**Luciana R. Tallini 1, Jaume Bastida 1, Natalie Cortes 2, Edison H. Osorio 3, Cristina Theoduloz <sup>4</sup> and Guillermo Schmeda-Hirschmann 5,6,\***


Academic Editor: John C. D'Auria

Received: 5 June 2018; Accepted: 19 June 2018; Published: 26 June 2018

**Abstract:** Amaryllidaceae plants are the commercial source of galanthamine, an alkaloid approved for the clinical treatment of Alzheimer's disease. The chemistry and bioactivity of Chilean representatives of *Rhodophiala* genus from the family of Amaryllidaceae have not been widely studied so far. Ten collections of five different Chilean *Rhodophiala* were analyzed in vitro for activity against enzymes such as acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) as well as for their alkaloid composition by GC-MS. To obtain an insight into the potential AChE and BuChE inhibitory activity of the alkaloids identified in the most active samples, docking experiments were carried out. Although galanthamine was found neither in aerial parts nor in bulbs of *R. splendens*, these plant materials were the most active inhibitors of AChE (IC50: 5.78 and 3.62 μg/mL, respectively) and BuChE (IC50: 16.26 and 14.37 μg/mL, respectively). Some 37 known alkaloids and 40 still unidentified compounds were detected in the samples, suggesting high potential in the Chilean Amaryllidaceae plants as sources of both novel bioactive agents and new alkaloids.

**Keywords:** *Rhodophiala*; alkaloids; molecular docking; AChE; BuChE; GC-MS

#### **1. Introduction**

The vast structural and chemical diversity of natural products gives them a significant role in drug discovery [1]. Alkaloids are of particular interest in biomedicine and drug discovery research [2] due to their structural diversity and specific biological potential [3]. The Amaryllidaceae is a plant family that contains an exclusive, large and still expanding alkaloid group known as the Amaryllidaceae alkaloids, which are characterized by unique skeleton arrangements and a broad spectrum of biological activities [4,5]. Amaryllidaceae plants have been used in folk medicine for their therapeutic and toxic properties [6]. Hippocrates of Kos (460–370 BP), considered the father of modern medicine, recommended the oil of *Narcissus* (Amaryllidaceae) species for the treatment of symptoms that today would be recognized as cancer [7].

The Amaryllidaceae alkaloids are classified mainly into nine skeleton types: norbelladine, lycorine, homolycorine, crinine, haemanthamine, narciclasine, tazettine, montanine and galanthamine (Figure 1) [4]. The most important Amaryllidaceae alkaloid is galanthamine, which was isolated for the first time from *Galanthus woronowii* in the 1950s and was approved for the clinical treatment of mild to moderate Alzheimer's disease (AD) by the Food and Drug Administration (FDA) at the beginning of this century [8,9]. Alzheimer's disease (AD), characterized by severe and progressive memory loss, is the most common form of dementia and is becoming increasingly prevalent in people older than 65 years [10,11]. It is estimated that 47 million people live with dementia in the world today, with an economic impact estimated at 818 billion dollars [12]. Known factors involved in the development of AD include a reduced cholinergic neurotransmission level, oxidative stress, amyloid-β-peptide (Aβ) and tau protein aggregation [13].

**Figure 1.** Amaryllidaceae alkaloid types.

Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) are involved in the hydrolysis of the neurotransmitter acetylcholine (ACh) [14]. Acetylcholinesterase is highly selective for ACh hydrolysis, and BuChE can metabolize different substrates [15]. In the brain of AD patients, AChE activity tends to decrease while that of BuChE increases [16]. Consequently, cholinesterase inhibitors that suppress both AChE and BuChE may provide a better therapeutic response rather than AChE-selective agents [17].

Little is known on the chemistry and bioactivity of the South American endemic Amaryllidaceae genus *Rhodophiala*. The *Rhodophiala* species have high ornamental potential due to its attractive red, yellow, white or orange flowers [18–22]. The approximately 40 species described at present occur in Argentina, Bolivia, southern Brazil, Chile and Uruguay [22]. *Rhodophiala* plants have a tunicate bulb of 4–6 cm diameter, which is set 20–30 cm underground, a single umbel holding up to six flowers, each flower being 4–6 cm wide and a flower stem 35 to 50 cm long [18].

*Rhodophiala* species are well known ornamental plants. Propagation of the Chilean species was reported [19] as well as phylogenetic [20] and morphological studies [21,22]. The renewed interest on galanthamine sources including the search for additional alkaloids with inhibitory activity towards the enzymes AChE and BuChE has prompted research work on the South American species of this family. Brazilian [23] and Argentinean wild Amaryllidaceae [24] showed high structural diversity in the alkaloid patterns and encouraged work on the Chilean species of this family. The knowledge of the chemical composition and anti-cholinesterase activity of the Chilean species belonging to genus *Rhodophiala* is limited. The comparative studies of their chemical composition are needed to identify the best potential sources of bioactive alkaloids in the South American species.

The aim of this work was to disclose the potential of Chilean *Rhodophiala* species as inhibitors of the enzymes AChE and BuChE as well as to analyze the alkaloid content and composition of native species by gas chromatography coupled to mass spectrometry (GC-MS) looking for galanthamine sources and searching for other compounds with effect of AChE and BuChE. Molecular docking studies were also carried out to investigate the affinity of the alkaloids identified in the most promising sample at the active sites of AChE and BuChE.
