Isolation of positive, allosteric GABA(A) receptor modulators from Chinese herbal drugs traditionally used in the treatment of anxiety and insomnia

In der Traditionell Chinesischen Medizin gibt es eine Reihe von Arzneidrogen die gegen Angst- (weltweite Prävalenz ~ 16 %) und Schlafstörungen (~ 30 %) eingesetzt werden. Dennoch gibt es kaum wissenschaftliche Beweise für deren Wirkung und den zugrundeliegenden Wirkmechanismen, auch wenn pflanzliche Drogen als effektiv und besser verträglich als synthetische Arzneistoffe angesehen werden. In der vorliegenden Arbeit wurden 14 verschiedene zur Behandlung von Angst- und Schlafstörungen eingesetzte chinesische Arzneidrogen auf ihre Aktivität am GABAA Rezeptor untersucht, wobei dieser Rezeptor ein wichtiges Angriffsziel für angstlösende und sedierende Arzneistoffe darstellt. Weiters ist GABA der wichtigste inhibitorische Neurotransmitter im Säugetiergehirn. Für die in-vitro Untersuchungen wurden α1β2γ2S GABAA Rezeptoren in Xenopus laevis Oozyten exprimiert und die Potenzierung des GABA-induzierten Chloridionenstroms (IGABA) mittels der Zwei-Mikroelektroden Spannungs-Klemm Technik und einem automatisierten Perfusionssystem gemessen. In einem ersten Aktivitäts-Screening von 4x14 Extrakten unterschiedlicher Polarität zeigten die lipophilen Extrakte von Atractylodes macrocephala, Cnidium monnieri L. und Juncus effusus L. die höchste Aktivität. Von diesen wurden die für die Wirkung verantwortlichen Inhaltsstoffe bioaktivitäts-geleitet isoliert und als Sesquiterpenlaktone, Coumarine und Phenanthrenderivate identifiziert. Alle Komponenten konnten IGABA konzentrationsabhängig modulieren, ohne dabei über die Benzodiazepinebindungsstelle zu wirken. Diese ist eine wichtige allosterische Bindungsstelle am GABAA Rezeptor für gebräuchliche sedierende oder anxiolytische Wirkstoffe wie Benzodiazepine oder die sogennanten "z-drugs". Weiters wurden die isolierten Coumarine mit ähnlichen Coumarinderivaten in ihrer Wirkung verglichen, und erste Einsichten in die strukturellen Voraussetzungen für eine Modulation von IGABA durch Coumarine gewonnen. In dieser Dissertation wurde ein wissenschaftlicher Nachweis für die ethnomedizinische Anwendung der drei Arzneipflanzen erbracht. Des Weiteren wurden neue GABAA Rezeptor Modulatoren gefunden, die als Grundstrukturen zur Entwicklung von neuen anxiolytischen und sedierenden Arzneistoffen dienen könnten.In Chinese Medicine several herbal drugs are frequently applied against anxiety or insomnia, two highly prevalent mental disorders affecting around 16 and 30 % of the population worldwide. Although herbal remedies are considered effective as well as better-tolerated compared to synthetic drugs like benzodiazepines, scientific proof of their activity and the molecular mechanisms underlying it are rare. In the present work 14 different Chinese herbal drugs commonly applied in the treatment of insomnia, restlessness or anxiety were screened for their activity on the GABAA receptor, which is the major inhibitory neurotransmitter receptor in the mammalian brain and a key target for anxiolytic as well as sedative drugs. For the in-vitro assay α1β2γ2S GABAA receptors were expressed in Xenopus laevis oocytes and modulation of the GABA-induced chloride current (IGABA) was measured using the two-microelectrode voltage clamp technique and an automated fast perfusion system. In a preliminary screening of 4x14 extracts of different polarity the lipophilic extracts from Atractylodes macrocephala Koidz., Cnidium monnieri L. and Juncus effusus L. displayed highest activity. From these extracts the active principles were isolated in a bioactivity-guided manner and identified as sesquiterpene lactones, coumarins and phenanthrene derivatives, respectively. All components potentiated IGABA concentration-dependently without involvement of the benzodiazepine binding site, which is an important allosteric target site on the GABAA receptor for typical sedative and anxiolytic drugs eg. benzodiazepines and "z-drugs". Furthermore, the isolated coumarins were compared to other coumarin derivatives regarding their activity on the GABAA receptor and first insights into the structural requirements for IGABA modulation by this compound class could be gained. Thus in the present thesis scientific evidence for the ethnomedicinal use of three herbal drugs was found. Moreover, new GABAA receptor modulators were discovered which could function as novel scaffolds for the development of sedative and anxiolytic drugs

Towards Modernization of the Formulation of the Traditional Uighur Medicine Herbal Preparation Abnormal Savda Munziq

Abnormal Savda Munziq (ASMq) is a herbal preparation used in Traditional Uighur Medicine for the treatment and prevention of diabetes, cardiovascular diseases, chronic asthma and cancer. The recommended dose of this decoction for cancer patients is 500 mL administered orally three times a day. Our approach aimed at reducing the high amount of fluid intake required by fractionation of ASMq guided by the antiproliferative activity on HL-60 cells. The fractionation of ASMq resulted in the preparation of an active extract, Extr-4. Using solid phase extraction, Extr-4 was further fractionated into five fractions (SPE-0, SPE-20, SPE-40, SPE-60 and SPE-80), with SPE-40 showing the strongest antiproliferative activity. Caffeic acid, rutin, isoquercitrin, isorhamnetin 3-O-rutinoside, apigenin 7-O-glucoside, rosmarinic acid, luteolin and formononetin were identified in Extr-4 and fractions thereof by means of TLC, HPLC-DAD and LC-MS. SPE-40 contained the main compounds responsible for the antiproliferative activity on HL-60 cells. Thus, a phenolic fraction with high antiproliferative activity on HL-60 cells was obtained from ASMq through the bioassay-guided fractionation process. This could provide a better pharmaceutical formulation that minimizes the administration inconveniencies of a high volume (1.5 L per day) of ASMq decoction for cancer patients

Fractionation of an Extract of Pluchea odorata Separates a Property Indicative for the Induction of Cell Plasticity from One That Inhibits a Neoplastic Phenotype

Introduction. Several studies demonstrated that anti-inflammatory remedies exhibit excellent anti-neoplastic properties. An extract of Pluchea odorata (Asteraceae), which is used for wound healing and against inflammatory conditions, was fractionated and properties correlating to anti-neoplastic and wound healing effects were separated. Methods. Up to six fractionation steps using silica gel, Sephadex columns, and distinct solvent systems were used, and eluted fractions were analysed by thin layer chromatography, apoptosis, and proliferation assays. The expression of oncogenes and proteins regulating cell migration was investigated by immunoblotting after treating HL60 cells with the most active fractions. Results. Sequential fractionations enriched anti-neoplastic activities which suppressed oncogene expression of JunB, c-Jun, c-Myc, and Stat3. Furthermore, a fraction (F4.6.3) inducing or keeping up expression of the mobility markers MYPT, ROCK1, and paxillin could be separated from another fraction (F4.3.7), which inhibited these markers. Conclusions. Wound healing builds up scar or specific tissue, and hence, compounds enhancing cell migration support this process. In contrast, successful anti-neoplastic therapy combats tumour progression, and thus, suppression of cell migration is mandatory

In vitro anti-neoplastic activity of the ethno-pharmaceutical plant Hypericum adenotrichum Spach endemic to western Turkey

Hypericum perforatum (St. John's wort) is well-established for its antidepressant activity throughout the world and also various other species within this genus are used in different folk medicines. Hyperforin of St. John's wort inhibited growth of cancer cell lines and the use of hypericin (another compound of H. perforatum) in cancer photodynamic therapy is proposed. Therefore, we investigated the anti-cancer properties of H. adenotrichum Spach (Guttiferae), an endemic species in Turkey called ‘kantaron’, which is used for wound healing and antiseptic effects. Freeze-dried plant was extracted with petroleum ether, dichloromethane, ethyl acetate, and methanol and the bioactivity of these extracts was analysed by proliferation assay, cell death determination, by investigating protein expression profiles specific for cell cycle arrest and apoptosis as well as composition by HPLC. The strongest anti-proliferative activity was determined for the petroleum ether extract with an IpC50 of H"5.8 µg/ml medium (referring to 1 mg dried plant) which correlated with cyclin D1 suppression and p21 induction. This extract also induced phosphorylation of H2AX, and activated caspase-3 followed by signature-type cleavage of PARP resulting in H"50% apoptosis at 23.2 µg/ml after 24 h of treatment. Neither hyperforin, hypericin, or amentoflavone contributed to these properties. To the best of our knowledge, we report for the first time that the endemic plant H. adenotrichum Spach exhibits potent p53-independent anti-neoplastic properties due to yet unexplored Hypericum constituents