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 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 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.

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.

[An analysis of the ingredients in decoctions and extracts of Kampo medicines: Amounts of baicalin and baicalein in Kampo medicines containing Scutellariae Radix].

AIM: Kampo medicines containing Scutellariae Radix (the root of Scutellaria baicalensis Georgi; SR) sometimes cause serious adverse effects, including interstitial pneumonia and liver dysfunction. Baicalin (BL) is the major active component of SR and is presumed to be responsible for the adverse effects. We analyzed the amounts of BL in Kampo medicines to better understand how they can be used safely. METHODS: We determined the amounts of BL in 28 Kampo decoctions containing SR (recommended daily dose: 1.5-4 g/day) and corresponding Kampo extract products by high-performance liquid chromatography. RESULTS: The amounts of BL in the Kampo decoctions were 1.7-4.0-fold higher than those of the corresponding Kampo extract products. Inter-product variations in the amounts of BL in Shosaikoto, Otsujito, Daisaikoto, Saibokuto, Orengedokuto, and Saireito Kampo extracts from various companies were also examined. Significant differences in the amounts of BL were observed for Shosaikoto, Otsujito, Daisaikoto, and Saibokuto extract products (up to 2.6, 1.6, 1.5, and 1.3-fold differences, respectively), whereas no significant differences were observed for Orengedokuto and Saireito extract products. CONCLUSIONS: Because the Kampo decoctions containing SR that we examined contained 1.7-4.0 times as much BL as the corresponding Kampo extract products, medical doctors and pharmacists should be aware that Kampo decoctions containing SR might cause more severe side effects than corresponding Kampo extract products. Furthermore, we recommend that the amounts of BL and its aglycone, baicalein (BA), in Kampo extract products be made known to practitioners and patients.

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.

Pregnane glycosides from the bark of Marsdenia cundurango and their cytotoxic activity.

Seven new pregnane glycosides (1-7) and eight known compounds (8-15) were isolated from the bark of Marsdenia cundurango (Asclepiadaceae). The structures of 1-7 were determined by spectroscopic analysis, including two-dimension NMR spectroscopy, chemical transformations, and chromatographic analysis of the hydrolyzed products. The isolated compounds 1-15 were evaluated for their cytotoxic activity against HL-60 human leukemia cells, A549 human lung adenocarcinoma cells, and TIG-3 normal human lung cells, including apoptosis-inducing activity of a representative pregnane glycoside in HL-60 cells.

Daisaikoto Inhibits Pancreatic Lipase Activity and Decreases Serum Triglyceride Levels in Mice.

Daisaikoto Extract, a Kampo medicine listed in the Japanese pharmacopoeia 17th edition, is clinically used to treat obesity and related symptoms. Lipid metabolism is closely related to obesity, and pancreatic lipase inhibitors are therefore regarded as effective for the treatment of obesity. Although Daisaikoto has shown promise in the treatment of obesity, its mechanism of action has yet to be elucidated. In the present study, we found that Daisaikoto extract inhibits pancreatic lipase activity in a dose-dependent manner and decreases serum triglyceride levels in mice. To determine the crude drugs responsible for lipase inhibition, 8 variants of Daisaikoto extract without one crude drug were prepared and evaluated for lipase inhibitory activity. The lipase inhibitory activity of the Daisaikoto extract was reduced by excluding Scutellariae Radix (SR), which was found to inhibit lipase activity with an IC50 value of 1.70 mg/mL. In conclusion, Daisaikoto represents a natural medicine, in particular SR, capable of inhibiting pancreatic lipase and lipid absorption.

Aestivalosides A-L, twelve pregnane glycosides from the seeds of Adonis aestivalis.

Eight adonilide (14,20α-epoxy-3ß,20-dihydroxy-14ß-pregn-5-en-18-oic acid γ-lactone) glycosides, named aestivalosides A-H, and four glycosides of the adonilide derivatives, named aestivalosides I-L, were isolated from the MeOH extract of seeds of Adonis aestivalis. Aestivalosides A-L were previously undescribed compounds, and were structurally characterized using spectroscopic techniques, including two-dimensional NMR, and chemical methods.

Discrimination of Schisandrae Chinensis Fructus and Schisandrae Sphenantherae Fructus based on fingerprint profiles of hydrophilic components by high-performance liquid chromatography with ultraviolet detection.

High-performance liquid chromatography with ultraviolet detection (HPLC-UV) using 20 mM phosphate mobile phase and an octadecylsilyl column (Triart C18, 150 × 3.0 mm i.d., 3 µm) has been developed for the analysis of hydrophilic compounds in the water extract of Schisandrae Fructus samples. The present HPLC-UV method permits the accurate and precise determination of malic, citric, and protocatechuic acids in the Japanese Pharmacopoeia (JP) Schisandrae Fructus, Schisandrae Chinensis Fructus and Schisandrae Sphenantherae Fructus. The JP Schisandrae Fructus studied contains 27.98 mg/g malic, 107.08 mg/g citric, and 0.42 mg/g protocatechuic acids, with a relative standard deviation (RSD) of repeatability of <0.9% (n = 6). The content of malic acids in Schisandrae Chinensis Fructus is approximately ten times that in Schisandrae Sphenantherae Fructus. To examine whether the HPLC-UV method is applicable to the fingerprint-based discrimination of Schisandrae Fructus samples obtained from Chinese markets, principal component analysis (PCA) was performed using the determined contents of organic acids and the ratio of six characteristic unknown peaks derived from hydrophilic components to internal standard peak areas. On the score plots, Schisandrae Chinensis Fructus and Schisandrae Sphenantherae Fructus samples are clearly discriminated. Therefore, the HPLC-UV method for the analysis of hydrophilic components coupled with PCA has been shown to be practical and useful in the quality control of Schisandrae Fructus.

AU-1 from Agavaceae plants downregulates the expression of glycolytic enzyme phosphoglycerate mutase.

The spirostanol saponin AU-1 from Agavaceae plants stimulates the expression of the glycolytic enzyme phosphoglycerate mutase (PGAM) in ACHN cells. We hypothesized that this may arise from the downregulation of the NAD+-dependent deacetylase SIRT1. In this article, we showed that, unlike in renal adenocarcinoma cells, AU-1 does not affect the expression of SIRT1 in the normal renal cell-derived cell line HK-2. Consistent with the lack of a downregulation of SIRT1, AU-1 did not upregulate, but rather decreased PGAM expression. Moreover, AU-1 inhibited the increase in PGAM levels that results from the knock-down of SIRT1. Our results suggest that AU-1 may prevent carcinogenesis caused by increased cellular PGAM.

Fermented Citrus reticulata (ponkan) fruit squeezed draff that contains a large amount of 4'-demethylnobiletin prevents MK801-induced memory impairment.

A previous study reported biotransformation of a citrus peel polymethoxyflavone, nobiletin, by Aspergillus enabling production of 4'-demethylnobiletin, and the product's antimutagenic activity. However, the effects of fermented citrus peel on the basal forebrain-hippocampal system remain unidentified. Citrus reticulata (ponkan) fruit squeezed draffs are generated as mass waste in beverage factories. In this study using PC12D cells and cultured central nervous system neurons, we therefore examined whether Aspergillus kawachii-fermented citrus fruit squeezed draff could affect cAMP response element (CRE)- and choline acetyltransferase gene (ChAT) promoter region-mediated transcriptional activities relevant to memory formation and cholinergic function. Our current fermentation yielded approximately 80% nobiletin bioconversion, and a sample of hot-water extract of the fermented fruit squeezed draff was stronger than that of the unfermented one in facilitating CRE-mediated transcription in cultured hippocampal neurons as well as in PC12D cells. A sample of 0-80% ethanol-eluted fraction of Diaion HP-20 column-adsorbed components of the preparation obtained by the fermentation concentration-dependently and more strongly facilitated CRE-mediated transcription than did the fraction of the unfermented one in both cell culture systems. In a separate study, this polymethoxyflavone-rich fraction of the fermented fruit squeezed draff showed a potent ability to facilitate CRE-mediated and ChAT transcription in a co-culture of hippocampal neurons and basal forebrain neurons. Repeated oral gavage of mice with the fermented fraction sample prevented MK801-impaired memory formation in mice. These findings suggest that the 4'-demethylnobiletin-rich fraction prepared from the Aspergillus-fermented ponkan squeezed draff has a potential anti-dementia effect.

AU-1 from Agavaceae plants causes transient increase in p21/Cip1 expression in renal adenocarcinoma ACHN cells in an miR-34-dependent manner.

Here, we show that AU-1, spirostanol saponin isolated from Agavaceae plants, causes a transient increase in cyclin-dependent kinase inhibitor (CDKI) p21/Cip1 through the upregulation of miRNAs, miR-34 and miR-21. AU-1 stimulated p21/Cip1 expression without exerting cytotoxicity against different types of carcinoma cell lines. In renal adenocarcinoma ACHN cells, AU-1 transiently elevated the expression level of p21/Cip1 protein without marked increases in p21/Cip1 mRNA levels. Rapid and transient increases in miR-34 and miR-21, both of which are known to upregulate p21/Cip1, were observed in AU-1-treated cells. Inhibitor for miR-34 and for miR-21 significantly blocked the AU-1-caused increase in p21/Cip1, indicating that elevation of p21/Cip1 protein by AU-1 is dependent on these microRNAs. We further clarified that NAD-dependent deacetylase SIRT1, a direct target of miR-34, is decreased by the treatment with AU-1. Furthermore, we found that SIRT1-knockdown increases p21/Cip1 protein levels in an miR-21-dependent manner. On the other hand, ectopic expression of p21/Cip1 resulted in the lowered expression of miR-34 and miR-21, suggesting that reciprocal regulation exists between p21/Cip1 and these miRNAs. We propose that the following feedback network composed of miR-34/SIRT1/miR-21/p21 is triggered by the treatment with AU-1: in cells treated with AU-1, transient elevation of miR-34 leads to the downregulation of SIRT1, thereby miR-21 is freed from SIRT1-dependent suppression. Then, elevated miR-21 upregulates p21/Cip1 protein, followed by the suppression of miR-34 expression.

Chemical Constituents of the Roots and Rhizomes of Saposhnikovia divaricata and their Cytotoxic Activity.

Phytochemical investigation of the MeOH extract of the roots and rhizomes of Saposhnikovia divaricata (Umbelliferae) resulted in the isolation of six chromons (1-6)-and five polyacetylene derivatives (7-11). Compounds 9 and 11 were isolated from S. divaricate for the first time. The chromon derivatives -(1-6) were evaluated for their cytotoxic activity against HL-60 human promyclocytic leukemia cells. Compound 1 (3'-O-angeloylhamaudol) showed the most potent cytotoxic activity with an IC50 value of 4.41 µM and was found to induce apoptotic cell death in HL-60 cells. The loss of mitochondrial membrane potential, release of cytochrome c into the cytoplasm, and activation of caspase-9 in the 1-treated HL-60 cells suggests that I induces apoptosis through the mitochondial-dependent apoptotic pathway.

Nerve growth factor enhances the CRE-dependent transcriptional activity activated by nobiletin in PC12 cells.

Prevention and treatment of Alzheimer disease are urgent problems for elderly people in developed countries. We previously reported that nobiletin, a poly-methoxylated flavone from the citrus peel, improved the symptoms in various types of animal models of memory loss and activated the cAMP responsive element (CRE)-dependent transcription in PC12 cells. Nobiletin activated the cAMP/PKA/MEK/Erk/MAPK signaling pathway without using the TrkA signaling activated by nerve growth factor (NGF). Here, we examined the effect of combination of nobiletin and NGF on the CRE-dependent transcription in PC12 cells. Although NGF alone had little effect on the CRE-dependent transcription, NGF markedly enhanced the CRE-dependent transcription induced by nobiletin. The NGF-induced enhancement was neutralized by a TrkA antagonist, K252a. This effect of NGF was effective on the early signaling event elicited by nobiletin. These results suggested that there was crosstalk between NGF and nobiletin signaling in activating the CRE-dependent transcription in PC12 cells.

A New Neolignan Glycoside from Vetiveria zizanioides Roots.

A new neolignan glycoside (1) and four known aromatic compounds (2-5) were isolated. from the roots of Vetiveria zizanioides. The structure of compound 1 was determined based on spectroscopic analysis and hydrolysis. The structure of known flavonoid glycoside 3 was confirmed by X-ray crystallography. Compound 5 showed weak cytotoxic activity against HL-60 cells with an IC50 value of 13.1 ± 0.04 µM.

Chemical Constituents of the Leaves of Tussilago farfara and their Aldose Reductase Inhibitory Activity.

The inhibitory activity of 37 medicinal plant extracts against aldose reductase (AR) activity was evaluated. The most potent AR inhibitory activity was found in the MeOH extract of the leaves of Tussilagofarfara (Compositae). Enzyme assay-guided fractionation of the extract led to the isolation of a new flavonoid glycoside, kaempferol 3-0-[3,4-0-(isopropylidene)-α-L-arabinopyranoside] (1), along with 15 known compounds (2-16), of which 3, 5, 13, 15, and 16 were isolated from T. faifara for the first time. The structures of 1-16 were elucidated based on MS and NMR data. Dicaffeoylquinic acid derivatives (7-12) showed potent AR inhibitory activity with IC(50)values ranging from 0.58 to 5.38 µM, whereas flavonoid glycosides 1, 3, 5, and 6 showed weak inhibitory activity with IC(50) values of 13.9, 15.1, 13.3, and 14.1 µM, respectively.

Novel Steroidal Glycosides from the Bulbs of Lilium pumilum.

Examination of the bulbs of Lilium pumilum (Liliaceae) led to the isolation of four novel steroidal glycosides (1-4) with a 2,3,4-trisubstituted ß-d-glucopyranosyl unit. In 1 and 3, the α-L-arabinopyranosyl moiety is linked to C-3 of the inner trisubstituted ß-D-glucopyranosyl group and is present as an usual 4C1 conformation. In contrast, in 2 and 4, the α-L-arabinopyranosyl moiety, which is attached to C-4 of the inner trisubstituted ß-D-glucopyranosyl group, is present as a ¹C4 conformation. The structures of the new steroidal glycosides were determined based on the results of spectroscopic analyses, including two-dimensional (2D) NMR data and hydrolysis.