Abstract
Medicinal plants represent a rich source of bioactive compounds, namely primary and secondary metabolites (e.g. PMs and SMs), and have been widely used for their therapeutic properties since ancient times. In recent years, there has been a growing interest in expanding the range of pharmaceutical applications of SMs. As natural products represent a huge structural and functional diversity, nowadays SMs have become well established active ingredients beyond pharmaceuticals, in the field of cosmetics and food supplements.This continuous increasing demand of plant-based products lead to the investigation of modern and large-scale techniques for producing higher quantities of bioactive compounds from medicinal plants. The production of SMs in the plants and thus, plant metabolome, are highly affected according to environmental conditions and cultivation regime, that can hinder their commercial exploitation. Furthermore, plant-microorganism symbioses, such as the treatment with arbuscular m ...
Medicinal plants represent a rich source of bioactive compounds, namely primary and secondary metabolites (e.g. PMs and SMs), and have been widely used for their therapeutic properties since ancient times. In recent years, there has been a growing interest in expanding the range of pharmaceutical applications of SMs. As natural products represent a huge structural and functional diversity, nowadays SMs have become well established active ingredients beyond pharmaceuticals, in the field of cosmetics and food supplements.This continuous increasing demand of plant-based products lead to the investigation of modern and large-scale techniques for producing higher quantities of bioactive compounds from medicinal plants. The production of SMs in the plants and thus, plant metabolome, are highly affected according to environmental conditions and cultivation regime, that can hinder their commercial exploitation. Furthermore, plant-microorganism symbioses, such as the treatment with arbuscular mycorrhizal fungi (AMF) or endophytic bacteria, have showed growth- promoting and SM yield-enhancement in different cultivation systems.Among the different plant families recorded, Boraginaceae family is known from Paleocene and presents an important worldwide distribution of different plants species, mainly in Europe and Asia, in the Northern hemisphere. The family is composed from more than a hundred of different genera and more than 2000 plant species are recognized, among which the well- known Myosotis, Lithospermum, Alkanna and Anchusa spp. The different species are well- documented for their therapeutic proprieties attributed to their SM content such as flavonoids, phenylpropanoids, saponins or naphtoquinones (e.g. cardiotonic, antimicrobial, antipyretic, and anti-asthmatic). Among the most relevant compounds of the family are the lipophilic isohexenylnaphthazarins derivatives, Alkannin and Shikonin, known as red dyes sources with a plethora of biological properties (e.g. wound healing, antimicrobial, anti-inflammatory).In the current thesis, we dedicated our research on Anchusa officinalis due to their wide distribution in the Mediterranean basin, especially in Greece, and to the beneficial properties attributed to this plant species reported in the literature.To this end, in the two first chapters of the current study, we developed different symbiotic associations between Anchusa officinalis and various AMF species, belonging to the genus Rhizophagus, to evaluate the effect of the AMF treatments on plant metabolome. For thispurpose, both experiments were conducted under highly-controlled cultivations conditions and the plants were grown in a semi-hydroponic cultivation system for a maximum period of 30 days. At the end of the experiments, shoots and roots parts of the plants were subjected to an in-house extraction procedure for the assessment of their metabolome reprogramming. An untargeted UHPLC-HRMS metabolomic workflow coupled with a multivariate data analysis (PCA, PLS-DA and Volcano-plots analysis) was designed to identify the most impacted mass signals occurred from the symbioses. The huge amount of mass signals resulted from the analysis lead to the annotation of several up-regulated primary and secondary metabolites, identified by comparing MS/MS spectra with literature data and customized in-house libraries. To the best of our knowledge, this is the first time that a semi-hydroponic cultivation system was proposed for the growth of A. officinalis associated with AMF strains, in combination with a deep metabolic analysis for the identification of the most promising up-regulated compounds in both parts of the plant. These studies emphasized the impact of the AMF association on modulating plant metabolism and on their possible usage for increasing the production of valuable bioactive compounds and for the discovery of novel active agents.In Chapter I, an untargeted metabolomic approach combined with multivariate data analysis was conducted on two independent plant growth experiments associating A. officinalis to the AMF Rhizophagus irregularis MUCL 41833. The plants, mycorrhized (M) and non- mycorrhized (NM), were grown under a semi-hydroponic cultivation system during 9 and 30 days. AMF impact on PMs and SMs production was evaluated in shoots, roots and exudates of M and NM plants at the end of the two periods. In total, 35 different PMs (10) and SMs (25) significantly up-regulated from the plant-microbe symbiosis were tentatively identified. Our results demonstrated how specific biosynthetic pathways (e.g., phenylpropanoid and mevalonate pathways, biosynthesis of organic acids and key amino acids) are influenced by the AMF colonization thought a qualitative and quantitative production of different metabolites, especially after 9 days of growth in the semi-hydroponic cultivation system. Among the identified compounds, several promising structures, already known for their bioactives properties (e.g., rosmarinic acid, caffeic acid, ferulic acid syringic acid), as well as their methylated derivatives (e.g. methylrosmarinic acid, methylsyringin), were significantly affected in mycorrhized plants. In addition, two new salvianolic acid B derivatives and one new rosmarinic acid derivative were described for the first time in the current study, all presenting a common substitution pattern.In Chapter II, we moved our efforts forward and proposed an untargeted metabolomics approach combined with multivariate data analysis, associating A. officinalis to four different AMF strains belonging to the same genus (Rhizophagus irregularis MUCL 41833, R. intraradices MUCL 49410, R. clarus MUCL 46238, R. aggregatus MUCL 49408) to test the hypothesis that different AMF strains affect the plant metabolome differently. Plants were grown 9 days under a semi-hydroponic cultivation system and the metabolic reprogramming in plants was assessed in roots and shoots of A. officinalis associated with the four different AMF strains. Our data showed a strong influence of specific primary (e.g. organic and amino acids) and secondary metabolites (e.g., phenylpropanoid derivatives, oleanane-type glycosides), which was in line with our previous recorded data. In total, 6 PMs and 36 SMs were significantly affected from the different AMF-plant symbiosis. Almost all the identified compounds (39) were up-regulated from the treatments with Rhizophagus irregularis MUCL 41833 and R. intraradices MUCL 49410, suggesting a stronger effect on plant metabolome as compared to the two other strains, R. clarus MUCL 46238 and R. aggregatus MUCL 49408. The experiment shed light on specific substitution pattern as a result of the symbiosis which were shared among the up-regulated compounds. In total, we described four new mevalonate derivatives and one new acetylated saponin as well as a new acetylated derivative of methylsyringin. All the compounds showed a common substitution at C-6 of the glucose moiety.Besides the valuable bioactive ingredients produced by the aforementioned plant species for the development of innovative cosmeceutical and pharmaceutical formulations, it is worth mentioning that the last decade there is an increased interest for the detection of some SMs (e.g. natural toxins) which can potentially be a source of severe adverse effects for human health. Among them, pyrrolizidine alkaloids (PA) are the most widely distributed naturally occurring SMs of high toxicological relevance. Their high toxicity have triggered the development of different PA quantitative methodologies based on commercially available PAs. However, the limited amount of certified standards currently available, hinders the actual detection of many PAs in several medicinal plants of commercial interest.For this reason, in Chapter III, it was of extreme importance to shed light on the huge structural variability of toxic PAs occurring in nature and on their distribution in herbal extracts of industrial interest. In addition, we developed a validated quantification methodology for the evaluation of the total PA content as retronecine-equivalents excluding any time-consuming pre-purification step and the use of commercial standards. In more details, in the current studywe performed a UHPLC-TOF/MSe comparative PA screening on eleven crude extracts obtained from different plants belonging to Boraginaceae, Fabaceae and Asteraceae families, which was combined with a hierarchical clustering approach of the extracts. In total, 105 compounds were tentatively annotated by using a diagnostic fragment filtering (DFF) strategy, and by comparing their MS/MS pattern with those from the literature and customized in-house libraries. Our data were divided in four categories as followed: (1) open-chain monoesters of PA; (2) open-chain di-esters of PA; (3) macrocyclic PAs; (4) glycosidic derivatives of PAs. The findings of the current study pointed out that the distribution of PAs is family-dependent. Interestingly, we described the presence of 18 new glycosidic derivatives and gave some additional structural information concerning the position of the glycosidic moiety in the structures, scarcely reported in literature.For the quantitative part, the total PA-content was expressed as retronecine-equivalents (RE), directly from their crude matrices assuring a good sensitivity (LLOD and LLOQ values of 3.46 pg mL-1 and 11.52 pg mL-1), accuracy (96.96 – 105.19 %) and precision (2.81 – 7.60 % RSD). Our data showed a total PA concentration varying from 8.64 ± 0.08 to 3096.28 ± 273.72 μg RE/g extract dry weight (EDW) in Alkanna graeca and Crotalaria retusa. The study highlighted a retronecine-based PA quantification methodology which may advantageously be exploited for the analysis of extracts of industrial interest. In addition, we showed that the effectiveness of the pre-purification steps, which are widely recommended in the literature for its benefits in both reducing matrix load and concentrating PAs from complex matrices, resulted ambiguous in relation to major PAs in the extracts.Τo conclude, our efforts were mainly concentrated in developing a strong metabolic workflow combined with multivariate data analysis which can be easily applied for the evaluation of the plant-AMF association for the enhancement of PMs and SMs production in medicinal plants. Secondly, we focused on the development of a highly sensitive analytical methodology for the annotation and the rapid quantification of the total PAs content in commercial plant extracts bypassing any additional purification step and the use of commercial standards.
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