Herbal Extract from Codonopsis pilosula (Franch.) Nannf. Enhances Cardiogenic Differentiation and Improves the Function of Infarcted Rat Hearts.

BACKGROUND: The roots of Codonopsis pilosula (Franch.) Nannf. have been used in traditional Chinese medicine for treating cardiovascular disease. In the current study, we aimed to discover herbal extracts from C. pilosula that are capable of improving cardiac function of infarcted hearts to develop a potential therapeutic approach. METHODS: A mouse embryonic stem (ES) cell-based model with an enhanced green fluorescent protein (eGFP) reporter driven by a cardiomyocyte-specific promoter, the α-myosin heavy chain, was constructed to evaluate the cardiogenic activity of herbal extracts. Then, herbal extracts from C. pilosula with cardiogenic activity based on an increase in eGFP expression during ES cell differentiation were further tested in a rat myocardial infarction model with left anterior descending artery (LAD) ligation. Cardiac function assessments were performed using echocardiography, 1, 3, and 6 weeks post LAD ligation. RESULTS: The herbal extract 417W from C. pilosula was capable of enhancing cardiogenic differentiation in mouse ES cells in vitro. Echocardiography results in the LAD-ligated rat model revealed significant improvements in the infarcted hearts at least 6 weeks after 417W treatment that were determined based on left ventricle fractional shortening (FS), fractional area contraction (FAC), and ejection fraction (EF). CONCLUSIONS: The herbal extract 417W can enhance the cardiogenic differentiation of ES cells and improve the cardiac function of infarcted hearts.

Efficient differentiation of mouse embryonic stem cells into insulin-producing cells.

Embryonic stem (ES) cells are a potential source of a variety of differentiated cells for cell therapy, drug discovery, and toxicology screening. Here, we present an efficacy strategy for the differentiation of mouse ES cells into insulin-producing cells (IPCs) by a two-step differentiation protocol comprising of (i) the formation of definitive endoderm in monolayer culture by activin A, and (ii) this monolayer endoderm being induced to differentiate into IPCs by nicotinamide, insulin, and laminin. Differentiated cells can be obtained within approximately 7 days. The differentiation IPCs combined application of RT-PCR, ELISA, and immunofluorescence to characterize phenotypic and functional properties. In our study, we demonstrated that IPCs produced pancreatic transcription factors, endocrine progenitor marker, definitive endoderm, pancreatic ß-cell markers, and Langerhans α and δ cells. The IPCs released insulin in a manner that was dose dependent upon the amount of glucose added. These techniques may be able to be applied to human ES cells, which would have very important ramifications for treating human disease.

Effects of alpha-mangostin on the expression of anti-inflammatory genes in U937 cells.

BACKGROUND: α-Mangostin (α-MG) is a main constituent of the fruit hull of the mangosteen. Previous studies have shown that α-MG has pharmacological activities such as antioxidant, antitumor, anti-inflammatory, antiallergic, antibacterial, antifungal and antiviral effects. This study aims to investigate the anti-inflammatory molecular action of α-MG on gene expression profiles. METHODS: U937 and EL4 cells were treated with different concentrations of α-MG in the presence of 0.1 ng/mL lipopolysaccharide (LPS) for 4 h. The anti-inflammatory effects of α-MG were measured by the levels of tumor necrosis factor (TNF)-α and interleukin (IL)-4 in cell culture media, which were determined with enzyme-linked immunosorbent assay kits. The gene expression profiles of all samples were analyzed with a whole human genome microarray, Illumina BeadChip WG-6 version 3, containing 48804 probes. The protein levels were determined by Western blotting analyses. RESULTS: α-MG decreased the LPS induction of the inflammatory cytokines TNF-α (P = 0.038) and IL-4 (P = 0.04). α-MG decreased the gene expressions in oncostatin M signaling via mitogen-activated protein kinase (MAPK) pathways, including extracellular signal-regulated kinases (P = 0.016), c-Jun N-terminal kinase (P = 0.01) , and p38 (P = 0.008). α-MG treatment of U937 cells reduced the phosphorylation of MAPK kinase 3 / MAPK kinase 6 (P = 0.0441), MAPK-activated protein kinase-2 (P = 0.0453), signal transducers and activators of transcription-1 (STAT1) (P = 0.0012), c-Fos (P = 0.04), c-Jun (P = 0.019) and Ets-like molecule 1 (Elk-1) (P = 0.038). CONCLUSION: This study demonstrates that α-MG attenuates LPS-mediated activation of MAPK, STAT1, c-Fos, c-Jun and EIK-1, inhibiting TNF-α and IL-4 production in U937 cells.

Development and validation of molecular markers for characterization of Boehmeria nivea var. nivea and Boehmeria nivea var. tenacissima.

BACKGROUND: The root of Boehmeria spp (ramie) is a hepatoprotective Chinese herbal medicine. Medicinal properties vary between Boehmeria nivea var. nivea and Boehmeria nivea var. tenacissima, which are local species found in Taiwan. As commercial preparations may use either species, there is a need for a rapid and simple assay to identify variants for quality control. METHODS: Four methods were developed and tested for their applicability in differentiating the two species. These methods were random amplified polymorphic DNA (RAPD); sequence characterized amplified regions (SCAR); single nucleotide polymorphisms (SNP) and cleaved amplified polymorphic sequences (CAPS). RESULTS: Three RAPD markers were developed that produced unique bands in B. nivea var. tenacissima and B. nivea var. nivea. Based on sequenced RAPD bands, one SCAR marker was developed that produced a single DNA band in B. nivea var. nivea. Two SNP markers differentiated between B. nivea var. nivea and B. nivea var. tenacissima based on single nucleotide substitutions. A pair of CAPS oligonucleotides was developed by amplifying a 0.55-kb DNA fragment that exhibited species-specific digestion patterns with restriction enzymes Alf III and Nde I. Consistent results were obtained with all the four markers on all tested Boehmeria lines. CONCLUSION: The present study demonstrates the use of the RAPD, SCAR, SNP and CAPS markers for rapid identification of two closely related Boehmeria species.

Melanogenesis inhibition by gallotannins from Chinese galls in B16 mouse melanoma cells.

Nowadays herbal medicines of skin-whitening cosmetics are popular with women. We attempted to find the whitening activity compounds present in many herbal medicines used for this purpose and discuss their mechanisms in melanin biosynthesis. The 70% acetone extracts of 10 kinds of herbs were investigated for their mushroom tyrosinase activity inhibition. Among these 10 extracts, Chinese galls showed inhibitory activity against tyrosinase, with a 50% inhibitory concentration (IC(50)) value of 22 microg/ml. In a B16 mouse melanoma cell culture assay, Chinese galls dose-dependently inhibited melanin biosynthesis. Using ultraviolet A (UVA) or alpha-melanocyte-stimulating hormone (alpha-MSH) to stimulate B16 cells after Chinese gall treatment, the melanin biosynthesis of B16 cells was inhibited in a dose-dependent manner. The active compounds of Chinese galls were isolated by column chromatography, and the melanin biosynthesis inhibition in B16 melanoma cells was measured. Three gallotannins, 2,3,4,6-tetra-O-galloyl-D-glucopyranose, 1,2,3,6-tetra-O-galloyl-beta-D-glucopyranose, and 1,2,3,4,6-penta-O-galloyl-beta-D-glucopyranose, were isolated from Chinese gall extract, and their IC(50) values of tyrosinase inhibition activity were 54, 30, and 15 muM, respectively. By the mushroom tyrosinase inhibition kinetics assay, the three gallotannins were all determined to be non-competitive inhibitors. These results indicated that Chinese galls inhibit melanin biosynthesis, associated with hyperpigmentation and can be used as skin-whitening cosmetics for skin care.

Ellagitannins from Terminalia calamansanai induced apoptosis in HL-60 cells.

Terminalia calamansanai (Blanco) Rolf. (Combretaceae) is used medicinally as lithontriptic in Philippines. The 70% acetone extracts of T. calamansanai leaves inhibited the viability of human promyelocytic leukemia HL-60 cells. 1-Alpha-O-galloylpunicalagin, punicalagin, 2-O-galloylpunicalin, sanguiin H-4, and methyl gallate were the main components isolated from T. calamansanai with the IC(50) values of 65.2, 74.8, 42.2, 38.0 and >100 microM, respectively, for HL-60 cells. Apoptosis of HL-60 cells treated with 1-Alpha-O-galloylpunicalagin, punicalagin, 2-O-galloylpunicalin, and sanguiin H-4 was noted by the appearance of a sub-G(1) peak in flow cytometric analysis and DNA fragmentation by gel electrophoresis. 2-O-Galloylpunicalin and sanguiin H-4 induced a decrease of the human poly(ADP-ribose)polymerase (PARP) cleavage-related procaspase-3 and elevated activity of caspase-3 in HL-60 cells, but not normal human peripheral blood mononuclear cells (PBMCs), suggesting that both compounds may be new candidates for drug development in the prevention and treatment of cancer.

Sennoside B inhibits PDGF receptor signaling and cell proliferation induced by PDGF-BB in human osteosarcoma cells.

AIMS: To address the possibility that sennoside B inhibition of cell proliferation is mediated via interference with platelet-derived growth factor (PDGF) signaling. MAIN METHODS: Human osteosarcoma MG63 cells were treated with PDGF in the presence or absence of sennoside B. Activation of the PDGF signaling pathway was monitored using western immunoblotting with specific antibodies against the PDGF receptor, phosphotyrosine and components of the downstream signaling cascade. Activation of cell metabolism and proliferation was assessed by chromogenic reduction of MTT. KEY FINDINGS: Sennoside B was found to inhibit PDGF-BB-induced phosphorylation of the PDGF receptor (PDGFR) in human MG63 osteosarcoma cells. Downstream signaling was also affected; pre-incubation of PDGF-BB with sennoside B inhibited the phosphorylation of pathway components including Ak strain transforming protein (AKT), signal transducer and activator of transcription 5 (STAT-5) and extracellular signal-regulated kinase 1/2 (ERK1/2). Further, we found that sennoside B can bind directly to the extracellular domains of both PDGF-BB and the PDGF-beta receptor (PDGFR-beta). The effect was specific for sennoside B; other similar compounds including aloe-emodin, rhein and the meso isomer (sennoside A) failed to inhibit PDGFR activation or downstream signaling. Sennoside B also inhibited PDGF-BB stimulation of MG63 cell proliferation. SIGNIFICANCE: These results indicate that sennoside B can inhibit PDGF-stimulated cell proliferation by binding to PDGF-BB and its receptor and by down-regulating the PDGFR-beta signaling pathway. Sennoside B is therefore of potential utility in the treatment of proliferative diseases in which PDGF signaling plays a central role.

Effects of 6-gingerol, an antioxidant from ginger, on inducing apoptosis in human leukemic HL-60 cells.

6-Gingerol, a naturally occurring plant phenol, is one of the major components of fresh ginger. In this paper, the antioxidative effects of 6-gingerol were detected by DPPH and DCFH assays and, as predicted, 6-gingerol as an antioxidant was shown to protect HL-60 cells from oxidative stress. Moreover, it induced cell death in promyelocytic leukemia HL-60 cells, caused DNA fragmentation and inhibited Bcl-2 expression in HL-60 cells. These results suggested that the inhibition of Bcl-2 expression in HL-60 cells might account for the mechanism of 6-gingerol-induced apoptosis. In the inhibitory assay, the cytotoxic effect of 6-gingerol could be prevented by catalase. We suggest that 6-gingerol induced cell death by mediating reactive oxygen species such as hydrogen peroxide and the superoxide anion. Therefore, the results showed that 6-gingerol induced apoptosis in HL-60 cells, not due to its antioxidative activity.

Aqueous two-phase extraction as an effective tool for isolation of geniposide from gardenia fruit.

Natural products are normally obtained by organic solvent extraction and many subsequent chromatographic separations. Compounds of interest are often isolated with very low yield and limited purity. An aqueous two-phase extraction process combined with a simple ethanol treatment, for removing excess inorganic salt, has been developed for preparation of geniposide from gardenia. The system was comprised of PE62, a random copolymer composed of 20% ethylene oxide and 80% propylene oxide, KH2PO4 and ethanol. To find optimal conditions, the partition behavior of geniposide under an aqueous two-phase system was investigated. Various factors were considered, including the concentration of salt, the concentration of polymer, the sample loading, and the addition of ethanol. The experimental results demonstrated that increasing salt concentration or decreasing PE62 concentration results in enhancement of the geniposide partition in the salt-rich phase. The addition of ethanol and higher sample loading also promoted the partition efficiency of geniposide. Based on this study, an optimized system containing 5% PE62, 7.5% KH2PO4, and 10% ethanol was tested on a large-scale extraction. A 39.0-g aliquot of final product (in powder form) with 77% purity of geniposide can be effectively extracted from 500 g of gardenia fruit. This process is proved to be useful for industrial application of geniposide preparation.

Inducible nitric oxide synthase inhibitors of Chinese herbs III. Rheum palmatum.

In this paper, the effects of bioactive compounds of Rheum palmatum L. on the inhibition of NO production from RAW 264.7 cells were explored. Seven main anthraquinone derivatives were isolated from the root of R. palmatum, and of these, emodin and rhein significantly inhibited nitrite production from lipopolysaccharide (LPS)-activated RAW 264.7 cells. The IC(50) values for inhibition of nitrite production by emodin and rhein were 60.7 and 67.3 microM, respectively. After iNOS enzyme activity was stimulated by LPS for 12 h, treatment with emodin or rhein at 20 microg/ml for 18 h did not significantly inhibit NO production. The data show that the inhibitory activity of emodin and rhein is not due to direct inhibition of iNOS enzyme activity. However, expression of iNOS and the COX-2 protein was inhibited by emodin in LPS-activated RAW 264.7 cells, and PGE(2) production was reduced. Rhein also inhibited LPS-induced iNOS protein expression, but not COX-2 or PGE(2) production. On the other hand, inhibition effects on NO production from RAW 264.7 cells were enhanced and cytotoxic effects decreased by co-treatment with emodin and rhein. In conclusion, emodin and rhein are major iNOS inhibitors of R. palmatum and may possibly serve as bioactive substances for anti-inflammation effects.