Cytotoxic Phenylpropanoid Derivatives and Alkaloids from the Flowers of Pancratium maritimum L.

Regarding our growing interest in identifying biologically active leads from Amaryllidaceous plants, the flowers of Pancratium maritimum L. (Amaryllidaceae) were investigated. Purification of the cytotoxic fractions of the alcoholic extract of the flowers gave a new glycoside, 3-[4-(β-D-glucopyranosyloxy)phenyl]-2-(Z)-propenoic acid methyl ester (1), together with the previously reported compounds 3-methoxy-4-(β-D-glucopyranosyloxy)benzoic acid methyl ester (2), 3-(4-methoxyphenyl)propan-1-ol-1-O-β-D-glucopyranoside (3), (E)-3-(4-hydroxyphenyl)acrylic acid methyl ester (4), caffeic acid (5), dihydrocaffeic acid methyl ester (6), and pancratistatin (7). Interestingly, compounds 1 and 2 are phenolic-O-glycosides, while the glucose moiety in 3 is attached to the propanol side chain. This is the first report about the existence of 1–6 in the genus Pancratium. Further, glycosides 1–3 from the Amaryllidaceae family are reported on here for the first time. The structures of 1–7 were determined by analyses of their 1D (1H and 13C) and 2D (COSY, HMQC, HMBC) NMR spectra, and by high-resolution mass spectral measurements. Pancratistatin displayed potent and selective growth inhibitory effects against MDA-MB-231, HeLa, and HCT 116 cells with an IC50 value down to 0.058 µM, while it possessed lower selectivity towards the normal human dermal fibroblasts with IC50 of 6.6 µM.


Introduction
The Amaryllidaceae family consist of about 85 genera and 1100 species; they are well known for their ornamental values and biologically-active alkaloids [1]. Plants of the genus Pancratium are bulbous monocotyledons, which grow on sand banks and sandy coastal environments [2]. Although Pancratium includes about 15 species, distributed throughout the Mediterranean, Africa, and Asia, only three species of this genus, P. maximum, P. sickenbergeri, and P. tortuosum, are indigenous to Saudi Arabia [3]. In Egypt, there are four species belonging to the genus Pancratium, namely P. arabicum, P. maritimum L., P. sickenbergeri, and P. tortuosum [4]. The extracts of the bulbs and flowers of P. maritimum possess analgesic, antifungal, and anticancer activities. Furthermore, these extracts possess purgative, hypotensive, emetic, and anti-inflammatory effects [5].
Cancer is a worldwide health concern and accounts for 8 million deaths worldwide each year, with almost 600,000 deaths in the United States only [6]. Plant-derived alkaloids, such as vinblastine, vincristine, and paclitaxel, are valuable and important antitumor drugs [7].
As a continuation of our work in exploring the secondary metabolites of Amaryllidaceous plants [26][27][28][29][30], the cytotoxic fractions of the alcoholic extracts of the fresh flowers of P. maritimum L. (Sea Daffodil) were investigated. One new glycoside (1), together with the previously reported compounds 2-7, were purified from the active fractions of the flower extracts. In this work, we describe the purification, structural assignments, and the cytotoxic effects of the compounds.
The attachment of the Z-propenoic acid methyl ester to the benzene ring at C-1 was supported by HMBC correlations from H-7 and H-8 to C-1, and from H-2,6 to C-7. Similarly, the placement of the β-D-glucopyranoside moiety at C-4 of the benzene moiety was supported by HMBC cross-peaks from H-3,5 to C-1 and from H-1 to C-4. Thus, 1 was assigned as 4-(β-D-glucopyranosyloxy)phenyl]-2-(Z)-propenoic acid methyl ester, and it is reported here as a new natural product.

Structure of Compound 2
Compound 2 ( Figure 1) showed the molecular formula C 15 H 20 O 9 as obtained from the pseudomolecular ion peak at m/z 367.1003 [M + Na] + in the (+)-HRESIMS ( Figure S9), suggesting six degrees of unsaturation. The structure of 2 was assigned from interpretation of its 1D (Figures S10-S13) and 2D NMR spectra (Figures S14-S16). The 1 H and 13 C NMR data of 2 ( Table 2) showed resonances for 3,4-disubstituted benzoic acid methyl ester. proposed the existence of 3,4-disubstituted benzoic acid methyl ester. The COSY correlation between H-6 and H-2 and between H-6 and H-5 supported the existence of an ABX system and the assignment of these protons ( Figure 2). In addition, the protonated carbons were assigned from HSQC correlations ( Table 2).   Figure 2). In addition, the protonated carbons were assigned from HSQC correlations ( Table 2).  The substituents at C-3 and C-4 were assigned as a methoxyl (δH/C 3.92 (s)/56.2) group and an O-β-glucopyranoside unit, respectively. The presence of O-β-glucopyranoside moiety was established from the 1 H and 13 (Table 2 and Figure 2).
The placement of the OCH 3 group at C-3 was supported by HMBC correlations from H-5 to C-3 and from H 3 -9 to C-3, while the HMBC from H-1 to C-4 and from H-5 to C-1 secured the placement of the glucopyranoside moiety at C-4. The 1 H and 13 C NMR data of 2 are similar to those of 3-methoxy-4-(β-D-glucopyranosyloxy)benzoic acid methyl ester [31], which was previously reported from the Solanaceous plant Lycium schweinfurthii (family: Solanaceae) [31]. Thus, compound 2 was assigned as 3-methoxy-4-(β-D-glucopyranosyloxy)benzoic acid methyl ester. This is the first report about the existence of compound 2 in the Amaryllidaceae family.
The COSY experiment displayed three coupling systems, including the coupling between H-2,6 and H-3,5 in the aromatic moiety, the vicinal couplings in the aliphatic side chain from H 2 -7 to H 2 -8 and from H 2 -8 to H 2 -9, and the contiguous coupling system within the glucopyranoside moiety from H-1 to H 2 -6 ( Figure 2). The placement of the OCH 3 group at C-1 was assigned from HMBC of H 3 -10 to C-1 (Table 3 and Figure 2). Similarly, the attachment of the glucose moiety at the terminal OH of the propanol moiety was assigned from HMBC correlations from H 2 -9 to C-1 and from H-1 to C-9. The NMR data of 3 are similar to those of 3-(4 -methoxyphenyl)-propanol 1-O-β-glucopyranoside, which was previously isolated from the plant Mediasia macrophylla (family: Apiaceae) [32]. Thus, 3 was assigned 3-(4-methoxyphenyl)propan-1-ol-1-O-β-D-glucopyranoside. This is the first report about the occurrence of compound 3 in the Amaryllidaceae family.

Antiproliferation Activities of the Compounds
Compounds 1-7 were evaluated for their antiproliferative and growth inhibition activities against MDA-MB-231, HeLa, and HCT 116 cell lines (Table 5). Pancratistatin displayed a potent growth inhibitory activity towards these cell lines with IC 50 values 0.14, 0.058, and 0.10 µM, respectively. On the other hand, compounds 1-3 and 5 were inactive at the level of 10 µM against these cells. Due to the high potency of compound 7, a concentration of 10 µM was set as a cutoff value in this assay.

Plants 2022, 11, x FOR PEER REVIEW
Moreover, it causes flipping of phosphatidyl-serine, activation of caspase-3, generation of reactive oxygen species (ROS), and loss of the mitochondrial membrane leading to apoptosis [41].
However, it is worth mentioning that this is the first report, to the best of our knowledge, about the evaluation of pancratistatin against MDA-MB-231, HeLa, HCT 116, and NHDF cell lines.

General Experimental Procedures
Optical rotations of the compounds were measured on a JASCO DIP-370 digital polarimeter at 25 • C at the sodium D-line (589 nm). The IR spectra were recorded on a Shimadzu Infrared-400 spectrophotometer (Shimadzu, Kyoto, Japan). The 1D and 2D NMR spectra (chemical shifts in ppm, coupling constants in Hz) were recorded on Bruker Avance DRX 600 MHz (600 MHz for 1 H and 150 MHz for 13

Botanical Materials
The fresh entire flowers of P. maritimum L. with most of their stalks ( Figure 4)

Purification of Compounds 1-7
The fresh flowers with their stalks (1.8 kg) were crushed into small pie directly in 70% ethanol (1.5 L) (Scheme 1). After receiving the materials in the mixture was sonicated for an additional 6 h at room temperature an solution was filtered. This process was repeated thrice and the combined h extracts were evaporated under reduced pressure. The resulting suspe solved in 300 mL H2O and successively extracted with n-hexane (3 × 200 m 200 mL), and ethyl acetate (3 × 200 mL). Finally, 500 mL of H2O were added

Evaluation of the Antiproliferative Activity
The evaluation of the antiproliferative effects compounds 1-7 were performed using MTT assay, as reported earlier [42][43][44]. In short, the cells were incubated at 37 • C overnight in 5% CO 2 /air. After that, the compounds were added at the top row of a 96-well microtiter plate; a descendant serial dilution (1:4) of the concentration was performed followed by incubation of the cells with the compounds for 72 h. Using the Cell Titer 96 AQueous non-radioactive cell proliferation protocol, the cell viability was estimated at 490 nm on a Molecular Devices Emax microplate reader. The IC 50 values of the compounds (expressed in µM) were evaluated using the program SoftMax Pro. A concentration of 10 µM was set as a cutoff value in this assay.

Conclusions
Partition of the cytotoxic fractions of the alcoholic extract of the fresh flowers of P. maritimum L. afforded a potent cytotoxic alkaloid, pancratistatin (7), along with three glycosides (1-3) and three phenylpropanoid derivatives (4)(5)(6). Their structures were determined by analyses of their NMR and HRESIMS spectral data. We should note that compound 1 is reported here as a new natural product, while compounds 2 and 3 are reported here for the first time from the Amaryllidaceae family. On the contrary, 4-6 are reported here for the first time from the genus Pancratium.
Pancratistatin displayed highly potent activity against the HCT116, HeLa, and MDA-MB-231 cell lines down to 0.058 µM. Furthermore, pancratistatin showed less selectivity towards the normal human dermal fibroblasts (NHDF) with a IC 50 value of 6.6 µM compared to the average IC 50 value of 0.099 µM against the cancerous cell lines HCT116, HeLa, and MDA-MB-231, suggesting a higher selectivity (66-fold) towards these cancerous cells.
On the other hand, compounds 1-3 and 5 were inactive against these cell lines at a concentration of 10 µM. The results clearly indicate that pancratistatin is a powerful lead with potent antiproliferative and growth inhibitory activities against different cancer cell lines.
The results of the current study highlight the importance of medicinal plants as a great source of bioactive compounds. In the long-term, the results of this study and similar studies will have broader and long-term impacts, including opening new channels of cooperation with the private sector in the areas of biotechnology and drug discovery, and exploring indigenous plants for their chemical diversity and biomedical importance.