Bioactivity of plants secondary metabolites: Estrogenic, cytotoxic and anabolic effects on estrogen target organs of an extract of Erythrina excelsa and Ecdysterone

Sadrine Tchoukouegno Ngueu

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Medicinal plants serve as primary health care in most Asian and African countries. In developed countries where allopathic medicine is the most practiced form of medicine, chemicals of plant origin are increasingly consumed either as herbal formulations, diet, or complement to existing therapies in order to prevent or treat diseases. There is a general belief that because of their natural origin, herbal remedies are safe. Therefore they are commonly used in self therapeutic measures and are not prescribed in Germany. This work deals with the bioactivity and potential side effects of such herbal remedies. Namely the extract from Erythrina excelsa and the pure compound Ecdysterone were the focus of our attention. Erythrina excelsa is a plant used in the Cameroonian traditional medicine to alleviate menopausal symptoms and to treat female gynecological tumors. However, no test for biological activities evidencing these health benefit claims has been made so far. Ecdysterone (Ecdy) is the principal constituent of several plants used in the Chinese, Ayurvedic, and Russian traditional medicines to treat various ailments. Ecdy has been shown to exert anabolic properties in vitro and in vivo. Although the mechanisms underlying these effects are not yet elucidated, it is advertised as a food supplement to increase muscle mass and physical performance and is used by bodybuilders. A misuse for doping purpose in sports is suspected. The aims of this thesis were on the one hand to investigate the biological activities likely to explain the traditional use of Erythrina excelsa and to characterize its biologically active ingredients. On the other hand, this work also aims at examining the mechanisms underlying the anabolic effect of ecdysterone Using the yeast estrogen screen, the uterotrophic assay, and the MTT assay, we showed that an ethanol extract of E. excelsa exhibits estrogenic/anti-estrogenic and cytotoxic effects. A three-day subcutaneous administration of the extract at a dose of 50 mg/kg BW/day to ovariectomized Wistar rats produced significant estrogenic effects on uterine morphometric parameters (wet weight and epithelial height) and on the gene expression of estrogen responsive genes in the uterus (C3 and Clusterin) and in the liver (CaBP9K and IGFBP-1). Unexpectedly, at a dose of 100 mg/kg BW/day the extract significantly decreased the same parameters below the control level indicating anti-estrogenic effects. The extract exhibited a biphasic profile on the growth of ER positive MCF-7 cells with a slight increase of cell viability at low concentrations and induction of cell death at higher concentrations. But on ER negative HT-29, cytotoxicity was observed at all concentrations tested. To get more insights into the active principles of the extract, a bioactivity-guided fractionation was performed and the bioactive fractions were identified by means of both YES and XTT assays. The effect of the most active fraction [Dichloromethane-Methanol (50:50, v/v) (F4)] on the proliferation and apoptosis of MCF-7 breast cancer cell line was assessed by flow cytometry, gene expression analysis and immunohistochemistry. F4 exhibited a non-monotonic concentration-response effect on the proliferation and apoptosis of MCF-7 breast cells. At low concentrations and in the absence of E2, F4 promoted cell cycle progression through ER signaling and modulated gene expression in favor of cell survival (upregulation of cyclin D1 and and downregulation of the Bax/Bcl-2 ratio mRNA). Interestingly, when E2 was present, F4 at the same concentrations acted as an antagonist and prevented E2-induced cell growth. At higher concentrations, F4 modulated the cell cycle in favor of increased apoptotic cell death (increase of caspase cleaved cytokeratin 18, upregulation of Bax/Bcl-2 ratio and downregulation of cyclin D1 mRNA). In collaboration with the group of Dr. Halabalaki from the Department of Pharmacognosy and Natural Products Chemistry of the Unviversity of Athens, a LC-HRMS and LC-HRMS/MS analysis was performed to identify the isoflavonoids contained in the extract. This analysis showed that pyran and prenyl derivatives of isoflavonoids are the most characteristic chemical groups of the extract and may explain the observed biological activities. In the second part of this work, the mechanisms of the anabolic effect of ecdysterone, Erythrina excelsa extract and other plant secondary metabolites such as genistein were studied in a cell culture model of differentiated C2C12 myoblasts. This test system is a well established model to study the ability of anabolic substances to induce skeletal muscle hypertrophy as measured by the increase in the diameter of myotubes. Several comparative substances such as androgen receptor agonist (DHT) and antagonist (flutamide), anabolic peptide hormone (IGF-1), and different estrogen receptor binding compounds with agonistic (E2), antagonistic (ZK), and ERα and ERβ selective binding properties were used for the characterization of the involved molecular mechanisms. With this model, we confirmed the skeletal muscle growth-promoting effects of IGF1, DHT, E2, Ecdy, and the phytoestrogen genistein. Co-treatment of myotubes with ZK and Ecdy led to an inhibition of Ecdy-induced hypertrophy. In addition, a co-treatment of myotubes with E2 or Ecdy and the selective ERβ antagonist prevented both substances to induce myotube hypertrophy, what unequivocally shows an ERβ-mediated effect. In general, the results of this work show that the extract of Erythrina excelsa has biological activities that are in close agreement with its reported traditional use to manage menopausal complaints and to treat female gynecological tumors. They also highlight the fact that this plant is a source of substances of potential interest for further pharmacological investigations. In addition, our findings also show that substances with ERβ binding affinity promote skeletal muscle growth and may provide new insights into therapeutic strategies for the treatment of skeletal muscle atrophic conditions such as the age-related decline in muscle mass (sarcopenia). However, this effect may be misused for doping purpose and is therefore also of importance for Anti-doping regulation.
Original languageGerman
Place of PublicationKöln
PublisherDeutsche Sporthochschule Köln
Number of pages143
Publication statusPublished - 2013