Characterization of alkaloid profile of Hyoscyamus reticulatus L. and Atropa belladonna subsp. caucasica (Kreyer) Avet by LC-MS and NMR

Abstract The alkaloid profile of Hyoscyamus reticulatus L. and Atropa belladonna subsp. caucasica (Kreyer) Avet have not been characterized yet. UHPLC-PDA-Q-TOF-MS/MS and LC-DAD-QqQ-MS/MS methods were used herein to characterize the metabolite profiles of these plants. Flash chromatography in combination with preparative- and semi preparative HPLC were utilized for the isolation of the compounds of interest. The structure of the isolated compounds was proposed based on their MS/MS fragmentation and NMR characteristics. As a total of 19 tropane derivatives, two tyramine derivatives (N-cis- and N-trans- feruloyl tyramine), a lignanamide (grossamide), an alkylamide (pellitorine) were identified in the root and herb extracts of H. reticulatus. Moreover, rutin and caffeoylquinic acids were found in the leaves and fruits, while kaempferol-3-O-glucoside-7-O-rhamnoside and quercetin-3-O-glucoside-rhamnoside-rhamnoside were exclusively characteristic for the leaves of H. reticulatus. The root and herb extracts of A. caucasica contained 16 tropane alkaloid derivatives along with methyl tropate and two tyramine derivatives. Graphical Abstract


Introduction
Tropane alkaloidscharacteristic compounds of the Solanaceae familyare well known for their anticholinergic and central nervous system effects (Mar ın-S aez et al. 2018). Hyoscyamus reticulatus L. is one of the most important plants in the Solanaceae family (Ionkova 2002), containing valuable tropane alkaloids. These secondary metabolites are biosynthesized mainly in the root cells of Hyoscyamus species (Hashimoto et al. 1991). Previously, gas-liquid chromatography was used for the characterization of its hyoscyamine, scopolamine, apoatropine and littorine constituents (Kartal et al. 2003), while hyoscyamine was determined quantitatively by GC-MS (Ionkova 1992). Non-alkaloid secondary metabolites (Begum 2010) including flavonoids, lignans (Begum et al. 2009), saponins (Lunga et al. 2008) and fatty acid components (Guler 2012) were also isolated from Hyoscyamus species. Traditionally H. reticulatus was used for its mydriatic, analgesic and sedative properties (Oto et al. 2013) however, the erroneous use of the plant may lead to severe toxicity (Urkin et al. 1991;Aslan et al. 2013).
As a medicinal plant Atropa belladonna subsp. caucasica (Kreyer) Avet (syn.: Atropa caucasica Kreyer) is included in the Russian Pharmacopoeia together with A. belladonna (Shikov et al. 2021). Atropa caucasica is endemic to Caucasus and Southwest Asia (Salmanova 2020). Depending on environmental stress factors, at least 20 tropane alkaloids (e.g. atropine, scopolamine and hyoscyamine) can be found in A. belladonna (Malik et al. 2021). Tropane alkaloids were characterized by ultra-high pressure liquid chromatography (UHPLC) coupled to high-resolution mass spectrometry using Exactive-Orbitrap analyzer (Mar ın-S aez et al. 2018), and HPLC-DAD (Hank et al. 2004) methods. Beside LC, gas chromatography (GC) can also be utilized in plant analyses (Sukhadiya et al. 2021). According to Dr€ ager, GCflame ionization detector (GC-FID) or GC-MS is the first choice technique for tropane alkaloid characterization (Dr€ ager 2002). S-hyoscyamine as well as the racemic mixture of Sand R-hyoscyamineatropine and S-scopolamine are also included in the European Pharmacopoeia in their salt form (Ph. Eur. 10.3a, b, c, 2021). Moreover, Streptomyces sp. was isolated from the shoots of A. belladonna, producing piperazic acid containing peptides (Bekiesch et al. 2021).
In this study the phytochemical profile of H. reticulatus and A. caucasica were characterized in order to explore new alternative sources of tropane alkaloid derivatives.

Phytochemical characterization of H. reticulatus
In order to unambiguously identify their structures, seven tropane alkaloids (H7, H9, H10, H11, H12, H19 and H22), one tyramine component (H17) were isolated ( Figure  S1). Their structures were established by 1D and 2D NMR experiments (see Supplementary material) as well as MS/MS analyses. Besides these isolated components, further compounds were detected by UHPLC-MS/MS in the alkaloid-rich fraction of H. reticulatus herb and root. In line with literature data, 23 compoundsmainly tropane derivativeswere tentatively identified (see Table S1 with references). TIC chromatograms of the extracts are shown in Figures S2 and S3.

Experimental
All details are provided in the supplementary material.

Conclusion
In the present work the alkaloid content of H. reticulatus and A. caucasica from the Solanaceae family were investigated by LC-MS/MS and NMR spectroscopic methods. 9 compounds were successfully isolated from the aerial parts of H. reticulatus and 14 further compounds were tentatively identified based on MS/MS fragmentation studies. To the best of our knowledge, vanillyltropine (H11), norhyoscyamine (H13), 3-(2 0 -phenylpropionyloxy)tropane (H22), 2 0 -hydroxyatropine (H8), and the N-cis/trans-feruloyltyramines (H15 and H17), grossamide (H20), as well as the alkylamide pellitorine (H23) were described in H. reticulatus for the first time.

Disclosure statement
No potential conflict of interest was reported by the author(s)