Militarinone A induces differentiation in PC12 cells via MAP and Akt kinase signal transduction pathways

AbstractThe fungal metabolite militarinone A (MILI A) promotes neurite outgrowth in PC12 cells. This study was conducted to investigate the signaling pathways involved in the cellular differentiation processes induced by the compound, with a focus on cascades implicated with nerve growth factor (NGF)-mediated neuritogenesis. MILI A possessed pronounced amphiphilic properties. The compound rapidly accumulated in the cell membrane and was slowly released into the cytoplasma. In primed PC12 cells, an early activation of protein kinase B (Akt), representing a downstream target of phosphoinositol 3 (PI3) kinase, and a delayed phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), and of transcription factor cAMP responsive element binding protein (CREB) was found. The NGF-dependent activation of c-Jun amino terminal kinase (SAPK/JNK1) was potentiated. Morphological differentiation of cells and the phosphorylation of specific signal molecules were blocked by the MAP kinase (MEK1) inhibitor PD098059, the PI3-kinase (PI3K) inhibitor wortmannin and the adenylyl cyclase inhibitor 9-cyclopentyladenine

Promotion of cell death or neurite outgrowth in PC-12 and N2a cells by the fungal alkaloid militarinone A depends on basal expression of p53

The fungal alkaloid militarinone A (MiliA) was recently found to stimulate neuronal outgrowth in PC-12 cells by persistant activation of pathways that are also involved in NGF-mediated differentiation, namely the PI3-K/PKB and the MEK/ERK pathways. Application of equal concentrations of MiliA to other cells such as the murine neuroblastoma cell line N2a resulted in immediate onset of apoptosis by nuclear translocation of apoptosis inducing factor (AIF), activation of caspases and c-Jun/AP-1 transcription factor without an intermediate differentiated phenotype, although minor transient phosphorylation of PKB and MAPK as well as activation of NF-κB were also observed. Translocation of AIF was preceded by p53 phosphorylation at Ser15 and blocked by pifithrin α, a known inhibitor of p53-transcriptional activity. We here show that both cell types activate the same pathways albeit in different time scales. This is mainly due to contrasting basal expression levels of p53, which in turn regulates expression of AIF. In PC-12 cells, continuous activation of these pathways after prolonged treatment with 40μM MiliA first led to up-regulation of p53, phosphorylation of p53, release of AIF from mitochondria and its translocation into the nucleus. Additionally, also activation of the c-Jun/AP-1 transcription factor was observed, and PC-12 cells subsequently underwent apoptosis 48-72h post-treatment. We report that similar pathways working on different levels are able to initially shape very divergent cellular response

HPLC-based activity profiling for GABAA receptor modulators from the traditional Chinese herbal drug Kushen ( Sophora flavescens root)

An EtOAc extract from the roots of Sophora flavescens (Kushen) potentiated γ-aminobutyric acid (GABA)-induced chloride influx in Xenopus oocytes transiently expressing GABAA receptors with subunit composition, α 1 β 2 γ 2S. HPLC-based activity profiling of the extract led to the identification of 8-lavandulyl flavonoids, kushenol I, sophoraflavanone G, (−)-kurarinone, and kuraridine as GABAA receptor modulators. In addition, a series of inactive structurally related flavonoids were characterized. Among these, kushenol Y (4) was identified as a new natural product. The 8-lavandulyl flavonoids are first representatives of a novel scaffold for the targe

Screening and HPLC-Based Activity Profiling for New Antiprotozoal Leads from European Plants

Based on a survey of remedies used in Renaissance Europe to treat malaria, we prepared and screened a library of 254 extracts from 61 plants for antiplasmodial activity in vitro. HPLC-based activity profiling was performed for targeted identification of active constituents in extracts. One of the most remarkable results was the identification of onopordopicrin, a germacranolide sesquiterpene lactone isolated from Arctium nemorosum as a potent inhibitor of P. falciparum with an IC50 of 6.9 μM. It was tested similarly against Trypanosoma brucei rhodesiense, the parasite which causes African sleeping sickness. With an IC50 of 0.37 μM, onopordopicrin was one of the most potent natural products reported so far. Cytotoxicity was determined against rat myoblast L6 cells (IC50: 3.06)

Mining sudanese medicinal plants for antiprotozoal agents

Neglected tropical diseases are major health hazards in developing countries. Annually, up to 30 million people are affected by either Chagas disease, African trypansomiasis or leishmaniasis, and more than 200 million by malaria. Most of the currently available drugs have drawbacks in terms of toxicity, limited oral availability, development of resistance, or non-affordability. Tropical plants of the arid zones are a treasure chest for the discovery of bioactive secondary metabolites. This study aims to compile Sudanese medicinal plants, validate their antiprotozoal activities, and identify active molecules. We have performed a survey of medicinal plants of Sudan and selected 62 that are being used in Sudanese traditional medicine. From these, we collected materials such as leaves, stem, bark, or fruit. The plant materials were extracted in 70% ethanol and further fractionated by liquid-liquid partitioning using solvents of increasing polarity. This resulted in a library of 235 fractions. The library was tested; in vitro; against; Plasmodium falciparum; (erythrocytic stages),; Trypanosoma brucei rhodesiense; (bloodstream forms),; Trypanosoma cruzi; (intracellular amastigotes), and; Leishmania donovani; (axenic amastigotes). Active fractions were also tested for cytotoxicity. Of the 235 fractions, 125 showed growth inhibitory activity >80% at 10 μg/ml, and >50% at 2 μg/ml against at least one of the protozoan parasites.; Plasmodium falciparum; was the most sensitive of the parasites, followed by; T. b. rhodesiense; and; L. donovani; . Only few hits were identified for; T. cruzi; , and these were not selective. Contrary to expectation based on phylogeny, but in agreement with previous results, a large number of extracts displayed mutual activity against; T. brucei; and; P. falciparum; . HPLC-based activity profiling for selected active extracts was performed to identify the bioactive principles. Active compounds identified by dereplication were guieranone A from; Guiera senegalensis; J.F.Gmel.; pseudosemiglabrin from; Tephrosia apollinea; (Delile) DC; ellagic acid and quercetin from; Terminalia leiocarpa; (DC.) Baill.; and catechin, ethyl gallate, and epicatechin gallate from; Vachellia nilotica; (L.) P.J.H.Hurter & Mabb. Also the extracts of; Croton gratissimus; var.; gratissimus; and; Cuscuta hyalina; Roth ex Schult. exhibited promising antitrypanosomatid activity. This assessment provides a comprehensive overview of Sudanese medicinal plants and supports the notion that they are a potential source of bioactive molecules against protozoan parasites

Pheophorbide a identified in an Eupatorium perfoliatum extract is a novel lymphatic vascular activator

The lymphatic vascular system is crucial for maintaining tissue fluid homeostasis and immune surveillance. Promoting lymphatic function represents a new strategy to treat several diseases including lymphedema, chronic inflammation and impaired wound healing. By screening a plant extract library, a petroleum ether extract from the aerial parts of Eupatorium perfoliatum (E. perfoliatum) was found to possess lymphangiogenic properties. With the aid of HPLC activity profiling the active compound was identified as pheophorbide a. Both plant extract and pheophorbide a induced the sprouting and tube formation of human primary lymphatic endothelial cells (LECs). The proliferation of the LECs was increased upon treatment with pheophorbide a but not the E. perfoliatum extract. Treatment with the MEK1/2 inhibitor U0126 reduced the LEC sprouting activity, indicating a potential mechanism of action. These studies suggest that pheophorbide a could represent novel natural therapeutic agent to treat human lymphatic vascular insufficiencies

Comparative study of four immortalised human brain capillary endothelial cell lines, hCMEC/D3, hBMED, TY10, and BB19, and optimization of culture conditions, for an in vitro blood-brain barrier model for drug permeability studies

BACKGROUND: Reliable human in vitro blood–brain barrier (BBB) models suitable for high-throughput screening are urgently needed in early drug discovery and development for assessing the ability of promising bioactive compounds to overcome the BBB. To establish an improved human in vitro BBB model, we compared four currently available and well characterized immortalized human brain capillary endothelial cell lines, hCMEC/D3, hBMEC, TY10, and BB19, with respect to barrier tightness and paracellular permeability. Co-culture systems using immortalized human astrocytes (SVG-A cell line) and immortalized human pericytes (HBPCT cell line) were designed with the aim of positively influencing barrier tightness. METHODS: Tight junction (TJ) formation was assessed by transendothelial electrical resistance (TEER) measurements using a conventional epithelial voltohmmeter (EVOM) and an automated CellZscope system which records TEER and cell layer capacitance (C(CL)) in real-time. Paracellular permeability was assessed using two fluorescent marker compounds with low BBB penetration (sodium fluorescein (Na-F) and lucifer yellow (LY)). Conditions were optimized for each endothelial cell line by screening a series of 24-well tissue culture inserts from different providers. For hBMEC cells, further optimization was carried out by varying coating material, coating procedure, cell seeding density, and growth media composition. Biochemical characterization of cell type-specific transmembrane adherens junction protein VE-cadherin and of TJ proteins ZO-1 and claudin-5 were carried out for each endothelial cell line. In addition, immunostaining for ZO-1 in hBMEC cell line was performed. RESULTS: The four cell lines all expressed the endothelial cell type-specific adherens junction protein VE-cadherin. The TJ protein ZO-1 was expressed in hCMEC/D3 and in hBMEC cells. ZO-1 expression could be confirmed in hBMEC cells by immunocytochemical staining. Claudin-5 expression was detected in hCMEC/D3, TY10, and at a very low level in hBMEC cells. Highest TEER values and lowest paracellular permeability for Na-F and LY were obtained with mono-cultures of hBMEC cell line when cultivated on 24-well tissue culture inserts from Greiner Bio-one® (transparent PET membrane, 3.0 μm pore size). In co-culture models with SVG-A and HBPCT cells, no increase of TEER could be observed, suggesting that none of the investigated endothelial cell lines responded positively to stimuli from immortalized astrocytic or pericytic cells. CONCLUSIONS: Under the conditions examined in our experiments, hBMEC proved to be the most suitable human cell line for an in vitro BBB model concerning barrier tightness in a 24-well mono-culture system intended for higher throughput. This BBB model is being validated with several compounds (known to cross or not to cross the BBB), and will potentially be selected for the assessment of BBB permeation of bioactive natural products

Wound Healing Potential of Chlorogenic Acid and Myricetin-3-O-β-Rhamnoside Isolated from Parrotia persica

Wound healing is a complex physiological process that is controlled by a well-orchestrated cascade of interdependent biochemical and cellular events, which has spurred the development of therapeutics that simultaneously target these active cellular constituents. We assessed the potential of Parrotia persica (Hamamelidaceae) in wound repair by analyzing the regenerative effects of its two main phenolic compounds, myricetin-3-O-β-rhamnoside and chlorogenic acid. To accomplish this, we performed phytochemical profiling and characterized the chemical structure of pure compounds isolated from P. persica, followed by an analysis of the biological effects of myricetin-3-O-β-rhamnoside and chlorogenic acid on three cell types, including keratinocytes, fibroblasts, and endothelial cells. Myricetin-3-O-β-rhamnoside and chlorogenic acid exhibited complementary pro-healing properties. The percentage of keratinocyte wound closure as measured by a scratch assay was four fold faster in the presence of 10 µg/mL chlorogenic acid, as compared to the negative control. On the other hand, myricetin-3-O-β-rhamnoside at 10 µg/mL was more effective in promoting fibroblast migration, demonstrating a two-fold higher rate of closure compared to the negative control group. Both compounds enhanced the capillary-like tube formation of endothelial cells in an in vitro angiogenesis assay. Our results altogether delineate the potential to synergistically accelerate the fibroblastic and remodelling phases of wound repair by administering appropriate amounts of myricetin-3-O-β-rhamnoside and chlorogenic acid

Traditional Use of Herbal Remedies in Livestock by Farmers in 3 Swiss Cantons (Aargau, Zurich, Schaffhausen)

Background: This study investigated the extent of traditional knowledge and use of homemade herbal remedies for livestock by farmers in 3 Swiss cantons (Aargau, Zurich, Schaffhausen). The study focused on organic farms. Methods: At 21 farms, 24 farmers aged 36–83 years were interviewed with a semi-structured, detailed questionnaire. For each homemade herbal remedy, the plant species, mode of preparation, source of knowledge, and application were gathered. Satisfaction of the farmers with the application was estimated with the aid of a visual analogue scale. Results: Information on a total of 165 homemade remedies was collected of which 123 contained a single plant species only (homemade mono-species herbal remedies, HMHR). The 123 HMHR were selected for this paper. They corresponded to 150 different applications and originated from 43 plant species from 30 families. Plants belonging to the families of Asteraceae, Lamiaceae, and Apiaceae were used most frequently. The single most applied species were Matricaria recutita L., Calendula officinalis L., Symphytum officinale L., and Coffea arabica L. For each formulation, 1–4 different applications were mentioned, most of them for cattle. The main applications were skin alterations and sores, gastrointestinal and metabolic diseases as well as infertility and diseases of the female genitalia. Approximately half of the applications were used during the last 12 months prior to the interview. Conclusion: This study shows that HMHR are used by Swiss farmers for the treatment of different livestock diseases. In general, the farmers were satisfied with the outcome of the applications