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.

[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.

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.

Cardenolide glycosides from the seeds of Digitalis purpurea exhibit carcinoma-specific cytotoxicity toward renal adenocarcinoma and hepatocellular carcinoma cells.

Four cardenolide glycosides, glucodigifucoside (2), 3'-O-acetylglucoevatromonoside (9), digitoxigenin 3-O-ß-D-glucopyranosyl-(1 → 4)-ß-D-glucopyranosyl-(1 → 4)-3-O-acetyl-ß-D-digitoxopyranoside (11), and purpureaglycoside A (12), isolated from the seeds of Digitalis purpurea, exhibited potent cytotoxicity against human renal adenocarcinoma cell line ACHN. These compounds exhibited significantly lower IC50 values against ACHN than that against normal human renal proximal tubule-derived cell line HK-2. In particular, 2 exhibited the most potent and carcinoma-specific cytotoxicity, with a sixfold lower IC50 value against ACHN than that against HK-2. Measurement of cyclin-dependent kinase inhibitor levels revealed that upregulation of p21/Cip1 expression was involved in the carcinoma-specific cytotoxicity of 2. Further, compound 2 also exhibited the carcinoma-specific cytotoxicity toward hepatocellular carcinoma cell line.

Triterpene glycosides from the stems and leaves of Lonicera japonica.

Five new triterpene glycosides, namely lonicerosides F-J (1-5), together with five known ones, were isolated from the stems and leaves of Lonicera japonica. Based on extensive spectroscopic analysis, including two-dimensional (2D)-NMR experiments, and the results of hydrolysis, the structures of the new compounds were determined to be 3ß-[(ß-D-glucopyranosyl)oxy]-23-hydroxyolean-12-en-28-oic acid O-ß-D-glucopyranosyl-(1→6)-[α-L-rhamnopyranosyl-(1→2)]-ß-D-glucopyranosyl ester (loniceroside F), 3ß-[(ß-D-glucopyranosyl)oxy]-23-hydroxyolean-12-en-28-oic acid O-(3-O-acetyl-ß-D-xylopyranosyl)-(1→6)-[α-L-rhamnopyranosyl-(1→2)]-ß-D-glucopyranosyl ester (loniceroside G), 3ß-[(ß-D-glucopyranosyl)oxy]-23-hydroxyolean-12-en-28-oic acid O-(4-O-acetyl-ß-D-xylopyranosyl)-(1→6)-[α-L-rhamnopyranosyl-(1→2)]-ß-D-glucopyranosyl ester (loniceroside H), 3ß-[(α-L-arabinopyranosyl)oxy]-23-hydroxyolean-12-en-28-oic acid O-(3-O-acetyl-ß-D-xylopyranosyl)-(1→6)-[α-L-rhamnopyranosyl-(1→2)]-ß-D-glucopyranosyl ester (loniceroside I), and 3ß-[(α-L-rhamnopyranosyl-(1→2)-ß-D-glucopyranosyl)oxy]-olean-12-en-28-oic acid O-α-L-rhamnopyranosyl-(1→2)-[ß-D-xylopyranosyl-(1→6)]-ß-D-glucopyranosyl ester (loniceroside J).

Natural products reveal cancer cell dependence on oxysterol-binding proteins.

Cephalostatin 1, OSW-1, ritterazine B and schweinfurthin A are natural products that potently, and in some cases selectively, inhibit the growth of cultured human cancer cell lines. The cellular targets of these small molecules have yet to be identified. We have discovered that these molecules target oxysterol binding protein (OSBP) and its closest paralog, OSBP-related protein 4L (ORP4L)--proteins not known to be involved in cancer cell survival. OSBP and the ORPs constitute an evolutionarily conserved protein superfamily, members of which have been implicated in signal transduction, lipid transport and lipid metabolism. The functions of OSBP and the ORPs, however, remain largely enigmatic. Based on our findings, we have named the aforementioned natural products ORPphilins. Here we used ORPphilins to reveal new cellular activities of OSBP. The ORPphilins are powerful probes of OSBP and ORP4L that will be useful in uncovering their cellular functions and their roles in human diseases.

New cardenolide glycosides from the seeds of Digitalis purpurea and their cytotoxic activity.

A chemical investigation of Digitalis purpurea seeds led to the isolation of three new cardenolide glycosides (1, 8 and 11), together with 12 known cardenolide glycosides (2-7, 9, 10 and 12-15). The structures of 1, 8 and 11 were determined by 1D and 2D NMR spectroscopic analyses and the results of an acid or enzymatic hydrolysis. The cytotoxic activity of the isolated compounds (1-15) against HL-60 leukemia cells was examined. Compounds 2, 9, 11 and 12 showed potent cytotoxicity against HL-60 cells with respective 50% inhibition concentration (IC50) values of 0.060, 0.069, 0.038, and 0.034 µM. Compounds 2, 9 and 11 also exhibited potent cytotoxic activity against HepG2 human liver cancer cells with respective IC50 values of 0.38, 0.79, and 0.71 µM. An investigation of the structure-activity relationship showed that the cytotoxic activity was reduced by the introduction of a hydroxy group at C-16 of the digitoxigenin aglycone, methylation of the C-3' hydroxy group at the fucopyranosyl moiety, and acetylation of the C-3' hydroxy group at the digitoxopyranoyl moiety.

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.

New cardenolides from the seeds of Adonis aestivalis.

Chemical investigation of the seeds of Adonis aestivalis has led to the isolation of a new cardenolide (3ß,5α,14ß,17ß-tetrahydroxycard-20,22-enolide) (1), two new glycosides (2, 3) of 1, and a new strophanthidin hexaglycoside (4), together with a known compound, strophanthidin 3-O-ß-D-glucopyranoside (5). The structures of 1-4 were determined by 1D- and 2D-NMR spectroscopic analysis and the results of hydrolytic cleavage. The isolated compounds (1-5) were examined for their cytotoxic activity against neoplastic HSC-2, HSC-3, HSC-4, and HL-60 cells, as well as HGF, HPLF, and HPC normal cell lines. Compounds 2, 4, and 5 were found to display selective cytotoxicity toward malignant tumor cell lines. Although the morphological observations of HL-60 and HSC-2 cell deaths by 2, 4, and 5 revealed changes characteristic of apoptosis, neither DNA degradation nor activation of caspase-3 was observed. Our findings demonstrated that 2, 4, and 5 may trigger caspase-3-independent apoptotic cell death in HL-60 and HSC-2 cells.

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.

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.

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 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.

Phenolics from Glycyrrhiza glabra roots and their PPAR-gamma ligand-binding activity.

Bioassay-guided fractionation of the EtOH extract of licorice (Glycyrrhiza glabra roots), using a GAL-4-PPAR-gamma chimera assay method, resulted in the isolation of 39 phenolics, including 10 new compounds (1-10). The structures of the new compounds were determined by analysis of their spectroscopic data. Among the isolated compounds, 5'-formylglabridin (5), (2R,3R)-3,4',7-trihydroxy-3'-prenylflavane (7), echinatin, (3R)-2',3',7-trihydroxy-4'-methoxyisoflavan, kanzonol X, kanzonol W, shinpterocarpin, licoflavanone A, glabrol, shinflavanone, gancaonin L, and glabrone all exhibited significant PPAR-gamma ligand-binding activity. The activity of these compounds at a sample concentration of 10microg/mL was three times more potent than that of 0.5microM troglitazone.

Chromones from the tubers of Eranthis cilicica and their antioxidant activity.

Chemical studies on the constituents of Eranthis cilicica led to isolation of ten chromone derivatives, two of which were previously known. Comprehensive spectroscopic analysis, including extensive 1D and 2D NMR data, and the results of enzymatic hydrolysis allowed the chemical structures of the compounds to be assigned as 8,11-dihydro-5-hydroxy-2,9-dihydroxymethyl-4H-pyrano[2,3-g][1]benzoxepin-4-one, 5,7-dihydroxy-8-[(2E)-4-hydroxy-3-methylbut-2-enyl]-2-methyl-4H-1-benzopyran-4-one, 5,7-dihydroxy-2-hydroxymethyl-8-[(2E)-4-hydroxy-3-methylbut-2-enyl]-4H-1-benzopyran-4-one, 7-[(beta-d-glucopyranosyl)oxy]-5-hydroxy-8-[(2E)-4-hydroxy-3-methylbut-2-enyl]-2-methyl-4H-1-benzopyran-4-one, 7-[(beta-d-glucopyranosyl)oxy]-5-hydroxy-2-hydroxymethyl-8-[(2E)-4-hydroxy-3-methylbut-2-enyl]-4H-1-benzopyran-4-one, 9-[(O-beta-d-glucopyranosyl-(1-->6)-beta-d-glucopyranosyl)oxy]methyl-8,11-dihydro-5,9-dihydroxy-2-methyl-4H-pyrano[2,3-g][1]benzoxepin-4-one, 8,11-dihydro-5,9-dihydroxy-9-hydroxymethyl-2-methyl-4H-pyrano[2,3-g][1]benzoxepin-4-one, and 7-[(O-beta-d-glucopyranosyl-(1-->6)-beta-d-glucopyranosyl)oxy]methyl-4-hydroxy-5H-furo[3,2-g][1]benzopyran-5-one, respectively. The isolated compounds were evaluated for their antioxidant activity.

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.

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.