Study on the metabolism of natural sesquiterpene lactones in human liver microsomes using LC-Q-TOF-MS/MS

Abstract Sesquiterpene lactones are naturally occurring, highly active specialised metabolites, which are biosynthesized by important medicinal plants, fulfilling many functions. The in vitro metabolism of parthenolide (1), grosheimin (2), carbetolide C (3), 8α-O-(3,4-dihydroxy-methylenebutanoyloxy)-dehydromelitensin (4) and arteludovicinolide A (5) was examined using human liver microsomes. Phase I, phase II (glucuronidation), as well as combined phase I + II metabolism were studied. Metabolites were identified via liquid chromatography-high resolution quadrupole time-of-flight mass spectrometry. Monohydroxylated, hydrated, carboxylated, methylated derivatives, together with corresponding monoglucuronides were detected, suggesting that the metabolism of sesquiterpene lactones is changeable due to structural features and scaffold diversity, though the lactone ring is the main site of metabolism. Graphical Abstract


Introduction
Sesquiterpene lactones (SLs) are highly active specialised metabolites, which are produced by widely used medicinal plants, such as Arnica montana L., Tanacetum parthenium L., and Matricaria chamomilla L. (Bohlmann and Zdero 1982;Douglas et al. 2004;Zaiter et al. 2007;Merfort 2011;Moujir et al. 2020).These compounds are biosynthesized via the mevalonic pathway, resulting in a 15-carbon skeleton, which contains a lactone ring.Such compounds are categorised, relative to their carbocyclic skeleton, into over 40 structural types, to date, the most widespread are germacrene, guaiane, eudesmane, and pseudoguaiane derivatives.The SL ring is reacting through Michael-type additions with biological nucleophiles such as sulfhydryl groups (Schmidt 2018), which explain their broad range of human-relevant biological effects (Sokovic et al. 2017).SLs interact with the human body as both pharmaceutical agents and as part of a balanced diet.Reasonable, they are well known for their potential as cytotoxic drug leads, with some of them being in cancer clinical or preclinical trials (thapsigargin, parthenolide, or their semi-synthetic derivatives) (Ghantous et al. 2010), while others could probably reach the clinical phases in the near future (alantolactone, custonolide) (Moujir et al. 2020).Some representative plant-derived SLs as typical cases are parthenolide (1), grosheimin (2), carbetolide C (3), 8α-O-(3,4-dihydroxy-methylenebutanoyloxy)-dehydromelitensin (4) and arteludovicinolide A (5), covering structurally different SL groups.In detail, parthenolide (1) is a SL of germacranolide class, isolated from Tanacetum parthenium (feverfew), which has a long-term use in folk medicine.A plethora of studies have been conducted both on its biosynthesis (Majdi et al. 2011, Darbahani et al. 2022) and its biological effects, and its semi-synthetic derivative was the subject of cancer clinical trials (Freund et al. 2020;Moujir et al. 2020;Saosathan et al. 2021).Moreover, feverfew is a traditional herbal medicinal product for the prophylaxis of migraine headaches, due to its SLs load (www.ema.eu).Grosheimin (2) is a guaiane-type SL found highly abundant in artichokes and other Asteraceae members.This compound is now receiving attention as a result of its reputed pharmacological properties beyond influenza or as a hypolipidemic agent, etc. (Kasenova et al. 2016;Schepetkin et al. 2018).Carbetolide C (3), together with analogous heliangolides, are the bio-active specialised metabolites in Calea L. species, many of which are used in tropical and sub-tropical America for gastrointestinal, dermatological, respiratory ailments, diabetes, etc.In our previous studies, carbetolide C (3) was proven to possess cytotoxic and anti-inflammatory effects similar to that of parthenolide (1) (Grafakou et al. 2021).The genus Centaurea L. is characterised by the biosynthesis of elemanolides (Grafakou et al. 2018), such as 8α-O-(3,4-dihydroxy-methylenebutanoyloxy)-dehydromelitensin (4), and its taxa are being widely used throughout the world as diuretic, digestive, antipyretic agents, etc.This compound showed both strong cytotoxic and anti-inflammatory activities (Koukoulitsa et al. 2002;Grafakou et al. 2022).Arteludovicinolide A (5) and such seco-guaianolide derivatives mainly occur in Achillea L. and Artemisia L. species, both genera encompass powerful medicinal plants with a wide range of therapeutic purposes.To be mentioned seco-guaianolides are highly active specialised metabolites and the strong anti-inflammatory and cytotoxic potential of arteludovicinolide A (5) has been previously reported in the literature (Kreuzer et al. 2013;Papakosta et al. 2020;Barda et al. 2021).
Despite increasing data regarding bioactivity, to date, there are only a few reports on the metabolic pathways and metabolites of some SLs (Yu et al. 2021), reporting the identification of phase I and II metabolites using different metabolism systems, mainly through in vivo rat models or liver microsomes.In vitro drug metabolism studies account for a useful tool in basic research, as well as preclinical screening of drug-like properties.Human liver microsomes (HLM) constitute a popular and affordable model for drug metabolic profiling (Sowjanya et al. 2019).As it is known, metabolism is carried out in two general phases: in phase I polar groups are either introduced by oxidation, reduction, hydrolysis, or uncovered by dealkylation.In phase II polar groups are conjugated with glucuronic acid, sulphate, glycine, glutamine, glutathione, acetate, or methyl groups in order to render the molecule more polar and facilitate excretion (Gillette 1971).In the present study, we, therefore, analysed the in vitro metabolism of five naturally occurring SLs (Table S1) using HLM: parthenolide (1), grosheimin (2), carbetolide C (3), 8α-O-(3,4-dihydroxy-methylenebutanoyloxy)-dehydromelitensin (4) and arteludovicinolide A (5). Different microsomal incubation systems were applied to study phase I metabolism and phase II glucuronidation by the addition of uridine diphosphoglucuronic acid, as well as combined phase I and II metabolism.Metabolites were identified by liquid chromatography-high resolution quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS/MS).

Results and discussion
By screening reactive compound metabolites of phase I, as well as, phase II acyl-glucuronide conjugate reactivities we can achieve some understanding of such complex procedures that are taking place in metabolic systems.On this basis, we set under-investigation structurally diverse plant-derived SLs to be characterised on a detailed level by HPLC-MS/MS after in vitro metabolism on HLM.The proposed metabolites are presented in Figure 1 and the corresponding MS data are reported in Tables S2-S6.
Step by step, we underwent different microsomal incubation systems, starting with phase I reactions and phase II glucuronidation by the addition of UDPGA, to a combined system that encompasses both activations of phase I enzymes and UDP-glucuronosyl transferases.In parallel, all compounds were subjected to stability test in DPBS, while control incubation systems without cofactors or microsomes were examined (Table S7).
More specifically, regarding parthenolide (1), six phase I metabolites were found.According to retention times (t R ) and LC-MS data (Table S2), they were identified as one hydroxylated (t R = 3.12 min; m/z [M + H] + 265.1444), and three methylated derivatives (one lost the double bond at Δ 10-1 ) (t R = 4.60 min; m/z [M + H] + 263.1652; t R = 5.13 min; m/z [M + H] + 263.1649; t R = 6.23 min; m/z [M + H] + 265.1801).Methylation of the main skeleton has been described before in SL metabolism (Cai et al. 2015;Jiang et al. 2019) and the additional carbon atom is supposed to be attached enzymatically, similar to biosynthetic pathways mediated by methyltransferases (Liscombe et al. 2012).Moreover, no metabolites with an opened lactone ring could be identified, however, the exomethylenic double bond at Δ 11-13 was subject of reduction or carboxylation, giving rise to two phase I metabolites (t R = 5.45 min; m/z [M + H] + 251.1652, t R = 6.75 min; m/z [M + H] + 281.1387).Combined phase I and II metabolism led to the formation of a glucuronide on the lactone moiety (t R = 5.17 min, m/z [M − H] − 443.1892).Following the literature data, the lactone ring, which acts as a strong Michael acceptor (Schmidt 2018), is the main location for metabolism and conjugation (Yu et al. 2021).
Regarding grosheimin (2), four metabolites were detected (Table S3).In phase I similar events were conducted on the lactone ring (carboxylation, t R = 1.21 min, m/z [M + H] + 295.1191), while in that case another metabolic site proved to be the carbonyl group of C-3 (t R = 1.94 min, m/z [M + H] + 265.144).Carbonyl groups in other SLs are also found to be subject of metabolism (Yu et el., 2021) S4).
Regarding 8α-O-(3,4-dihydroxymethylenebutanoyloxy)-dehydromelitensin (4), four metabolites were detected (Table S5).More specifically, in phase I, a derivative with a free hydroxyl group was present as a consequence of the replacement of 8-substitution (t R = 3.86 min, m/z [M + H] + 265.1456).Similarly, a metabolite detected from Ixerin Z was formed by the loss of a glucose substitution on the main SL skeleton (Cai et al.

2015)
. Two further metabolites were formed by hydrolysis events, supposing that the addition of H 2 O is taking place at the double bond 1-2 or the lactone ring (t R = 4.16 min, m/z [M + H] + 397.1882; t R = 4.76 min, m/z [M − H] -393.1561).Thus, contrary to the previous metabolites (1-3), in this case, the opening of the lactone ring was observed, similar to the reported metabolite of the SL 11α,13-dihydrohelenaline acetate formed in pig liver microsomes (Jürgens et al. 2022).Moreover, in phase II the glucuronidation is taking place on position 15 (t R = 3.79 min, m/z [M − H] -553.1933).
Concerning arteludovicinolide A ( 5), we detected one metabolite in each phase I, II and I + II (t R = 2.37 min, m/z [M + H] + 311.1099; t R = 3.53 min, m/z [M − H] -453.1415;t R = 3.71 min, m/z [M − H] -471.1499),respectively.In accordance with the above-mentioned compounds, a carboxylate formation is taking place on the lactone ring at position Δ 11-13 , which further formed a glucuronide conjugate during phase I + II.Additionally to the glucuronidation of the combined metabolism, in phase II, the former free hydroxyl group led to another glucuronide conjugation.These data are depicted in Table S6.
Due to structural polymorphism, metabolic sites and pathways for SLs are warranted to be changeable.Such metabolic processes may occur simultaneously or sequentially and undergo phase I to convert lipophilic drugs into more polar commonly by CYP450s (or reduction and hydrolysis).In phase I, metabolites are often still active and further proceed to phase II to yield larger polar metabolites by enzymatic reactions such as glucuronidation (most common), methylation, acetylation, sulphation, or conjugation with glutathione and amino acids.Outlining our results, we detected monohydroxylated, hydrated, carboxylated, and methylated derivatives, as well as corresponding monoglucuronides of phase II and combined metabolism.Under the given condition of our in vitro system, we can observe the formation of glucuronides that are likely also occurring in vivo, however, these metabolites represent just a part of the total metabolisation throughout the body's cells.Sulphate, cysteine, acetylcysteine, and glucuronide conjugates have been described from SLs using different metabolism systems (Yu et al. 2021).For an overall view and to cover all possible phase II bioproducts, further studies using liver cells and in vivo systems should be utilised.It is also worth mentioning that the reactivity of the basic SL backbone is relatively weak, while the lactone ring is a Michael centre, which, similar to bioactivity, is the main site of metabolic reactions.The exomethylenic double bond is subject to reduction, oxidation, or conjugation, while some cases are reporting the opening of the lactone ring.The inherent structures play an integral role in the metabolism outcome, thus even similar compounds result in different pathways.

Microsomal incubation systems for phase I, II, combined I and II metabolism
The incubation systems were prepared as described before (Zenger et al. 2015) and are presented in Table S7.Briefly, phase I incubation mixtures (NADPH regeneration system) with a total volume of 1 mL in DPBS contained 0.5 mg HLM, 3.3 mM magnesium chloride (MgCl 2 ), 3.3 mM G6P, 0.4 U/mL G6PDH, 1.29 mM NADP, and 10 μM test compound.Phase II glucuronidation is based on the addition of UPDGA and the mixtures comprised of 0.5 mg HLM, 3.3 mM MgCl 2 , 2 mM UDPGA, 25 μg/mL Ala, and 10 μM test compound.Combined phase I and II metabolism mixtures included all of the above-mentioned reagents of phase I and II.Mixtures without microsomes, or cofactors (NADP or UDPGA) were used as negative controls, matrix samples without test compound were used as blank controls and mixtures with the addition only of the tested compound in DPBS were used to investigate the compound's stability in the given conditions of the assay (see Table S7).Incubation was carried out in a stirred water bath at 37 °C for 3 h, and was terminated by the addition of the same volume of ice-cold EtOH.Mixtures were vigorously vortexed for 5 min, and centrifuged for another 5 min (14,000 rpm, 4 °C).The supertants were dried under N 2 and stored at −20 °C until further analysis.All experiments were repeated at least three times.

Conclusions
The metabolisation of natural products is much less studied than their biosynthetic pathways.With structural diverse sesquiterpene lactones as a case study, we herein report the produced bioavailable metabolites arising through phase I and phase II (glucuronidation) or combined I and II, in vitro, metabolism on human liver microsomes.Such bioproducts are structurally unreported and their biological existence-effects are fully unidentified.In summary, this study shows that the sesquiterpene lactones are subject to changeable metabolism although the highly active lactone ring is the most common metabolic site.Natural products metabolism can be considered a blue ocean for future research studies, suggesting a potential new strategy not only for the identification of specified metabolites, but also to give an inside view and record the in vitro and in vivo pathways of natural product metabolism.Further detailed studies are warranted to define actions or interactions that result in the final formations of the component in a living system.
. The latter position, as well as C-8 hydroxyl group, were further conjugated by glucuronides on phase II and I + II (t R = 4.30 min, m/z [M − H] -437.1449;t R = 4.01 min, m/z [M − H] -439.1604), while it is worth mentioning that no glucuronide conjugates could be detected on Δ 11-13 , contrary to compound 1.Furthermore, two metabolites of carbetolide C (3) were found after phase I metabolism (t R = 1.70 min, m/z [M + H] + 281.1385; t R = 3.16 min, m/z [M − H] -363.1803).The first was observed as a result of the replacement of 8-substitution with a hydroxyl group, while the second metabolite was formed by saturation on Δ 11-13 .Glucuronide conjugations were observed on one of the free hydroxyl groups, as well as the lactone ring, based on phase II and phase I + II reactions (t R = 4.83 min, m/z [M − H] -537.1986;t R = 6.92 min, m/z [M − H] -555.2182)(Table

Figure 1 .
Figure 1.Metabolic sites of tested sls.Pink: formation of one of these metabolites; green: conversion between single and double bond; blue: opening of the lactone ring; red: Ph.II metabolite formed under given conditions (glucuronidation).