Search for new steroidal glycosides with anti-cancer potential from natural resources

Chemical investigations of higher plants, with particular attention paid to their steroidal glycosides, present a promising approach for generating anti-cancer agents from natural products. We conducted a systematic phytochemical investigation of nine higher plants—whole plants and rhizomes of Convallaria majalis, whole plants of Agave utahensis, roots of Adonis amurensis, seeds of Adonis aestivalis, bulbs of Bessera elegans, bulbs of Fritillaria meleagris, seeds of Digitalis purpurea, underground parts of Yucca glauca, and bulbs of Lilium pumilum—which led to the discovery of novel steroidal glycosides. The structures of these new constituents were determined based on spectroscopic data and chemical transformations. The identification of the monosaccharides including their absolute configurations was carried out by direct HPLC analysis of their hydrolysates using an optical rotation detector. Cytotoxicity of the isolated steroidal glycosides was evaluated against various tumor cells (A549, ACHN, HepG-2, HL-60, HSC-2, HSC-3, HSC-4, HSG, and SBC-3) and normal cells (Fa2 N-4, HK-2, and TIG-3 cells). Certain steroidal glycosides exhibit selective cytotoxicity and synergistic effects, making them potential lead compounds for use as anti-cancer agents. We document the isolation of 139 steroidal glycosides from higher plants and assessment their cytotoxic activities. Graphical abstract


Introduction
Steroidal glycosides constitute a group of natural products found in a limited range of plant families, including Agavaceae, Leguminosae, Liliaceae, Plantaginaceae, Ranunculaceae, and Solanaceae.Predominantly present as glycosides in nature, steroidal glycosides display a diverse array of chemical structures.These compounds are categorized as spirostanol, furostanol, pregnane, cardenolide, and cardiac glycosides, based on their aglycone structures.Furthermore, steroidal glycosides from higher plants have demonstrated various biological activities, including antitumor, antidiabetic, antitussive, and inhibition of platelet aggregation [1,2].In vitro studies have identified cytotoxic activities against tumor cell lines including A549, Caco-2, Hela, HepG-2, HL-60, HT-29, MCF-7, PC-3, SGC-7901, and U251, showing mechanisms, such as apoptosis, necrosis, autophagy, ferroptosis, and cell cycle arrest [3][4][5][6].Terrestrosin D, Fig. 3 Chemical transformations of 6 a spirostanol glycoside isolated from Tribulus terrestris, has shown to induce cell cycle arrest in the G 1 phase and stimulate the caspase-independent apoptosis of PC-3 cells [5].A novel spirostanol glycoside T-17, isolated from Tupistra chinensis, induces apoptosis and triggers cytoprotective autophagy in human gastric cancer cell lines by activating the JNK pathway [7].Thus, a large amount of cytotoxic data against a variety of tumor cells were performed and the molecular mechanism of steroidal glycosides was elucidated.Although in vivo experiments on their anti-cancer activities are limited, certain steroidal glycosides have been shown to exhibit anti-cancer effects.For instance, oleandrin (administered at 0.3 or 0.6 mg/ kg, i.p., 7 days) suppressed tumor growth in mice bearing EMT6 murine mammary carcinoma [8].In another study, cotreatment of glioma bearing mice with temozolomide (TMZ) and oleandrin (administered at 0.3 mg/kg, i.p., 100 days) strongly prolongs mice survival compared with TMZ treatment alone [9].Garofalo et al. demonstrated the potential of oleandrin as a co-adjuvant drug in standard chemotherapeutics.Additionally, OSW-1, a steroidal cholestane diglycoside isolated from Ornithogalum saundersiae, significantly boosted the anti-metastatic ability of doxorubicin in 4T1 mice and enhanced CD8 + T cell infiltration into the immune microenvironment of the lungs [10].Immune checkpoint inhibitors (ICIs) and molecular targeted therapeutic agents have emerged as promising tools for cancer therapy.However, resistance to ICIs can develop in some patients, leading to a lack of an objective response [11].Between 1981 and 2019, 25% of all the anti-cancer drugs approved by the Food and Drug Administration were derived from natural products or natural product derivatives [12], highlighting the potential of novel anti-cancer agents in chemotherapy.Steroidal This review presents our previous phytochemical studies on steroidal glycosides from nine plant sources: the whole plants and rhizomes of Convallaria majalis (Liliaceae) [13][14][15], whole plants of Agave utahensis (Agavaceae) [16][17][18], roots of Adonis amurensis (Ranunculaceae) [19,20], seeds of Adonis aestivalis (Ranunculaceae) [21,22], bulbs of Bessera elegans (Liliaceae) [23], bulbs of Fritillaria meleagris (Liliaceae) [24], seeds of Digitalis purpurea (Scrophulariaceae) [25][26][27][28], underground parts of Yucca glauca (Agavaceae) [29], and bulbs of Lilium pumilum (Liliaceae) [30].These studies also investigated the cytotoxic activities of the isolated compounds against tumor cells and normal cells.Plants belonging to the families Liliaceae and Ranunculaceae are particularly rich sources of bioactive steroidal glycosides, such as OSW-1 or galtonioside A, which exhibit cytotoxic activity against tumor cells [31][32][33].Furthermore, potent cytotoxic compounds have been discovered in popular ornamental garden plants, such as Bessera elegans and Fritillaria meleagris [23,24].These results indicate that garden plants with no proven medicinal backgrounds may also be potential sources of novel drugs.

Pregnane glycosides
Pregnane glycosides, initially isolated from Digitalis purpurea and termed "digitanol glycosides", are now found in various Apocynaceae, Asclepiadaceae, Malpighiaceae, Ranunculaceae, and Zygophyllaceae species [34].Structurally, pregnane glycosides consist of six-membered A-C rings and a five-membered D ring in the C21 steroid skeleton, commonly featuring deoxy sugars linked to C-3 of the aglycone.The C/D cis structure of pregnane glycoside was synthesized via the cardenolide route.Based on the substituents and degree of oxidative cracking of the aglycone ring, the skeleton of the aglycone structure is divided into diverse structures.Various types of pregnane skeletons show various biological activities, including antibacterial, antidiabetic, anti-inflammatory, anti-obesity, and antitumor activities [34].
Chemical transformations were conducted to support the chemical structure determination of steroidal glycosides, including spirostanol, furostanol, pseudo-furostanol, and pregnane (Fig. 3).The new pregnane glycoside (6), isolated from the whole plants of Convallaria majalis, was formed from a pseudo-furostanol glycoside through the oxidative cleavage of the C-20 (22) double bond.This was confirmed by the fact that the peracetate (6b) of 6 was identical to the product obtained by treating furostanol glycoside (124) with acetic anhydride (Ac 2 O) in pyridine at 110 ℃ for 3 h followed by treatment with CrO 3 in acetic acid (AcOH).Compound 6 was subjected to alkaline methanolysis with 7% sodium methoxide to obtain pregnane glycoside (7) and 5-[(β-D-glucopyranosyl)oxy]-4-methyl pentanoic acid (6a).Conversely, acid hydrolysis of 124 with 1 M HCl yielded a spirostanol steroid (107a) as the aglycone and D-galactose, D-glucose, and D-xylose as carbohydrate moieties [14].Identification of the monosaccharides was conducted by direct HPLC analysis of the hydrolysate using a combination of refractive index and optical rotation detector.Compounds 6 and 7, isolated from the whole plants of Convallaria majalis, did not show cytotoxic activity against HL-60 human promyelocytic leukemia cells, A549 human lung adenocarcinoma cells, HSC-4, and HSC-2 human oral squamous cell carcinoma cells (IC 50 > 12 μM).

Cardenolide glycosides
Cardenolide glycosides are a group of naturally occurring compounds found in several plant families including Apocynaceae, Liliaceae, Ranunculaceae, and Scrophulariaceae.Cardenolide has α,β-unsaturated butyrolactone ring at C-17 of the aglycone, a 14β-hydroxy group at C-14, and a C/D cis ring connection in the C23 steroid skeleton.The oligoglycosides attached to the C-3 of the aglycones comprise deoxysugars, which are typical of plant pregnane and cardiotonic glycosides.Cardenolide glycosides selectively inhibit Na + /K + -ATPase pumps.Recent studies demonstrated the specific cytotoxicity of cardenolide glycosides against renal adenocarcinoma and hepatocellular carcinoma cells, indicating their potential as candidates for anti-cancer drug development.Strophanthidin, a cardiac glycoside isolated from Strophanthus kombe, induces apoptosis by attenuating multiple biochemical signaling pathways and arresting the cell cycle at the G2/M phase through p53-dependent and -independent mechanism in A549, HepG-2, and MCF-7 cells [40].Furthermore, strophanthidin inhibited autophagy by inhibiting the expression of the LC3 and p62 complexes in A549 cells [3].Oleandrin, a cardiac glycoside isolated from Nerium oleander, induces apoptosis in SW480 human colorectal cancer cells via the mitochondrial pathway, without significantly reducing the viability of NCM460 normal human colonic epithelial cells [4].
Compounds 60 and 62 showed high selectivity index (SI) values (> 5.9 and 10) between the SBC-3 tumor cells and TIG-3 normal cells.Compound 60 induced apoptotic cell death along with caspase-3 activation in SBC-3 cells, whereas 62 did not induce any apoptotic features in SBC-3 cells.Combining each of 58 and 60-62 (0.1 and 1.0 μM) with etoposide (0.01 and/or 0.1 μM) showed a synergistic effect (combination index (CI) values 0.17-0.50)against SBC-3 cells.Notably, the combination of gitonin (58) and etoposide achieved a strong synergistic effect (CI 0.27-0.43),although this synergistic effect was reduced by the introduction of a hydroxy group at the C-15 position (59).Among 58-63, gitonin (58) notably released high mobility group box (HMGB)-1 in the preliminary screening assay.HMGB-1 is a DNA-binding protein in the nucleus; when released by dying cells, it acts as a damage-associated molecular pattern (DAMP) to trigger a strong inflammatory response [46].Furthermore, the combination of 58 and etoposide resulted in an increase in the extracellular release of DAMPs, including the release of HMGB-1, secretion of ATP, and exposure of calreticulin (CALR) in SBC-3 cells.DAMPs can induce immunogenic cell death (ICD), a type of tumor cell death, and play a major role in stimulating the immune system in cancer therapy.These data indicate that 58, either alone or in combination with etoposide, could induce immunogenic cell death (Fig. 9).Recently, Li et al. achieved the total synthesis of gitonin (58) for the first time from readily commercially available tigogenin and evaluated the cytotoxicity of gitonin and its structural analogs against A549, HepG-2, and MCF-7 cells [47].The trisaccharide analog of gitonin exhibited cytotoxicity comparable to that of gitonin, indicating that the molecular structure of the branched tetrasaccharide of gitonin may not be essential for its cytotoxic activity.
Eight steroidal glycosides (64-71) isolated from the bulbs of B. elegans and F. meleagris, including five new compounds (64-68), were evaluated for their cytotoxic activities against HL-60 and A549 tumor cells, as well as TIG-3 normal cells [23,24] (Fig. 8).Compounds 64, 68, and 69-70 showed cytotoxic activity against both HL-60 and A549 cells, demonstrating selective cytotoxicity toward tumor cells.Consistent with previous findings [48,49], aglycones 64a, 65a, 67a, 68a, and 69a lacked cytotoxic activity against all tested cell lines.Comparisons of their cytotoxic activities indicated that introducing a hydroxy group to the C-14α position reduced cytotoxicity (64 vs. 65 and 66 vs. 70), whereas the presence of a C-2α hydroxy group or C-12 ketone group did not affect cytotoxic activity (61 vs. 62, 68 vs. 69).Preliminary analysis suggests that the presence of a C-6 hydroxy group or C-6 O-glucosyl group significantly reduces the cytotoxic activity of spirostanol glycosides [50,51].This study further supports the notion that the introduction of a hydroxy group to steroidal aglycones reduces their cytotoxic activity (Fig. 10).
Compounds 130-136 were isolated from the bulbs of B. elegans (Fig. 17  exhibited selective cytotoxicity toward HL-60 and A549 cells (IC 50 0.5-6.2μM) while demonstrating no significant impact on cell growth in TIG-3 normal cells (IC 50 > 10 μM) [23].The findings suggest that the presence of a C-2α hydroxy group does not influence cytotoxic activity, whereas the introduction of a hydroxy group to the C-14α position decreases cytotoxicity.Notably, pseudo-furostanol glycoside 136 selectively inhibited tumor cell growth in a timedependent manner and induced apoptosis in HL-60 and A549 cells.Furthermore, 136 induced cell cycle arrest at the G0/G1 phase in A549 cells.

Conclusions and discussions
We conducted chemical investigations of higher plants, focusing on steroidal glycosides.Our ongoing research has led to the discovery of several steroidal glycosides in ornamental garden plants and medicinal herbs that exhibit cytotoxic activity or possess novel skeletons.Screening and systematic chemical analysis of plant extracts are thus promising avenues for uncovering the structural diversity of steroidal glycoside, despite the time investment required.Structure-activity relationships (SAR) are frequently observed for spirostanol and furostanol glycosides.The introduction of polar substituents, such as hydroxy, carbonyl, and glucosyl groups, to the aglycone moiety has been observed to diminish cytotoxicity.Notably, the presence of a hydroxy group in the axial position of the aglycone moiety may influence the cytotoxicity.While replacement of the terminal sugar in the sugar sequence at the C-3β hydroxy group of the aglycone had minimal impact on activity, the inner sugar attached to C-3 of the aglycone played a significant role in cytotoxicity.In our study, steroidal glycosides demonstrated cytotoxicity against various tumor cells (A549, ACHN, HepG-2, HL-60, HSC-2, HSC-3, HSC-4, HSG, and SBC-3) through diverse mechanisms, including necrosis, caspase-dependent or -independent apoptosis, cell proliferation arrest, and the induction of DAMPs release.Notably, the spirostanol glycosides showed synergistic cytotoxicity when combined with etoposide, whereas this synergistic effect was not observed for the corresponding furostanol glycosides.The identification of potential novel anti-cancer agents requires precise SAR determination and in vivo evaluation of steroidal glycosides.In addition, cytotoxicity assays against normal cell lines have not been sufficiently reported, and the selectivity of steroidal glycosides toward cancer cells needs to be studied.Further detailed studies on the cytotoxicity mechanisms, such as autophagy and ferroptosis, molecular targets, and chemical structures, are warranted.

Fig.
Fig. Structures of pregnane glycosides 1-5 isolated from the seeds of Digitalis purpurea and 6 and 7 from the whole plants of Convallaria majalis

Fig. 7
Fig. 7 Structures of cardenolide glycosides 46-49 and 55 from the roots of Adonis amurensis, 50-54 from the seeds of Adonis aestivalis, and 56 and 57 from the rhizomes of Convallaria majalis

Fig. 8
Fig. 8 Structures of 5α-spirostanol glycosides 58-63 from the seeds of Digitalis purpurea, 64-70 isolated from the bulbs of Bessera elegans, and 71 from the bulbs of Fritillaria meleagris

Fig. 11
Fig. 11 Structures of 5β-spirostanol glycosides 72-79 isolated from the whole plants of Agave utahensis and 80-91 isolated from the underground parts of Yucca glauca ◂