A Brief Up-to-Date Overview of Amaryllidaceae Alkaloids: Phytochemical Studies of Narcissus tazetta subsp. tazetta L.,Collected in Turkey

Abstract

A brief up-to-date overview on the isolation, and chemical and biological characterization of new and known alkaloids from different Amaryllidaceae species, including Brunsvigia, Crinum, Cyrtanthus, Narcissus, and Nerine genera, was reported. Furthermore, the isolation and chemical characterization of alkaloids extracted from bulbs of Narcissus tazetta subsp. tazetta L. collected from Muğla, Turkey were described.

The plants belonging to the Amaryllidaceae family are known all over the world for their use in folk medicine and as decorations in public gardens and parks due to their beautiful flowers. About 1600 species are grouped in this family and divided into about 75 genera, which are distributed throughout both tropical and subtropical regions of the world. ¹ The majority of these species are common to the Mediterranean basin, Andean South America, and southern Africa. The Amaryllidaceae plants are well-known producers of numerous alkaloids that are divided into 12 structural families ² with a wide range of biological effects, including antitumor, antiviral, antibacterial, antifungal, antimalarial, analgesic, and cytotoxic activities. ^{3 -6} Galanthamine is the first example of their practical application in medicine. It is a potent and selective inhibitor of the enzyme acetylcholinesterase and is used to treat the Alzheimer's disease. ⁶

The most abundant Amaryllidaceae alkaloid is lycorine (1, Figure 1), which belongs to the pyrrolo[de]phenanthridine subgroup. Although its biological effects have been known for many years, it is still being investigated for a variety of medicinal applications, in particular as an anticancer agent showing promising activity against tumors with dismal prognoses. ^7,8 Its anticancer activity prompted extensive structure-activity relationship studies. ⁹ The Amarylidaceae alkaloids belonging to the 5,10-ethanophenanthridine subgroup (crinine-type), such as haemanthamine (2, Figure 1), also exhibited potent in vitro anticancer activities, regardless of cancer cell sensitivity to the apoptotic cell death. ¹⁰ Bulbispermine (3, Figure 1), a crinine-type Amaryllidaceae alkaloid, was first isolated from Zephyranthes robustus and later from Crinum bulbispermum. ¹¹ Bulbispermine was used to synthesize a small group of analogs, which were tested in vitro against a panel of cancer cell lines with various levels of resistance to apoptotic cell death. It was found that the C1,C2-dicarbamate derivative of bulbispermine exhibited notable growth inhibitory properties. Bulbispermine inhibits the proliferation of glioblastoma cells through cytostatic effects, possibly arising from rigidification of the actin cytoskeleton. These results suggested that crinine-type alkaloids have a potential to be used as drug leads for the treatment of apoptosis-resistant cancers and, in particular, glioblastoma. ¹¹ Also, the multicyclic framework of haemanthamine was transformed into a novel natural product-like skeleton ¹² as well as to compounds that contain the montanine ring system. These derivatives inhibited proliferation of cancer cells resistant to apoptosis at micromolar concentrations and some of them were also active against patient-derived glioblastoma cells expressing stem-cell markers. ¹³ Furthermore, studies on haemanthamine’s mode of action involving the ribosome receptor were also recently reported. ¹⁴

Figure 1

Amaryllidaceae alkaloids belonging to different subgroups isolated from Brunsvigia, Crinum, Cyrtanthus, Narcissus, and Nerine species.

A new alkaloid, jonquailine (4, Figure 1), belonging to the pretazettine group of Amaryllidaceae alkaloids, was isolated from dried bulbs of Narcissus jonquilla (quail) collected in Middlesex county of southeast England. ¹⁵ The main alkaloids of N. jonquilla quail were galanthamine and haemanthamine. ¹⁶ Jonquailine revealed significant antiproliferative effects against glioblastoma, melanoma, uterine sarcoma, and non-small-cell lung cancer cells displaying various forms of drug resistance. When used in combination with paclitaxel, jonquailine showed a synergic antiproliferative action against drug-resistant lung cancer cells. ¹⁵ Jonquailine belongs to the ² benzopyrano[3,4 c]indole subgroup and it is the C-8 epimer of 8-O-methylpretazettine (7, Figure 1) isolated from Eucharis amazonica. ¹⁶ Noteworthily, in this subgroup, tazettine (5, Figure 1), the most abundant alkaloid, was devoid of anticancer activity. ^{17 -19} However, a structurally related pretazettine (6, Figure 1) ²⁰ has shown promising activity in numerous models of murine cancer. ²¹ It undergoes an easy conversion under mild basic conditions to the inactive tazettine. ^22,23 As recently reported, the absolute stereochemistry is important for the bioactivity of Amaryllidaceae alkaloids ⁶ and the presence of an oxygenated chiral carbon at C-8 is crucial for anticancer activity of pretazettine and jonquailine, while tazettine lacking this stereocenter is inactive. The absolute configuration of tazettine was determined by X-ray analysis of its N-methyl iodide ²⁴ and confirmed by chemical correlation ²⁵ and its total synthesis. ^26,27 As mentioned above, under mild basic conditions pretazettine was converted into tazettine, ²¹ which allowed to assign the stereochemistry at C-3, C-4a, and C-6a in pretazettine (i.e., the junction of the B, C, D tricyclic system). This was also confirmed by its total synthesis. ^28,29 Later, the relative and absolute stereochemistry at the junction between rings B and D, and C and D, as well as that at C-3 in jonquailne (4) and 8-O-methyl-pretazettine (7), were assigned by comparing their nuclear magnetic resonance and electronic circular dichroism (ECD) spectra with those of pretazettine (6). Finally, the R absolute configuration at C-8 of jonquailine was only recently assigned by chiroptical (optical rotatory dispersion, ECD, and vibrational circular dichroism) and computational methods and thus also the S one at C-8 of pretazettine and its 8-O-methyl analog. ³⁰

As the search for new alkaloids often leads to new bioactive natural products, a recent screening was carried out by some of the authors, on the alkaloids produced by indigenous South African Amaryllidaceae plants, which are largely unexplored. The studies, performed on Nerine sarniesis, Crinum buphanoides, Crinum graminicola, Cyrtanthus mackenii, and Brunsvigia grandiflora, led to the isolation of lycorine as one metabolite produced by all the species studied. Furthermore, 2 mesembrine-type alkaloids, named sarniensine and sarniensinol (8 and 9, Figure 1), and a new crinine-type alkaloid, named crinsarnine (10, Figure 1), were isolated together with lycorine (1), tazettine (5), bowdensine, hippadine, and 1-O-acetyl-lycorine (11, 12 and 14, Figure 1) from the dried bulbs of N. sarniensis. ^31,32 The alkaloids isolated were evaluated against the Aedes aegypti, which is the major vector for dengue, yellow fever, and the Zika viruses. None of the compounds showed mortality against first instar A. aegypti larvae at the concentrations tested. In adult topical bioassays, only crinsarnine (10) displayed adulticidal activity with an LD₅₀ = 2.29 ± 0.05 mg/mosquito. As regards the structure–activity relationship, the pretazettine- and crinine-type compounds had produced different results. Among the pretazettine group compounds, 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 appear to be important for activity, while in crinine-type alkaloids, the substituent at C-2 seems to play a role. ^31,32

C. graminicola produced lycorine in the best yield (2.1 g/kg). Moreover, 1-O-acetyl-(14) and 2-O-acetyl-lycorine (15, Figure 1), pratorimine (16, Figure 1), hippadine (12), and tazettine (5) were isolated from C. buphanoide. Haemanthamine (2), haemanthidine, and criwelline (17 and 18, Figure 1) were isolated from C. graminicola, while tazettine and 11-hydroxyvittatine (19, Figure 1) were produced by C. mackenii. Finally, crinamine (20, Figure 1) and 11-hydroxyvittatine were isolated from B. grandiflora. This is the first report on the alkaloids from these 4 South African Amaryllidaceae. ²⁸

Among the Narcissus genus, Narcissus tazetta subsp. tazetta is a widely distributed bulbous plant known for its biologically active alkaloids. ²⁹ The isolation and chemical characterization of alkaloids extracted from bulbs of this Amaryllidaceae species collected from Muğla/Turkey have been reported in this article for the first time. Phytochemical studies on N. tazetta subsp. tazetta resulted in the isolation of 4 Amaryllidaceae alkaloids including lycorine (1, Figure 1), pseudolycorine, galanthamine, and 11-hydroxygalanthine (21 -23, Figure 2). The isolated compounds, excluding galanthamine, belong to lycorine subgroup of the Amaryllidaceae alkaloids. ³³ Galanthamine is an Amaryllidaceae alkaloid with acetylcholinesterase inhibitory activity used in the treatment of mild-to-moderate Alzheimer’s disease as mentioned earlier. ³⁴ In particular, the ethanolic extract of N. tazetta subsp. tazetta L. was acidified using 2% HCl and then basified using ammonia. This aqueous solution was extracted with CHCl₃ yielding 4 alkaloids, identified as lycorine, pseudolycorine, galanthamine, and 11-hydroxygalanthine, by comparing their spectroscopic and optical rotation data with those reported in the literature. ^{35 -41}

Figure 2

Pseudolycorine, galanthamine, and 11-hydroxygalanthine isolated from Narcissus tazetta subsp. tazetta.

Previously, a limited number of alkaloids including lycorine have been isolated from a different specimen of N. tazetta subsp. tazetta L. collected from South Turkey. ²⁹ According to that report, haemanthamine, tazettine, lycorine, N-norgalanthamine, and 3-epi-hydroxybulbispermine were isolated from the subspecies growing in Antalya province, which has more humidity and higher air temperature comparing to Mugla. Additionally another isolation study was carried out on the subspecies provided from an export firm. ⁴² Buphanisine, 9-O-demethylhomolycorine, haemanthamine, galanthamine, tazettine, and 3-epihydroxy-bulbispermine were reported from this plant. Also, previous reports point to isolation of plenty of alkaloids with various skeleton types from N. tazetta L. ^43,44 Apart from Turkey, alkaloidal profile of N. tazetta from different Mediterranean countries such as Egypt and Iran have been previously investigated. ^{45 -48} Lycorine, pseudolycorine, tazettine, pretazettine, galanthamine, haemanthamine, 9-O-demethyl-2-α-hydroxyhomolycorine, homo-lycorine, and ismine were isolated from the plant grown in those countries. To the best of our knowledge, this is the first report of pseudolycorine, 11-hydroxygalanthine, and galanthamine from this Amaryllidaceae subspecies naturally growing in Turkey. A report on the Amarylladaceae species growing in Turkey has been provided by Baytop and Mathew. ⁴⁹ In addition, to the biological activities already reported for lycorine and galanthamine, for the other 2 alkaloids isolated from N. tazetta subsp. tazetta, only for pseudolycorine (21) have been reported some interesting activities while for 11-hydroxygalanthine (23) no biological activity was described during its first isolation from Narcissus serotinus. ³⁶ In particular, 21 exhibited cytotoxic activity when tested on 5 human cancer cell lines and 1 murine mouse cancer cell together with other 22 Amaryllidaceae alkaloids belonging to different subgroups. ¹⁰ Previously it was also reported that pseudolycorine inhibited protein synthesis and manifested activity against murine Rauscher leukemia virus and neurotropic RNA viruses, ⁵⁰ including herpes simplex virus. ⁵¹

Experimental

Plant Material

The bulbs of Narcissus tazetta subsp. tazetta were collected from Akçapınar/Muğla during flowering period and voucher specimens of the plant (No:1596) have been deposited in the Herbarium of the Ege University, Faculty of Pharmacy, Department of Pharmacognosy, Izmir, Turkey.

Extraction and Isolation

The organic extract of N. tazetta subsp. tazetta L. dried bulbs was obtained following the procedure described previously. ⁵² Briefly, fresh leaves and bulbs (~60 kg) were minced, air-dried, and pulverized. This powdered plant material (10.5 kg) was macerated with 95% EtOH for 48 hours and the solvent was evaporated in vacuo. The residue was macerated with 2% HCl (9 × 350 mL per liter) overnight at 20°C, filtered through filter paper and basified to pH 8 with NH₄OH. The aqueous solution was extracted with CHCl₃ (8 × 500 mL per liter ), the organic extracts were combined, dried (Na₂SO₄), and evaporated under vacuum to afford a crude alkaloid extract (26.45 g). The latter was fractionated by column chromatography (110 cm × 7 cm) using silica gel 60 (70-230 mesh) eluted with a gradient system of CHCl₃ and MeOH (0-100% MeOH). Fractions were collected, checked by thin-layer chromatography (TLC), exposed to iodine vapor, and the homogeneous fractions were combined. The residue of fractions 51 to 53 (275.1 mg) was further purified by 2 sequential column chromatography (CC) using EtOAc:CHCl₃:Me₂O:MeOH:NH₄OH (6:2:1.5:0.5:0.1) and EtOAc:CHCl₃:MeOH:MeCN (5:2.5:2:0.5) as solvent systems, respectively, to afford an homogeneous compound, identified as galanthamine (22, 7 mg). The residue of 58 to 61 (607 mg) was purified by CC, using C₆H₆:CHCl₃:MeOH:NH₄OH (7:2:1:1) and EtOAc:CHCl₃:MeOH:NH₄OH (6:2:2:2) as eluent, yielding a fraction that was crystallized from Et₂O. The crystallized compound was identified as lycorine (1, 38 mg). The residue of fractions 81 to 83 (618 mg) of the original column was further purified by CC, eluted with C₆H₆:CHCl₃:MeOH:NH₄OH (6.25:3:0.75:0.1), and then by preparative TLC, eluted with EtOAc:CHCl₃:MeOH:NH₄OH (6:2:2:2), to afford another homogeneous compound identified as 11-hydroxygalanthine (23, 10 mg). Finally, from the residue (5.36 g) of 84 to 99 a homogeneous compound (12.5 mg) was obtained by precipitation in CHCl₃ and was identified as pseudolycorine (21).

Supplemental Material

Supplementary data - Supplemental material for A Brief Up-to-Date Overview of Amaryllidaceae Alkaloids: Phytochemical Studies of Narcissus tazetta subsp. tazetta L., Collected in Turkey

Supplemental material, Supplementary data, for A Brief Up-to-Date Overview of Amaryllidaceae Alkaloids: Phytochemical Studies of Narcissus tazetta subsp. tazetta L., Collected in Turkey by Çiğdem Karakoyun, Marco Masi, Alessio Cimmino, Mustafa Ali Önür, Nehir Unver Somer, Alexander Kornienko, and Antonio Evidente in Natural Product Communications