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l Planta Medica An International Journal of Natural Products and Medicinal Plant Research Editor-in-Chief Luc Pieters, Antwerp, Belgium Advisory
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l Planta Medica An International Journal of Natural Products and Medicinal Plant Research Editor-in-Chief Luc Pieters, Antwerp, Belgium Advisory Board Giovanni Appendino, Novara, Italy John T. Arnason, Ottawa, Canada Yoshinori Asakawa, Tokushima, Japan Lars Bohlin, Uppsala, Sweden Gerhard Bringmann, Würzburg, Germany Reto Brun, Basel, Switzerland Mark S. Butler, Singapore, R. of Singapore Ihsan Calis, Ankara, Turkey Salvador Caæigueral, Barcelona, Spain Hartmut Derendorf, Gainesville, USA Verena Dirsch, Vienna, Austria Jürgen Drewe, Basel, Switzerland Roberto Maffei Facino, Milan, Italy Alfonso Garcia-Piæeres, Frederick MD, USA Rolf Gebhardt, Leipzig, Germany Clarissa Gerhäuser, Heidelberg, Germany Jürg Gertsch, Zürich, Switzerland Simon Gibbons, London, UK De-An Guo, Beijing, China Leslie Gunatilaka, Tuscon, USA Solomon Habtemariam, London, UK Andreas Hensel, Münster, Germany Werner Herz, Tallahassee, USA Kurt Hostettmann, Geneva, Switzerland Peter J. Houghton, London, UK Jinwoong Kim, Seoul, Korea Gabriele M. König, Bonn, Germany Ulrich Matern, Marburg, Germany Matthias Melzig, Berlin, Germany Dulcie Mulholland, Guildford, UK Eduardo Munoz, Cordoba, Spain Kirsi-Maria Oksman-Caldentey, Espoo, Finland Ana Maria de Oliveira, Sˆo Paulo, Brazil Nigel B. Perry, Dunedin, New Zealand Joseph Pfeilschifter, Frankfurt, Germany Peter Proksch, Düsseldorf, Germany Thomas Schmidt, Münster, Germany Volker Schulz, Berlin, Germany Hans-Uwe Simon, Bern, Switzerland Leandros Skaltsounis, Athens, Greece Han-Dong Sun, Kunming, China Benny K. H. Tan, Singapore, R. of Singapore Ren Xiang Tan, Nanjing, China Deniz Tasdemir, London, UK Nunziatina de Tommasi, Salerno, Italy Arnold Vlietinck, Antwerp, Belgium Angelika M. Vollmar, München, Germany Heikki Vuorela, Helsinki, Finland Jean-Luc Wolfender, Geneva, Switzerland De-Quan Yu, Beijing, China Publishers Georg Thieme Verlag KG Stuttgart New York Rüdigerstraûe 14 D Stuttgart Postfach D Stuttgart Senior Editor Adolf Nahrstedt, Münster, Germany Review Editor Matthias Hamburger, Basel, Switzerland Editors Wolfgang Barz, Münster, Germany Rudolf Bauer, Graz, Austria Veronika Butterweck, Gainesville FL, USA Joˆo Batista Calixto, Florianopolis, Brazil Thomas Efferth, Heidelberg, Germany Jerzy W. Jaroszewski, Copenhagen, Denmark Ikhlas Khan, Oxford MS, USA Wolfgang Kreis, Erlangen, Germany Irmgard Merfort, Freiburg, Germany Kurt Schmidt, Graz, Austria Thomas Simmet, Ulm, Germany Hermann Stuppner, Innsbruck, Austria Yang-Chang Wu, Kaohsiung, Taiwan Yang Ye, Shanghai, China Editorial Offices Claudia Schärer, Basel, Switzerland Tess De Bruyne, Antwerp, Belgium Thieme Publishers 333 Seventh Avenue New York, NY 10001, USA Reprint Georg Thieme Verlag KG Stuttgart New York Reprint with the permission of the publishers only l This is a copy of the author's personal reprint l 6 Original Paper Ginkgo biloba Extract and its Flavonol and Terpenelactone Fractions do not Affect β-secretase mrna and Enzyme Activity Levels in Cultured Neurons and in Mice Author Sabine Augustin 1, Patricia Huebbe 2, Nicole Matzner 2, Kay Augustin 2, Reinhard Schliebs 3, Rainer Cermak 4, Siegfried Wolffram 1, Gerald Rimbach 2 Affiliation Affiliation addresses are listed at the end of the article Key words Ginkgo biloba Ginkgoaceae EGb761 β-secretase Alzheimer's disease amyloid-beta Tg2576 received June 28, 2007 revised November 21, 2007 accepted November 24, 2007 Bibliography DOI /s Planta Med 2008; 74: 6 13 Georg Thieme Verlag KG Stuttgart New York Published online January 10, 2008 ISSN Correspondence Prof. Gerald Rimbach Institute of Human Nutrition and Food Science Christian Albrechts University of Kiel Hermann-Rodewald-Strasse Kiel Germany Tel.: Fax: Introduction Alzheimer's disease (AD) is a widespread neurodegenerative disorder. The clinical manifestation of AD is associated with memory loss, impairment of language and behaviour and disorientation in time and space. Pathological hallmarks of AD are extracellular deposits of amyloid-beta peptides and intracellular neurofibrillary tangles (NFTs) in brain regions such as the cortex and hippocampus [1], [2], [3]. The extract of Ginkgo biloba L., Ginkgoaceae, EGb761, is a standardised, concentrated extract from the leaves of the ginkgo tree (drug extract ratio: 35 67: 1). Its main constituents are glycosides of the flavonols quercetin, isorhamnetin and kaempferol (24 %) and the terpenelactones bilobalide and ginkgolides A, B and C (6 %) [4]. In recent years, the leaf extracts of Ginkgo biloba have been widely sold as a phytomedicine in Europe and as a dietary supplement worldwide. Human clinical trials have reported potential beneficial effects of EGb761 in the prevention and therapy of mild to moderate AD. Several studies documented a significant enhancement in concentration and cognitive and social functions in Abstract Numerous clinical trials have reported beneficial effects of the Ginkgo biloba extract EGb761 in the prevention and therapy of cognitive disorders including Alzheimer's disease (AD). Although neuroprotective properties of EGb761 have been consistently reported, the molecular mechanisms of EGb761 and the specific role of its major constituents, the flavonols and terpenlactones, are largely unknown. One major hallmark of AD is the deposition of amyloid-beta (Aβ) as amyloid plaques in the brain. Aβ is a cleavage product of amyloid precursor protein (APP). Certain proteases, called β-secretases (BACE), are crucial in the formation of Aβ.The purpose of the present study was to investigate the efficacy of EGb761 and its flavonol and terpenelactone fraction to modulate BACE-1 enzyme activity and mrna levels in vitro and in vivo. Neither EGb761 nor its fractions affected BACE-1 activity in vitro. Furthermore, also in Neuro-2a cells and wild-type as well as transgenic (Tg2576) laboratory mice, no significant effect of EGb761 on BACE-1 enzyme activity and mrna levels were observed. Current findings suggest that BACE-1 may not be a major molecular target of EGb761 and its flavonol and terpenelactone fraction. response to Ginkgo biloba treatment [5], [6], [7], [8]. EGb761 alters neuronal excitability, synaptic efficacy and plasticity in the hippocampus of aged mice, which is a possible explanation for its age-related effects on memory [9]. The ability of EGb761 to enhance acquisition and retention of memory was observed in several animal studies [10]. The molecular mechanism of the neuroprotective properties of EGb761 and the role of its constituents are largely unknown. Besides postulated effects as a vasoregulatory and antioxidant agent, neuroprotective and gene regulatory properties of EGb761 may mediate its positive health effects [11], [12]. Watanabe and co-workers revealed that EGb761 has notable effects on the expression of several genes encoding for proteins with potential neuroprotective properties in the brain of mice [12]. Amyloid plaques, one of the pathogenic hallmarks in the brain of AD patients, are extracellular deposits of amyloid-β peptides (Aβ). Aβ are a cleavage product of amyloid precursor protein (APP), a neuronal transmembrane protein. Thus, enzymes and substrates involved in the processing of APP as well as APP itself are potential targets in the prevention and treatment of AD. APP Augustin S et al. Ginkgo biloba Extract Planta Med 2008; 74: 6 13 Original Paper 7 is cleaved by several proteases, so-called α-, β-, and γ-secretases. The β-secretase BACE (N-terminal β-site APP cleaving enzyme) is a type I transmembrane protein of the pepsin and renin family of aspartyl proteinases, localised in the Golgi system [3]. Two homologue proteases with β-secretase-activity are known, BACE-1 (EC ) and BACE-2 (EC ) [13], [14]. BACE-1 is the major β-secretase in vivo, mainly expressed in the CNS and the pancreas and was found in neurons, but not in glia cells. However, the enzyme activity of BACE-1 is enriched in the brain, but hardly detectable in the pancreas [14], [15], [16]. BACE-1 is required for the cleavage of APP and the generating of Aβ [17], [18]. BACE-1 cleaves APP at the so-called β-site and generates the two products βapp and C99, a COOH-terminal fragment which remains membrane bound. Further cleavage of the C99 fragment by γ-secretases generates pathogenic Aβ (40 43 amino acids). Aβ monomers are soluble but in adequately high concentration Aβ aggregates extracellularly to amyloid fibrils and deposits, so-called senile plaques [19]. An alternative pathway for APP degradation is initiated by α-secretases. Further cleavage by γ-secretases generates the non-pathogenic p3 peptide [16]. Thus, cleavage of APP by the BACE-1 is the critical step in generating the pathogenic Aβ fragment. Both pathways for APP degradation occur in vivo, but cleavage by β-secretase is physiologically subordinate to α-secretase [1], [20]. Individuals bearing the so-called Swedish mutation in the gene encoding for APP are subject to increased formation of amyloid plaques in the brain. This mutation was found in a large Swedish family with early onset of Alzheimer's disease and leads to an enhanced formation of double mutated APP with an increased affinity towards BACE-1. Thus, the substrate of BACE-1 is augmented and, in turn, the amount of Aβ deposits increases equally [20], [21], [22]. The transgenic Tg2576 mice which express the Swedish mutation of human APP695 (K670N, M671L) develop Alzheimer-like β-amyloid deposits in the brain and thus represent an appropriate animal model to study the β-secretase under pathological conditions [23], [24]. Since BACE-1 is centrally involved in the development of senile plaques, a potential modulation of BACE-1 mrna and activity levels by dietary or pharmacological treatment is one focus in current AD research. The purpose of the present study was to investigate the effect of Ginkgo biloba extract EGb761 and its main constituents on gene expression and enzyme activity of BACE-1 Table 1 Amount of flavonols, ginkgolides and bilobalides in EGb761 and its flavonol and terpenelactone fractions EGb761, DM Flavonol fraction, HE 171/ Amount ( %w/w) * Flavonols 25 Quercetin Kaempferol Isorhamnetin 2.3 n. d. Terpenoids 1 Sesquiterpenoids Bilobalide Diterperpenoids Ginkgolide A Ginkgolide B Ginkgolide C * Data provided by Dr. Schwabe pharmaceuticals (Karlsruhe, Germany). Data are shown as % w/w to the whole extract. in vitro, in cultured neuronal cells and in vivo in laboratory mice. Additionally, in the transgenic mouse model Tg2576 the effect of a long-term treatment with EGb761 on BACE-1 enzyme activity was investigated. Material and Methods Materials Neutral red dye, dimethyl sulfoxide (DMSO), agarose, β-mercaptoethanol and ethidium bromide dilution were obtained from Carl Roth (Karlsruhe, Germany). Cell culture medium, foetal bovine serum, phosphate buffered saline (PBS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), trypsin/ethylenediaminetetraacetic acid (EDTA) and penicillin/streptomycin were all from PAA Laboratories (Coelbe, Germany). Phenylmethanesulfonyl fluoride (PMSF), E-64, Pepstatin A, EDTA and Triton X-100 were obtained from Sigma (Steinheim, Germany). Neuro-2a cells were purchased from DSMZ (Braunschweig, Germany). Total cell and tissue protein was measured with the BCA kit (Pierce Biotechnology;, Rockford, IL, USA). Source of extracts Three different extracts of Ginkgo biloba leaves were used in the present study: the standardised Ginkgo biloba extract (EGb761), a flavonol fraction and a terpenelactone fraction of Ginkgo biloba. All extracts were a kind gift from Dr. Schwabe Pharmaceuticals (Karlsruhe, Germany). EGb761, the flavonol and terpenelactone fractions were manufactured according to the German Federal Health Authority (BGA/BfArM Kommission E, 1994) using acetone/water as extraction solvent and subsequent purification steps. The drug extract ratio is 35 67: 1, on average 50 :1 [25]. In Table 1, the main constituents of each extract are shown. The amounts of the main flavonols and terpenelactones were measured by HPLC and GC/MS, respectively [26], [27]. BACE-1 enzyme activity in vitro To study the effect of Ginkgo biloba extract and its fractions on the beta-secretase enzyme activity in vitro, human recombinant BACE-1 (R&D Systems; Wiesbaden-Nordenstadt, Germany) activity was measured in the presence of EGb761 (1 50 μg/ml) Terpenelactone fraction, HE 186C/10 231/ Bz n.d.: not detectable. Augustin S et al. Ginkgo biloba Extract Planta Med 2008; 74: 6 13 8 Original Paper and its flavone and terpenelactone fraction (1 50 μg/ml). Measurements were conducted according to the protocol of the β-secretase activity kit (R&D Systems). A BACE-1 inhibitor (OM99 2, 500 nmol/l; Mobitec; Goettingen, Germany) was used as positive control. Measurement occurred in the Infinite TM F200 microplate reader (Tecan; Crailsheim, Germany). Cell culture Neuro-2a cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10 % foetal bovine serum, 4 mmol/l L- glutamine, 100 μg/ml penicillin and 100 μg/ml streptomycin. Cells were grown in a humidified incubator at 37 8C and 5 % CO 2. For ribonucleic acid (RNA) isolation, Neuro-2a cells ( cells/well) were seeded in 6-well plates (Sarstedt; Nuembrecht, Germany). After 24 hours, test substances were applied and cells were incubated for another 24 hours until harvesting. Cells were harvested by trypsin/edta treatment and lysed with RLT buffer (Qiagen; Hilden, Germany) containing β-mercaptoethanol as described in the manufacturer's protocol. Total RNA was isolated with the RNeasy Mini Kit (Qiagen) according to the corresponding protocol. Deoxyribonucleic acid (DNA) digestion was done with RNase-Free DNase Set (Qiagen). RNA integrity was checked with denaturating agarose gel electrophoresis and ethidium bromide staining. The concentration and purity of isolated RNA was determined by measuring the absorbance (AB λ ) at 260 nm and 280 nm in a spectrophotometer (Beckmann Instruments; Munich, Germany). A ratio of 1.9 between AB 260 nm and AB 280 nm was considered as acceptable. RNA aliquots were stored at 80 8C until analyses. Cytotoxicity test In order to avoid toxic concentrations of the test substance in further experiments, cells ( cells/well) were cultured in 12-well plates for 24 hours before treatment. Cells were incubated with test substances at increasing concentrations (5 250 μg/ml medium) for another 24 hours. Cell viability was measured by using the neutral red assay according to Borenfreund and Puerner [28]. Cell viability (neutral red uptake) was determined by reading AB λ at 540 nm (Labsystems iems Reader; Helsinki, Finland). BACE-1 enzyme activity in Neuro-2a cells For assessing BACE-1 enzyme activity, Neuro-2a cells ( cells/well) were cultured in 6-well plates for 24 hours before EGb761 (25 μg/ml medium) was supplemented. 24, 36 and 48 hours after addition of EGb761 cells were harvested and lysed with a special lysis buffer (1 mmol/l HEPES, 1 mmol/l ETDA, 0.2 % Triton X-100, 1 mmol/l PMSF, 10 μg/ml Pepstatin A, 10 μg/ml E-64). As a positive control, Neuro-2a cells were cultured with α-tocopherol (BASF; Ludwigshafen, Germany) at 25 μmol/l for 24 h, which resulted in 23.2 ± 2.6 % inhibition of BACE-1 activity. Enzyme activity measurements were conducted according to the manufacturer'protocol of the β-secretase activity kit (R&D Systems). Fluorescence intensity was measured at 488/520 nm using an Infinite TM F200 microplate reader (Tecan). Animals Eighty female C57B6 mice aged 6 months (Charles River Breeding Laboratories; Sulzfeld, Germany) were randomly allocated to four dietary groups (n = 20 each, body weight 24.5 ± 1.2 g; means ± SEM). Five mice per cage were housed and maintained under standard conditions (23 ± 1 8C, 55 % relative humidity, 14- h light/10-h dark cycle). The mice were fed a commercial semisynthetic low-flavonoid diet (C1000 code #100E; Altromin; Lage, Germany) enriched with no extract, 300 mg/kg EGb761, 300 mg/kg flavonol fraction or 100 mg/kg terpenelactone fraction. Prior to dietary intervention mice had an adaptation period with the control diet for one week. The animals had ad libitum access to the diet and tap water. Body weights were recorded weekly. After 4 weeks of intervention, mice were decapitated and blood was collected in heparinised tubes. Pooled blood samples were taken from all animals per cage. Plasma was obtained after centrifugation (2000 g for 10 min, 4 8C) and stored at -80 8C until analyses. Brain cortices and hippocampi for PCR analyses were rapidly dissected and immediately suspended in RNAlater TM RNA stabilisation reagent (Qiagen) and incubated overnight. Total RNA was extracted according to the RNeasy Lipid Tissue Protocol (Qiagen). RNA integrity, concentration and purity were checked as described above. RNA aliquots were stored at -80 8C until analyses. Tissues (10 per group) for measurement of enzyme activity were frozen in liquid nitrogen after dissection and stored at 80 8C until analyses. Animal care and experimental procedures were conducted according to the German Guidelines and Regulation on Animal Care (Deutsches Tierschutzgesetz, 2006) and were approved by the University of Kiel Committee on Animal Care. Transgenic animals The transgenic mouse model used in this study has been established by Hsiao et al. [23], [24]. The transgene is expressed in C57B6/SJL F1 mice (kindly provided by Dr. Karen Hsiao, University of Minnesota). Transgenic mice were generated by crossing wild-type female (C57B6/SJL F1) with Tg2576 males (C57B6/SJL F2). The transgenity was monitored in 3-week-old animals in tail biopsy material by PCR [24]. At the age of 4 months, female animals were randomly allocated into 8 groups as shown in Table 2. Mice were fed a commercial semi-synthetic low-flavonoid diet (C1000, Altromin) with no addition (control) or with the addition of 300 mg/kg EGb761 over a period of 4 weeks or 16 months ( Table 2). After the intervention period, mice aged 5 and 20 months, respectively, were decapitated. Brain cortices for measurement of Table 2 Groups, durations, treatments and group abbreviations (n = 3 each, body weight 21.5 ± 1.2 g; means ± SEM) Short-term treatment (4 weeks) transgenic wild-type transgenic + EGb761 Long-term treatment (16 months) tg - control - young wt - control - young tg - EGb761 - young wild-type + EGb761 wt - EGb761 - young transgenic wild-type transgenic + EGb761 tg - control - old wt - control - old tg - EGb761 - old wild-type + EGb761 wt - EGb761 - old Augustin S et al. Ginkgo biloba Extract Planta Med 2008; 74: 6 13 Original Paper 9 enzyme activity were rapidly dissected and frozen in liquid nitrogen and stored at -80 8C until analyses. Animal care and experimental procedures were conducted according to the German Guidelines and Regulation on Animal Care (Deutsches Tierschutzgesetz, 2006) and were approved by the University of Kiel Committee on Animal Care. Determination of BACE-1 gene expression BACE-1 and β-actin primer pairs were designed to the corresponding sequence of Mus musculus mrna sequences (NM_ for BACE-1 and according NM_ for β-actin) with primer3 software (; ). Primer sequences are listed in Table 3. Primer pairs were obtained from MWG (Ebersberg, Germany). QuantiTect Primer Assay (Qiagen) was used for 18S rrna amplification, with a product of 149 bp. For one-step quantitative reverse transcriptase polymerase chain reaction (One-Step qrt- PCR) two aliquots of RNA were amplified. External relative standard curves of total RNA were determined with each run. Data were normalised by dividing the expression level of BACE- 1 by the level of β-actin or 18S rrna. Each PCR reaction (final volume 20.0 μl) contained 0.5 μm of each primer, 10.0 μl of 2 QuantiTect SYBR Green RT-PCR Master Mix, 0.2 μl Quanti- Tect RT-Mix, 8.0 μl of RNA dilution and 1.4 μl water. Real-time cycler conditions were processed accordi
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