Steroidal glycosides from Ornithogalum thyrsoides bulbs and their cytotoxicity toward HL-60 human promyelocytic leukemia cells and SBC-3 human small-cell lung cancer cells.

Ornithogalum thyrsoides Jacq belongs to the Asparagaceae family and is cultivated for ornamental purposes. The authors have previously reported several cholestane- and spirostan-type steroidal glycosides from O. thyrsoides. Conventional TLC analysis of the methanolic bulb extract of O. thyrsoides suggested the presence of unprecedented compounds; therefore, a detailed phytochemical investigation of the extract was performed and 35 steroidal glycosides (1-35), including 21 previously undescribed ones (1-21) were collected. The structures of 1-21 were determined mainly by analyses of their 1H and 13C NMR spectra with the aid of two-dimensional NMR spectroscopy. The isolated compounds were classified into three distinct groups: furostan-type (1, 2, 8-12, and 22), spirostan-type (3-7 and 23-26), and cholestane-type (13-21 and 27-35). Although the C/D-ring junction of the steroidal skeleton is typically trans-oriented, except for some cardiotonic and pregnane-type steroidal derivatives, 7 possess a cis C/D-ring junction. This is the first reported instance of such a configuration in spirostan-type steroidal derivatives, marking it as a finding of significant interest. Compounds 1-35 were evaluated for cytotoxicity against HL-60 human promyelocytic leukemia cells and SBC-3 human small-cell lung cancer cells. Compounds 3-6, 9, 17-21, 23-25, and 30-35 demonstrated cytotoxicity in a dose-dependent manner with IC50 values ranging from 0.000086 to 18 µM and from 0.00014 to 37 µM toward HL-60 and SBC-3 cells, respectively. Compound 19, which is obtained in a good yield and shows relatively potent cytotoxicity among the undescribed compounds, induces apoptosis in HL-60 cells, accompanied by arresting the cell cycle of HL-60 cells at the G2/M phase. In contrast, 19 causes oxidative stress-associated necrosis in SBC-3 cells. The cytotoxic mechanism of 19 is different between HL-60 and SBC-3 cells.

OSW-1 inhibits tumor growth and metastasis by NFATc2 on triple-negative breast cancer.

OSW-1 is a natural compound extracted from the bulbs of Ornithogalum saundersiae in 1992. It has been shown strong antitumor activities in various cancer cells. However, the effects of OSW-1 on tumor growth and metastasis in breast cancer are still poorly understood. In our research, we showed that OSW-1 had a strong anticancer effect on breast cancer cells, but lower toxicity to normal cells. Accordingly, it also revealed significant inhibition of tumor growth by OSW-1 in xenograft model. In addition, we performed Annexin V/PI-labeled flow cytometric assay and TUNEL assay and showed that OSW-1 inhibited tumor growth by inducing apoptosis. Furthermore, we carried out transwell assays and found that OSW-1 significantly repressed the migratory and invasive capabilities of triple-negative breast cancer (TNBC) cells via mediating epithelial-mesenchymal transition. Besides, OSW-1 also could inhibit metastasis in an orthotopic model and resulted in a longer survival compared with control group. Finally, we performed RNA-sequencing and cellular functions to investigate the molecular mechanism of how OSW-1 inhibits TNBC, and identified NFATc2 may as a pivotal factor for OSW-1-mediated effects on cell death, tumor growth, invasion, and migration.

Optimization of polysaccharides extraction from a wild species of Ornithogalum combining ultrasound and maceration and their anti-oxidant properties.

Polysaccharides were extracted from a wild species of Ornithogalum by using three methods: maceration, ultrasound-assisted extraction, and combination of maceration and ultrasound. Extraction conditions were optimized by using response surface method (RSM) with a central composite design (CCD). The optimal extraction yield was 81.7%, 82.5% and 85.7%, and the optimal polysaccharides yield was 74.7%, 75.7%, and 82.8% under the optimum conditions of maceration, ultrasound-assisted extraction and combined extraction, respectively. These results indicate that the combination method significantly improves the extraction and polysaccharides yields compared to traditional extraction methods. The combination method also allows reducing the time of ultrasound treatment and thus its adverse effects on polysaccharides. In addition, these results well corroborate with the theoretically predicted values. The NMR (1H,13C, HSQC, HMBC, and COSY) analysis shows that the extract is composed of fructo-polysaccharides with a backbone of (2 â†’ 6)-linked ß-d-fructofuranosyl (Fruf) and (2 â†’ 1)-linked ß-d-Fruf branched chains, and terminated with glucose and fructose residues. The antioxidant activities of the extract were evaluated from ABTS radical scavenging activity, total antioxidant capacity, metal-chelating power and ß-carotene bleaching test. Data show that the extract presents outstanding antioxidant activities.

OcUGT1-Catalyzing Glycodiversification of Steroids through Glucosylation and Transglucosylation Actions.

Steroidal glycosides are important sources of innovative drugs. The increased diversification of steroidal glycosides will expand the probability of discovering active molecules. It is an efficient approach to diversify steroidal glycosides by using steroidal glycosyltransferases. OcUGT1, a uridine diphosphate-d-glucose (UDP-Glc)-dependent glycosyltransferase from Ornithogalum caudatum, is a multifunctional enzyme, and its glycodiversification potential towards steroids has never been fully explored. Herein, the glycodiversification capability of OcUGT1 towards 25 steroids through glucosylation and transglucosylation reactions were explored. Firstly, each of 25 compounds was glucosylated with UDP-Glc. Under the action of OcUGT1, five steroids (testosterone, deoxycorticosterone, hydrocortisone, estradiol, and 4-androstenediol) were glucosylated to form corresponding mono-glucosides and biosides. Next, OcUGT1-mediated transglucosylation activity of these compounds with another sugar donor ortho-nitrophenyl-ß-d-glucopyranoside (oNPGlc) was investigated. Results revealed that the same five steroids could be glucosylated to generate mono-glucosides and biosides by OcUGT1 through transglucosylation reactions. These data indicated that OcUGT1-assisted glycodiversification of steroids could be achieved through glucosylation and transglucosylation reactions. These results provide a way to diversify steroidal glycosides, which lays the foundation for the increase of the probability of obtaining active lead compounds.

Steroidal glycosides from Ornithogalum dubium Houtt.

The phytochemical study of Ornithogalum dubium Houtt. (Asparagaceae) led to the isolation of five undescribed steroidal glycosides together with two known ones. Their structures were established by using NMR analysis and mass spectrometry as (25R)-3ß-hydroxyspirost-5-en-1ß-yl O-α-L-arabinopyranosyl-(1 → 2)-α-L-rhamnopyranoside, (25S)-3ß-hydroxyspirost-5-en-1ß-yl O-ß-D-glucopyranosyl-(1 → 6)-ß-D-glucopyranoside, (22S)-16ß-[(α-L-rhamnopyranosyl)oxy]-22-hydroxycholest-5-en-3ß-yl O-ß-D-glucopyranosyl-(1 → 4)-ß-D-glucopyranoside, (22S,23S)-1ß,3ß,11α,16ß,23-pentahydroxy-5α-cholest-24-en-22ß-yl ß-D-glucopyranoside, (22S,23S)-3ß-[(ß-D-glucopyranosyl)oxy]-22,23-dihydroxy-5α-cholest-24-en-16ß-yl O-α-L-rhamnopyranosyl)-(1 → 4)-ß-D-glucopyranoside. Their cytotoxic activities against two human cells, a lung carcinoma A-549 and a promyelocytic leukemia HL-60 cell lines, were evaluated by using the XTT method. The results showed no significant cytotoxicity on the tested cells. The influence of the potentiation of cisplatin cytotoxicity in A-549 cells was also investigated and a slight effect was observed only for the (25R) spirostane-type derivative.

Cholestane glycosides from Ornithogalum saundersiae bulbs and the induction of apoptosis in HL-60 cells by OSW-1 through a mitochondrial-independent signaling pathway.

A search for cytotoxic cholestane glycosides from Ornithogalum saundersiae bulbs resulted in the isolation of three new OSW-1 analogues (1-3), a new cholestane bisdesmoside (4), a 5ß-cholestane diglycoside (5), and four new 24(23 → 22)-abeo-cholestane glycosides (6-9), together with 11 known cholestane glycosides (10-20), including OSW-1 (11). The structures of 1-9 were determined based on conventional spectroscopic analysis and chemical evidence. As expected, based on previous data, 1-3 exhibited potent cytotoxic activity against HL-60 human promyelocytic leukemia cells and A549 human lung adenocarcinoma cells. Furthermore, the ability of OSW-1 to induce apoptosis in HL-60 cells was examined. Aggregation of nuclear chromatin, accumulation of the sub-G1 cells, DNA fragmentation, and caspase-3 activation were assessed in HL-60 cells treated with OSW-1, providing evidence for OSW-1-induced apoptosis in HL-60 cells. No mitochondrial membrane potential or release of cytochrome c into the cytoplasm were observed in the OSW-1-treated apoptotic HL-60 cells, indicating that a mitochondria-independent signaling pathway is involved in apoptotic cell death.

Identification of phenolic compounds, antioxidant activity and anti-cancer effects of the extract obtained from the shoots of Ornithogalum narbonense L.

This study aimed to examine the anti-cancer and antioxidant properties and identify the phenolic content of methanolic extract obtained from the shoots of the Ornithogalum narbonense L. (OR) species, which is used for folk-medicine and food in the Sanliurfa region of Turkey. The antioxidant activity of the extract was investigated using total phenolics, flavonoids, ABTS and CUPRAC methods. Phenolic component analysis of the plant extract was performed by LC-MS/MS. The anti-cancer property of OR extract was investigated on human colon (DLD-1), endometrium (ECC-1) cancer cells and embryonic kidney (HEK-293) cells. Cytotoxic effects were defined with MTT, apoptotic activity with DNA fragmentation ELISA and AO/EB fluorescent staining, the genotoxic effect with the comet assay and the intracellular oxidative status with TAS and TOS methods. As a result of the study, it was determined that OR extract showed an antioxidant effect, and as a result of the content analysis made with LC-MS/MS, phenolic components were determined, the most abundant being cosmosiin, followed by cinnamic acid, p-coumaric acid and quinic acid. OR extract showed cytotoxic activity on DLD-1 and ECC-1 cancer cells, while the cytotoxic effect on HEK-293 cells was determined to be low. It was determined that through OR extract, by increasing the intracellular amount of free radicals on cancer cells, led to DNA damage, which consequently led to apoptosis of the cancer cells.

Sterol-recognition ability and membrane-disrupting activity of Ornithogalum saponin OSW-1 and usual 3-O-glycosyl saponins.

OSW-1 is a structurally unique steroidal saponin isolated from the bulbs of Ornithogalum saundersiae, and has exhibited highly potent and selective cytotoxicity in tumor cell lines. This study aimed to investigate the molecular mechanism for the membrane-permeabilizing activity of OSW-1 in comparison with those of other saponins by using various spectroscopic approaches. The membrane effects and hemolytic activity of OSW-1 were markedly enhanced in the presence of membrane cholesterol. Binding affinity measurements using fluorescent cholestatrienol and solid-state NMR spectroscopy of a 3-d-cholesterol probe suggested that OSW-1 interacts with membrane cholesterol without forming large aggregates while 3-O-glycosyl saponin, digitonin, forms cholesterol-containing aggregates. The results suggest that OSW-1/cholesterol interaction is likely to cause membrane permeabilization and pore formation without destroying the whole membrane integrity, which could partly be responsible for its highly potent cell toxicity.

Structural Characterization of Cholestane Rhamnosides from Ornithogalum saundersiae Bulbs and Their Cytotoxic Activity against Cultured Tumor Cells.

Previous phytochemical studies of the bulbs of Ornithogalum saundersiae, an ornamental perennial plant native to South Africa, resulted in the isolation of 29 new cholestane glycosides, some of which were structurally unique and showed potent cytotoxic activity against cultured tumor cell lines. Therefore, we aimed to perform further phytochemical examinations of methanolic extracts obtained from Ornithogalum saundersiae bulbs, isolating 12 new cholestane rhamnosides (1-12) and seven known compounds (13-19). The structures of the new compounds (1-12) were identified via NMR-based structural characterization methods, and through a sequence of chemical transformations followed by spectroscopic and chromatographic analysis. The cytotoxic activity of the isolated compounds (1-19) and the derivatives (1a and 6a) against HL-60 human promyelocytic leukemia cells and A549 human lung adenocarcinoma cells was evaluated. Compounds 10-12, 16, and 17 showed cytotoxicity against both HL-60 and A549 cells. Compound 11 showed potent cytotoxicity with an IC50 value of 0.16 µM against HL-60 cells and induced apoptotic cell death via a mitochondrion-independent pathway.

[Research ontherapeutics effect of extract of Ornithogalum caudatum on liver fibrosis].

Rat models of liver fibrosis were made by carbon tetrachloride, and the serum levels of AST, ALT, γ-GT, MDA, GSH-px, SOD were detected, serum markers of PCⅢ, IV-C, LN, HA were detected by ELISA method. HE and Masson staining were conducted in hepatic tissues to observe pathological variations. Collagen Ⅲ, TGF-ß, α-SMA, E-cadherin were detected by Western blot. The curative effect of the extract of Ornithogalum caudatum on rat liver fibrosis induced by CCl4was observed and the mechanism was discussed. The experiment results showed that the extract of O. caudatum (50, 150, 500 mg•kg⁻¹) obviously decreased the serum levels of AST, ALT, γ-GT, MDA, increased the serum levels of GSH-px, SOD, decreased the expression of serum markers of PCⅢ, IV-C, LN, HA, and improved the liver pathological variations of fibrotic rats. The experiment proved that the extract of O. caudatum could treat the liver fibrogenesis induced by CCl4 in rats. The positive medicine may inhibit accumulation of extracellular and activate hepatic stellate cell and epithelial-mesenchymal transition.

Concise synthesis of the core structures of saundersiosides.

A divergent synthesis of three core pentacyclic lactones of nine rearranged cholestane sapogenins, saundersiosides A-H (1-8) and candicanoside A (9), is reported. Key features include a one-flask CBS reduction/Brown hydroboration-oxidation, a SmI2-mediated intramolecular Reformatskii reaction, and an intramolecular transesterification. This synthesis provides a general strategy and key precursors for the collective synthesis of natural and designed saundersiosides. An efficient formal synthesis of candicanoside A is also achieved.

Effect of OSW-1 on microRNA expression profiles of hepatoma cells and functions of novel microRNAs.

3ß,16ß,17α-trihydroxycholest-5-en-22-one 16-O-(2-O-4-methoxybenzoyl-ß-D-xylopyranosyl)-(1→3)- (2-O-acet​yl-α-L-arabinopyranoside) (OSW-1) is a member of the cholestane saponin family, which was first isolated from the bulbs of Ornithogalum saundersiae and previously reported to be cytotoxic against several types of malignant cells. However, its antitumor mechanism remains unclear. Therefore, we investigated microRNA (miRNA) expression profiles in order to explore the antitumor activities of OSW-1. Furthermore, following study of differentially expressed miRNAs, the function of novel miRNAs and OSW-1 was determined using known miRNAs and anticarcinogens. The present study demonstrated that treatment with OSW-1 leads to the upregulation and downregulation of a large set of tumor-related miRNAs, including miR-299, miR-1908, miR-125b, miR-187a, miR-1275, hav1-miR-H6-3p, miR-181, miR-210, miR-483, miR-126, miR-208 and others. Notably, miR-141, miR-142, miR-200C and miR-1275 were found to be upregulated by OSW-1 and doxorubicine, as compared with doxorubicine alone. Additionally, the expression fold-change of miR-142-3P was ~58 times higher than its expression with a different treatment. These miRNAs are linked to cancer, including proliferation, differentiation, apoptosis, cell adhesion, migration, polarity and epithelial to mesenchymal transition (EMT).

Synthesis of cholestane glycosides bearing OSW-1 disaccharide or its 1-->4-linked analogue and their antitumor activities.

For further structure-activity relationship (SAR) research of OSW saponins, a cholestane glycoside, namely 3beta, 16beta, 26-trihydroxycholest-5-en-22-one 16-O-(2-O-4-methoxybenzoyl-beta-D-xylopyranosyl)-(1-->3)-2-O-acetyl-alpha-L-arabinopyranoside (1) together with two 1-->4-linked disaccharide analogues (2 and 3) were synthesized. Their cytotoxic activities were evaluated by the standard MTT assay. Compound 1 showed potent cytotoxicity against five types of human tumor cells, with IC50 ranging between 1.3 and 73 nM.

Diameters of microtubules change during rotation of the lipotubuloids of Ornithogalum umbellatum stipule epidermis as a result of varying protofilament monomers sizes and distance between them.

Microtubules in lipotubuloids of the Ornithogalum umbellatum stipule epidermis cells change their diameters depending on the motion of the cytoplasmic domains rich in microtubules and lipid bodies. Microtubules fixed during rotary and progressive motion of the lipotubuloids composed of the same number of protofilaments fall into two populations - wide (43-58 nm) and narrow (24-39 nm) in size. Following blockage of the motion with 2,4-dinitrophenol (DNP), the range of this diversity is smaller, microtubules become a medium-sized population (34-48 nm). When DNP is removed and the motion reactivated, 2 populations of microtubules reappear. Analysis of the structure of the microtubule wall revealed that changes in the microtubule diameters resulted from varying distances between the adjacent protofilaments, and stretching and compression of tubulin subunits in the protofilaments. A supposition has been put forward that the changes in the sizes of O. umbellatum microtubule diameters: 1) are connected with the interactions between microtubules and actin microfilaments lying along these microtubules; 2) can be the driving force of the rotary motion of lipotubuloids.

Synthesis of OSW saponin analogs with modified sugar residues and their antiproliferative activities.

Eight monosaccharide analogs of the potent antitumor OSW saponins (2-9) were synthesized. One analog, 2-O-acetyl-alpha-l-arabinopyranoside 3, showed antiproliferative activity against the Jurkat cells (IC(50)=0.078microM) comparable to that of the disaccharide derivative (1).

Accumulation of apocarotenoids in mycorrhizal roots of Ornithogalum umbellatum.

Colonization of roots of Ornithogalum umbellatum by the arbuscular mycorrhizal fungus Glomus intraradices induced the accumulation of different types of apocarotenoids. In addition to the mycorrhiza-specific occurrence of cyclohexenone derivatives and the "yellow pigment" described earlier, free mycorradicin and numerous mycorradicin derivatives were detected in a complex apocarotenoid mixture for the first time. From the accumulation pattern of the mycorradicin derivatives their possible integration into the continuously accumulating "yellow pigment" is suggested. Structure analyses of the cyclohexenone derivatives by MS and NMR revealed that they are mono-, di- and branched triglycosides of blumenol C, 13-hydroxyblumenol C, and 13-nor-5-carboxy-blumenol C, some of which contain terminal rhamnose as sugar moiety.

Ornithosaponins A-D, four new polyoxygenated steroidal glycosides from the bulbs of Ornithogalumthyrsoides.

By analyzing the steroidal glycoside content of fresh bulbs of Ornithogalum thyrsoides (Liliaceae), we were able to isolate four new polyoxygenated steroidal glycosides, which we named ornithosaponins A-D (1-4). The structures of 1-4 were elucidated on the basis of extensive spectroscopic analysis, including that of 2D NMR data, and the results of acidic or alkaline hydrolysis. The aglycone structure of 1-4 has not been previously reported. It is also notable that ornithosaponins B-D (2-4) have been found to contain 6-deoxy-beta-D-gulopyranose as a sugar component, which is rarely encountered in plant glycosides.

Steroidal glycosides from the bulbs of Ornithogalum thyrsoides.

Phytochemical analyses have been carried out on the fresh bulbs of Ornithogalum thyrsoides with particular attention to the steroidal glycoside constituents, resulting in the isolation of four new spirostanol saponins and seven new cholestane glycosides, together with three known steroidal compounds. The structures of the new glycosides were determined on the basis of their spectroscopic data, including 2D NMR spectroscopy, and the results of hydrolytic cleavage. The isolated compounds were evaluated for their cytotoxic activities against HL-60 human promyelocytic leukemia cells and HSC-2 human oral squamous cell carcinoma cells.