Advances on the Amaryllidacea Alkaloids Collected in South Africa, Andean South America and the Mediterranean Basin

The alkaloids are one of the most represented family of natural occurring biological active compounds. Amaryllidaceae are also very well known for their beautiful flower and are thus used as ornamental plants in historic and public gardens. The Amaryllidacea alkaloids constitute an important group that is subdivided into different subfamilies with different carbon skeletons. They are well known from ancient times for their long application in folk medicine, and in particular, Narcissus poeticus L. was known to Hippocrates of Cos (ca. B.C. 460–370), who treated uterine tumors with a formulate prepared from narcissus oil. To date, more than 600 alkaloids of 15 chemical groups exhibiting various biological activities have been isolated from the Amaryllidaceae plants. This plant genus is diffused in regions of Southern Africa, Andean South America and the Mediterranean basin. Thus, this review describes the chemical and biological activity of the alkaloids collected in these regions in the last two decades as weel those of isocarbostyls isolated from Amaryllidaceae in the same regions and same period.


Introduction
Amaryllidaceae plants grow as wild species in several countries and are also cultivated for their beautiful flowers and for the production of volatile oils ( Figure 1). They are classified into 60 genera [1] that are diffused in different world regions but that are dominant within three distinct geographical locations, i.e., Andean South America, Southern Africa and the Mediterranean basin [2,3]. About one third of one thousand Amaryllidaceae species grow in South Africa and are commonly used in folk medicine [2]. They include well-known ornamental varieties such as daffodils (Narcissus), snowdrops (Galanthus) and snowflakes (Leucojum) [3]. Consequently, they have a high commercial value and are important for the floriculture industry [4,5]. The use of Narcissus in the Mediterranean basin begins at the time of Hippocrates and Pliny [6], while in South America in the archeological Inca ruins, there were found floral depictions of Ismene, Pyolirion and Stenomesson [7]. Paintings of Brunsvigia species were found in Lesotho [8].
Studies on Amaryllidaceae alkaloids (AA) began in 1877 with lycorine (1, Figure 2), which is the main Amaryllidaceae alkaloid that was isolated from Narcissus pseudonarcissus [9]. From that time, investigations of this group of alkaloids increased considering their wide range of biological activities. These include antitumor, antiviral, antibacterial, antifungal, antimalarial, analgesic, and cytotoxic activities [10]. The most important application in medicine of Amaryllidaceae alkaloids is represented by the use of galanthamine (2, Figure 2) to treat Alzheimer's disease and is already commercialized as a drug. Galanthamine (2) is able to selectively inhibit the enzyme acetylcholinesterase (AChE), which plays a fundamental role in the disease [1,11]. Amaryllidaceae plants also synthesize poisons such as lycorine and galanthamine, and this toxicity should be always considered [12]. Studies on Amaryllidaceae alkaloids (AA) began in 1877 with lycorine (1, Figure 2), which is the main Amaryllidaceae alkaloid that was isolated from Narcissus pseudonarcissus [9]. From that time, investigations of this group of alkaloids increased considering their wide range of biological activities. These include antitumor, antiviral, antibacterial, antifungal, antimalarial, analgesic, and cytotoxic activities [10]. The most important application in medicine of Amaryllidaceae alkaloids is represented by the use of galanthamine (2, Figure 2) to treat Alzheimer's disease and is already commercialized as a drug. Galanthamine (2) is able to selectively inhibit the enzyme acetylcholinesterase (AChE), which plays a fundamental role in the disease [1,11]. Amaryllidaceae plants also synthesize poisons such as lycorine and galanthamine, and this toxicity should be always considered [12]. Hundreds of scientific articles have reported on the biosynthesis, source, isolation and biological activities of AA, which are grouped into more than 12 subfamilies considering their carbon skeleton, including norbelladine, rystilline, α-crinanes, β-crinanes, lycorane, galanthamine, pretazettine, homolycorine, montanine, cherylline, crinasiadine, clivimine, ismine analogues and miscellaneous alkaloids [1,6].
Considering the number of scientific articles describing the different aspects of AA, several articles were published on this topic regarding the phytochemical, biological and pharmacological properties of Crinum bulbispermum [13] Nerine [14], Haemantheae [15] and Crinum, Ammocharis, Amaryllis and Cyrtanthus [16]. The relation between the biological activity of AA and their absolute configuration was also extensively described [17] as well as the acetylcholinesterase inhibition of extracts of Amaryllidaceae collected in South Africa with potential for Alzheimer's disease treatment [18]. Recently, only AA isolated in the decade 2009-2020 were reported [19].
This review reports on the plant sources, isolation, and chemical and biological characterization of alkaloids produced by Amaryllideceae that are native to Andean South Hundreds of scientific articles have reported on the biosynthesis, source, isolation and biological activities of AA, which are grouped into more than 12 subfamilies considering their carbon skeleton, including norbelladine, rystilline, α-crinanes, β-
Lycorine (1), which is the main alkaloid, as well as dehydroanhydrolycorine (20) and 1-O-acetyllycorine (35, Figure 2) were identified in the leaves and bulbs of Crinum amabile, Crinum erubescens, Crinum moorei, Amaryllis belladonna and Zephyranthes carinata, which are all Amaryllidaceae species collected in different regions of Merida State-Venezuela [34]. In addition, buphanisine (36, Figure 3) in the C. amabile and C. moorei bulb extracts and undulatine (33) in the bulb extract of A. belladonna were found [34]. The extract of C. amabile leaves showed the strongest inhibition of AChE and BuChE followed by C. erubescens leaves [34].
A. belladonna has a geographical distribution covering mainly southern Africa [40], where it has significant usage in the traditional medicine of the native people but which was also isolated in Brazil [40]. The chemical analysis of the bulb organic extract of the samples collected in Brazil helped to identify twenty-six different AA, and three of them, namely, 1-O-acetylcaranine (44, Figure 2), buphanamine, and 3-O-acetylhamayne (45 and 46, Figure 3) were isolated [40]. The AA and the crude bulb organic extracts were tested against four different parasitic protozoa (Trypanosoma cruzi, Trypanosoma brucei rhodesiense, Leishmania donovani, and Plasmodium falciparum [40]). The crude organic extract and 3-Oacetylhamayne exhibited good antiprotozoal activity in vitro, although both had a high cytotoxic index [40].

Alkaloids Isolated from Amaryllidacae Plants Collected in South Africa
Lycorine, galanthamine, tazettine haemanthamine, homolycorine, ismine, trisphaeridine, undulatine, buphanisine, and crinine montanine (1-3, 5, 10, 12, 13, 33, 36, 39 and 40) and buphanidrine, ambelline, norbelladine, augustine, disticahamine, distichaminol, crinamine, haemanthidine, and buphanamine (52-60, Figure 4) were isolated together with pancratistatine, an isocarbostyryl (see below Section 5), from Boophone haemanthoides [44]. The bulbs of this Amaryllidacea were collected during the flowering season in the Nieuwoudtville area of the Northern Cape Province of South Africa [44]. B. haemanthoides belong to the African genus Boophone Herb, which also includes Boophone disticha. B. disticha is widely distributed in Africa, ranging from Sudan to the Western Cape Province, while B. haemanthoides is a rare and endangered species found in a more limited range in the winter rainy region of South Africa, which is confined to a relatively small area in the southwest, the Western Cape area, where gentle rain falls from May to August, but the summers are dry, and in parts of southern Namibia [45]. Both Amaryllidaceae are widely used in folk medicine [46,47]. Among the alkaloids identified, lycorine (1) and distichamine (56) showed cytotoxic activity demonstrated in acute lymphoblastic leukemia (CEM), breast adenocarcinoma (MCF7) and cervical adenocarcinoma (HeLa) cells with IC 50 s in the range from 1.8 to 9.2 µM [44]. Previously from B. heamanthoides, collected in Saldhana Bay area in South Africa, also buphanisine crinine and buaphanidrine (36, 38 and 52) were isolated together with distichamine (56) [48].  Nineteen AA belonging to different subgroups were isolated from Clivia miniata, which is an herbaceous evergreen plant endemic to South Africa and Swaziland [76]. The alkaloids were identified as lycorine, galanthamine, tazettine, vittatine, haemanthamine 11-hydroxyvittatine, sternbergine, hippeastrine, 1-O-acetylcaranine, haemanthidine, Crinsarnine and sarniensinol (61 and 62, Figure 4), belonging, respectively, to the crinine and mesembrine-types subgroups, were isolated together with bowdensine, sarniensine (63 and 64, Figure 4), and 1-O-acetyl-lycorine (35), from the dried bulbs of Nerine sarniensis [49], a species restricted to the Western Cape of the South Africa region [2]. All the alkaloids were assayed together with tazettine and 3-epi-macronine (3 and 21) against the Orlando reference strain of Aedes aegypti, which is the primary vector of dengue and yellow fever and Zika viruses [49]. The latter causes microcephaly and other serious brain anomalies during pregnancy, and it could easily become a potential threat to international public health safety [50]. Mosquito control is one of the main methods used to reduce the spread of these viruses. None of the compounds tested showed mortality against the first instar Ae. aegypti larvae at the concentrations tested. In adult topical bioassays, only crinsarnine (61) exhibited adulticidal activity with an LD 50 value of 2.29 ± 0.049 µg/mosquito [49]. Regarding the structure-activity relationship, the pretazettine and crinine scaffold in alkaloids 62 and 64 and in 61 and 63, respectively, proved to be important for their activity, while the pyrrole[de]phenanthridine scaffold present in alkaloid 35 appeared to not be important for toxicity [49]. Among the pretazettine group compounds, 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 are important features for activity, while in crinine-type alkaloids, the substituent at C-2 seems to play a role in their toxicity [49].
Channaine (70, Figure 4), which is an AA with an unusual cage-like ring structure at the interface of two aryl-hydroindole subunits, was isolated from Sceletium tortuosum [64]. This species was collected from St. Helena in the Western Cape Province of South Africa and belongs to the Sceletium genus, which is endemic to South Africa and which is a well-known producer of alkaloids [65,66]. Alkaloid 70 was previously isolated from the same Amaryllidaceae [67], but only its empirical formula and some functional groups were assigned. Later, Popelak and Lettenbauer [68] described that channaine (66) contained two veratrole rings, being a dimer of two subunits and racemic of both channaine compounds.
Amaryllidacea species in folk medicine in South Africa [76]. All the isolated alkaloids were tested for their AChE/BuChE inhibition, using galanthamine and eserine as reference compounds. Alkaloids belong to the homolycorine structure type as clivimine, cliniatine C, clivonine, nobilisitine B and clivimine B, which show low AChE/BUChE inhibitory activity. Among the lycorine-type alkaloids such as lycorine, sternbergine, 1-Oacetylcaranine, and caranine was noted that the activity could be due to the presence of free hydroxyl groups in positions C1 and C2, which are not present in those of the homolycorine type. This diol system is probably a functional group that improves binding in the active site of AChE/BuChE. However, lycorine and several of its analogues did not show significant activity against AChE and BuChE [76]. The 'lily borer' moth Brithys crini is a very dangerous parasite of Amaryllidaceae plants, having a great effect during the larval stage. The organic extract of Crinum moorei, collected in the botanical garden of the University of KwaZulu-Natal, was analyzed, and alkaloids belonging to different subgroups of AA were identified as ambelline (53) and cherylline (74, Figure 5). The presence of ambelline represents a surprise, as it was not previously isolated in the organic extract obtained from this Amaryllidaceae [70].
Nineteen AA belonging to different subgroups were isolated from Clivia miniata, which is an herbaceous evergreen plant endemic to South Africa and Swaziland [76]. The alkaloids were identified as lycorine, galanthamine, tazettine, vittatine, haemanthamine 11-hydroxyvittatine, sternbergine, hippeastrine, 1-O-acetylcaranine, haemanthidine, caranine, (1-5, 8, 42-44, 59 and 75) Figure 4, and 89, Figure 4) [76]. The main alkaloids were lycorine, haemanthamine, and clivimine (1, 5 and 82). C. miniata is the most used Amaryllidacea species in folk medicine in South Africa [76]. All the isolated alkaloids were tested for their AChE/BuChE inhibition, using galanthamine and eserine as reference compounds. Alkaloids belong to the homolycorine structure type as clivimine, cliniatine C, clivonine, nobilisitine B and clivimine B, which show low AChE/BUChE inhibitory activity. Among the lycorine-type alkaloids such as lycorine, sternbergine, 1-O-acetylcaranine, and caranine was noted that the activity could be due to the presence of free hydroxyl groups in positions C1 and C2, which are not present in those of the homolycorine type. This diol system is probably a functional group that improves binding in the active site of AChE/BuChE. However, lycorine and several of its analogues did not show significant activity against AChE and BuChE [76].

Alkaloids Isolated from Amaryllidacae Plants Collected in Mediterranean Basin
Ungeremine and zefbetaine (90 and 91, Figure 6), which are two 2-oxyphenanthridinium alkaloids, were isolated from the bulb organic extract of Egyptian Pancratium maritimum collected from sandy hills on the northern coast during the flowering and fruit-producing stages [77]. From the same organic extract, lycorine, galanthamine, tazettine, haemanthamine, 11-hydroxyvittatine, homolycorine, trispheridine, pancracine, 9-O-demethylhomolycorine and haemanthidine, (1-3, 5, 8, 10, 13, 41, 50 and 59), and lycorenine (92, Figure 6) were isolated and identified [78,79]. Ungeremine showed toxicity against Flavobacterium columnare and Edwardsiella ictaluri, respectively, with IC 50 and MIC values of 58 ± 0 and 3 ± 0 and (0.8 ± 0 and 0.9 ± 0.2 mg/L [80]. This activity was compared to that of lycorine (1) and pseudolycorine (93, Figure 6), isolated from Narcisuss tazetta subsp. tazetta [81], ungeremine isomer, zefbetaine (90), and anhdrolycorine (17). The aromatization of the C ring and the oxidation to an azomethine group of C-7 of the B ring are structural features important for antibacterial activity [82]. Furthermore, the presence of the 1,3-dioxole ring joined to the A ring and oxygen location of the C ring of the pyrrolo[de]phenanthridine skeleton also play significant roles on the antibacterial activity [82]. Ungeremine (90) showed to be a promising biofungicide against Penicillium roqueforti and Aspergillus niger with MIC90 values of 0.003 and 0.2 mg/mL [83]. These two fungi are very dangerous food contaminants and can cause bakery product deterioration. They show significant potential to be included as an appropriate biofilm that can be used in intelligent food packaging [83]. Alkaloid 90 was incorporated in chitosan-based microbeads, which were prepared by external gelation by using sodium tripolyphosphate (TPP) as a crosslinking agent. All the microbeads evidenced antimicrobial activity against P. roqueforti [84]. These microbeads were included in a thermoplastic starch-based polymer Mater-Bi (MBi), and MBi/CTUn and bioactive biocomposites were obtained. The films showed bioactivity against P. roqueforti [85].
Jonquailine (128, Figure 8), an alkaloid belonging to the pretazettine group, was isolated from dried bulbs of Narcissus jonquilla quail, widespread in Spain and Portugal [96]. Alkaloid 128 showed significant antiproliferative effects against glioblastoma, melanoma, uterine sarcoma and non-small-cell lung cancer cells, which exhibit various forms of drug resistance, including resistance to apoptosis and multi-drug resistance [97]. Jonquailine (128) was able to synergize, with paclitaxel, its antiproliferative action against drug-resistant lung cancer cells [97]. The hydroxylation at C-8 is an important feature for anticancer activity, but this seems to be affected by both the stereochemistry and acetalization of lactol [97].
Lycorine and 8-O-demethylmaritidine (1 and 89) were isolated together with clivatine and nobilisine (129 and 130, Figure 8) from the flower extract of Clivia nobilis, cultivated in Egypt [98]. The crude flower organic extract as well as all the alkaloids isolated were tested for their antibiotic activity against Gram-positive Staphylococcus aureus and Gramnegative Pseudomonas aeroginosea bacteria. The crude flower extract showed antibacterial activity against both microorganisms, while nobilisine (130) showed very good activity against the Gram-negative P. aeroginosea [98].
compound, as well as lycorine (1). L. aestivum is a salt-tolerant plant, and treatment with 4 g/L of CaCl2 increased the amount of galanthamine and the antioxidant activities [107]. Bulbs collected in six different locations in Turkey (Gölcük-Bolu, Yeniçağa-Bolu, Kaynarca-Sakarya, Delmece-Yalova, Uluabat-Bursa and Terkos-Istanbul) at three different growing periods showed that genetic factor is important in alkaloid biosynthesis in the bulbs collected in Gölcük-Bolu, which showed to be the most productive in both alkaloids 1 and 2. The same alkaloids were obtained in abundant amounts from both the bulbs and leaves collected in Delmece-Yalova. The vegetative period followed by ripening are the two periods in which the amount of both alkaloids is high [108].
Lycorine (1) was isolated from the bulb extract of Pancratium foetidum, collected in Saïdia, Oujda region, Morocco [102]. Lycorine showed moderate antibacterial activity and had more efficacy than streptomycin and ampicillin against P. aeruginosa. A virtual docking ligand-lycorine protein screening showed that compound 1 can interact with target amino residues studied by hydrogen and metal-ion bonds [102].
Leucojum aestivum, commonly named summer snowflake, is a bulbous plant in the Euro-Mediterranean region and is a well-known source of pharmacologically important alkaloids. Among all the alkaloids produced, galanthamine (2) is the major bioactive compound, as well as lycorine (1). L. aestivum is a salt-tolerant plant, and treatment with 4 g/L of CaCl 2 increased the amount of galanthamine and the antioxidant activities [107]. Bulbs collected in six different locations in Turkey (Gölcük-Bolu, Yeniçaga-Bolu, Kaynarca-Sakarya, Delmece-Yalova, Uluabat-Bursa and Terkos-Istanbul) at three different growing periods showed that genetic factor is important in alkaloid biosynthesis in the bulbs collected in Gölcük-Bolu, which showed to be the most productive in both alkaloids 1 and 2. The same alkaloids were obtained in abundant amounts from both the bulbs and leaves collected in Delmece-Yalova. The vegetative period followed by ripening are the two periods in which the amount of both alkaloids is high [108].

Conclusions
This review describes the chemical and biological properties of alkaloids isolated in the last two decades from different Amaryllidaceae species. The different sections (Sections 2-4) report, in detail, the characterization of alkaloids isolated from Amaryllidaceae species diffused in regions of Southern Africa, Andean South America Among isocarbostyryls, narciclasine and pancratistatin (152 and 153, Figure 10) are amide analogues of lycorine that are very well known for they anticancer activity [6,[110][111][112]. Narciclasine (152) was isolated together with haemamthamine (5) from the bulbs of Narcissus pseudonarcissus [113]. Previously, isocarbostyryl 152 was also isolated from Sternbergia lutea, its new process of extraction and purification was optimized, and its NMR spectroscopic full data were assigned [114]. Pancratistatin was extracted for the first time from Hymenocallis littoralis bulbs [115], and it was also isolated from B. haemanthoides bulbs, as reported above in Section 2 [1].

Data Availability Statement:
All the data reported in this review were based on SciFinder research using appropriate key words.