Identification of compounds responsible for the anthelmintic effects of chicory (Cichorium intybus) by molecular networking and bio-guided fractionation

10 páginas, 7 figuras.Increasing resistance towards anthelmintic drugs has necessitated the search for alternative treatments for the control of gastrointestinal nematode parasites. Animals fed on chicory (Cichorium intybus L.), a temperate (pasture) crop, have reduced parasite burdens, hence making C. intybus a potentially useful source for novel anthelmintic compounds or a diet-based preventive/therapeutic option. Here, we utilized in vitro bioassays with the parasitic nematode Ascaris suum and molecular networking techniques with five chicory cultivars to identify putative active compounds. Network analysis predicted sesquiterpene lactones (SL) as the most likely group of anthelmintic compounds. Further bioassay-guided fractionation supported these predictions, and isolation of pure compounds demonstrated that the SL 8-deoxylactucin (8-DOL) is the compound most strongly associated with anti-parasitic activity. Furthermore, we showed that 8-DOL acts in a synergistic combination with other SL to exert the anti-parasitic effects. Finally, we established that chicory-derived extracts also showed activity against two ruminant nematodes (Teladorsagia circumcincta and Cooperia oncophora) in in vitro assays. Collectively, our results confirm the anti-parasitic activity of chicory against a range of nematodes, and pave the way for targeted extraction of active compounds or selective breeding of specific cultivars to optimize its future use in human and veterinary medicine.We are very grateful for the guidance and support by Dr. Thomas Ostenfeld Larsen and Christopher Phippen, Technical University of Denmark, Natural product discovery, and the laboratory assistance of Mette Schjelde, University of Copenhagen. This work was funded by the Danish Council for Independent Research (Grant DFF–6111-00394). Fractionation and purification of compounds were further supported by the Green Development and Demonstration Program (GUDP) (Project No. 34009-17-1220). MPE was supported by CONICYT Chile (FONDE-CYT Postdoctorado #317087

A comparison of headspace solid-phase microextraction and classic hydrodistillation for the identification of volatile constituents from Thapsia spp. provides insights into guaianolide biosynthesis in Apiaceae

ntroduction - Thapsia spp. (Apiaceae) are the major natural source of polyoxygenated guaianolide sesquiterpene lactones known as thapsigargins, which induce apoptosis in mammalian cells via a high affinity inhibition of the sarco/endoplasmic reticulum Ca(2+) ATPase. The mechanism of biosynthesis of thapsigargins has not been elucidated, and probable biochemical precursors such as hydrocarbon or oxygenated sesquiterpenes have not been identified in previous phytochemical analyses of essential oils from this genus. Objective - To investigate the utility of solid phase micro-extraction (SPME), when compared with classical essential oil distillates, for identifying potential precursors of guaianolide sesquiterpene lactones from Thapsia garganica L. and Thapsia villosa L. type II. Methodology - A systematic description of the volatile components of roots, flowers, stems and fruits of T. villosa and of root, flower and fruits of T. garganica was constructed via GC-MS analyses of SPME-adsorbed compounds and of essential oils obtained through hydrodistillation of the same tissues. Results - The sesquiterpenoids delta-cadinene, alpha- and delta-guaiene, elemol and guaiols were found to be major volatile constituents of the roots of T. garganica and T. villosa trapped using SPME. In contrast, these sesquiterpenoids were not detected or were at negligible levels in essential oils, where sesquiterpenoids are potentially converted to azulenes during hydrodistillation. Conclusion - The new data reported in this study demonstrates that SPME is a valuable tool for the identification of volatile sesquiterpenes when compared with analysis of essential oils, and we postulate that guaiene is the likely precursor of guaianolide sesquiterpenes from Thapsia

Domesticating deadly carrots: Predicting the biosynthetic pathway of thapsigargins for the treatment of solid tumors

Thapsigargin, a guaianolide sesquiterpene lactone, is the active component of a pro-drug currently in phase 2 of clinical trials for the treatment of solid tumours. Thapsigargin is currently only being isolated from Thapsia garganica L. (Apiaceae); a member of the taxonomically complex genus Thapsia L. found in the Mediterranean. Thapsia species do not grow well out of their natural habitat and on-going research seeks to solve the potential resource problem for Mipsagargin, a new thapsigargin derived drug, by transferring the biosynthetic pathway to other heterologous hosts for efficient production [1]. Plant material of Thapsia garganica has been collected from around the species' Mediterranean range, as part of a revision of the genus. The amount of thapsigargin and other closely related guaianolides was quantified between individuals across its range, in planta and during the growing season. Secondly the expression levels of the terpene synthases TgTPS1 and TgTPS2, as well as a cytochrome P450, thought to be involved in the biosynthesis of thapsigargin, were investigated. These tests have also been carried out in conjunction with soil analyses to test if fluctuations in the levels of thapsigargin can be linked to environmental factors. Thapsigargin has been found to be extremely variable over its geographical distribution even over small areas. Lastly, to see whether candidates for the biosynthesis of thapsigargin could shed light on where the compound is made within the plant, root sections have been investigated from greenhouse plants provided by ThapsIbiza. Using light microscopy and MALDI-imaging epithelial cells containing thapsigargin and surrounding secretory channels within the vascular cambium were observed, indicating for the first time that this is the localisation of thapsigargin biosynthesis. It is hoped that these results will fast track the discovery of new enzymes involved in the biosynthetic pathway of thapsigargin

Stable Production of the Antimalarial Drug Artemisinin in the Moss Physcomitrella patens

Malaria is a real and constant danger to nearly half of the world’s population of 7.4 billion people. In 2015, 212 million cases were reported along with 429,000 estimated deaths. The World Health Organization recommends artemisinin-based combinatorial therapies, and the artemisinin for this purpose is mainly isolated from the plant Artemisia annua. However, the plant supply of artemisinin is irregular, leading to fluctuation in prices. Here, we report the development of a simple, sustainable, and scalable production platform of artemisinin. The five genes involved in artemisinin biosynthesis were engineered into the moss Physcomitrella patens via direct in vivo assembly of multiple DNA fragments. In vivo biosynthesis of artemisinin was obtained without further modifications. A high initial production of 0.21 mg/g dry weight artemisinin was observed after only 3 days of cultivation. Our study shows that P. patens can be a sustainable and efficient production platform of artemisinin that without further modifications allow for industrial-scale production. A stable supply of artemisinin will lower the price of artemisinin-based treatments, hence become more affordable to the lower income communities most affected by malaria; an important step toward containment of this deadly disease threatening millions every year

Short-term scientific mission at CSIC-Universidad de León: Potent in vitro anthelmintic effects of the seaweed Saccharina latissima against Teladorsagia circumcinta

Trabajo presentado al: JOINT COMBAR - ACSRPC meeting: Anthelmintic Resistance in Ruminants: Who cares? Abstract Book, Short Term Scientific Missions, pp. 36. Gante (Bélgica). 27-29 agosto 2019.Seaweed contains an abundance of bioactive compounds, and some seaweed species have been used for livestock feeding and as natural deworming agents for centuries. However, there is a lack of scientific evidence. In this study, we investigated the in vitro anthelmintic (AH) activity of extracts of seaweed from cold (Nordic) waters. We prepared three different extracts with hexane, dichlormethan:methanol (DCM), or water:methanol (WM) of dried and milled seaweed from four species: Saccharina latissima, Laminaria digitata, Ascophyllum nodosum, and Palmaria palmata. Both fermented (addition of Lactobacillus spp.) and non-fermented material (N=24) was included. The AH activity was assessed using an 48 hour Teladorsagia circumcincta first stage larval (L1) mortality assay (1 mg DM/mL dissolved in DMSO). The results showed a high AH activity of all six S. latissima extracts and fermented L. digitata extracts, and no activity of A. nodosum extracts. An egg hatch assay (EHA), using the same extracts (1 mg/mL, 48 h) and T. circumcincta eggs, showed >95% inhibition by the WM extracts of fermented and non-fermented S. latissima, and fermented L. digitata and P. palmata. We conclude that the seaweed S. latissima has a strong in vitro AH effect against eggs and L1 of the common sheep nematode T. circumcincta, and that fermentation of the other seaweeds may increase their ability to inhibit egg hatching

Localization and in-Vivo Characterization of Thapsia garganica CYP76AE2 Indicates a Role in Thapsigargin Biosynthesis

The Mediterranean plant Thapsia garganica (dicot, Apiaceae), also known as deadly carrot, produces the highly toxic compound thapsigargin. This compound is a potent inhibitor of the sarcoplasmic-endoplasmic reticulum Ca2+-ATPase calcium pump in mammals and is of industrial importance as the active moiety of the anticancer drug mipsagargin, currently in clinical trials. Knowledge of thapsigargin in planta storage and biosynthesis has been limited. Here, we present the putative second step in thapsigargin biosynthesis, by showing that the cytochrome P450 TgCYP76AE2, transiently expressed in Nicotiana benthamiana, converts epikunzeaol into epidihydrocostunolide. Furthermore, we show that thapsigargin is likely to be stored in secretory ducts in the roots. Transcripts from TgTPS2 (epikunzeaol synthase) and TgCYP76AE2 in roots were found only in the epithelial cells lining these secretory ducts. This emphasizes the involvement of these cells in the biosynthesis of thapsigargin. This study paves the way for further studies of thapsigargin biosynthesis