A new spirostanol steroid and a new spirostanol steroidal saponin from Aspidistra triradiata and their cytotoxic activities

Abstract A new spirostanol steroid, aspidiata A (1), and a new spirostanol steroidal saponin, aspidiata B (2), along with three known compounds, paris saponin VII (3), daucosterol (4), and (25R)-spirostane-1β,2β,3β,4β,5β,6β-hexol (5), were isolated from whole plants of Aspidistra triradiata collected in Vietnam. Their chemical structures were established by spectroscopic analysis and comparison with previously published data. Compound 3 showed strong cytotoxicity against LU-1, Hep-G2, MCF-7, and KB human cancer cell lines with half maximal inhibitory concentration (IC50) values ranging from 0.57 to 1.23 µM. Compound 5 exhibited weak cytotoxic activity against the LU-1 cell line, with an IC50 value of 95.81 µM. Graphical Abstract


Introduction
Aspidistra is a large genus in the family Asparagaceae, including herbs that are mainly distributed in the subtropical and tropical monsoon regions of Asia.Over the past decade, botanical studies on Aspidistra have made great progress, significantly increasing the number of species of this genus to approximately 170 in March 2019 (Vislobokov et al. 2019).Among the recently announced Aspidistra species, many have been discovered in Vietnam.In particular, several Aspidistra species in Vietnam have been identified as endemic and only distributed locally, suggesting that Vietnam is one of the most diverse centres of the Aspidistra genus in the world.
In many countries, primarily Asia, several species of Aspidistra have been used in traditional and folk medicine to treat back pain, hypercholesterolemia, weakness, fatigue, diarrhoea, injury, rheumatic fever, congestion, and snakebites (Cui et al. 2016;Singhatong 2017;Liang et al. 2018).Previous studies on the chemical composition and pharmacological effects of this genus have mainly focused on four species: A. elatior, A. typica, A. sichuanensis, and A. letreae.In recent years, saponins have been identified as the main constituents of the genus Aspidistra, with approximately 90 steroidal saponins being isolated (Mori and Kawasaki 1973;Hirai et al. 1982;Konishi et al. 1984;Cui et al. 2013;Peng et al. 2017;Zuo et al. 2018).Notably, many extracts or compounds isolated from Aspidistra species (such as steroid saponins, flavonoids, and lectins) exhibit various valuable biological activities, including antibacterial (Liang et al. 2016) (Liang et al. 2018), antiviral, antitumor (Xu et al. 2015;Cui et al. 2016), antifungal (Koketsu et al. 1996), antioxidant (Singhatong 2017), cytotoxicity (Liang et al. 2016;Zuo et al. 2018), and anti-inflammatory activity (Sun et al. 2019).Our previous study reported on the cytotoxic activities of three new saponins, aspiletreins A-C, isolated from A. letreae, which was collected in Vietnam (Ho et al. 2020).However, a new species, Aspidistra triradiata was recently discovered in Vietnam in 2015 (Vislobokov 2015).Therefore, in this study, a new spirostanol steroid (1), and a new spirostanol steroidal saponin (2), along with three known compounds, including a saponin (3), and two steroids (4 and 5), are isolated from whole plants of A. triradiata collected in Vietnam.The chemical structures and cytotoxic activities of 1-5 are also reported herein (Figure 1).

Results and discussion
Compound 1 was obtained as a white, amorphous powder.Its molecular formula was determined to be C 27 H 44 O 7 based on the HRESIMS ion peak at m/z 503.2990 in conjunction with NMR data.The IR spectrum of 1 revealed absorption bands corresponding to hydroxy (3404 cm À1 ) and ether groups (1057 cm À1 ).
Compound 2 was isolated as a white, amorphous powder.The HRESIMS of 2 revealed a pseudo-molecular ion peak at m/z 871.4688 [M þ H] þ , and the resulting molecular formula was C 44 H 70 O 17 , in conjunction with NMR data analysis.The IR spectrum of 2 suggests the presence of hydroxy (3431 cm À1 ), double bond (1641 cm À1 ), and ether groups (1057 cm À1 ).
The monosaccharide composition of 2 was elucidated by acid hydrolysis of 2 followed by reduction, per-acetylated derivatization, and GCMS analysis.The results indicate that 2 contained three sugar units: L-rhamnose, L-arabinose, and D-glucose.These monoses were further determined to be L-rhamnopyranose, L-arabinofuranose, and D-glucopyranose moieties based on the corresponding resonances in the 1 H and 13 C NMR spectra (Table S1) and the correlations observed in the 1 H-1 H COSY and HMBC spectra (Figure S3).The coupling constant of J ¼ 8.5 Hz between H-1 0 (d H 4.90, d, J ¼ 8.5 Hz) and H-2 0 (d H 3.93, t, J ¼ 8.5 Hz) indicates a di-axial relationship between the two protons, and the b-configuration at the anomeric position of the D-glucopyranosyl unit.A comparison of the chemical shifts of C-2 00 (d C 82.8) and C-3 00 (d C 78.2) in 2 with those of the corresponding carbons of methyl aand b-arabinofuranoside successfully assigned the a-configuration to the anomeric proton of the arabinofuranosyl moiety of 2. Similarly, the a-configuration was assigned to rhamnopyranosyl by comparing the d C values of C-3 000 (d C 71.7) and C-5 000 (d C 69.6) of 2 with those of methyl aand b-L-rhamnopyranoside (Agrawal 1992).The HMBC correlations from H-1 0 to C-3, H-1ʺ (d H 5.87, s) to C-4 0 (d C 77.2), and H-1 0 '' (d H 5.29, s) to C-5 0 ' (d C 67.6), together with the ROESY correlations of H-1 0 to H-3 (d H 3.81) establishing the attachment of the a-Lrhamnopyranosyl-(1!5)-a-L-arabinofuranosyl-(1!4)-b-D-glucopyranosyl moiety at C-3 of the aglycone via a glycosidic linkage.Thus, the structure of 2 was determined to be (25 R)-17a-hydroxyspirost-5-en-3b-yl a-L-rhamnopyranosyl-(1!5)-a-L-arabinofuranosyl-(1!4)-b-D-glucopyranoside, which was named aspidiata B.
The cytotoxicity of 1-5 against LU-1, Hep-G2, MCF-7, and KB human cancer cell lines were assessed using the sulforhodamine B (SRB) assay.Compound 3 exhibited potent cytotoxicity against the tested human cancer cell lines, with IC 50 values ranging from 0.57 ± 0.05 to 1.23 ± 0.18 mM (Table S2).Compound 5 demonstrated a weak inhibitory effect against the LU-1 cell line with an IC 50 value of 95.81 ± 5.32 mM.Compounds 1, 2, and 4 did not show any significant cytotoxicity with IC 50 values greater than 100 mM.These results suggested that 3 should be further investigated for its anticancer activities.

Plant material
The whole plants of A. triradiata were collected from Quang Tri province, Vietnam (16 29 0 20.6 00 N 107 00 0 33.1 00 E) in March 2020, and were identified by Dr. Nguyen The Cuong, Institute of Ecology and Biological Resources, VAST, Vietnam.A voucher specimen (AT-01) was deposited at the Faculty of Pharmacy, Hue University of Medicine and Pharmacy, Vietnam.

Determination the monosaccharide compositions of compound 2
The monosaccharide constituents of 2 were determined using the method described by Pettolino et al. [Pettolino et al. 2012] with slight modifications.The preparation of alditol per-acetate derivatives was described in our previous studies [Ho et al. 2020].
Comparison of the GCMS retention times of the alditol per-acetate derivatives with the corresponding derivatives synthesized from standard sugars under similar conditions confirmed the presence of L-rhamnose, L-arabinose, and D-glucose in 2.

Sulforhodamine B assay for evaluating cytotoxic activity
The cytotoxic activities of the isolated compounds from whole plants of A. triradiata against the growth of four monolayer human cancer cell lines, LU-1, Hep-G2, MCF-7, and KB were determined using the SRB assay (Monks et al. 1991).Stock cultures were grown in T-75 flasks containing 50 mL of Dulbecco's modified Eagle's medium (DMEM) with 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, and 10% fetal bovine serum (FBS); the media were changed at 48 h intervals.The cells were dissociated with 0.05% trypsin-ethylenediaminetetraacetic acid (EDTA), sub-cultured every 3-5 days at a 1:3 ratio and incubated under a humidified 5% CO 2 atmosphere at 37 C. Tumour cells were cultivated in a humidified atmosphere of 5% CO 2 at 37 C for 48 h.Cell viability was examined using the SRB method to determine the cell density, depending on the cellular protein content.Viable cells were seeded into 96-well microplates containing growth medium (180 lL) at a density of 4 Â 10 4 cells per well and allowed to attach for 12 h.Test samples were carefully added to each well of the 96-well plates, and the cultivation was continued under the same conditions for another 72 h.Subsequently, the medium was removed, and the remaining cell monolayers were fixed with cold 20% (w/v) trichloroacetic acid at 4 C for 1 h and stained with 1X SRB staining solution at room temperature for 30 min.The unbound dye was eliminated by repeated washing with 1% (v/v) acetic acid.The protein-bound dye was dissolved in 10 mM Tris base to determine the optical density at 515 nm using an ELISA plate reader (BioTech, New York, NY, USA).Ellipticine was used as the positive control and 10% dimethyl sulfoxide (DMSO) was used as the blank sample.
The concentrations of the tested samples were in the range of 100-0.8lg/mL and estimated as the half-maximal inhibitory concentration (IC 50 ), which was calculated by the program TableCurve 2Dv4.All experiments were performed in triplicates.The inhibition rate (IR) of cells was calculated using the following formula: where IR is the inhibition rate of cell growth, OD t is the average optical density at day 3, OD 0 is the average optical density at time zero, and OD c is the average optical density of the blank DMSO control sample.

Supplementary material
HRESIMS, UV, IR and NMR spectra for 1 and 2 and their cytotoxicities are available as Supporting Information.

Figure 1 .
Figure 1.Structures of 1-5 isolated from the whole plants of Aspidistra triradiata.