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An integrated strategy to characterize active constituents and their metabolites in Herniaria hirsuta and Nauclea pobeguinii

Book - Dissertation

The world still faces diseases which lack adequate treatment or prevention. Amongst them are urolithiasis and malaria. Urolithiasis causes a high burden to healthcare systems all over the world, with a lifetime risk of about 10-15% in the developed world and recurrence rates as high as 50% in ten years. Although urinary stones can be treated, these treatments cause many side effects compromising their long-term consumption. This leads to an increasing interest in phytotherapy as an alternative for treatment and/or prevention of urinary stones. Malaria is still one of the most devastating infectious diseases in the world, causing over 400,000 deaths on yearly basis in 2019. Over 3 billion people are living in areas at risk of malaria transmission. Taking limited access to synthetic drugs in developing countries into account together with an increasing resistance against currently available medicines, there is a high need for new antiplasmodial treatments. Traditional medicine plays an important role in treatment of malaria as 80% of the population in developing countries relies on the use of plant extracts, in particular against parasitic diseases. During the past decades, researchers have shifted their focus towards natural products as they offer a rich source of inspiration for potential new active compounds against diseases for which adequate treatment or prevention is lacking. Nevertheless, the fact that many natural products act as prodrugs which are biotransformed and activated after oral administration is usually overlooked when using classical approaches. Therefore, an integrated strategy to characterize new active compounds is developed. As a first step, comprehensive extraction followed by phytochemical profile elucidation is conducted. Then, gastrointestinal and hepatic biotransformation is simulated in vitro, avoiding extensive in vivo studies. The longitudinal multiclass data are subjected to an automated data analysis workflow, allowing rapid scoring of large amounts of data in an unbiased manner: a limited number of biotransformation time profiles is rated by an expert scientist, which is used to train a machine learning model in the background to analyze the data. Ultimately, the metabolites are further tested in in vitro activity assays, allowing to assess antilithiatic and antiplasmodial activity of Herniaria hirsuta and Nauclea pobeguinii, respectively. Extracts of the plant H. hirsuta are traditionally used against kidney stones while bark extracts of the tree N. pobeguinii are extensively used in Central Africa against malaria. Comprehensive extraction of the aerial parts of Herniaria hirsuta by combining H2O:EtOAc and CHCl3:MeOH:H2O extraction resulted in extraction of the full range of constituents. The phytochemical composition of H. hirsuta was characterized using UHPLC-UV-HRMS data and led to the tentative identification of 63 compounds, mainly flavonoids and saponins, including 15 saponins that have not been reported before in H. hirsuta. Several flavonoid compounds were identified, including rutin, narcissin and quercetin-3-O-(2"-O-α-L-rhamnopyranosyl)-β-D-glucuronopyranoside. A rich diversity of saponins was detected, differing in the presence of various aglycones, a different composition of sugar chains and varied linkages of sugar moieties. Three different aglycone moieties were observed including acetylated medicagenic acid, zanhic acid and medicagenic acid, with medicagenic acid as the most abundant one. In vitro gastrointestinal biotransformation followed by metabolomics profiling revealed the biotransformation pathway of several compounds present in H. hirsuta. A decrease in relative abundance over time was observed for the majority of all identified compounds, including saponins and flavonoids, especially during the colon phase. Additionally, the relative abundance of saponin aglycones increased, illustrating the biotransformation of possible saponin prodrugs to their respective aglycones. As medicagenic acid is the major metabolite after in vitro gastrointestinal biotransformation of the H. hirsuta extract, in vitro hepatic biotransformation was simulated using this compound to avoid gastrointestinal matrix hampering hepatic biotransformation reactions. Hepatic biotransformation of medicagenic acid using human S9 fractions resulted in a mixture of metabolites. The results of a suspect screening method combined with non-target screening indicated formation of thirteen metabolites, four of which have never been reported before, with medicagenic acid glucuronide as the most abundant one. For the first time, a tentative effect on CaC2O4 crystal aggregation of the hepatic biotransformation products of medicagenic acid could be demonstrated in vitro, resulting in smaller CaC2O4 crystals in comparison with standard conditions. Smaller crystals are more easily excreted by urine, preventing urinary stone formation. Moreover, the hepatic biotransformation products of medicagenic acid showed significant inhibition of crystal binding to MDCK I cells. As these effects affect both formation of crystals by inhibiting aggregation and retention of crystals to renal tubular cells by inhibiting crystal binding, it can be stated that the beneficial effect of H. hirsuta against urinary stones may be attributed at least in part to metabolites of medicagenic acid, indicating that saponins containing medicagenic acid may act as prodrugs. The MeOH 80% Nauclea pogebuinii bark extract was subjected to extensive dereplication studies to obtain a complete overview of the phytochemical composition of the extract. Alkaloids were mainly observed as glycoconjugates, consisting of an alkaloid moiety bound to one or more sugar moieties. Among others, strictosamide, 5α-carboxystrictosidine, desoxycordifoline, pobeguinine and angustoline were identified. Derivatives of angustoline, such as 19-O-methylangustoline, 19-O-ethylangustoline, 3,14-dihydroangustoline and 19-O-methyl-3,14-dihydroangustoline were reported for the first time in N. pobeguinii. A diversity of saponins was detected, differing in the presence of various aglycones, a different composition of sugar chains and varied linkages of the sugar moieties. In total, phytochemical profiling led to the tentative identification of 54 compounds, of which 32 are reported for the first time in N. pobeguinii. In vitro gastrointestinal biotransformation showed that non-glycosylated alkaloids showed no gastrointestinal biotransformation. On the other hand, glycosylated alkaloids showed a decrease in intensity over time. Nevertheless, biotransformation rates of glycosylated compounds, both alkaloids and saponins, were unexpectedly low, illustrated by a slower and incomplete biotransformation of strictosamide in the extract compared to strictosamide as a pure compound. This suggests that compound(s) present in the extract of N. pobeguinii hamper biotransformation. As the extract had no significant effect on the number of viable bacteria, it is suggested that the extract is not toxic to fecal bacteria in this concentration. Nevertheless, 16S rDNA-targeting PCR showed changes in relative ratios of the numbers of some dominant bacteria to the total number in presence of the extract over time. The altered bacterial composition might explain the slower biotransformation rate of glycosylated compounds. Although strictosamide was previously hypothesized to be a precursor of antiplasmodial metabolites, in vitro gastrointestinal biotransformation did not result in a single major metabolite which might be responsible for the antiplasmodial effects of N. pobeguinii. Multivariate data analysis using OPLS-DA showed no in vitro activity of strictosamide or its metabolites suggesting that other compounds are responsible for the antiplasmodial effect of the N. pobeguinii extract. The OPLS-DA proposed alkaloids with a β-carboline moiety as active principles, including angustoline or 3,14-dihydroangustoline, nauclefiline, 19-O-methylangustoline, nauclefidine and 19-O-methyl-3,14-dihydroangustoline, suggesting that antiplasmodial activity of N. pobeguinii derives from an additive or synergistic effect of multiple minor alkaloids present in the bark extract and their metabolites. To conclude, the integrated strategy offers added value in screening phases, allowing phytochemical identification of compounds and their metabolites after in vitro biotransformation, followed by preliminary in vitro activity testing, avoiding in vivo studies in early stages of research.
Number of pages: 377
Publication year:2022
Keywords:Doctoral thesis
Accessibility:Open