Cytotoxic effects of aporphine alkaloids from the stems and leaves of Stephania dielsiana Y.C.Wu

Abstract Phytochemical studies of the stems and leaves of Stephania dielsiana Y.C.Wu yielded two new aporphine alkaloids (1 and 5), along with six known alkaloids (2–4 and 6–8). Their structures were characterised based on analyses of spectroscopic data, including one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy and high-resolution electrospray ionisation mass spectrometry (HR-ESI-MS). The cytotoxic activities of the isolated compounds against a small panel of tumour cell lines were assessed by MTS assay. Interestingly, compound 2 exhibited particularly strong cytotoxic activities against HepG2, MCF7 and OVCAR8 cancer cell lines, with IC50 values of 3.20 ± 0.18, 3.10 ± 0.06 and 3.40 ± 0.007 µM, respectively. Furthermore, molecular docking simulations were carried out to explore the interactions and binding mechanisms of the most active compound (compound 2) with proteins. Our results contribute to understanding the secondary metabolites produced by S. dielsiana and provide a scientific rationale for further investigations of cytotoxicity of this valuable medicinal plant. Graphical Abstract


Introduction
According to the World Health Organization, cancer and cardiovascular disease are the two major causes of mortality worldwide.An overview summarised cancer incidence and mortality rates by sex and age in 2020, for 38 cancer sites in 185 nations and territories worldwide (Ferlay et al. 2021).Numerous disorders, including allergies, oxidative stress, chronic inflammation, cardiovascular diseases and disorders that promote aberrant cell growth, are linked to cancer (Khansari et al. 2009).Although there are a variety of treatment options for cancer, including surgery, chemotherapy and radiotherapy, there is a great deal of research interest in more affordable options using natural ingredients to both prevent and treat cancer (Vinh et al. 2020;Li et al. 2022).Therefore, identification of novel therapeutic components in folk medicines is crucial in the battle against cancer (Thang Hoang et al. 2021;duyen et al. 2022).
Alkaloids are a large class of organic molecules containing at least one nitrogen atom that exist naturally in both plants and marine organisms.Alkaloids have diverse pharmacological properties, including anti-inflammatory, anticancer, antibacterial and antioxidant properties (Li et al. 2022).Numerous alkaloids have been identified and used in traditional and modern medicine, or have served as the basis for new drug development (e.g.morphine and other opium alkaloids found in opium poppies) (Matos et al. 2022).In addition, berberine, an alkaloid derived from the Berberis genus, has historically been employed in Ayurvedic, Chinese and Middle Eastern folk medicines for its effects against a range of pathogens, including bacteria, viruses, fungi, protozoa and helminths (Kong et al. 2022).
Stephania dielsiana Y.C.Wu is a plant species classified under the family Menispermaceae.It is commonly found in Vietnam and China and is valued for its medicinal properties, particularly for its potential cytotoxic effects (Qiming et al. 2008).Phytochemical investigations of this plant have shown diverse alkaloids.In particular, over twenty alkaloids were reported, such as sinoacutin, stephanin, ayuthianin, dehydrostephanin, cephamorphinanin, aknadinin, liriodenin, and sinomenin (Zhou et al. 2018;Knockleby et al. 2020).Consequently, further investigation into the chemical constituents of S. dielsiana may enhance our understanding of the novel alkaloids and provide a sound rationale for undertaking further research into the cytotoxic properties of this valuable medicinal plant.
Especially, alkaloids from Stephania dielsiana Y.C.Wu exhibited diverse pharmacological effects, such as cytotoxic, anticancer, anthelmintic and antimicrobial activities (Zhou et al. 2018;Knockleby et al. 2020).As part of an ongoing effort to discover bioactive components from herbal medicine as possible anticancer treatments (Vinh et al. 2019a;2019b;2020;Tuan Anh et al. 2021), we describe the structure, extraction, and isolation of two new aporphine alkaloids (1 and 5), along with six known alkaloids (2-4 and 6-8), from the leaves of S. dielsiana.The anticancer properties of isolated compounds were also evaluated by the 3-(4,5-dim e t h y l t h i a z o l -2 -y l ) -5 -( 3 -c a r b o x y m e t h o x y p h e n y l ) -2 -( 4 -s u l f o p h e n y l ) -2H-tetrazolium (MTS) protocol using the HepG2, MCF7 and OVCAR8 human cancer cell lines.The results showed that compound 2 exhibited particularly strong cytotoxic activities against HepG2, MCF7 and OVCAR8 cancer cell lines, with IC 50 values of 3.20 ± 0.18, 3.10 ± 0.06 and 3.40 ± 0.007 µM, respectively.Furthermore, molecular docking simulations of active compounds were performed to further support our in vitro findings.

Structure and identification of new compounds
dried stems and leaves of S. dielsiana (7.0 kg) were extracted with 95% MeOH (15 L × 3 times) at ambient temperature.MeOH residue was produced after the solvent evaporated under reduced pressure (680 g).
Stedieltine B ( 5) was obtained as a brown solid, and the molecular formula C 17 H 11 NO 4 was determined by HRESIMS from the protonated molecular ion observed at m/z 294.0766 [M + H] + (calcd C 17 H 12 NO 4 + , 294.0761) (Figure S15).In the 1 H NMR and 1 H-1 H 3), revealed the existence of one pentasubstituted and tetrasubstituted benzene moiety (Figure S13).In addition, a singlet downfield hydrogen signal at δ H 6.31 (2H, s, 1-OCH 2 O-2) indicated the presence of a methylenedioxy moiety, and a methoxy group at C-8 by {δ H 3.90 (s; δ C 55.9; -OCH 3 )}.The 13 C NMR and HSQC spectra of 5 indicated the occurrence of 17 carbons, including 1 sp 3 methylene, 15 sp 2 carbons and 1 methoxy group (Figures S10 and S11).The 15 sp 2 carbons could be ascribed to the presence of one double bond and two benzene ring moieties that accounted for 9 of the 13 degrees of unsaturation.The remaining three indices of hydrogen deficiency were assigned to three additional ring systems in 5.The above spectroscopic data analysis revealed that the structure of 5 resembled that of oxostephanine (2) .The difference between 5 and oxostephanine (2) was revealed by comparing the NMR spectral data of 5 (Table S1) with that of oxostephanine, which showed that the carbonyl group at C-7 in oxostephanine was replaced by a bond to an oxygen atom in 5.This assignment was confirmed by the chemical shifts at δ C 156.3 (C-6a) and 140.6 (C-7a) as well as by high-resolution electrospray ionisation mass spectrometry (HR-ESI-MS) data (Figure S16).Additionally, HMBC has showed a signal between H-9 (δ H 7.14) and C-9 (δ C 113.3), that has meant for the lack of one -OCH 3 at position 9 in comparing with oxostephanine (Figure S12).The NOESY of 5 data showed long interaction from δ H 7.18 (1H, t, J = 8.0 Hz, H-10) to 7.14 (1H, dd, J = 8.0, 1.0 Hz, H-9) and 7.68 (1H, dd, J = 8.0, 1.0 Hz, H-11); and from δ H 7.84 (1H, d, J = 5.5 Hz, H-5) to 7.22 (1H, d, J = 5.5 Hz, H-4) and 7.20 (1H, s, H-3).Additionally, the methoxy group at C-8 {δ H 3.90 (s; δ C 55.9; -OCH 3 )} gave; a long correlation with H-9; and between H-11 and H-5; between H-9 and H-4.That suggested the structure of compound 5 may be planar (Figure S14).Thus, the structure of 5 was determined as an aporphine alkaloid in Figure 1, and named stedieltine B.

Biological evaluation
The potential cytotoxicity of compounds 1-8 was examined using three human cancer cell lines: HepG2, MCF7 and OVCAR8 (Tuan Anh et al. 2021).Compound 2 exhibited particularly strong cytotoxic activities against HepG2, MCF7 and OVCAR8 cells, with IC 50 values of 3.20 ± 0.18, 3.10 ± 0.06 and 3.40 ± 0.007 µM, respectively (Table S2).Taxol was used as positive control.In addition, compound 4 (oxostephanosine) showed weak cytotoxic activities on HepG2 and OVCAR8 cell lines with sIC 50 values of 122.9 ± 5.7 and 30.0 ± 1.31 µM, respectively.These were higher than the values of compound 2 about 40 to 10 times.Besides that, this compound did not reveal the activity on MCF-7.The difference in structure between compounds 4 and 2 were hydroxy and methoxy group at position 8, which may cause the distinctive power of cytotoxicity on the cancer cell lines.
Based on the potential cytotoxic effects of compound 2, a computational study was performed to support the results of the in vitro experiment.The interaction and binding mechanism of active compound 2 with proteins related to cancer were investigated by molecular docking simulations.The results showed that compound 2 had good binding energies of −9.8, −8.0 and −9.8 kcal/mol for HepG2 (PdB Id: 5EQG), MCF7 (PdB Id: 3ERT) and OVCAR8 (PdB Id: 3OG7), respectively (Figure S16).Furthermore, compound 2 exhibited a hydrogen bond with TRP412 in the active site for HepG2 (PdB Id: 5EQG).Therefore, compound 2 from S. dielsiana is a potential candidate for the development of novel anticancer therapeutic agents.
Cancer cell lines: HepG2, MCF7 and OVCAR8 cell lines were provided by the American Type Culture Collection (ATCC), then stored in nitrogen liquid in Biology department, Natural Sciences univeristy, Hanoi National university.The cells were grown in dulbecco's modified Eagle's medium (dMEM; Gibco; Thermo Fisher Scientific, Inc).

Identification of plant S. dielsiana
The study materials, which had harvested whole aerial parts from the first branch of trunk of Stephania dielsiana, were collected at cordinates (21°06′22.9″N105°24′40.2″E) in Tan Linh commune, Ba Vi district, Hanoi, Vietnam, in October 2019 and a voucher specimen (Sd10/2019) was kept at the department of Botany and Pharmacognosy, Vietnam university of Traditional Medicine, Hanoi, Vietnam (Figure S17).The plant materials were identified by dr.Le Thi Kim Van.

Cytotoxicity assay
The MTS assay.Cells HepG2, MCF7 and OVCAR8 were seeded at a concentration of 1 × 10 5 cells/mL, 200 mL/well, into 96-well flat-bottomed tissue culture plates in eight replicates.The MTS assay was carried out using the MTS Cell Proliferation Colorimetric Assay Kit (BioVision, Inc., Milpitas, CA) following the manufacturer's instruction.In brief, after the cells were cultured with medium for 24 h, then washed out and replaced with 200 µL fresh warmed the culture medium before adding 20 µL of MTS reagent (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) into each well.The cells were further incubated for 2-4 h at 37 °C in standard culture conditions.Then the absorbance was detected at 490 nm with a microplate reader.

Molecular docking simulation
The docking study was carried out as described previously (duyen et al. 2022).Briefly, molecular docking studies were carried out using Autodock Vina 1.1.2to determine the binding affinity and interaction of the most active compound (compound 2) with proteins related to cancer, using protocols reported previously (Cao et al. 2021;duyen et al. 2022;Vinh et al. 2023).The X-ray crystallographic structures of proteins related to cancer, i.e.HepG2 (PdB Id: 5EQG), MCF7 (PdB Id: 3ERT) and OVCAR8 (PdB Id: 3OG7), were obtained from the RCSB Protein data Bank.Energy minimisation of the active compound was accomplished using Chem 3d ultra version 20.The molecular docking results were visualised using discovery Studio 20.1 (dassault Systemes Biovia, San diego, CA, uSA).

Conclusion
Previously, alkaloids isolated from the genus Stephania were reported to show remarkable anti-inflammatory, antinociceptive and anticancer activities (deng et al. 2011;Knockleby et al. 2020).Indeed, some alkaloids from Stephania were reported for their cytotoxic activities against some kinds of human cancer cell lines such as BT471, KB, and HepG2.Stephanine and crebanine (7) demonstrated potent cytotoxicity against BT474 cell with IC 50 of 1.55 and 1.58 g/mL, respectively.In this study, eight compounds (1-8), including two new aporphine alkaloids (1 and 5), were identified in the methanol (MeOH) extract of S. dielsiana by combined column chromatography (CC).Based on in vitro and in silico experiments, compound 2 exhibited strong cytotoxicity effect.Our results suggested that the alkaloids in S. dielsiana might have potential for the treatment of cancer and related diseases.