Isolation and characterisation of altissimin: a novel cytotoxic flavonoid C-apioglucoside from Drimia altissima (Asparagaceae)

Abstract Flavonoids are a class of biologically active compounds with various proven nutraceutical benefits. In flavonoid C-glycosides, the aglycones are attached to sugar residues via cleavage-resistant C-C bonds which alter typical flavonoid pharmacokinetic properties. In these compounds, the combination of biological activities from the flavonoid moieties and sugar residues create unique and more diverse biological functions than those of O-glycosylated and unsubstituted flavonoids. Through a series of reverse phase chromatography techniques and various spectroscopic methods, the phytochemical investigation of Drimia altissima (L.F.) Ker Gawl., a specie from the Asparagaceae family, led to the isolation and chemical characterisation of a novel C-glucosylflavonoid, altissimin, with a unique apioglucoside arrangement to the apigenin aglycone. Altissimin was found to possess strong in vitro anti-proliferative activity against HeLa cervical cancer cells.


Introduction
Drimia altissima (L.F.) Ker Gawl., commonly known as the 'African Squill' or the 'Tall White Squill', is a terrestrial bulbous plant that is widely distributed in tropical and southern Africa from Senegal all the way to South Africa (Williams et al. 2016). D. altissima is a member of the Asparagaceae, a family of monocotyledonous flowering plants from the Asparagales order (APG III 2009). Within Asparagaceae, D. altissima comes from the sub-family Schilloidae, which is sometimes considered as a separate family under the name Hyacinthaceae. The Hyacinthaceae family has been divided into four tribes; Oziro€ eeae, Urgineeae, Hyacintheae (which is further divided into sub-tribes Pseudoprospero, Massoliinae and Hyacinthinae) and Ornithogaleae (Manning et al. 2003).
From the Urgineeae tribe arises the genera Bowiea, Drimia, Schizobasis and Fusifilum, with the latter two occasionally included in Drimia. Among these, species from Drimia have the most extensive use as medicinal plants (Nath et al. 2014). The main types of compounds isolated from genera within Urgineeae are flavonoids and bufadienolides. Bufadienolides are considered as chemotaxonomic markers for the Urgineeae tribe (Pohl et al. 2000). Phytochemical investigations of Drimia species have resulted in the isolation of several bio-active C-glycosylflavones and scillaridin-based cardiotonic glycosides. For instance, the C-glycosylflavones vitexin, isovitexin, orientin, isoorientin, scoparin, vicenin-2 and possibly an isovitexin-O-xyloside (Fernandez et al. 1975), as well as the bufadienolides proscillaridin A and Scillaren A have been isolated from D. maritima (Iizuka et al. 2001;Kedra and Kedrowa 1968). Vitexin is among the most extensively studied C-glycosylflavonoids isolated from Drimia species. It has been reported to possess anticancer activity against various cancer cell lines such as HepG2 (Wang et al. 2014), EC-109 (An et al. 2015), OC2 (Yang et al. 2013) and MCF-7 (Czemplik et al. 2016;Mohammed et al. 2014), mainly through the induction of apoptosis. For instance, by inhibiting Bcl-2, vitexin induces apoptosis and activates caspases 3, 7 and 9 in U937 human leukaemia cells (Lee et al. 2012). Vitexin has also been found to inhibit the proliferation of hepatocellular carcinoma (HCC) cells and induce apoptosis in HepG2 hepatocellular carcinoma cells (Wang et al. 2014).
Species from the genus Drimia have been used for over centuries as ethnomedicines for the treatment of various ailments such as dropsy, respiratory conditions, bone and articulation disorders, dermatological diseases, epileptic seizures and cancers (Bozorgi et al. 2017). Phytochemical studies involving D. altissima have so far only resulted in the isolation of bufadienolides. Bufadienolides are well known for their anticancer properties, though the mechanisms by which they elicit these actions are often unknown (Gao et al. 2011). Bufadienolides that have been isolated from D. altissima (Figure 1) include hellebrigenin (1), urginin (2), arenobufagin-3-O-a-L-rhamnopyranoside (3) and gamabufotalin-3-O-a-L-rhamnopyranoside (4) (Ermias et al. 1994;Shimada et al. 1979). Hellebrigenin (1) has been reported to induce cell cycle arrest and apoptosis in HepG2 cells (Deng et al. 2014), with the anti-proliferative activity possibly attributed to the inhibition of integrin a1b1 (Moreno et al. 2013). To our knowledge, there are currently no literature reports of any type of flavonoids isolated from D. altissima.  Figure S3) exhibited a typical two maxima flavonoid absorption pattern with a pronounced Band I at k 333 nm indicating the presence of hydroxylation on the B-ring and a less pronounced Band II at k 272 nm. The shorter wavelength of Band I confirmed absence of an -OH substituent at position C-3 while absence of a bathochromic shift in Band I and lack of splitting in Band II indicated a single -OH substituent at C-4 0 of the B-ring. Compared to the absorption patterns of non-hydroxylated A-rings, the 22 nm bathochromic shift observed in Band II of compound 5 confirmed the A-ring -OH substituents at positions C-5 and C-7 (Mabry et al. 1970).

Results and discussion
The CD spectrum of compound 5 ( Figure S4) exhibited a negative pp Ã Cotton effect at k 235 and a positive pp Ã Cotton effect at 271 nm corresponding with a 2S configuration at chiral position C-2 (Gaffield, 1970).The FT-IR spectrum of compound 5 presented a sharp carbonyl stretching vibration at 1649.89 cm-1, confirming the flavonoid carbonyl functional group at C-4. The FT-IR spectrum also exhibited a broad -OH stretching vibration at 3338.91 cm-1 and a sharp aromatic ring vibration at 1608.89 cm-1. Para-substitution of the flavonoid B-ring was confirmed by a conspicuous out-of-plane deformation vibration at 833.27 cm À1 .
The 1 H NMR spectrum of compound 5 exhibited overlapping signals in the sugar region (d H 3.0-4.0) with two anomeric protons appearing as doublets at d H 4.93 and 5.0. Within the aromatic region, there were two singlets at d H 6.55 and 6.64 and two doublets at d H 6.96 and 7.88, creating an AMX spin system characteristic of flavonoid compounds (Maltese et al. 2009).
The 13 C NMR spectrum of compound 5 showed 24 carbon resonances with peak intensities at d C 117.1 and 129.5 showing the presence of symmetry in the aromatic region and a carbonyl signal at d C 184.1. The presence of sugars was confirmed by carbon signals in the region d C 60-85. Comparison of 1 H and 13 C NMR spectroscopic data of compound 5 with that of isovitexin from literature allowed for the identification of the flavonoid aglycone as apigenin (Table S1) (Ramarathnam et al. 1989).
From HSQC-TOCSY results, a single spin system was observed in one of the sugar moieties. The anomeric proton H-1 00 at d H 4.93 correlated with protons of carbons C-2 00 , C-3 00 , C-4 00 and C-5 00 at d C 72.86, 79.9, 71.65 and 81.26 respectively. Further correlations were observed between H-5 00 and the protons of carbons C-1 00 and C-6 00 at d C 75.45 and 68.95 respectively. These results, coupled with comparison of 1 H and 13 C NMR chemical shifts belonging to the C-glucose of isovitexin helped to identify the sugar unit as glucopyranoside. Three separate spin systems were observed in the HSQC-TOCSY correlations of the second sugar. The anomeric proton H-1 000 at d H 5.0 shared the same spin system with H-2 000 (d H 3.94) whereas H-4 000 (d H 3.79, 4.0) and H-5 000 (d H 3.61) geminal protons were in two separate spin systems, a characteristic of furanose moieties. This led to the identification of the second sugar unit as apio-D-furanoside. From 1 H NMR results, the H-1 000 to H-2 000 coupling constant (J ¼ 2.5 Hz) corresponded to an aanomeric configuration.
From HMBC results of compound 5, attachment of the glucopyranoside to the aglycone via a C-glycosidic bond was determined through a correlation between C-6 at d C 109 and the anomeric proton H-1 00 at d H 4.93 ( Figure S5). Based on the large H-1 00 to H-2 00 coupling constant (J ¼ 10.2 Hz) observed in the 1 H NMR, the anomeric configuration of the C-sugar was determined as b-glucopyranoside. The fact that no HMBC correlations were observed between the aglycone and the furanose anomeric proton H-1 000 was suggestive of a glycosidic linkage between the b-glucopyranoside and the apio-a-D-furanoside. 13 C NMR chemical shift of C-6 00 in the unsubstituted C-glucopyranoside of isovitexin was reported in literature at d C 62.9 while in compound 5, C-6 00 had a significantly higher chemical shift, resonating at d C 68.95 (Table S1). It was therefore concluded that in compound 5, the apio-a-D-furanoside is linked to the C-b-glucopyranoside at C-6 00 via an Oglycosidic bond. The structure of compound 5 was thus established as 6-C-[-apio-a-D-furanosyl-(1!6)-b-glucopyranosyl]-4 0 , 5, 7-trihydroxyflavone or simply 6 00 -O-a-apio-D-furanosylisovitexin ( Figure 1). Compound 5 has since been accorded the trivial name altissimin. Several flavonoids with C-glucopyranosyl and furanosyl residues have been previously reported in literature. However, altissimin (5) presents a unique structural arrangement of these two sugar residues to the apigenin aglycone.
Altissimin (5) is also the first C-glycosylflavone to be isolated from D. altissima. A similar compound consisting of C-b-D-glucopyranosyl and apio-b-D-furanosyl residue attachments to an 8-hydroxyapigenin aglycone was isolated from the aerial parts of Gaillardia grandiflora (Moharram et al. 2017). NOESY correlation between the furanose anomeric proton H-1 000 and the glucopyranose geminal protons H-6 00 confirmed O-inter-glycosidic attachment between the furanose and the C-glucose. A correlation between the geminal protons H-4 000 could also be observed. Correlations between the vicinal protons H-2 00 , 6 00 and H-3 00 , 5 00 confirming a para-substitution of the flavonoid B-ring.

Anti-proliferative activity
The anti-proliferative activity of partition P-4 and altissimin (5) against HeLa cells was evaluated via Hoechst 33342/propidium iodide dual staining with melphalan (40 lM) used as positive control. From the results obtained after 48 h of treatment, partition P-4 exhibited a dose dependent inhibition of cell proliferation with an IC 50 of ± 0.25 lg/ mL ( Figure S13). Similarly, altissimin (5) exhibited a dose dependant inhibition of cell proliferation with an IC 50 of ± 2.44 lM (1.37 lg/mL) ( Figure S13). The fact that partition P-4 exhibited a lower IC 50 than altissimin (5) implies the presence of other cytotoxic compounds within the D. altissima extract. Thus, altissimin (5) is, at least in part, responsible for the anti-proliferative activity of D. altissima.

General experimental
All extractions and chromatography utilised LiChrosolv V R (Merck, Germany) solvents. Open column reverse phase chromatography techniques were carried out using Diaion V R HP-20 (Supelco, USA) and Sephadex V R LH-20 resins (Merck, South Africa). Samples were freeze dried using a Virtis SP Scientific sentry 2.0 lyophilizer with an Alcatel Pascal vacuum pump and/or dried in vacuo using a Buchi R-210 Rotavapor. NMR experiments were performed with an Ultrashield Plus Bruker Avance III 400 MHz NMR spectrometer using standard pulse sequences. FT-IR results were obtained using a Bruker Tensor 27 FT-IR spectrometer and analysed using Opus data collection program. LC/MS and HR-MS results were obtained on a Waters Synapt G2 quadrupole time-of-flight mass spectrometer from the Central Analytical Facilities (CAF) at Stellenbosch University, South Africa. The UV and Circular Dichroism (CD) results were recorded on a Chirascan Plus Spectrapolarimeter. HeLa cervical cancer cells were obtained from Cellonex, South Africa and grown in Roswell Park Memorial Institute 1640 (RPMI 1640) medium from GE Healthcare Life Sciences (South Logan, Utah, USA) supplemented with gamma irradiated Fetal Bovine Serum (FBS) from Biowest (South America). HeLa cells were cultured in BioFlow-II Labotec laminar flow cabinets and incubated in a ThermoForma CO 2 incubator. Propidium iodide and Hoechst 33342 were sourced from Sigma-Aldrich (St. Louis, MO, USA).

Plant material
The plant bulbs of Drimia altissima (L.F.) Ker Gawl were collected in Kwa-Nobuhle, (Uitenhage, Eastern Cape, South Africa). Plant authentication was performed by a curator and taxonomist at Selmar Schonland Herbarium (GRA) in Makhanda, where a specimen of Drimia altissima (L.F.) Ker Gawl. with voucher number Hlangothi011(GRA) has been deposited.

Extraction and isolation
The bulbs of D. altissima were washed under running water, peeled and dried at 40 C after which the plant material was shredded into smaller pieces and submerged in liquid nitrogen until brittle. The resulting biomass was crushed with a mortar and pestle to powder in readiness for soxhlet extraction. About 100 g of dry powdered biomass was loaded onto the soxhlet apparatus and extracted at 65 C under reflux using MeOH/CH 2 Cl 2 (8:2, 150 mL). The resulting extract was dried in vacuo to yield a brown crude extract (10.85 g) which was solubilized in 160 mL de-ionised H 2 O and transferred into a 2 L separating funnel. The crude extract then underwent a succession of liquidliquid partition extractions using 160 mL each of Hex, EtOAc and n-BuOH to obtain partitions P-1 (H 2 O, 4.85 g), P-2 (Hex, 1.78 g), P-3 (EtOAc, 2.86 g) and P-4 (n-BuOH, 1.77 g). Each partition was partially dried in vacuo, diluted with sufficient amounts of HPLC-grade H 2 O, frozen at -80 C and lyophilized. 100 g of dry Diaion V R HP-20 resin was transferred into a 500 mL beaker to which sufficient amounts of MeOH were added to cover the resin bed by 5 cm. The resin was gently stirred for 1 min and allowed to stand for 15 min after which the MeOH was decanted and replaced with de-ionized H 2 O. The mixture was stirred and allowed to stand for 10 min before it was carefully packed into an open column of diameter 30 mm to a resin height of 12.5 cm. Partition P-4 (1.77 g) was applied to the column and eluted using a series of solvents (200 mL each) in decreasing polarity to yield fractions Fr-1 (H 2 O/MeOH, 7:3, n.d.), Fr-2 (H 2 O/MeOH, 5:5, 270.8 mg), Fr-3 (H 2 O/MeOH, 3:7, 216.3 mg), Fr-4 (MeOH, 144 mg), Fr-5 (EtOH, 5 mg) and Fr-6 (EtOAc, 15.8 mg). After lyophilisation, 160 mg of fraction Fr-3 was solubilized in 2 mL MeOH, applied onto an open column of diameter 20 mm prepacked with 12 g of Sephadex V R LH-20 gel in de-ionized H 2 O (swelling ratio; 1 g ¼ 4 mL) and eluted with 50% MeOH. 10 mL fractions were collected and dried to afford subfractions SF-1 to SF-12. Sub-fractions SF-7 to SF-12 were combined to afford compound 5 (26.7 mg) as a yellow powder.

Hoechst 33342/propidium iodide (PI) cytotoxicity assay
High Content Analysis (HCA) was used for cytotoxicity screening of partition P-4 and altissimin (5). HeLa cells were seeded at a density of 5000 cells/well into 96 well culture plates and incubated for 24 h at 36.7 C in a humidified 5% CO 2 incubator. The cells were treated at different concentrations of the partition and compound. For partition P-4, cells were treated at 0.001, 0.002, 0.01, 0.02, 0.1, 0.2, 2, 20 and 100 lg/mL. For altissimin (5), cells were treated at 0.3125, 0.625, 1.25, 2.5, 5 and 20 lM. Treatments were incubated for 48 h at 36.7 C after which the medium was aspirated and cells washed with PBS containing calcium and magnesium. The cells underwent Hoechst/PI staining with bisBenzamide H 33342 trihydrochloride (Hoechst 33342) at 5 mg/mL and propridium iodide (PI) at 10 mg/mL. After 15 min incubation at 36.7 C, cellular images were acquired with a Molecular DevicesV R ImageXpress Micro XLS Widefield High-Content Analysis System using a DAPI filter for Hoechst 33342 and a Texas Red filter for PI. Images were acquired from nine sites per well of a treated 96-well plate at 10x objective with values reported as the average of nine sites from each well. The acquired images were analysed using the Multiwavelength Cell Scoring analysis module of MetaXpressV R High-Content Image Acquisition and Analysis Software. The generated data was analysed using GraphPad Prism version 6.

Conclusion
Despite previous efforts only yielding bufadienolides, phytochemical investigation of the methanolic bulb extract of D. altissima led to the isolation of a flavonoid C-apioglucoside, 6-C-[-apio-a-D-furanosyl-(1!6)-b-glucopyranosyl]-4 0 , 5, 7-trihydroxyflavone (altissimin). There are few reports of natural flavonoid C-glycosides containing apiofuranose residues. Most of the existing ones contain an attachment of the C-glucose residue to the flavonoid aglycone via position C-8 as well as the presence of (1!2) interglycosidic linkages. This makes the combination of a 6-C-apioglucoside attachment and a (1!6) inter-glycosidic linkage found in altissimin (5) a unique structural arrangement to the apigenin aglycone. To this effect, altissimin (5) has become the first C-glycosylflavone to be isolated from D. altissima. In light of this finding, D. altissima may contain other bio-active flavonoid-type compounds that still need to be isolated. As opposed to bufadienolides which have potential cardiotoxic effects and a narrow therapeutic index, flavone-type compounds are generally considered to be safe and well tolerated. Through in vitro cytotoxicity evaluation, altissimin (5) was found to be partly responsible for the cytotoxic activity of D. altissima against HeLa cells. The observed in vitro cytotoxicity of altissimin (5) in HeLa cells provides basis for its further evaluation as a potential anticancer lead. An investigation of the biochemical mechanism of action through which altissimin (5) elicits this anti-proliferative activity is currently underway.