Furostanol Saponins from Asparagus cochinchinensis and Their Cytotoxicity

Graphic Abstract Phytochemical investigation on the roots of Asparagus cochinchinensis led to the isolation of one new furostanol saponin, named 26-O-β-d-glucopyranosyl-22α-hydroxyl-(25R)-Δ5(6)-furost-3β,26-diol-3-O-α-l-rhamnopyranosyl-(1 → 2)-[β-d-glucopyranosyl-(1 → 4)-α-l-rhamnopyranosyl-(1 → 4)]-β-d-glucopyranoside (1), along with three known congeners (2‒4). The structure of new saponin was elucidated via comprehensive inspection of its HRMS and NMR spectral data as well as chemical technology, whereas those of known ones were identified by comparison of their NMR and MS spectral data with those reported in literatures. All isolated saponins were evaluated for their cytotoxic effects on two human liver (MHCC97H) and lung adenocarcinoma (H1299) cancer cells in vitro. Among them, both 1 and 2 showed significant cytotoxicity against above mentioned cell lines. Further studies revealed that these two saponins could significantly inhibit their proliferation of MHCC97H and H1299 cells. Supplementary Information The online version contains supplementary material available at 10.1007/s13659-021-00321-0.


Introduction
Steroid saponins, whose aglycones were usually a spirostanol or its derivatives [1], were commonly found from roots, tubers, leaves, blooms or seeds in more than 100 families of plants [2,3]. Compared with other glycosides, the strong foam-forming property in aqueous solution of steroidal saponins was their main feature [2,4]. Previous researches revealed steroidal saponins possessed various pharmacological activities, such as antifungal [5], hypocholesterolemic [6], antimitotic [7] and cAMP phosphodiesterase inhibitory [8] effects. Among them, a large number of publications have revealed steroid saponins shared different 1 3 cytotoxic properties that promoted their potential as anticancer drugs or adjuvants [9,10].
Asparagus cochinchinensis, belonging to the genus Asparagus (Liliaceae), is well-known as "Tianmendong" in China. Its roots have been historically used in Chinese folk medicine for the treatment of cough, acute and chronic bronchitis, chronic pharyngitis, hemorrhoids, and tumors for thousands of years [11]. Apart from steroidal saponins [12], phenolic compounds [13], norlignans [14] and alkaloids [15] have been isolated from this plant as revealed by previous phytochemical studies. However, steroidal saponins obtained from title species were proved to be its major and bioactive components responsible for its cytotoxic [16], antiinflammatory [17], hepatotoxic and nephrotoxic [18], and anti-neuroinflammatory [11] properties. In continuation of a search for bioactive constituents from plants of the Yunnan province [19], a chemical investigation was performed on the roots of A. cochinchinens. As a result, a total of steroidal saponins (1-4) were isolated and identified including one new and three previously described furostan-type steroidal saponins. Their cytotoxic effects on two human cancer cells MHCC97H and H1299 were also evaluated ( Fig. 1). It showed a positive reaction to the Ehrlich's reagent (red color), suggesting a furostanol skeleton [20]. The 13 C NMR spectrum (Table 1) -16); an olefinic group at δ H 5.26 (1H, brs) as well as δ C 121.6 (CH-6) and 140.6 (C-5); and a ketal carbon at δ C 110.4 (C-22). The abovementioned data indicated that the aglycone of 1 should be a furostanol one as that of protodioscin (2) [21]. Moreover, the aglycone of 1 was further confirmed a by the following diagnostic 1 16) indicated these protons were located at the other side. Additionally, the 25R configuration of 1 was assigned according to the small chemical shift difference between Ha-26 and Hb-26 at Δab = 0.34 ppm (Δab > 0.57 ppm for 25S, and Δab < 0.48 ppm for 25R) [22]. In view of aforementioned  evidence, the aglycone of 1 was thus elucidated as 22α-hydroxyl-(25R)-furost-Δ 5(6) -3β,26-diol. As for the sugar units of 1, its 1 H NMR spectrum (Table 1)  Additionally, three known steroidal glycosides were identified as protodioscin (2) [23], and dioscoreside H (4) [24] by comparison of their spectroscopic data with those reported in the literatures.

Results and Discussion
The steroid saponins obtained from species of Liliaceae have shown the potential to significantly inhibit the proliferations of various human tumor cell lines in vitro [25][26][27][28][29]. Therefore, all isolated compounds were evaluated for their cytotoxicity against MHCC97H and H1299 by the MTT method. More specifically, compared with the IC 50 values of positive control doxorubicin hydrochloride, and both 1 and 2 displayed strong cytotoxicity against MHCC97H and H1299 cells with IC 50 values of 3.56 ± 0.45/4.18 ± 0.43 μg/mL and  Table 3) revealed that saponins 1-4 showed moderate antimicrobial activity against C. albicans and B. subtilis, while only saponin 3 showed weak antimicrobial activity against S. aureus (63.30 ± 0.55 μg/mL).

General Experiment Procedures
Optical rotation was measured on a Autopol VI automatic polarimeter. The IR spectrum were measured on a Thermo Nicolet iS10 infrared spectrophotometer with KBr disk. The NMR spectra were obtained on Bruker DRX-400 and DRX-600 spectrometers. Chemical shifts (δ) were expressed in ppm with reference to the solvent signals. Both ESI and HRESIMS spectra were performed on an UPLC-IT-TOF spectrometer. Semi-preparative HPLC was performed on a Waters 600 with a COSMOSIL C18 (10 × 250 mm, Nacalai Tesque Corporation, Japan) column. Analytical HPLC was performed on a Shimadzu SIL-20A Series HPLC system equipped with a reverse-phase COSMOSIL C18 column (4.6 mm × 250 mm, 5 μm, Nacalai Tesque Corporation, Japan). Column chromatography was carried out using silica gel (100-200 mesh, Qingdao Haiyang Chemical, Qingdao, Co., Ltd., People's Republic of China) and macro-porous absorption resin (D101, Donghong Chemical Co., Ltd., People's Republic of China). The PMP (Chengdu Aikeda Chemical Reagent Co., Ltd., China) was purchased from Beijing 4A Biotech Co., Ltd. (Beijing, China). Fractions were monitored by TLC, and spots were visualized by heating silica gel plates sprayed with Ehrlich's reagent.

Extraction and Isolation
The air-dried roots of A. cochinchinensis (5.0 kg) were extracted with 90% aqueous EtOH at 80 ℃ (15 L × 4, each time for 3 h). The solvent was removed under reduced pressure to yield an amber residue (2.5 kg). The residue was subjected to column chromatography over an macroporous resin column eluted first with H 2 O then successively with 25%, 70%, and 90% EtOH, respectively. The 70% EtOH partition was evaporated under reduced pressure to obtain a total

Acid Hydrolysis of 1
The acid hydrolysis of compound 1 was carried out by a previously reported procedure [19]. Compound 1 (2.0 mg) was refluxed at 120 °C for 2 h with 2 M TFA on an oil bath. The aglycone was removed by the extraction with CHCl 3 (5.0 mL) for three times. The reaction residue was filtered after neutralizing with 60.0 μL of NaOH (0.3 M). After removing the solvent under reduced pressure, the residue was refluxed at 75 °C for 1 h with 60.0 μL of PMP (0.5 M in methanol). Moreover, the reaction was quenched with 60.0 μL of HCl (0.3 M) and the reaction mixture was extracted with CHCl 3 (5.0 mL, three times). Then, the aqueous layer was analyzed over HPLC (18% acetonitrile: 82% sodium phosphate (pH 6.8; 1.5 mL/min). Likewise, the standard monosaccharides d-glucose (1.0 mg) and l-rhamnose (1.0 mg) were derivatized with PMP by the same way as 1, and HPLC analyses were performed under the same conditions as 1. The sugar units in 1 were identified as d-glucose (t R = 14.5 min) and l-rhamnose (t R = 17.0 min) by comparison of the retention times of the corresponding derivatives.

Antimicrobial Activity Assay
The antimicrobial activity of isolated steroid saponins against 10 strains using the microdilution broth susceptibility assay [31]. The strains frozen in the refrigerator at -80 ℃ were activated and inoculated on standard tryptone soy broth agar (TSA) plates at 37 ℃ for 8 h to observe the bacterial growth. Subsequently, single colonies were selected and inoculated in tryptone soy broth (TSB) plates. After cultivated at 37 ℃ in shaker (120 rpm) for 8 h, the absorbance of bacterial solution was measured and its concentration was adjusted to 10 5 CFU/mL. Whereafter, an inoculum of 10 5 CFU/mL was made to sterile 96-well plate containing tested compounds at different concentrations (100.00, 50.00, 25.00, 12.50, 6.25 and 3.13 μg/mL) at 37 ℃ for 8 h. The wells containing only broth served as growth control. The absorbance of bacterial solution was recorded on a microplate reader at 600 nm.