Aspidiatas C and D, two new spirostanol saponins from Aspidistra triradiata and their cytotoxic activities

Abstract Aspidiatas C and D (1 and 2), two new spirostanol saponins, were isolated along with two known compounds, (25 R*)-spirost-5-en-3β-yl α-L-rhamnopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→4)-[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside (3), (25 R*)-spirost-5-en-3β-yl α-L-rhamnopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranoside (4) from the whole plant of Aspidistra triradiata collected in Vietnam. The chemical structures were determined by HRESIMS, 1D- and 2D-NMR analysis, and comparison with published data. Compound 3 exhibited potent cytotoxicity against MCF7, HepG2, SK-LU-1, and HT-29 human cancer cell lines with IC50 values ranging from 0.19 to 0.65 µM. Compounds 1, 2, and 4 displayed moderate cytotoxic effects with IC50 values ranging from 12.32 to 82.27 µM. Compounds 1–4 were isolated from the genus Aspidistra for the first time. GRAPHICAL ABSTRACT


Introduction
Aspidistra species are widespread herbs in the tropical and subtropical forests of Southeast Asia.The genus Aspidistra ranges from the Assam region of India to southern Japan and from central China as far as the Malay peninsula, but the center of diversity is Guangxi province of China and northern Vietnam (Tillich and Averyanov 2012).Botanical studies of this genus began in 1822 (Vislobokov et al. 2019).These studies have developed with the rapid increase in the number of species in the past two decades.Vietnam has a variety of Aspidistra with at least 25 recently reported species from 2014 to 2016 (Averyanov et al. 2017).As of 2019, the genus Aspidistra includes approximately 170 species, comprising about 60 endemic species to Vietnam (Vislobokov et al. 2019).
The chemical composition of the genus Aspidistra contains saponins, lectins, xanthones, coumarins, and flavonoids.Recently steroid saponins gained research attention with approximately 90 secondary metabolites (Cui et al. 2013;Peng et al. 2014Peng et al. , 2017;;Zuo et al. 2018).Previous studies have also revealed that the Aspidistra species such as A. elatior, A. typica, A. sichuanensis, and A. letreae have been known to be biologically active plants with various significant biological activities such as antibacterial (Xiaoxia Liang et al. 2016;Liang et al. 2018), antifungal (Koketsu et al. 1996), antiviral, antitumor (Xu et al. 2015;Nguyen et al. 2021), antioxidant (Singhatong 2017;Ho et al. 2021), cytotoxicity (Zuo et al. 2018;Ho et al. 2020), and anti-inflammatory (Sun et al. 2019) activities.Traditionally, these species have been used for the treatment of weakness, fatigue, back pain, injury, hypercholesterolemia, diarrhea, and snakebites (Cui et al. 2016;Singhatong 2017;Liang et al. 2018).Previously, we reported the isolation of two new compounds aspidiata A and aspidiata B, together with three known compounds from A. triradiata and their cytotoxic activities against the four cancer cell lines (Bich Thi Le et al. 2022).In our continuous effort to search for novel anticancer agents from A. triradiata, we recently isolated two new spirostanol saponins (1 and 2), two known compounds (3-4) and evaluated the cytotoxicity of all isolates against four human cancer cell lines, including MCF7 (human breast carcinoma), HepG2 (human hepatocarcinoma), SK-LU-1 (human lung carcinoma) and HT-29 (human colorectal carcinoma) (Figure 1).

Results and discussion
Compound 1 was obtained as a white, amorphous powder.Based on the combination of NMR data analysis and the appearance of a molecular ion cluster at m/z 853.4600 [M-H] -on the HRESIMS, the molecular formula of 1 was determined to be C 44 H 70 O 16 .The IR absorption bands of 1 indicated the presence of hydroxyl, double bond, and ether functional groups at 3445, 1638, and 1074 cm −1 , respectively.

Plant material
The whole plant of A. triradiata was collected from Quang Tri province, Vietnam (16° 29′ 20.6"N 107° 00′ 33.1"E) in March 2020, and was 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, Hue University, Vietnam.

Determination of the monosaccharide compositions of 1 and 2
Detailed analyses of monosaccharide derivatization were evaluated using Pettolino et al. method (Pettolino et al. 2012) with minor modifications.The alditol per-acetate derivatives were prepared by reactions including hydrolysis, reduction, and acetylation.The procedure was performed as previously described (Ho et al. 2020).The structures of sugar units of 1 and 2 were determined using GCMS.The retention times of the alditol per-acetate derivatives consistently corresponded to derivatives synthesized from standard sugars.

Sulforhodamine B assay for determining cytotoxic activity
The cytotoxic activity of isolated compounds against the four human cancer cell lines, MCF7, HepG2, SK-LU-1, and HT-29 (Monks et al. 1991) was performed using Sulforhodamine B (SRB) assay.The cells were cultured with Dulbecco's modified eagle medium (DMEM) containing 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, and 10% fetal bovine serum (FBS) and incubated at 37 °C, 5% CO 2 until 80% confluency.The cells were detached using 0.05% (w/v) trypsin-EDTA.A density of 4 × 10 4 cells/well were seeded into each well.The plates were further incubated for 12 h.Then, the various concentrations of isolated compounds were added gently and incubated for 3 days.After removing the medium, the cold 20% (w/v) trichloroacetic acid was added to fix the cell for 1 h at 4 °C.1X SRB solution was added to each well and kept at room temperature for 30 min.The cancer cells were washed with 1% (v/v) acetic acid to remove the unbound dye while the protein-bound dye was dissolved in a 10 mM Tris base.The optical density was recorded at 515 nm using an ELISA Plate Reader.Ellipticine and 10% dimethyl sulfoxide (DMSO) were used as a positive control and the blank, respectively.The cytotoxic data was calculated and expressed as the half maximal inhibitory concentration (IC 50 ) using TableCurve 2Dv4 software.All experiments were performed in triplicates.The cell inhibition rate (IR) was calculated by the following formula

Conclusions
In this present study, the chemical investigation of the whole plant of In continuing our study on A.triradiata, these findings further elucidated the phytochemical analysis as well as cytotoxic activities of this species.

Figure 1 .
Figure 1.structures of 1-4 isolated from the whole plant of Aspidistra triradiata.
0 and OD t are the average optical density value at time-zero and day 3, respectively, and OD c is the average optical density value of the blank.