Amaryllidaceae Alkaloids of Different Structural Types from Narcissus L. cv. Professor Einstein and Their Cytotoxic Activity

In this detailed phytochemical study of Narcissus cv. Professor Einstein, we isolated 23 previously known Amaryllidaceae alkaloids (1–23) of several structural types and one previously undescribed alkaloid, 7-oxonorpluviine. The chemical structures were identified by various spectroscopic methods (GC-MS, LC-MS, 1D, and 2D NMR spectroscopy) and were compared with literature data. Alkaloids which had not previously been isolated and studied for cytotoxicity before and which were obtained in sufficient amounts were assayed for their cytotoxic activity on a panel of human cancer cell lines of different histotype. Above that, MRC-5 human fibroblasts were used as a control noncancerous cell line to determine the general toxicity of the tested compounds. The cytotoxicity of the tested alkaloids was evaluated using the WST-1 metabolic activity assay. The growth of all studied cancer cell lines was inhibited by pancracine (montanine-type alkaloid), with IC50 values which were in the range of 2.20 to 5.15 µM.


Introduction
Plants from the Amaryllidaceae family have been used for centuries in folk medicine due to their therapeutic properties [1]. Plants from this family contain a distinct and still not fully explored group of alkaloids called Amaryllidaceae alkaloids (AA). They are most well known for their broad spectrum of biological properties such as antitumor [2,3], antimalarial [4], anti-inflammatory [5], antimicrobial [6], and AChE-inhibiting activities [7,8]. The AA are classified into 9 main structural types (crinine, galanthamine, haemanthamine, homolycorine, lycorine, montanine, narciclasine, norbelladine, and tazettine) alongside more than 10 others (plicamine, galanthindole, augustamine, graciline, etc.). These minor structure types are usually found in trace amounts and are most often represented by single alkaloids [9,10]. Galanthamine, a selective, competitive acetylcholinesterase inhibitor with antioxidant properties is the most important AA identified so far. It was isolated for the first time from Galanthus woronowii in the 1950s. Since 2000, when it was approved, it has been used to treat mild
The structure of compound 21 is already known, but confirmation of it was not possible due to the absence of its NMR data in the available literature. This led us to publish the complete 1 H and 13 C NMR spectra for this alkaloid. The structural constitution was elucidated utilizing 2D experiments such as gCOSY, gHSQC, and gHMBCAD (see Figure 2).   Table 1). While two protons were not detected by NMR in methanol-d4, they should have been bound to the heteroatoms. The 13 C NMR spectrum exhibited 16 carbons. A 1,2,4,5-tetrasubstituted benzene ring was determined by gHMBCAD experiment. The identification was based on correlations from H-11 to C-7a (δC 117.4) and C-9 (δC 148.1), and from H-8 to C-10 (δC 154.5) and C-11a (δC 136.5), respectively. The deshielded aromatic carbons were substituted by a hydroxyl group (C-9) and a methoxy group (C-10). The carbonyl group C-7 (δC 167.6) was identified in the 13 C NMR spectrum; this signal was shown to be correlated with H-8 in gHMBCAD ( Figure 2). The tertiary carbon C-11b (δC 41.9), which was correlated to H-11, was determined as a junction point between the aromatic substructural fragment and the rest of the molecule. The gCOSY experiment of 24 revealed cross-peaks of H-11b with H-11c and H-1, H-2 with H-1, H-3 with H-2 and H-11c, and H-5 with H-4. These correlations determined the hexahydroindole   Table 1). While two protons were not detected by NMR in methanol-d 4 , they should have been bound to the heteroatoms. The 13 C NMR spectrum exhibited 16 carbons. A 1,2,4,5-tetrasubstituted benzene ring was determined by gHMBCAD experiment. The identification was based on correlations from H-11 to C-7a (δ C 117.4) and C-9 (δ C 148.1), and from H-8 to C-10 (δ C 154.5) and C-11a (δ C 136.5), respectively. The deshielded aromatic carbons were substituted by a hydroxyl group (C-9) and a methoxy group (C-10). The carbonyl group C-7 (δ C 167.6) was identified in the 13 C NMR spectrum; this signal was shown to be correlated with H-8 in gHMBCAD ( Figure 2). The tertiary carbon C-11b (δ C 41.9), which was correlated to H-11, was determined as a junction point between the aromatic substructural fragment and the rest of the molecule. The gCOSY experiment of 24 revealed cross-peaks of H-11b with H-11c and H-1, H-2 with H-1, H-3 with H-2 and H-11c, and H-5 with H-4. These correlations determined the hexahydroindole fragment in the structure ( Figure 2). In addition, the gHMBCAD experiment supported this assumption by correlations from H-1 to C-3 (δ C 118.3), from H-1 to C-11c (δ C 60.9), from H-1 to C-11a (δ C 137.7), and from H-5 to C-11c. The absolute configuration could not be determined due to insufficient amount of sample. The relative configuration was established and the result was supported by NOESY experiment (see Figure 2). This suggested configuration of either 1R, 11bS, 11cS or 1S, 11bR, 11cR is supported by the cross-peaks of H-11b and H-1. Moreover, no correlation of these protons with H-11c was detected.
Plants 2020, 9, x FOR PEER REVIEW 4 of 12 fragment in the structure ( Figure 2). In addition, the gHMBCAD experiment supported this assumption by correlations from H-1 to C-3 (δC 118.3), from H-1 to C-11c (δC 60.9), from H-1 to C-11a (δC 137.7), and from H-5 to C-11c. The absolute configuration could not be determined due to insufficient amount of sample. The relative configuration was established and the result was supported by NOESY experiment (see Figure 2). This suggested configuration of either 1R, 11bS, 11cS or 1S, 11bR, 11cR is supported by the cross-peaks of H-11b and H-1. Moreover, no correlation of these protons with H-11c was detected.

Cytotoxic Study of Isolated Amaryllidaceae Alkaloids
A set of cell lines derived from various human tumor histotypes (leukemia-Jurkat, MOLT-4; adenocarcinomas of lung-A549, colon-HT-29, pancreas-PANC-1, cervix-HeLa, breast-MCF-7; ovarian carcinoma-A2780, and osteogenic sarcoma-SAOS-2) was used to screen the cytotoxic activity of all isolated compounds which had not been previously studied and were obtained in sufficient quantities. Normal human fetal lung fibroblasts (MRC-5) were used to study effect on non-tumorigenic cells. Growth-inhibitory activity of tested alkaloids was determined at a dose of 10 µM (Table 2) using the WST-1 mitochondrial dehydrogenase activity assay. Table 2. Cytotoxic activity of alkaloidal extract of N. cv. Professor Einstein (concentration 50 µg·mL −1 ), and isolated Amaryllidaceae alkaloids 1, 2, 7, 13, 16, 20, 21, 22, and 23 using a single dose (concentration of 10 µM). WST-1 metabolic activity assay was used to analyze cell growth 48 h after treatment. The results are expressed as the mean values ± SD of at least three independent experiments (n = 3). Cells treated with 1 µM doxorubicin served as a positive control. The efficacy of each alkaloid in decreasing the growth of individual cell line, referred as the mean growth percentage (GP), was calculated as an average of all cell lines proliferation in percent. The threshold GP value for this screen was <50% (50% tumor growth inhibition), meaning good activity at 10 µM, as can be seen in the result for pancracine (22) in Table 2. Pancracine was able to inhibit all cancer cells used in the study, with the exception of PANC-1, where IC 50 values ranged from 2.20 to 5.15 µM (Table 3). Pancracine (22) is a montanine type of AA, in the same group as montanine, coccinine, and manthine. This structural type of AA seems to be a promising souce of compounds in the search for new anticancer drugs. They are characterized by a 5,11-methanomorphantridine ring system, the only differencies being the substitutions and configuration at C-2 and C-3 centers (Figure 3) [40]. Some of these compounds have been screened against different cancerous cells [12,41].  Montanine and manthine showed strong in vitro growth inhibitory effect on three apoptosisresistant cancer cell lines (A549, SKMEL-29, and U373) and three apoptosis-sensitive cancer cell lines (MCF7, Hs683, and B16F10) with IC50 values between 5 and 31 µM [12]. In another recent study, C-2α-/C-2β-methoxy isomers montanine and coccinine were found to significantly affect the proliferation of human breast, colon, lung, and melanoma cancer cell lines over 48 h of treatment. The obtained results revealed that montanine has a more promising cytotoxic activity (IC50 values were 1.9 ± 0.4 µM for A549 cells, 6.8 ± 0.5 µM for HCT-15 cells, 23.2 ± 1.9 µM for SK-MEL-28 cells, 4.4 ± 0.4 µM for MCF-7 cells, and 3.4 ± 0.9 µM for MDA-MB-231 cells, 3.6 ± 1.7 µM for Hs578T cells) when compared with coccinine (IC50 values were 5.9 ± 0.8 µM for A549 cells, 16.8 ± 1.8 µM for HCT-15 cells, >50 µM for SK-MEL-28 cells, 7.9 ± 0.9 µM for MCF-7 cells, 13.8 ± 0.8 µM for MDA-MB-231 cells, and 5.3 ± 0.4 µM for Hs578T cells) [42]. Although previous studies, as well as our work, have demonstrated that AA of the montanine type can effectively suppress viability and proliferation of human cancer cells, the molecular mechanism of this cytotoxic activity has not yet been fully explored and is still waiting to be described. Montanine and manthine showed strong in vitro growth inhibitory effect on three apoptosis-resistant cancer cell lines (A549, SKMEL-29, and U373) and three apoptosis-sensitive cancer cell lines (MCF7, Hs683, and B16F10) with IC 50 values between 5 and 31 µM [12]. In another recent study, C-2α-/C-2β-methoxy isomers montanine and coccinine were found to significantly affect the proliferation of human breast, colon, lung, and melanoma cancer cell lines over 48 h of treatment. The obtained results revealed that montanine has a more promising cytotoxic activity (IC 50 values were 1.9 ± 0.4 µM for A549 cells, 6.8 ± 0.5 µM for HCT-15 cells, 23.2 ± 1.9 µM for SK-MEL-28 cells, 4.4 ± 0.4 µM for MCF-7 cells, and 3.4 ± 0.9 µM for MDA-MB-231 cells, 3.6 ± 1.7 µM for Hs578T cells) when compared with coccinine (IC 50 values were 5.9 ± 0.8 µM for A549 cells, 16.8 ± 1.8 µM for HCT-15 cells, >50 µM for SK-MEL-28 cells, 7.9 ± 0.9 µM for MCF-7 cells, 13.8 ± 0.8 µM for MDA-MB-231 cells, and 5.3 ± 0.4 µM for Hs578T cells) [42]. Although previous studies, as well as our work, have demonstrated that AA of the montanine type can effectively suppress viability and proliferation of human cancer cells, the molecular mechanism of this cytotoxic activity has not yet been fully explored and is still waiting to be described.

General Experimental Procedures
All solvents were treated using standard techniques before use. All reagents were purchased from commercial sources (Sigma Aldrich, Prague, Czech Republic) and used without purification. The NMR spectra were obtained in CDCl 3 and CD 3 OD at ambient temperature on a VNMR S500 (Varian, Palo Alto, CA, USA) spectrometer operating at 500 MHz for 1 H and 125.7 MHz for 13 C. Chemical shifts were recorded as δ values in parts per million (ppm) and were indirectly referenced to tetramethylsilane (TMS) via the solvent signal (CDCl 3 -7.26 ppm for 1 H and 77.0 ppm for 13 C; CD 3 OD-3.30 ppm for 1 H and 49.0 ppm for 13 C). Coupling constants (J) are given in Hz. For unambiguous assignment of 1 H and 13 C signals, 2D NMR experiments, namely gCOSY, gHSQC, gHMBC, and NOESY were measured using standard parameter settings and standard pulse programs provided by the producer of the spectrometer. HRMS were obtained with a Waters Synapt G2-Si hybrid mass analyzer of a quadrupole-time-of-flight (Q-TOF) type, coupled to a Waters Acquity I-Class UHPLC system. The EI-MS were obtained on an Agilent 7890A GC 5975 inert MSD operating in EI mode at 70 eV (Agilent Technologies, Santa Clara, CA, USA). A DB-5 column (30 m × 0.25 mm × 0.25 µm, Agilent Technologies, USA) was used. The temperature program was: 100-180 • C at 15 • C/min, 1 min hold at 180 • C, 180-300 • C at 5 • C /min, and 5 min hold at 300 • C; detection range m/z 40-600. The injector temperature was 280 • C. The flow-rate of the carrier gas (helium) was 0.8 mL/min. A split ratio of 1:15 was used. TLC was carried out on Merck precoated silica gel 60 F254 plates. Compounds on the plate were observed under UV light (254 and 366 nm) and visualized by spraying with Dragendorff's reagent.

Plant Material
Fresh bulbs of Narcissus cv. Professor Einstein were obtained from the herbal dealer Lukon Glads (Sadská, Czech Republic). The botanical identification was performed by Professor L. Opletal, CSc. A voucher specimen is deposited in the Herbarium of the Faculty of Pharmacy in Hradec Králové under number CUFPH-16130/AL-447.

Extraction and Isolation of Alkaloids
Fresh bulbs (34 kg) were cut, minced, and underwent comprehensive extraction with ethanol (EtOH) (96%, v/v, 2×). Each portion (approximately 1.2 kg) was boiled twice for 30 min under reflux. The combined extracts were filtered and evaporated under reduced pressure. The crude extract (638 g) was dissolved in 1.5 L of 5% hydrochloric acid (HCl), filtered twice, and diluted with distilled water to 4.5 L at pH 1. The water solution was defatted with diethyl ether (Et 2 O; 2 × 4 L), alkalized with 10% Na 2 CO 3 to pH 10, and exhaustively extracted with chloroform (CHCl 3 ; 4 × 4 L). The organic phase was evaporated to give 58 g of dark brown residue. The purification process was repeated to give 32 g of concentrated alkaloidal residue (brown syrup). The obtained extract, which was Dragendorff positive, was fractionated by column chromatography on neutral Al 2 O 3 (ACROSS, 2100 g) deactivated with 6 % of distilled water. The extract was eluted with light petrol enriched with CHCl 3 (gradually from 30:70 to 80:20), then with CHCl 3 gradually enriched with EtOH (from 1:99 to 50:50), and finally with pure EtOH. Fractions of 250 mL were collected and monitored by TLC, yielding 495 fractions, which were combined into 15 final fractions and analyzed by TLC and GC-MS.

Cell Treatment
All the alkaloids evaluated and doxorubicin, used as positive control, were dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA) to prepare stock solutions with a concentration of 10-50 mM based on their solubility. Stock solutions were freshly prepared before use in the experiments. For the experiments, the stock solutions were diluted with the appropriate culture medium to create final concentrations (10 µM for a single-dose alkaloid cytotoxicity screen and 1 µM for doxorubicin, used as a reference compound) making sure that the concentration of DMSO was <0.1% to avoid toxic effects on the cells. Control cells were sham-treated with a DMSO vehicle only (0.1%; control).

WST-1 Cytotoxicity Assay
The WST-1 (Roche, Mannheim, Germany) reagent was used to determine the cytostatic effect of the test compounds. WST-1 is designed for the spectrophotometric quantification of cell proliferation, growth, viability, and chemosensitivity in cell populations using a 96 well plate format (Sigma, St. Louis, MO, USA). The principle of WST-1 is based on photometric detection of the reduction of tetrazolium salt to a colored formazan product. The cells were seeded at a previously established optimal density (30,000 Jurkat, 25,000 MOLT-4, 500 A549, 1500 HT-29, 2000 PANC-1, 5000 A2780, 500 HeLa, 1500 MCF-7, 2000 SAOS-2, and 2000 MRC-5 cells/well) in 100 µL of culture medium, and adherent cells were allowed to reattach overnight. Thereafter, the cells were treated with 100 µL of either corresponding alkaloids or doxorubicin stock solutions to obtain the desired concentrations and incubated in 5% CO 2 at 37 • C. WST-1 reagent diluted 4-fold with PBS (50 µL) was added 48 h after treatment. Absorbance was measured after 3 h incubation with WST-1 at 440 nm. The measurements were performed in a Tecan Infinite M200 spectrometer (Tecan Group, Männedorf, Switzerland). All experiments were performed at least three times with triplicate measurements at each drug concentration per experiment. The viability was quantified as described in Havelek et al. according to the following formula: (%) viability = (Asample − Ablank)/(Acontrol − Ablank) × 100, where A is the absorbance of the employed WST-1 formazan measured at 440 nm [43]. The viability of the treated cells was normalized to the viability of cells treated with 0.1% DMSO (Sigma-Aldrich, St. Louis, MO, USA) as a vehicle control.

Statistical Analysis
The descriptive statistics of the results were calculated and the charts made in either Microsoft Office Excel 2010 (Microsoft, Redmond, WA, USA) or GraphPad Prism 5 biostatistics (GraphPad Software, La Jolla, CA, USA). In this paper, all of the values have been expressed as arithmetic means with SD of triplicates (n = 3), unless otherwise noted. The significant differences between the groups were analyzed using Student's t-test and a p value ≤ 0.05 was considered statistically significant. IC 50 curves of pancracine and standard cytostatic treatment doxorubicin were generated and values of IC 50 were deducted with GraphPad Prism 5 Biostatistics.

Conclusions
In conclusion, 24 Amaryllidaceae alkaloids of various structural types were isolated from fresh bulbs of Narcissus cv. Professor Einstein. The constitution of a newly isolated alkaloid, 7-oxopluviine, was structurally determined. The complete NMR data for 9-O-demethylmaritidine are reported. Isolated compounds not previously studied for cytotoxicity were assayed for their cytotoxicity on a set of nine human cancer cells of different histotypes. This study revealed promising cytotoxic activity of pancracine, a montanine-type alkaloid, which was isolated in an amount that will allow more detailed cytotoxic studies in the future, and also the preparation of semisynthetic derivatives. Taken together, the plant cultivar Narcissus cv. Professor Einstein is as a rich source of diverse alkaloids, some of which show interesting activities for further pharmaceutical research.