In vitro selection of aptamer S1 against MCF-7 human breast cancer cells.

Breast cancer is the most common female cancer. However, the known effective specific biomarkers for breast cancer are still scarce. Abnormal membrane proteins serve as ideal biomarkers for disease diagnoses, therapeutics and prognosis. Thus aptamers (single-stranded oligonucleotide molecules) with molecular recognition properties can be used as efficient tools to sort cells based on differences in cell surface architecture between normal and tumor cells. In this study, we aimed to screen specific aptamer against MCF-7 human breast cancer cells. Cell-SELEX process was performed to isolate aptamers from a combinatorial single-stranded nucleic acid library that selectively targeting surface proteins of MCF-7 cells in contrast with MCF-10A human mammary epithelial cells. The process was repeated until the pool was enriched for sequences that specifically recognizing MCF-7 cells in monitoring by flow cytometry. Subsequently, the enriched pool was cloned into bacteria, and positive clones were sequenced to obtain individual sequences. Representative sequences were chemically synthesized and evaluated their binding affinities to MCF-7 cells. As a result, an aptamer S1 was finally identified to have high binding affinity with equilibrium dissociation constant (Kd) value of 29.9 ±â€¯6.0 nM. FAM-labeled aptamer S1 induced fluorescence shift in MCF-7 cells but not in MCF-10A human mammary epithelial cells, or MDA-MB-453 and MDA-MB-231 human breast cancer cells. Furthermore, result of cell imaging observed from laser confocal fluorescence microscope showed that MCF-7 cells exhibited stronger fluorescence signal resulted from Cy5-labeled aptamer S1 than MCF-10A cells. The above findings suggested that S1 may be a specificity and selectivity aptamer for MCF-7 cells and useful for the breast cancer detection and diagnosis.

Diarylpentanol constituents from the aerial part of Stelleropsis tianschanica.

Five diarylpentanol derivatives including two new compounds stellerasme A (1), stellerasme B (2) were isolated from the aerial parts of Stelleropsis tianschanica. Their structures were elucidated by various spectroscopic techniques (UV, IR, MS, CD, 1D and 2D NMR). All compounds were evaluated for their cytotoxicity activity against HeLa and KB cell lines, and compound 1 showed selective activities against HeLa cell line with an IC50 value of 7.4 µM.

Honokiol sensitizes breast cancer cells to TNF-α induction of apoptosis by inhibiting Nur77 expression.

BACKGROUND AND PURPOSE: The orphan nuclear receptor Nur77 is implicated in the survival and apoptosis of cancer cells. The purpose of this study was to determine whether and how Nur77 serves to mediate the effect of the inflammatory cytokine TNF-α in cancer cells and to identify and characterize new agents targeting Nur77 for cancer therapy. EXPERIMENTAL APPROACH: The effects of TNF-α on the expression and function of Nur77 were studied using in vitro and in vivo models. Nur77 expression was evaluated in tumour tissues from breast cancer patients. The anticancer effects of honokiol and its mechanism of action were assessed by in vitro, cell-based and animal studies. KEY RESULTS: TNF-α rapidly and potently induced the expression of Nur77 in breast cancer cells through activation of IκB kinase and JNK. Knocking down Nur77 resulted in TNF-α-dependent apoptosis, while ectopic Nur77 expression in MCF-7 cells promoted their growth in animals. Levels of Nur77 were higher in tumour tissues than the corresponding tissues surrounding the tumour in about 50% breast cancer patients studied. Our in vitro and animal studies also identified honokiol as an effective sensitizer of TNF-α-induced apoptosis by inhibiting TNF-α-induced Nur77 mRNA expression, which could be attributed to its interference of TNFR1's interaction with receptor-interacting protein 1 (RIPK1). CONCLUSIONS AND IMPLICATIONS: TNF-α-induced Nur77 serves as a survival factor to attenuate the death effect of TNF-α in cancer cells. With its proven human safety profile, honokiol represents a promising agent that warrants further clinical development.

Online polar two phase countercurrent chromatography×high performance liquid chromatography for preparative isolation of polar polyphenols from tea extract in a single step.

Herein, we report an on-line two-dimensional system constructed by counter-current chromatography (CCC) coupling with preparative high-performance liquid chromatography (prep-HPLC) for the separation and purification of polar natural products. The CCC was used as the first dimensional isolation column, where an environmental friendly polar two-phase solvent system of isopropanol and 16% sodium chloride aqueous solution (1:1.2, v/v) was introduced for low toxicity and favorable resolution. In addition, by applying the stop-and-go flow technique, effluents pre-fractionated by CCC was further purified by a preparative column packed with octadecyl silane (ODS) as the second dimension. The interface between the two dimensions was comprised of a 6-port switching valve and an electronically controlled 2-position 10-port switching valve connected with two equivalent holding columns. To be highlighted here, this rationally designed interface for the purpose of smooth desalination, absorption and desorption, successfully solved the solvent compatibility problem between the two dimensional separation systems. The present integrated system was successfully applied in a one-step preparative separation and identification of 10 pure compounds from the water extracts of Tieguanyin tea (Chinese oolong tea). In short, all the results demonstrated that the on-line 2D CCC×LC method is an efficient and green approach for harvesting polar targets in a single step, which showed great promise in drug discovery.

Apoptosis-inducing and antitumor activity of neolignans isolated from Magnolia officinalis in HeLa cancer cells.

Two neolignans, 4'-methoxymagndialdehyde (1) and magnaldehyde B (2), were isolated from the stem bark of Magnolia officinalis (Magnoliaceae), evaluated for apoptosis-inducing effects in human cervical epitheloid carcinoma HeLa cells. The apoptosis-inducing activity of compounds 1 and 2 were assessed by DNA content using flow cytometric analysis. In the immunoblotting analysis, the treatment with 1 and 2 resulted in the cleavage of procaspase-8 and -3 and poly(ADP-ribose)polymerase into active forms. In addition, in vivo, the administration of 2 to Lewis lung carcinoma-inoculated mice evidenced a significant inhibition of tumor growth (volume) with reduction of 28.7% at concentration of 20 mg/kg, as compared with the control mice. These findings suggest that 2 can inhibit the proliferation of tumor cells, and might be an anti-tumoric agent.

[Preliminary study on chemical constituents seperated from Cayratia japonica].

OBJECTIVE: To study effective active constituents of Cayratia japonica,a genuine herbal medicine from Fujian. METHOD: Such chromatographic methods as Macroporous, Sephadex LH-20, ODS and normal phase silica gel column chromatography were adopted to separate the chemical components of C. japonica. RESULT: Thirteen compounds were obtained, and their structures were identified by analyzing multiple spectral data as luteolin(1), apigenin(2), triethyl citrate-(3), 3-formylindole(4), esculetin(5), bis(2-ethylhexyl)-phthalate(6), calendin(7), ethyl-trans-3,4-dihydr-oxycinnamate(8), luteolin7-O-D-glucoside(9),5-hydroxy-3,4-dimethyl-5-pentyl-2(5H-furanone(10),ethyl-3,4-dihydroxybenzoate(11), eriodictyol(12) and daucosterol(13). CONCLUSION: Among them, compounds 3-8 and 10-12 were separated from the plant for the first time.

Anti-diabetic effect of ginsenoside Rb(3) in alloxan-induced diabetic mice.

As one of the main active component of protopanaxdiol type ginsenosides, ginsenoside Rb(3) is rarely reported in the treatment of diabetes. The anti-diabetic activity of ginsenoside Rb(3) was investigated in a model of alloxan-induced diabetic mice in the present study. The physiological parameter such as fasting blood glucose level, oral glucose tolerance, body weight, food intake and water intake were measured. Glucose consumption in C2C12 myotubes was also determined in order to investigate the molecular mechanism of ginsenoside Rb(3) in anti-diabetes. The alloxan-induced diabetic mice were treated with ginsenoside Rb(3) for 2 weeks at doses of 5 mg/kg, 15 mg/kg and 25 mg/kg. After 2 weeks treatment of ginsenoside Rb(3), the fasting blood glucose levels of DG 15 and DG 25 were respectively reduced by 36.70% and 37.50% compared to control group. At a dose of 25 mg/kg, oral glucose tolerance was significantly improved compared to control group (P < 0.05). The AUC decreased by 34.47% (from 2442 ± 291 mmol·min/L to 1600 ± 109 mmol·min/L). Both food intake and water intake were remarkably lowered. The injury of pancreas tissues was repaired, which was observed by using HE staining and optic microscope. In vitro, at concentrations of 100 and 200 µM, ginsenoside Rb(3) increased glucose consumption in C2C12 myotubes by 76.83% and 97.20%, respectively, as compared to the control group. However, the body weight of diabetic mice was not significantly altered. In conclusion, our results showed that ginsenoside Rb(3) reduced fasting blood glucose level, food intake, water intake, improved oral glucose tolerance, and repaired injured pancreas tissues of alloxan-induced diabetic mice. Therefore, it was suggested that ginsenoside possesses the potential of the clinical use in preventing and treating diabetes.

Structural modification of ginsenoside Rh(2) by fatty acid esterification and its detoxification property in antitumor.

Ginsenoside Rh(2), one of the most important ginsenosides with anticancer properties in red ginseng, has been developed as principal antitumor ingredient for clinical use. However, the cytotoxicity test in human hepatocyte cell line QSG-7701 (IC(50) 37.3µM) indicated that Rh(2) might show strong cytotoxic side-effect on the normal liver cells. For blunting the toxicity, Rh(2) was structurally modified by reacting with octanoyl chloride to give a dioctanoyl ester of Rh(2) (D-Rh(2)) in the present study. MTT assay in QSG-7701 cell line in vitro showed that the cytotoxicity of D-Rh(2) on human hepatocyte cells (IC(50) 80.5µM) was significantly lower than that of Rh(2). While antitumor xenograft assay in mice bearing H22 liver cancer cells in vivo showed that the antitumor activity of D-Rh(2) retained to be strong as that of Rh(2). According to previous pharmacokinetic studies, the fatty acid esterification of Rh(2) might be of detoxification reaction to cells. Additionally, D-Rh(2) showed significant enhancement on increasing thymus index at the dose of 10mg/kg compared with vehicle treated control group. Thus, D-Rh(2) might indirectly affect tumor growth by stimulating lymphocytes to become cytotoxic to tumor cells. Finally, our findings suggested that D-Rh(2), the fatty acid ester of Rh(2), might attenuate the side-effect by detoxification to human normal cell and could be a more potential candidate for developing as an antitumor drug.

Fatty acids as natural specific inhibitors of the proto-oncogenic protein Shp2.

Src homology-2 domain-containing protein tyrosine phosphatase (Shp2), a novel proto-oncogenic protein, is an important target in cancer therapy research. Approximately 2000 plant extracts were screened to find its natural specific inhibitors, with the ethyl acetate (EtOAc) active extract of the root of Angelica dahurica showing considerable inhibitory effects (IC(50)=21.6 mg/L). Bioguided isolation of EtOAc extract led to 13 compounds, including 10 fatty acids and derivatives. All these compounds were isolated from the plant for the first time. The inhibitory effects of these compounds on the enzyme activities of Shp2, VH1-related human protein (VHR), and hematopoietic protein tyrosine phosphatase (HePTP) were investigated. 8Z,11Z-Feptadecadienoic acid (4), 14Z,17Z-tricosadienoic acid (5), caffeic acid (9), and 2-hydroxy-3-[(1-oxododecyl) oxy]propyl-ß-d-glucopyranoside (11) showed considerable selective inhibition of Shp2 activity. After treatment of HepG2 cells with the compounds, only compound 5, a polyunsaturated fatty acid, strongly induced poly (ADP-ribose) polymerase (PARP) cleavage in a dose- and time-dependent manner and increased the activities of caspase-3, caspase-8, and caspase-9 at 100 µM. Compound 5 also inhibited colony formation of HepG2 cells in a dose-dependent manner. Thus, this study reported fatty acids as specific Shp2 inhibitors and provided the molecular basis of one active compound as novel potential anticancer drug.

Furocoumarin derivatives from radix Angelicae dahuricae and their effects on RXRα transcriptional regulation.

A novel furocoumarin derivative named oxyalloimperatorin (1), together with seventeen furocoumarins 2-18 were isolated from the radix of Angelica dahurica. The chemical structure of new metabolite was characterized by analysis of IR, NMR, and HR-ESI-MS spectroscopic data. Among the isolated compounds, 13, 16, and 18 (each at 20 µM) could significantly promote the gene transcriptional function of nuclear receptor RXRα. While 7-9, 13, 14, and the new structure 1 (each at 20 µM) showed significant reduction in RXRα gene transcriptional activities induced by 9-cis-retinoid acid. The findings indicated that these furocoumarin skeleton derivatives might hold beneficial effects on many intractable diseases, such as cancer and metabolic diseases, due to their potential activities on regulating the transcriptional activation function of RXRα.

Three new oblongolides from Phomopsis sp. XZ-01, an endophytic fungus from Camptotheca acuminate.

Four new metabolites, including three new oblongolides named C1, P1, and X1 (1-3) and 6-hydroxyphomodiol (10), along with eight known compounds--oblongolides B (4), C (5), D (6), O (7), P (8) and U (9), (3R,4aR,5S,6R)-6-hydroxy-5-methylramulosin (11), and (3R)-5-methylmellein (12)--were isolated from the endophytic fungal strain Phomopsis sp. XZ-01 of Camptotheca acuminate. Their structures were elucidated by spectroscopic analyses, including 1H- and 13C-NMR, 2D NMR (HSQC, HMBC, 1H-1H COSY and NOESY) and HR-FT-MS. Cytotoxic activities of these compounds were evaluated. Some of them showed weak selective activities.

The selected flavonol glycoside derived from Sophorae Flos improves glucose uptake and inhibits adipocyte differentiation via activation AMPK in 3T3-L1 cells.

Among nine flavonols (1-9) obtained from Sophorae Flos, we first isolated compounds 4, 5, 8, and 9. These isolates (1-9) were evaluated for the phosphorylation of AMPK and ACC. Administered at 10 µM, 9 possessed high potent activity. Compound 9 displayed a dose-dependent stimulation of glucose uptake in 3T3-L1 cells, and this increase was obviously attenuated by compound C, an AMPK inhibitor. In addition, 9 also phosphorylated AMPK and its downstream substrate ACC in 3T3-L1 cells in a time- and dose-dependent manner. Moreover, we discovered that compound C inhibits 9-stimulated ACC phosphorylation and motivated the 9-inhibited C/EBPα and PPARγ, and FAS gene expression, significantly. These results revealed the role of the AMPK downstream signaling pathway in 9-improved glucose metabolism in 3T3-L1 cells and 9-inhibited adipocyte differentiation. Differentiation was investigated by Oil Red O staining activity after 9 administration (0-20 µM) in 6 days. Compound 9 decreased mean droplet size in a dose-dependent manner. The results revealed that 9 blocked adipogenic conversion in 3T3-L1 cells together with several significant downregulating adipocyte-specific transcription factors, including PPARγ, C/EBPα, and SREBP1. It also reduced FAS gene expression in a dose-dependent manner, which is crucial for adipogenesis in vitro.

Effects of Gardeniae Fructus extract and geniposide on promoting ligament cell proliferation and collagen synthesis.

Gardeniae Fructus is a traditional medicine used for the treatment of contusion such as ankle sprain. Geniposide is one of the main components of Gardeniae Fructus with diverse biological activities. In order to gain further insight into the therapeutic action of Gardeniae Fructus extract (GFE) and geniposide on ligament injuries, a new in vitro model was developed in the present study. Rat hind ankle ligament fibroblasts (RHALFs) derived from Sprague-Dawley rats were cultured, and the cell proliferation and collagen content were examined by MTT and a Sirius Red-based colorimetric assay after stimulating with each drug. The cell growth of RHALFs was promoted by culturing with 37.5-150 microg/mL of GFE and 25-200 microM of geniposide. The content of collagen in the RHALFs was significantly increased up to 131.4% and 124.2% of the control value by culturing with the GFE and geniposide, respectively. By contrast, both cell growth and collagen content were impaired by adding 25-200 microM of diclofenac, one of the common medications for ligament injuries. The findings suggest that GFE and geniposide may ameliorate the treatment of ligament injuries by proliferating ligament fibroblasts and promoting the synthesis of collagen. However, the use of diclofenac to treat acute ligament injuries should be reassessed although it possesses a potential effect on relieving symptoms.

Flavonoids and isoflavonoids from Sophorae Flos improve glucose uptake in vitro.

Glucose uptake assay-guided fractionations on the methanol extract of Sophorae Flos led to the isolation of the flavonoids rutin (1), narcissin (2), quercetin (3), tamarixetin (4), and kaempferol (5) and the isoflavonoids cajanin (6), genistein (7), orobol (8), and pratensein (9). Among them, 1, 4, 5, 6, 8, and 9 significantly improved basal glucose uptake in HepG2 cells. Their improving effects were concentration dependent. Compounds 4, 5, 6, and 9 exhibited effects stronger than that of rosiglitazone, which has been used as an antidiabetic drug. However, 2, 3, and 7 did not show any improving effects. Stimulating glucose uptake into peripheral cells may be responsible for reducing the level of blood glucose in the circulation. Therefore, these findings demonstrate a potential to develop these flavonoids and isoflavonoids as hypoglycemic drugs.

Iridoid glycosides from Gardeniae Fructus for treatment of ankle sprain.

The iridoid glycosides, genipin 1-O-beta-D-isomaltoside (1) and genipin 1,10-di-O-beta-D-glucopyranoside (2), together with six known iridoid glycosides, genipin 1-O-beta-D-gentiobioside (3), geniposide (4), scandoside methyl ester (5), deacetylasperulosidic acid methyl ester (6), 6-O-methyldeacetylasperulosidic acid methyl ester (7), and gardenoside (8) were isolated from an EtOH extract of Gardeniae Fructus. The structures and relative stereochemistries of the metabolites were elucidated on the basis of 1D- and 2D-NMR spectroscopic techniques, high-resolution mass spectrometry, and chemical evidence. Geniposide (4), one of the main compounds of Gardeniae Fructus, was tested for treatment of ankle sprain using an ankle sprain model in rats. From the second to fifth day, the geniposide (4) (100mg/ml) treated group exhibited significant differences (p<0.01) with approximately 21-34% reduction in swelling ratio compared with those of the vehicle treated control group. This indicated the potential effect of geniposide (4) for the treatment of disorders such as ankle sprain.

Stilbenes and oligostilbenes from leaf and stem of Vitis amurensis and their cytotoxic activity.

Chromatographic separation of the EtOAc fraction from the leaf and stem of Vitis amurensis led to the isolation of six oligostilbenoids (i.e., r-2-viniferin (1), trans-amurensin B (2), trans-epsilon-viniferin (3), gnetin H (4), amurensin G (5), (+)-ampelopsin A (8)) and four stilbenoids (i.e., trans-resveratrol (6), (+)-ampelopsin F (7), piceatannol (9), and trans-piceid (10)). The structures have been identified on the basis of spectroscopic evidence and physicochemical properties. The isolates were investigated for cytotoxic activity against three cancer cell lines in vitro using the MTT assay method. Amurensin G (5) and trans-resveratrol (6) showed significant cytotoxic activity against L1210, K562 and HTC116 cancer cell lines with IC(50) values ranging from 15.7 +/- 2.1 to 30.9 +/- 1.8 microM. (+)-Ampelopsin A (8) and trans-piceid (10) exhibited considerable cytotoxic activity against L1210 (IC(50) values of 30.6 +/- 4.1 and 28.7 +/- 2.81 microM, respectively) and K562 (IC(50) values of 38.6 +/- 0.82 and 24.6 +/- 0.76 microM, respectively). Gnetin H (4) showed only weak cytotoxic activity against L1210 with an IC(50) value of 40.1 +/- 4.23 microM. On the other hand, r-2-viniverin (1), trans-amurensin B (2), trans-epsilon-viniferin (3), (+)-ampelopsin F (7), and piceatannol (9) exhibited no activity on three cancer cell lines.

Biphenylquinolizidine alkaloids from Lagerstroemia indica.

Two new biphenylquinolizidine alkaloids, 5-epi-dihydrolyfoline (1) and its stereoisomer, dihydrolyfoline (2), along with lagerine (3) were isolated from the aerial parts of Lagerstroemia indica. The structures of compounds 1-3 were elucidated by extensive spectroscopic techniques.

Enantioselective determination of chlorpheniramine in various formulations by HPLC using carboxymethyl-beta-cyclodextrin as a chiral additive.

A chiral mobile phase HPLC method is described for chiral separation and determination of chlorpheniramine (CP) enantiomers in various commercial preparations. Chromatographic separation was achieved on a conventional ODS column with a mixture of aqueous sodium phosphate (5 mM) containing 0.5 mM carboxymethyl-beta-cyclodextrin, methanol and triethylamine (73:25:2, v/v/v, pH 4.3) as the mobile phase. The flow rate of isocratic elution was 0.24 mL/min and peaks were detected at 224 nm. The method was applied to nine commercial CP preparations in six dosage forms and CP enantiomers were well separated without any disturbance of other ingredients or impurities present. The results showed that only one preparation was d-CP and the others were dl-CP preparations. The contents of all the preparations were found to be in the range of 97%-104% of labeled contents. This method was economical and convenient, affording sufficient accuracy, precision and reproducibility, as well as sensitivity and selectivity.

Pyronane monoterpenoids from the fruit of Gardenia jasminoides.

Five new pyronane-type monocyclic monoterpenoids, jasminodiol (1), jasminoside H (6), 6'-O-sinapoyljasminoside A (7), 6'-O-sinapoyljasminoside C (8), and jasminoside I (9), together with four known analogues, were isolated from the fruit of Gardenia jasminoides. The structures of the new metabolites were characterized using spectroscopic data, and the absolute configurations of 1, 6, and 7 were established using circular dichroism (CD) analysis. Compound 1 showed tyrosinase inhibitory activity (IC 50 2.2 mM).

Two new lignans from the stem bark of Magnolia obovata and their cytotoxic activity.

Two new lignans, 4-methoxymagnaldehyde B (1) and coumanolignan (2), were isolated from the stem bark of Magnolia obovata, together with 11 known compounds (3-13). The structures of compounds 1 and 2 were determined to be 5'-allyl-2'-hydroxyphenyl-4-methoxy-3-cinnamic aldehyde (1) and 6-allyl-8-(5'-allyl-2'-hydroxyphenyl)coumarin (2) on the basis of spectroscopic and physicochemical analyses including 2D NMR and high-resolution EI-MS. Compounds 1-8, 11, 12, and 13 were tested in vitro for their cytotoxic activities against the HeLa, A549, and HCT116 cancer cell lines. Among the compounds tested, compound 1 showed the strongest cytotoxic activity against the HCT116 cancer cell line, with an IC(50) value of 1.3 microg/ml.