Secoiridoids and other chemotaxonomically relevant compounds in Pedicularis: phytochemical analysis and comparison of Pedicularis rostratocapitata Crantz and Pedicularis verticillata L. from Dolomites

Abstract We compared the respective metabolite patterns of two Pedicularis species from Dolomites. Seven phenylethanoid glycosides, i.e., verbascoside (1), echinacoside (2), angoroside A (3), cistantubuloside B1 (4), wiedemannioside C (5), campneoside II (11) and cistantubuloside C1 (12), together with several iridoid glucosides as aucubin (6), euphroside (7), monomelittoside (8), mussaenosidic acid (9) and 8-epiloganic acid (13) were identified. Pedicularis verticillata showed also the presence of greatly unexpected secoiridoids, ligustroside (14) and excelside B (15), very rare compounds in Lamiales. Both PhGs and iridoids are considered of taxonomical relevance in the Asteridae and their occurrence in Pedicularis was discussed. In particular, the exclusive presence of several compounds such as 8-epiloganic acid (13), campneoside II (11), cistantubuloside C1 (12), ligustroside (14) and excelside B (15) in Pedicularis rostratocapitata, and angoroside A (3), cistantubuloside B1 (4) and wiedemannioside C (5) in P. verticillata could be considered specific markers for the two botanical entities.


Introduction
In this study, we analysed the secondary metabolite content of two Pedicularis species collected in the Dolomites Massif, and then, we compared their respective secondary metabolite patterns, focusing on constituents of medium polarity to be used as chemotaxonomic markers. the data collection from rare indigenous species or plants under protection is very important and merits special scientific attention to obtain all possible information, starting from the taxonomic one.
Pedicularis L. (orobanchaceae family) is one of the largest genera of hemiparasitic flowering plants accounting for over 500 described species mainly characteristic of cold mountain environments of the northern hemisphere. Several species belonging to this genus have been largely applied in traditional medicine in particular in China .
Pedicularis rostratocapitata Crantz is a little hemicryptophyte herbaceous plant with lanceolate pinnatisect leaves (6-9 cm) of suffused dark purple, divided into segments with dissected leaf margins. the flowers, presenting tubular calix and bilabiate purplish corolla, are gathered in little capitate inflorescences, the upper lip in darker colour is provided with rostrum. Pedicularis verticillata L. is very similar to the previous species, the main differences are on the caulinary leaves, which appear gathered in verticillaster of 3-4 elements, and in the trilobate lower lip (Pignatti 1982;Aeschimann et al. 2004;Conti et al. 2005).
Little data are available on the chemical composition of P. verticillata, while no previous work has been reported for P. rostratocapitata, probably due to relative inaccessibility of the habitat colonised by these species. In particular, from a Chinese accession of P. verticillata were isolated neolignans (verticillatosides A and B), phenylpropanoid glycosides (PhGs) (verbascoside, cistanosides C and D) and iridoid glycosides (7-deoxy-8-epi-loganic acid, 8-epi-loganic acid, plantarenaloside, geniposidic acid, euphroside, aucubin, boschnaloside and caryoptoside) (Su et al. 1997). In this paper, we report the first phytochemical analysis of P. rostratocapitata and a re-examination of the molecular pattern of P. verticillata collected from Italian Alps, using NMr and MS techniques which resulted to be very useful tools for the study of natural compounds (Sacchi et al. 1998;Mallamace et al. 2014;Dugo et al. 2015) and more in general for metabolites studies in complex matrices Corsaro et al. 2015).
A chiral polyol d-mannitol (10) was recognised in conspicuous amount from the more polar fractions of both species. Compound 10 has been evidenced in other hemiparasitic species as Parentucellia viscosa (Venditti, Ballero, et al. 2015) and Bellardia trixago (Venditti et al. 2013) resulting a very common polyol in this family. the more relevant differences between P. verticillata and P. rostratocapitata were showed in the PhGs pattern (table 1). From P. rostratocapitata, extracts were identified verbascoside (1), echinacoside (2) campneoside II (11) and cistantubuloside C 1 (12), while compounds (3-5) were not recognised. Campneoside II (11) was evidenced for the first time from a Pedicularis species in this study. Previously, this compound was isolated from other orobanchaceae as Cistanche tubulosa sinensis Beck (Liu et al. 2013) and Orobanche caerulescens K. Koch (Lin et al. 2004), but also from taxa included in Scrophulariaceae as Buddleja davidii Franch. (Ahmad et al. 2009) and Lamiaceae as Stachys riederi Cham. (Ikeda et al. 1994), Anisomeles indica (L.) Kuntze. (rao et al. 2009) and Clerodendrum bungei Steud. (Li et al. 2005), as well as in Bignoniaceae as Campsis chinensis (Lam.) Voss (Imakura et al. 1985) and in Plantaginaceae as Rehmannia glutinosa (Gaertn.) DC. (Zhang et al. 2014). All these species are comprised in families belonging to Lamiales, an order enclosed in the Asteridae subclass, and the cooccurrence of these PhGs is a chemical evidence of the systematic proximity among these orders and families.

Experimental
Details on isolation and identification of compounds are available as supplementary materials.
the following compounds were identified ( Figure 1) by comparison with data reported in literature or/and by comparison with pure compounds available in our laboratory.

Conclusions
the phytochemical analysis of P. verticillata and P. rostratocapitata from Dolomites revealed a euphroside/aucubin chemotype already evidenced in this genus and the presence of two mayor classes of taxonomically relevant natural compounds: iridoid glucosides and phenylethanoid glycosides (PhGs). Several isolated compounds were already identified in other entities of this genus thus confirming their taxonomical proximity some molecular peculiarities were also recognised i.e., the exclusive presence of 8-epiloganic acid (13), campneoside II (11) and cistantubuloside C 1 (12) in P. rostratocapitata, and angoroside A (3), cistantubuloside B 1 (4) and wiedemannioside C (5) in P. verticillata. these compounds may be considered specific markers for the two botanical entities. Moreover, the studied accession of P. verticillata showed also the unexpected presence of two secoiridoids, (14) and (15), which are extremely rare compounds in Lamiales, and were reported here for the first time as constituents of Pedicularis.