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.

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.

Determination of Structure and Cytotoxicity of Ten Undescribed Steroidal Glycosides from Allium cristophii × A. macleanii 'Globemaster'.

'Globemaster' is an ornamental hybrid cultivar whose parent plants are Allium cristophii and A. macleanii. The chemical constituents of 'Globemaster' bulbs have not yet been examined; thus, a systematic phytochemical investigation was undertaken herein. A series of chromatographic separations of the MeOH extract of 'Globemaster' bulbs afforded 27 steroidal glycosides (1-27), which are classified into 23 spirostanol glycosides (1-8 and 11-25), two furostanol glycosides (9 and 26), a pregnane glycoside (10), and a cholestane glycoside (27). The structures of the hitherto undescribed compounds (1-10) were determined from the two-dimensional NMR spectroscopic data and hydrolysis. The cytotoxicity of the isolated compounds (1-27) toward HL-60 human promyelocytic leukemia cells, A549 human adenocarcinoma lung cancer cells, and SBC-3 human small-cell lung cancer cells was evaluated. Compounds 8, 22, 23, 24, and 26 exhibited cytotoxicity toward all cell lines in a dose-dependent manner, with IC50 values in the 1.3-49 µM range.

Hesperetin Ameliorates Inhibition of Neuronal and Oligodendroglial Cell Differentiation Phenotypes Induced by Knockdown of Rab2b, an Autism Spectrum Disorder-Associated Gene Product.

Autism spectrum disorder (ASD) is a central nervous system (CNS) neurodevelopmental disorder that includes autism, pervasive developmental disorder, and Asperger's syndrome. ASD is characterized by repetitive behaviors and social communication deficits. ASD is thought to be a multifactorial disorder with a range of genetic and environmental factors/candidates. Among such factors is the rab2b gene, although it remains unclear how Rab2b itself is related to the CNS neuronal and glial developmental disorganization observed in ASD patients. Rab2 subfamily members regulate intracellular vesicle transport between the endoplasmic reticulum and the Golgi body. To the best of our knowledge, we are the first to report that Rab2b positively regulates neuronal and glial cell morphological differentiation. Knockdown of Rab2b inhibited morphological changes in N1E-115 cells, which are often used as the neuronal cell differentiation model. These changes were accomplished with decreased expression levels of marker proteins in neuronal cells. Similar results were obtained for FBD-102b cells, which are used as the model of oligodendroglial cell morphological differentiation. In contrast, knockdown of Rab2a, which is another Rab2 family member not known to be associated with ASD, affected only oligodendroglial and not neuronal morphological changes. In contrast, treatment with hesperetin, a citrus flavonoid with various cellular protective effects, in cells recovered the defective morphological changes induced by Rab2b knockdown. These results suggest that knockdown of Rab2b inhibits differentiation in neuronal and glial cells and may be associated with pathological cellular phenotypes in ASD and that hesperetin can recover their phenotypes at the in vitro level at least.

Structure Elucidation of 16 Undescribed Steroidal Glycosides from the Underground Parts of Agapanthus africanus and Apoptosis-Inducing Activity in Small-Cell Lung Cancer Cell.

To explore new candidates for anticancer agents from natural products, the underground parts of Agapanthus africanus, commonly used as an ornamental plant, were investigated phytochemically. As a result, 16 undescribed steroidal glycosides (1-16) were obtained, and their structures were determined mainly by NMR spectroscopic analysis and chemical transformations. The cytotoxic activities of the isolated compounds (1-16) against SBC-3 human small-cell lung cancer cells, A549 human adenocarcinoma cells, and HL-60 human promyelocytic leukemia cells were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2-tetrazolium bromide (MTT) assay. Compound 1, a bisdesmosidic furostanol glycoside, and 10, a bisdesmosidic spirostanol glycoside, were cytotoxic to all three cell lines with IC50 values ranging from 1.2 to 13 µM. As 1 exhibited the most potent cytotoxicity against SBC-3 cells among the isolated compounds, its apoptosis-inducing activity toward SBC-3 cells was examined. Compound 1 arrested SBC-3 cells at the G2/M phase of the cell cycle and effectively induced apoptosis via an intrinsic pathway accompanied by the dissipation of membrane potential and morphological changes in mitochondria.

Determination of oxalic acid in herbal medicines by semi-micro hydrophilic interaction liquid chromatography coupled with electrochemical detection.

In this study, the determination of oxalic acid in herbal medicines was performed by using a hydrophilic interaction liquid chromatography coupled with electrochemical detection (HILIC-ECD) method. A semi-micro column packed with amide-silica particles and an acetonitrile-30 mM phosphate buffer (pH 6.8) mixture (65:35, v/v) were used as the stationary and mobile phases, respectively, in the HILIC-ECD. A potential of + 1.1 V vs. Ag/AgCl was applied to a glassy carbon working electrode. The ratio of the peak height of oxalic acid to that of the internal standard (synephrine) was proportional to the concentration of 0.45 µg L-1 to 1.8 mg L-1 with a correlation coefficient of 0.999. The detection limit (signal-to-noise ratio, S/N = 3) of oxalic acid was 0.17 µg L-1. By the HILIC-ECD, the oxalic acid content in crude drugs and Kampo medicine extract granules (Zingiberis Rhizoma Processum, Pinelliae Tuber, Sho-seiryu-to, Hange-shashin-to, etc.) were determined with less than 2.9% relative standard deviation (RSD, n = 6), and their recoveries were more than 88.7% with less than 3.3% RSD (n = 6). In conclusion, we demonstrated that the HILIC-ECD performed measurements that were quite selective, accurate, and precise for the determination of oxalic acid in herbal medicines.

Lanceolanone A, a new biflavanone, and a chalcone glucoside from the flower heads of Coreopsis lanceolata and their aldose reductase inhibitory activity and AMPK activation.

The MeOH extract of the flower heads of Coreopsis lanceolata L. (Asteraceae) exhibited aldose reductase (AR) inhibitory activity (IC50 8.36 µg/mL). Bioassay-guided fractionation of the extract resulted in the isolation of a new biflavanone-named Lanceolanone A (1) and a chalcone glucoside (6), along with 12 known compounds (2-5 and 7-14), of which 4, 7, 9, 10, and 12 were isolated from C. lanceolata for the first time. The structures of the new compounds (1 and 6) were determined by extensive spectroscopic analysis, including two-dimensional (2D) NMR, and ECD calculation method. Compounds 2, 4, 11, 13, and 14 exhibited AR inhibitory activities with IC50 values between 2.40 and 9.99 µM. Furthermore, 8-13 at 1.0 mM activated AMPK expression in HepG2 human hepatoma cells compared to the control.

Chemical Constituents of the Leaves of Thujopsis dolabrata and Their Cytotoxicity.

Five podophyllotoxin derivatives (1-5), two diterpenoids (6 and 7), three diterpenoid xylosides (8-10), a flavanonol glycoside (11), flavonol (12), and biflavonoid (13) were isolated from the leaves of Thujopsis dolabrata (Cupressaceae). Compounds 1, 6, and 8 were named (-)-ß-isopeltatin, epi-nootkastatin 2, and acetoxyanticopalol 15-O-ß-D-xylopyranoside, respectively. The structures of the isolated compounds were determined based on a detailed analysis of NMR spectroscopic data and through chromatographic and spectroscopic analyses following specific chemical transformations. The isolated compounds (1-5 and 7-11) were evaluated for their cytotoxicity toward HL-60 human promyelocytic leukemia cells and Caki-1 human kidney carcinoma cells. The podophyllotoxin derivatives (1-5) exhibited cytotoxicity against both HL-60 and Caki-1 cells with IC50 values ranging from 0.00069 to 5.4 µM, and the diterpenoid derivatives (7-10) demonstrated cytotoxicity against HL-60 cells with IC50 values ranging from 4.5 to 11 µM. HL-60 cells treated with 8 exhibited apoptosis characteristics, such as accumulation of sub-G1 cells and nuclear chromatin condensation.

Cytotoxic triterpene and steroidal glycosides from the seeds of Digitalis purpurea and the synergistic cytotoxicity of steroidal glycosides and etoposide in SBC-3 cells.

The phytochemical investigations of the seeds of Digitalis purpurea have revealed their richness in cardenolide and pregnane glycosides exhibiting potent cytotoxicity; further chemical examinations of the D. purpurea seeds have achieved the isolation of six triterpene glycosides (1-6), six spirostanol glycosides (7-12), and three furostanol glycosides (13-15), including seven previously unidentified compounds (1-3, 10-12, and 14). Here, the structures of 1-3, 10-12, and 14 were determined via extensive spectroscopic analyses, including two-dimensional (2D) NMR; hydrolysis, followed by chromatographic and spectroscopic analyses; and X-ray crystallographic analysis. The cytotoxic activities of the isolated compounds (1-15) against SBC-3 small cell lung carcinoma and TIG-3 normal human diploid fibroblast cells were evaluated. Triterpene glycoside 3 and spirostanol glycoside 9 exhibited considerable cytotoxicity with IC50 values of 1.0 and 1.7 µM, respectively; they induced apoptotic cell death, which was accompanied by the activation of caspase-3 in SBC-3 cells. Spirostanol glycoside 7 exhibited cytotoxicity toward the SBC-3 cells (IC50 1.3 µM). Additionally, 7 at 0.1 and 1.0 µM synergistically enhanced the cytotoxicity of etoposide against SBC-3 cells; compound 7 induced the release of DAMPs; the release of HMGB1, the secretion of ATP, and the exposure of CALR in the SBC-3 cells. Furthermore, the combination of 7 and etoposide resulted in increasing the extracellular release of DAMPs. These data indicated that 7, as well as its combination with etoposide, might potentially cause immunogenic cell death.

Novel Oleanane-Type Triterpene Glycosides from the Saponaria officinalis L. Seeds and Apoptosis-Inducing Activity via Mitochondria.

Saponaria officinalis L., commonly known as "Soapwort", is a rich source of triterpene glycosides; however, the chemical constituents of S. officinalis seeds have not been fully identified. In this study, we conducted a systematic phytochemical investigation of the seeds of S. officinalis and obtained 17 oleanane-type triterpene glycosides (1-17), including seven new glycosides (1-7). The structures of 1-7 were determined based on a detailed analysis of NMR spectroscopic data and chromatographic and spectroscopic analyses following specific chemical transformation. The cytotoxicities of the isolated compounds were evaluated against HL-60 human promyelocytic leukemia cells, A549 human adenocarcinoma lung cancer cells, and SBC-3 human small-cell lung cancer cells. The cytotoxicities of 1, 4, and 10 toward HL-60 cells and SBC-3 cells were nearly as potent as that of cisplatin. Compound 1, a bisdesmosidic triterpene glycoside obtained in good yield, arrested the cell cycle of SBC-3 cells at the G2/M phase, and induced apoptosis through an intrinsic pathway, accompanied by ROS generation. As a result of the mitochondrial dysfunction induced by 1, mitochondria selective autophagy, termed mitophagy, occurred in SBC-3 cells.

Chemical Components in Hedera rhombea Leaves and Their Cytotoxicity.

Two novel triterpene glycosides (1 and 2), 17 known triterpene glycosides (3-19), two known flavonoid glycosides (20 and 21), and two known norsesquiterpene glucosides (22 and 23) were isolated from Hedera rhombea (Araliaceae) leaves. The structures of 1 and 2 were determined by spectroscopic analysis, including two-dimensional NMR spectroscopy, and chromatographic analysis of the hydrolyzed products. The cytotoxicity of the isolated triterpene glycosides (1-19) against HL-60 human promyelocytic leukemia cells was evaluated. Compounds 9, 10, and 11 were cytotoxic to HL-60 cells with IC50 values of 7.2, 21.9, and 32.8 µM, respectively. Other compounds isolated from the leaves were not cytotoxic at sample concentrations of 50 µM.

Chemical constituents and aldose reductase inhibitory activities of Betula alba bark and leaves.

Systematic phytochemical investigation of the bark and leaves of Betula alba was independently conducted. A new cyclic diarylheptanoid glucoside (1), five diarylheptanoids (2-6), a phenylethanoid (7), a methyl salicylate glycoside (8), a dihydrobenzofuran glucoside (9), an arylbutanoid glycoside (10), two lignan glycosides (11 and 12), a flavanone glucoside (13), and a triterpene (14) were isolated from the bark of B. alba. On the other hand, two cyclic diarylheptanoids (15 and 16), five flavonoids (17-21), a phenylpropanoid (22), a phenylbutanoid glucoside (23), and a monoterpene glucoside (24) were obtained from the leaves of B. alba. The structures of the isolated compounds (1-24) were identified on the basis of one- and two-dimensional NMR spectroscopic data. Compounds 1-24 were subsequently examined for aldose reductase (AR) inhibitory activity. Compounds 14 and 17-20 moderately inhibited AR activity with IC50 values ranging from 6.6 to 34 µM.

Three novel furospirostanol glycosides and a steroidal alkaloid glycoside from the bulbs of Fritillaria camtschatcensis (L.) Ker Gawl., and their cytotoxicity.

Three novel steroidal glycosides (1-3) and a previously described steroidal alkaloid glycoside (4) have been isolated from the bulbs of Fritillaria camtschatcensis (L.) Ker Gawl. (Liliaceae). The structures of novel compounds 1-3 were characterized based on NMR spectroscopy and chemical transformations. Compounds 1-3 are furospirostanol glycosides bearing a (3S)-3-hydroxy-3-methylglutaryl moiety at C-26 in the aglycone. Compounds 1-4 were evaluated in terms of their cytotoxic activities toward HL-60 human promyelocytic leukemia cells, A549 human lung adenocarcinoma cells, and SBC-3 human lung small cell carcinoma cells. Only 4 showed moderate cytotoxicity against HL-60, A549, and SBC-3 cells with IC50 values of 22.9, 13.3, and 11.9 µM, respectively. Compound 4 was found to cause necrotic-like cell death in HL-60 cells.

Steroidal Glycosides from the Aerial Parts of Avena sativa L. and Their Cytotoxic Activity.

Twelve steroidal glycosides (1-12) were isolated from the aerial parts of Avena sativa L. (Poaceae). Among the isolated compounds, 1 was directly isolated from the plant for the first time, and 2-6 were new steroidal glycosides. The structures of 1-6 were determined by analysis of their spectroscopic data, chemical transformations, and chromatographic and spectroscopic analyses of the hydrolyzed products. Compounds 5 and 6 were novel steroidal glycosides with a B-ring contracted skeleton (B-nor steroid). Compounds 1, 9, 11, and 12 were cytotoxic to HL-60 human promyelocytic leukemia cells, MIA PaCa-2 human pancreatic carcinoma cells, and A549 human lung adenocarcinoma cells with IC50 values ranging from 0.79 to 13.5 µM. HL-60 cells treated with 1 exhibited apoptotic characteristics, namely, condensed nuclear chromatin, accumulation of sub-G1 cells, and activation of caspase-3. Additionally, the loss of the mitochondrial membrane potential and the release of cytochrome c into the cytoplasm in 1-treated HL-60 cells suggested that 1 induced apoptosis through a mitochondrial-dependent apoptotic pathway.

Structure and Cytotoxicity of Novel Lignans and Lignan Glycosides from the Aerial Parts of Larrea tridentata.

Previously, the authors conducted phytochemical investigations of the aerial parts of Larrea tridentata and reported triterpene glycosides and lignan derivatives. In continuation of the preceding studies, 17 lignans and lignan glycosides (1-17) were isolated, including seven new compounds (1-7). Herein, the structure of the new compounds was determined based on spectroscopic analysis and enzymatic hydrolysis. The cytotoxicity of 1-17 against HL-60 human promyelocytic leukemia cells was examined. Compounds 4-11 and 14-16 were cytotoxic to HL-60 cells, with IC50 values in the range of 2.7-17 µM. Compound 6, which was the most cytotoxic among the unprecedented compounds, was shown to induce apoptotic cell death in HL-60 cells.

Bufadienolides and ecdysteroids from the whole plants of Helleborus niger and their cytotoxicity.

A new bufadienolide (1), two new bufadienolide glycosides (2 and 3), a new ecdysteroid (4), and four known compounds (5-8), were isolated from the whole plants of Helleborus niger L. (Ranunculaceae). The structures of the new compounds (1-4) were determined by spectroscopic analysis, including 2D NMR spectral data, and hydrolytic studies. Compounds 1-6 showed cytotoxicity against HL-60 human leukemia cells, A549 human lung adenocarcinoma cells, and SBC-3 human small-cell lung cancer cells, with IC50 values ranging from 0.0055 to 1.9 µM. HL-60 cells treated with either 3 or 4 showed apoptosis characteristics, such as nuclear chromatin condensation, accumulation of sub-G1 cells, and activation of caspase-3/7.

Chemical Constituents of the Bulbs of Scilla peruviana and Their Pancreatic Lipase Inhibitory Activity.

Scilla species are used as medicinal plants and contain lanosterol-type triterpene glycosides. The phytochemical investigation of the bulbs of Scilla peruviana led to the isolation of 17 compounds, including three new rearranged pentacyclic-lanosterol glycosides (1-3) and two new homoisoflavanone glycosides (12 and 13). The structures of the undescribed compounds were determined by extensive spectroscopic analyses, including two-dimensional (2D) NMR. Among the triterpene glycosides, 2, 3, and 6 showed significant pancreatic lipase inhibitory activity in a concentration-dependent manner in vitro. The oral administration of scillascilloside D-2 (6) reduced serum triglyceride levels in a dose-dependent manner in soybean oil-loaded mice.

A new and 23 known cardenolide glycosides from Thevetia neriifolia seeds and their cytotoxic activities against human oral carcinoma cell lines.

Phytochemical analysis of Thevetia neriifolia seeds resulted in the isolation of one new (1) and 23 known (2－24) cardenolide glycosides. The structure of 1 was determined based on one- and two-dimensional NMR spectroscopic analysis and acid hydrolytic cleavage reaction. The effect of the cytotoxic activity of 1－24 on three human oral carcinoma cell lines was assessed. The cell lines included Ca9-22 human gingival carcinoma cells, HSC-2 human mouth carcinoma cells, HSC-4 human tongue carcinoma cells, and HGF human gingival fibroblast cells. The isolated compounds had a cytotoxic effect on the carcinoma cells with IC50 values ranging from 0.004 µM to 64.9 µM. The structure-activity relationship is also discussed.

Steroidal constituents isolated from the seeds of Withania somnifera.

A new withanolide glycoside (1), two new ergostanol glycosides (2 and 3), and a new furostanol glycoside (4), along with nine known steroidal derivatives (5-12) were isolated from the seeds of Withania somnifera. The structures of the new compounds were determined using spectroscopic analysis and hydrolysis. The cytotoxic activities of the isolated compounds were evaluated against Ca9-22 human gingival carcinoma cells, HSC-2 human mouth carcinoma cells, and HL-60 human promyelocytic leukemia cells. Only 12 exhibited cytotoxic activity against these cell lines with IC50 values of 0.38, 0.54, and 1.5 µM, respectively.

A Total of Eight Novel Steroidal Glycosides Based on Spirostan, Furostan, Pseudofurostan, and Cholestane from the Leaves of Cestrum newellii.

Previously, various steroidal glycosides were reported from plants of Cestrum species. However, phytochemical investigation has not been conducted on Cestrum newellii. A systematic phytochemical investigation of the leaves of C. newellii resulted in the isolation of eight novel steroidal glycosides (1-8), which were classified into three spirostanol glycosides (1-3), two furostanol glycosides (4 and 5), two pseudofurostanol glycosides (6 and 7), and one cholestane glycoside (8). In addition, three known cholestane glycosides (9-11) were isolated and identified. The structures of the new compounds were determined based on spectroscopic data and chemical transformations. Compounds 1 and 2 are spirostanol glycosides having hydroxy groups at C-2, C-3, C-12, and C-24 of the aglycone moiety. Although C. newellii is known to be a poisonous plant, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay exhibited that none of the isolated compounds were cytotoxic to HL-60 human promyelocytic leukemia cells.