Biosynthesis and Biological Activities of Newly Discovered Amaryllidaceae Alkaloids

Alkaloids are an important group of specialized nitrogen metabolites with a wide range of biochemical and pharmacological effects. Since the first publication on lycorine in 1877, more than 650 alkaloids have been extracted from Amaryllidaceae bulbous plants and clustered together as the Amaryllidaceae alkaloids (AAs) family. AAs are specifically remarkable for their diverse pharmaceutical properties, as exemplified by the success of galantamine used to treat the symptoms of Alzheimer’s disease. This review addresses the isolation, biological, and structure activity of AAs discovered from January 2015 to August 2020, supporting their therapeutic interest.


Introduction
The Amaryllidaceae species, belonging to the Asparagales monocot order, are a class of herbaceous, perennial, and bulbous flowering plants. The Amaryllidaceae plant family contains 85 genera and 1100 species that are widely distributed in the tropic and warm temperate regions of the globe [1]. In addition, the Amaryllidaceae plants are cultivated and exploited as ornamental plants for their beautiful flowers [2]. For centuries, Amaryllidaceae have been used in traditional medicine, such as the oil extracted from the daffodil Narcissus poeticus, used to treat uterine tumors [3]. Since the isolation of lycorine in 1877 (initially named narcissia) from Narcissus pseudonarcissus and in 1897 from Lycoris radiata [4][5][6], the structures of hundreds of Amaryllidaceae alkaloids (AAs) have been elucidated [1,3,7]. They are exploited for their wide range of biological potentials including antitumor, antiviral, antibacterial, antifungal, antimalarial, anti-acetylcholinesterase (anti-AChE), analgesic, and cytotoxic activities [8,9]. Currently, in terms of commercial success, galantamine, widely occurring in the Amaryllidaceae plants, has been approved as an AChE inhibitor by the United States Food and Drug Administration to treat the symptoms of Alzheimer's disease (AD) [10]. Moreover, several other AAs, including lycorine, haemanthamine, and narciclasine have been used as lead molecules for anticancer research [11]. Thus, AAs represent an important resource for drug discovery.
This review addresses the isolation, biosynthesis, biological activities and structure activity of AAs discovered from January 2015 to August 2020.

Classification of Amaryllidaceae Alkaloids
To date, more than 650 AAs have been reported, and their chemical library is still expanding [1,[12][13][14][15][16][17][18][19][20][21][22][23][24]. Although diverse in structure, this plethora of AAs are categorized together as they share a common Molecules 2020, 25 initial synthesis pathway. In previous literature, large numbers of AAs have been classified into different groups according to chemical characteristics, e.g., molecular skeleton and ring structure [1,3,8,25]. For this review, AAs were classified into 10 main groups instead, following a biochemical classification based on biogenetic lineage and ring type, to easily track the biosynthetic pathways [26] (Table 1, Figure 1). For example, haemanthamine and crinine were grouped together with respect to their biosynthetic origin and ring type even if they were previously categorized separately [11]. Some AAs with ring types different than those of group I to IX were classified in group X (or other-types) because they follow distinct biogenetic pathway, or because we cannot clearly indicate their biosynthetic origin (Table 1). Galanthindole contains a non-fused indole ring and might represent an artifact of homolycorine-or of pretazettine-type derivatives [27]. Ismine is considered to be a catabolic product from the haemanthamine-type skeleton, thus not a specific type of AA [28]. The latter examples demand further investigation on biogenetic origin and are not yet included on any particular type of AA.  Some types of AA, such as plicamine and secoplicamine, are extracted in trace amounts exclusively from specific Amaryllidaceae species, such as Zephyranthes, but are classified in type X as they are rare, dinitrogenous members of AA, with a distinct biosynthetic linage [28][29][30][31]. Mesembrine alkaloids (also known as sceletium) have a distinct biosynthetic pathway, without norbelladine as key intermediate, they are usually extracted from Aizoaceae, but can be collected l-phenylalanine is converted to trans-cinnamic acid by the phenylalanine ammonia-lyase (PAL) ( Figure 2). Several PAL gene transcripts were identified and characterized from different species of Amaryllidaceae [41][42][43][47][48][49]. Interestingly, two main phylogenetic PAL clusters were identified; the first one contained PAL transcripts ubiquitously expressed in Amaryllidaceae whereas the second cluster contained PAL transcripts with expression highest and correlating with organs where AAs accumulated [26]. This indicates that different PAL transcripts encode enzymes with distinct functions in the phenylpropanoid pathway and it suggests the role of the latter cluster in AA biosynthesis. Next, trans-cinnamic acid is hydroxylated to form p-coumaric acid by the cinnamate 4-hydroxylase (C4H), a cytochrome P450 of the CYP73 subfamily (cinnamate 4-hydroxylase, C4H) [50,51]. Transcripts encoding C4H were reported from several Amaryllidaceae species [41][42][43]49] including C4H from L. radiate, which was characterized as producing a region-specific 4-hydroxylation of trans-cinnamic acid [49]. The reactions catalyzed by PAL and C4H are also crucial steps in the biosynthesis of many phenylpropanoids such as flavonoids, linins, coumarins and stilbenes. From there, the enzymes and order of reactions leading to 3,4-DHBA are not known however it is hypothesized that it may involve the CYP98A3 named coumarate 3-hydroxylase (C3H) and/or the ascorbate peroxidase (APX) and/or the 4-hydroxybenzaldehyde synthase (HBS) [26] (Figure 2). A few studies have reported on the presence of phenolic acids such as caffeic, p-coumaric, and ferulic acids in N. pseudonarcissus, N. poeticus and Galanthus nivalis [52][53][54]. In addition, 3,4-DHBA was detected in plants outside the Amaryllidaceae family [55]. Collectively, this suggest that the initial reactions and enzymes of the phenylpropanoid pathway participate in the synthesis of the AA precursor 3,4-DHBA. tyrosine decarboxylase (TYDC) (Figure 2). Two gene transcript variants of TYDC, named TYDC1 and TYDC2, were identified from the transcriptome of different Amaryllidaceae species including N. pseudonarcissus [41], Narcissus papyraceus [42], Lycoris radiata [43], and L. aureus [44]. Biosynthesis pathway to major types of Amaryllidaceae alkaloids. Arrows without labeling reflect chemical reactions that have not been enzymatically characterized. Enzymes that have been identified are labeled in blue. A solid arrow symbolizes one enzymatic step whereas a broken arrow shows multiple enzymatic reactions. Chemical structures of precursors were added to clarify the regioselective phenol-phenol' coupling reaction. Enzyme abbreviations: PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; C3H, coumarate 3-hydroxylase; APX, ascorbate peroxidase; HBS, 4-hydroxybenzaldehyde synthase; TYDC, tyrosine decarboxylase; NBS, norbelladine synthase; NR, noroxomaritidine reductase; CYP96T1, cytochrome P450 monooxygenase 96T1.
The pathway leading to 3,4-DHBA from L-phenylalanine involves a series of reactions known as the phenylpropanoid pathway which is phylogenetically spread in most plant species. In Amaryllidaceae, using precursor feeding experiments, it was reported that trans-cinnamic acid, pcoumaric acid, and caffeic acid were intermediate products that ultimately led to 3,4-DHBA [45,46]. Specifically, L-phenylalanine is converted to trans-cinnamic acid by the phenylalanine ammonia-lyase (PAL) (Figure 2). Several PAL gene transcripts were identified and characterized from different species of Amaryllidaceae [41][42][43][47][48][49]. Interestingly, two main phylogenetic PAL clusters were identified; the first one contained PAL transcripts ubiquitously expressed in Amaryllidaceae whereas the second cluster contained PAL transcripts with expression highest and correlating with organs . Biosynthesis pathway to major types of Amaryllidaceae alkaloids. Arrows without labeling reflect chemical reactions that have not been enzymatically characterized. Enzymes that have been identified are labeled in blue. A solid arrow symbolizes one enzymatic step whereas a broken arrow shows multiple enzymatic reactions. Chemical structures of precursors were added to clarify the regioselective phenol-phenol' coupling reaction. Enzyme abbreviations: PAL, phenylalanine ammonia-lyase; C4H, cinnamate 4-hydroxylase; C3H, coumarate 3-hydroxylase; APX, ascorbate peroxidase; HBS, 4-hydroxybenzaldehyde synthase; TYDC, tyrosine decarboxylase; NBS, norbelladine synthase; NR, noroxomaritidine reductase; CYP96T1, cytochrome P450 monooxygenase 96T1.
The core skeletons obtained from norbelladine, methylnorbelladine, and the phenol coupling steps form the basis of AA diversity. A complex network of enzyme catalyzing various types of reactions, such as C-C and C-O bond formations, Oand N-methylations, demethylations, hydroxylations, oxidations and reductions, yield the several hundred of structurally related AAs. Only a few of these enzymes are known to date and they are reported in Figure 2.

Occurrence of Amaryllidaceae Alkaloids
From January 2015 to August 2020, a total of 91 new AAs were isolated and identified from different plant species of the genus Crinum, Zephyranthes, Narcissus, Galanthus, Hymenocallis, Nerine, Lycoris, Brunsvigia, and Hippeastrum ( Table 2).
The Narcissus genus is composed of approximately 50 species that originate from the area of the Iberian Peninsula. Jonquailine (52), a pretazettine-type AA, was isolated from the dried bulbs of N. jonquilla quail and characterized for structural elucidation and absolute configuration using various spectroscopic techniques [83]. Narcipavline (6) and narcikachnine (87), two new alkaloids, were isolated, together with thirteen known alkaloids, from fresh bulbs of N. poeticus cv. Pink Parasol, their chemical structure was elucidated by MS, together with 1D and 2D NMR spectroscopic analyses, and by comparison with literature data [29]. From the fresh bulbs of N. pseudonarcissus L. cv. Dutch Master, a new AA, named narcimatuline (88), was isolated, together with twenty-one known AAs of various structural types. Their chemical structure was elucidated by a combination of MS, HR-MS, 1D and 2D NMR spectroscopic techniques and also by comparison with existing data [89].
The phytochemical investigation of fresh bulbs of Narcissus pseudonarcissus cv. Carlton led to the isolation of thirteen known AAs, and three new norbelladine-type AAs: carltonine A-C (4-6). Their structure was determined using spectroscopic methods including 1D NMR, 2D NMR, and HR-MS [62]. 7-Oxonorpluviine (24), a lycorine-type alkaloid was isolated from fresh bulbs of Narcissus L. cv. Professor Einstein, together with twenty three known AAs, and their structures were identified by using various spectroscopic methods like (GC-MS, LC-MS, 1D, and 2D NMR spectroscopy) [71]. Pseudolycorine N-oxide (25) was isolated from Narcissus tazetta whole plant, and the structural elucidation was determined by spectroscopic data analysis [72].
Hymenocallis plants are native of Central and South America and include more than 60 species. Novel AA hymenolitatine (82) was isolated together with trispheridine and tazettine from the dichloromethane extract of the bulbs of Hymenocallis littoralis (Jacq.) Salisb. Chemical characterization of these compounds was performed by spectroscopic methods including 1D NMR, 2D NMR, and HR-MS [88].
Amaryllis is a small genus comprised of two species (A. belladonna L. and A. paradisicola Snijman). In particular A. belladonna is native of South Africa [94]. A new crinine alkaloid 1,4-dihydroxy-3methoxy powellan (36), together with five known alkaloids, were isolated from the bulbs of A. belladonna Steud. by bioassay-guided isolation. Structures were elucidated by interpretation of combined HR-ESIMS, CD, and 2D NMR spectroscopic data [76].

Pharmacological Properties of Novel Amaryllidaceae Alkaloids
The pharmacological properties of the newly discovered AAs (1-91) were assessed when isolated in sufficient amount. AAs display a wide range of biological activities, including cytotoxicity, effects on the central nervous systems (CNS), anti-inflammatory, anti-microbial, anti-parasitic, larvicidal, and antioxidant activities.

Antitumoral Cytotoxic Activity
Lycorine is the most abundantly found AA, it belongs to the pyrrolo[de]phenanthridine subgroup. The biological effects of lycorine have been known for many years, and lycorine is still being investigated for a variety of therapeutic application, in particular as anticancer agent showing  Table 2.
Amaryllis is a small genus comprised of two species (A. belladonna L. and A. paradisicola Snijman). In particular A. belladonna is native of South Africa [94]. A new crinine alkaloid 1,4-dihydroxy-3-methoxy powellan (36), together with five known alkaloids, were isolated from the bulbs of A. belladonna Steud. by bioassay-guided isolation. Structures were elucidated by interpretation of combined HR-ESIMS, CD, and 2D NMR spectroscopic data [76].

Pharmacological Properties of Novel Amaryllidaceae Alkaloids
The pharmacological properties of the newly discovered AAs  were assessed when isolated in sufficient amount. AAs display a wide range of biological activities, including cytotoxicity, effects on the central nervous systems (CNS), anti-inflammatory, anti-microbial, anti-parasitic, larvicidal, and antioxidant activities.

Antitumoral Cytotoxic Activity
Lycorine is the most abundantly found AA, it belongs to the pyrrolo[de]phenanthridine subgroup. The biological effects of lycorine have been known for many years, and lycorine is still being investigated for a variety of therapeutic application, in particular as anticancer agent showing promising activity against tumors with dismal prognoses [96,97]. The structure-activity relationship (SAR) of lycorine and its derivatives has been evaluated using human leukemia T cells (Jurkat). The results showed that the free 1,2-diol functionality in the C-ring is required to induce apoptosis [98]. Furthermore, it has been demonstrated that the presence of the unaltered diol functionality in the C-ring in its original configuration in lycorine series, the stereochemistry of the C/D ring junction and the conformational freedom of the C-ring were required for the anticancer activity [96] Pseudolycorine N-oxide (25) was inactive against human cervical cancer (SiHa) and human epidermoid carcinoma (KB) cells [72].

Effects on the Central Nervous System (CNS)
Several enzymes of the CNS are interesting drug targets. AChE is a serine protease located at neuromuscular junctions, in cholinergic synapses of the central nervous system and in red blood cells [99][100][101]. The enzyme catalyzes the rapid hydrolysis and inactivation of the neurotransmitter acetylcholine into acetate and choline to enable cholinergic neurons to return to their resting state. Butyrylcholinesterase (BChE) can also hydrolyze acetylcholinesterase into acetate and choline. BChE is produced by the liver and detected in the plasma. Changes in its plasmatic levels can indicate of liver dysfunction. BChE is also expressed in neurons of the CNS [102].
In Alzheimer's disease (AD), AChE is overly active, and the consequential lower level of acetylcholine in the brain cause weakened neurotransmission [103]. Similarly, BChE deregulation is measured in the brain of individuals suffering from AD. Malfunction of the cholinergic system may be pharmacologically tackled via AChE inhibitors that ameliorate the cholinergic deficit at early stages of the disease and reduce progression. In addition, glycogen synthase kinase-3 (GSK-3) is a ubiquitous serine/threonine kinase, implicated in AD, which can trigger abnormal hyperphosphorylation of tau protein, which is believed to be a critical event in neurofibrilary tangle formation. Thus, GSK-3 inhibition represents an attractive drug target for AD and other neurodegenerative disorders [104]. Finally, prolyloligopeptidase (POP) is a cytosolic serine peptidase widely distributed in the organs of the body, including the brain, which cleaves peptide bonds at the carboxyl end of proline [105,106]. Previous studies have shown that POP inhibitors are efficient anti-dementia drugs [107,108].
The AA galantamine, donepezil and rivastigmine are potent reversible inhibitors of AChE approved for the symptomatic treatment of AD [109,110]. Since cholinesterase enzyme inhibitors are first generation drugs for AD, AChE and BChE are the most targeted enzymes at the moment.
Galantamine derivative sanguinine is ten times more active than galantamine whereas 11-hydroxygalantamine exhibits inhibitory activity similar to that of galantamine. The extra or protected hydroxyl group in its allylic position in (R 1 ) may be required for the activities [111]. SAR of galantamine and its derivatives was comprehensively reviewed elsewhere [8].
Among the six new galantamine-type alkaloids only 9-de-O-methyl-11β-hydroxygalantamine (13) showed a weak AChE inhibitory activity with IC 50 value 168.7 µM. The SAR of new galantamine derivatives alkaloids (11)(12)(13)(14)(15)(16) and known alkaloids isolated from the same plant species revealed that the 4,4a double bond and 9-OH are required for the AChE inhibitory activity, while the presence of the 11-OH group dramatically decreases AChE inhibitory activity [66].
Malaria (Plasmodium sp.), leishmaniasis (Leishmania sp.), and trypanosomiasis (Trypanosoma brucei and Trypanosoma cruzi) are the most common chronic protozoan diseases and occur mainly in poor rural and urban areas in tropical and subtropical regions of the world. Previously, several AAs were reported for their potent in vitro antiprotozoal activity [113]. The anti-plasmodial activity was recently reviewed elsewhere [114,115]. Newly isolated alkaloids such as cripowellin C (75) and D (76) were evaluated against the chloroquine/mefloquine-resistant Dd2 strain of Plasmodium falciparum and were found to have potent antiplasmodial activity, with IC 50 values of 180 ± 20, 26 ± 2, and 260 ± 20 nM, respectively [85].

Larvicidal and Insecticidal
Insects are important vectors of many diseases, controlling their proliferation is an efficient way of preventing disease spread. Aedes aegypti is the main vector for dengue, yellow fever and Zika infection. In an earlier study, organic extracts of the bulbs of Nerine sarniensis, demonstrated strong larvicidal and insecticidal activity with LC 50 of 0.008 µg.µL −1 against A. aegypti larvae and against grown-up females with LD 50 of 4.6 µg/mosquito. Sarniensine (78) was less efficient against larvae at the most minimal concentration of 0.1 µg/µL but displayed strong adulticidal activity with an LD 50 of 1.38 ± 0.056 µg/mosquito [86]. Mesembrine-class sarniensinol (77), sarniensine (78) and crinine-type crinsarnine (43) had no effect against A. aegypti larvae at all concentrations tested. In adult topical bioassays, only (43) displayed adulticidal activity, with an LD 50 = 2.29 ± 0.049 µg per mosquito. SAR studies revealed that the scaffold of pretazettine alkaloids in (77) and (78) and (43) and in bowdensine were important for their activity. Among the mesembrine group, the opening of the B-ring or the presence of a B-ring lactone as well as the trans-stereochemistry of the A/B-ring junction seem to be important for their activity, while in crinine-type alkaloids, the substituent at C-2 appears to be important [78,86,116].

Others Activities
The carbonic anhydrases (CAs) are metalloenzymes that catalyze the reversible hydration of carbon dioxide with water into a bicarbonate ion and a proton. In humans, sixteen isozymes have been identified including human cabonic isozyme II (hCAII) reported to be involved many diseases like glaucoma, epilepsy and cancer. Thus, hCAII is a target for therapeutic interventions [80]. Crinasiaticine A (46) and crinasiaticine B (47) were evaluated for their inhibitory potential against hCAII and they were inactive [80].

Chemical Extraction from Amaryllidaceae Plants
The acid-base extraction is the classical method used to extract alkaloids from Amaryllidaceae. The acid extraction usually uses 0.1% to 2% sulfuric acid (H 2 SO 4 ) or hydrochloric acid (HCl) as solvent by maceration. For example, in a recent study by our group, fresh bulbs of C. jagus were dried at room temperature and then finely powdered [63]. The resultant powder (1.35 kg) was extracted with 1% H 2 SO 4 , (2 × 2 L) overnight at room temperature. The suspension was filtered through a cloth and successively centrifuged at 10 • C at 7000 rpm for 30 min. The acid extract was alkalinized to pH 9-10 with 12 N NaOH. The aqueous solution was extracted with EtOAc (3 × 1.2 L), and the organic extracts were combined, dried (Na 2 SO 4 ) and evaporated under reduced pressure to give a brown oil residue (3.0 g) [63].
Alcoholic solvent such as methanol or ethanol can also be used to extract both free and salt of alkaloids. For example, fresh bulbs (1.5 kg) and leaves (400 g) of H. reticulatum were collected, crushed and extracted with MeOH (2 × 1.0 L) at room temperature for 4 days, and the combined macerate was filtered and evaporated under reduced pressure. The crude extracts of the bulbs and leaves (117.3 and 53.5 g, respectively) were acidified with H 2 SO 4 (2%, v/v) to pH 3 and extracted with Et 2 O (10 × 150 mL) and EtOAc (3 × 150 mL) to remove the neutral material. The aqueous solutions were basified with ammonia (25%, v/v) up to pH 9-10 and extracted with n-Hex (16 × 150 mL) to give the n-Hex extracts (0.14 and 0.02 g, respectively. This was followed by extraction with EtOAc (15 × 150 mL) to give the EtOAc extracts (1.6 and 0.3 g, respectively) and finally extracted with EtOAc:MeOH (3:1, v/v) (4 × 150 mL) to provide the EtOAc:MeOH extracts (2.10 and 1.56 g, respectively) [68].

Biotechnological Production of Amaryllidaceae Alkaloids
Usually, extraction of bioactive compound from its natural source is low, time-consuming and costly, which hinders further research and application. With the commercialization of galantamine as a drug, its demand from generic pharmaceutical companies increased, and production from native source became challenging to fulfill [117]. The diversity and quantity of AAs in plants also vary with plant species, developmental stage and types of tissue/cell. Plants growth can be affected by seasonal changes and environmental factors [25,42,118]. Therefore, mass production of AAs by cultivation of plants is not always sustainable.
Alternatively, several studies have depicted other approaches to produce specialized metabolites such as alkaloids by using in vitro culture of plant parts/tissues. Results from in vitro culture of Narcissus confusus and Leucojum aestivum show that alkaloids' amounts are low in undifferentiated cells as compared to differentiated cells, indicating that undifferentiated cells are unsuitable for such biotechnological process [119][120][121]. Growth and production of alkaloids and biomass in in vitro system often vary with source and concentration of carbohydrate (sucrose, fructose, glucose etc.), phosphorous and nitrogen. Optimization of these basal requirements in growth medium is crucial and can be achieved by using mathematical analysis to enhance production of target metabolites, increase biomass, or both. Another important factor that can regulate phytochemical profile of culture cells are phytohormones, i.e., auxins and cytokinins. Optimal ratio of phytohormones is essential and specific to maintain different stages of cultured cells, which ultimately can result in variation of phytochemicals [54,122,123]. Biosynthesis of galantamine was not detected in the in vitro culture of N. pseudonarcissus cv. Carlton in absence of phytohormone, whereas high amounts of galantamine were detected in differentiated tissues cultured in low auxin (4 mg/L of NAA) as compared to undifferentiated tissue cultured in high auxin (20 mg/L of NAA) [54]. Furthermore, in nature it was observed that plants synthesize alkaloids in response to different biotic and abiotic factors [124]. Therefore, these factors, or the associated signaling compounds produced in response to these factors, can be used as elicitors to enhance the biosynthesis of specialized metabolites in in vitro techniques [125,126]. For example, using methyl jasmonate as an elicitor in Leucojum shoot culture can enhance production of galantamine by two-fold [127]. Physical parameters further modulate production in in vitro system. Light, temperature and bioreactor types can greatly enhance the production of AAs in in vitro system. Finally, with a better understanding of the effect of the environment, and of microorganism-plant interactions, applying this knowledge to artificial culture system could boost the production of AAs and should be considered as a promising approach.
Like other specialized metabolites, AAs can be produced by heterologous expression of genes cluster that express the enzymes required for their biosynthesis. Precise and reliable construction of DNA fragments can be rapidly achieved by using modern recombination and DNA assembly techniques. Several strategies have been developed to engineer microbial hosts, such as bacteria, or yeast, for the heterologous production of alkaloids and their precursors [128][129][130][131][132][133][134]. Synthetic biology approaches for production of plant-derived specialized metabolites by metabolic engineering have been carried out primarily in yeast (Saccharomyces cerevisiae) so far and to a lesser extent in Escherichia coli; whereas tobacco species Nicotiana tabacum and Nicotiana benthamiana have emerged as hosts for the heterologous expression of biosynthetic genes and production of specialized metabolites in plants [135][136][137][138]. However, the lack of knowledge in biosynthetic pathway hinders this approach as of yet. Elucidation of the reactions will be achieved by using modern sequencing technology interrelated with metabolite studies, systems biology, and bioinformatic analysis. This will not only provide techniques to produce AAs but also help with biosynthesis of novel AAs derivatives.

Conclusions
A total of 91 new AAs have been isolated in the last 5 years, and some of their biological activities have been uncovered. AAs are a rich group of specialized metabolites with pleiotropic effects that represent an important resource for new drugs discovery. They should be deeper exploited, at the image of galantamine used to treat the symptoms of AD. Recent years of AAs research have been marked by the discovery of compounds with potent anticancer (e.g.