected in Spain were obtained through acid-base extraction, as described in Section 2.2.The yield of each species is available in Table 2.The average income value was 1.32%, with the highest value at 6.14% (sample N) and the lowest at 0.47% (sample B), which represent the species N. yepesii and N. jacetanus, respectively.In vitro assessments were carried out to examine the inhibitory potential of the fifteen alkaloid extracts from Narcissus leaves against the enzymes AChE and BuChE.Among these plant samples, fourteen demonstrated activity against AChE, while only nine exhibited activity against BuChE (see Figures 2 and 3).As was expected, all the species evaluated herein presented better results against AChE than BuChE.The species N. jacetanus (sample B) showed the best inhibition values against AchE, with IC 50 values of 0.75 ± 0.03 µg•mL −1 , while N. jonquilla (sample L) was the most active against BuChE, with IC 50 values of 11.72 ± 1.15 µg•mL −1 .The species N. jacetanus and N. jonquilla are illustrated in Figures 4 and 5, respectively.The samples N. assoanus, N. minor, N. confuses, and N. jonquilla also presented noteworthy activity against AChE, with values of IC 50 of Life 2024, 14, 536 7 of 15 0.99 ± 0.06, 0.81 ± 0.10, 1.04 ± 0.07, and 1.88 ± 0.05 µg•mL −1 , respectively.Regarding BuChE inhibition activity, N. confusus and N. genesii-lopezii also presented high and relevant activity, with IC 50 of 12.83 ± 0.87 and 11.98 ± 0.93 µg•mL −1 , respectively.Thus, N. jonquilla stands out for being the species of Narcissus with the highest global cholinesterase activity inhibition, followed by N. confusus and N. jonquilla.
Among other samples, Havlasová and co-authors [43] evaluated the inhibitory potential of N. jonquilla var.henriquesii against AChE and obtained IC50 values of 32.6 ± 4.3 µg•mL −1 , while for galanthamine, it was 1.7 ± 0.06 µg•mL −1 [43].As documented in the literature, the galanthamine-type skeleton, specifically the alkaloids galanthamine and sanguinine, are commonly active against AChE and BuChE [41].In the literature, a great amount of galanthamine-type structures is reported, such as galanthamine, lycoramine, and narwedine in the species N. jonquilla, representing about 65% of its alkaloid profiling [4,44].Furthermore, haemanthamine, tazettine, jonquailine, and narciclasine-and lycorine-type structures were also described in this plant species [4,44,45].Furthermore, the majority of the reported alkaloids of N. jonquilla have been studied in terms of molecular modelling in front of AChE and BuChE, and data reported in the literature support the fact that they could be responsible for the especially high activity of this species inhibiting BuChE and AChE.Galanthamine has been reported to have good docking scores for   Considering their biological potential, it is important to account for the potential synergistic interactions among Amaryllidaceae alkaloids in plant extracts [42].These interactions, previously reported to contribute to acetylcholinesterase (AChE) inhibition, should be taken into consideration when explaining the anticholinesterase potential of certain Amaryllidaceae species [42].
Among other samples, Havlasová and co-authors [43] evaluated the inhibitory potential of N. jonquilla var.henriquesii against AChE and obtained IC50 values of 32.6 ± 4.3 µg•mL −1 , while for galanthamine, it was 1.7 ± 0.06 µg•mL −1 [43].As documented in the literature, the galanthamine-type skeleton, specifically the alkaloids galanthamine and sanguinine, are commonly active against AChE and BuChE [41].In the literature, a great amount of galanthamine-type structures is reported, such as galanthamine, lycoramine, and narwedine in the species N. jonquilla, representing about 65% of its alkaloid profiling [4,44].Furthermore, haemanthamine, tazettine, jonquailine, and narciclasine-and lycorine-type structures were also described in this plant species [4,44,45].Furthermore, the majority of the reported alkaloids of N. jonquilla have been studied in terms of molecular modelling in front of AChE and BuChE, and data reported in the literature support the fact that they could be responsible for the especially high activity of this species inhibiting BuChE and AChE.Galanthamine has been reported to have good docking scores for Considering their biological potential, it is important to account for the potential synergistic interactions among Amaryllidaceae alkaloids in plant extracts [42].These interactions, previously reported to contribute to acetylcholinesterase (AChE) inhibition, should be taken into consideration when explaining the anticholinesterase potential of certain Amaryllidaceae species [42].
The AChE inhibition activity of the species N. assoanus, N. jacetanus, N. bujei, N. vasconicus, and N. pallidulus have also been described by López and co-authors.However, no activity has been described for the latter two species [41].Previous publications indicate that the alkaloid composition of the species N. assoanus includes assoanine, oxoassoanine, pseudolycorine, 1-O-acetylpseudolycorine, and 2-O-acetylpseudolycorine [51,52].The species N. jacetanus has been previously documented to contain the compounds assoanine, oxoassoanine, pseudolycorine, and lycorine [53].According to the literature, the compounds assoanine and oxoassoanine, which belong to a lycorine-type skeleton, are active against AChE, with IC 50 values of 3.87 ± 0.24 and 47.21 ± 1.13 µM, respectively, while pseudolycorine exhibits poor activity [41].According to prior publications, the alkaloid lycorine exhibits a very weak in vitro activity against AChE and BuChE, with IC 50 values higher than 200 µM [41,54] Regarding the molecular modelling of assoanine and oxoassoanine vs. AchE, they did not show great interaction with AchE in terms of electrophilicity, with values of 0.0026 and 0.0036 eV, respectively, (galanthamine electrophilicity values corresponded to 8.5725 eV).In relation to the molecular electrostatic potential (MEP) of assoanine and oxoassoanine, values of negative (−0.08705 and −0.06995 u.a.) and positive (0.03474 and 0.05755 u.a.) regions (which represent the probability to conduct and suffer, respectively, nucleophilic attacks) are not as high as other Amaryllidaceae alkaloids with AChE inhibition properties such as galanthamine (−0.06995 and 0.05755 u.a.negative and positive regions, respectively), hydroxygalanthamine (−0.06822 and 0.06648 u.a.), or sanguinine (−0.07504 and 0.06400 u.a.) [55].However, it must be considered that no other molecular modeling studies have been found in the literature regarding these two compounds; therefore, more studies should be performed to obtain a global idea of the characterization of the interaction of these compounds with AChE and BuChE.

Figure 3 .
Figure 3. Graph of BuChE inhibitory activity of the different samples of alkaloid extracts of Narcissus leaves collected in Spain.C = N. vasconicus; E = N. confusus; G = N. hedraeanthus; H = N. alcaracencis;

Figure 3 .
Figure 3. Graph of BuChE inhibitory activity of the different samples of alkaloid extracts of Narcissus leaves collected in Spain.C = N. vasconicus; E = N. confusus; G = N. hedraeanthus; H = N. alcaracencis;

Figure 4 .
Figure 4. Photo of Narcissus jacetanus, the sample with the best results for AChE inhibition.

Figure 5 .
Figure 5. Photo of Narcissus jonquilla, the sample with the best results for BuChE inhibition (also with noteworthy activity against AChE).

Figure 4 . 15 I
Figure 4. Photo of Narcissus jacetanus, the sample with the best results for AChE inhibition.

Figure 4 .
Figure 4. Photo of Narcissus jacetanus, the sample with the best results for AChE inhibition.

Figure 5 .
Figure 5. Photo of Narcissus jonquilla, the sample with the best results for BuChE inhibition (also with noteworthy activity against AChE).

Figure 5 .
Figure 5. Photo of Narcissus jonquilla, the sample with the best results for BuChE inhibition (also with noteworthy activity against AChE).

Table 1 .
Narcissus sample coding, biogeographical distribution, and province of collection.

Table 2 .
Yield of alkaloid extracted, obtained from the Narcissus species.