Secondary metabolites from Scrophularia canina L.

Abstract A re-examination of Scrophularia canina L. confirmed the presence of iridoid glucosides considered as chemotaxonomic markers for the Scrophulariaceae family, like aucubin, harpagide and 8-O-acetylharpagide, besides the further presence of 8-epiloganic acid, which is, indeed, considered the biogenetic precursor of iridoids normally found in Scrophulariaceae, and was recognised here for the first time in the studied species. Also verbascoside and (E)-phytol were evidenced for the first time in S. canina. The former compound is an almost ubiquitous glycosidic phenyl-ethanoid, which attains systematic importance when in co-occurrence with iridoids, and its taxonomical implications were discussed. The latter compound, even though it is omnipresent, is interestingly endowed with several biological activities, which may give an additional reason for the traditional uses of this plant.


Introduction
Scrophularia canina L. is a herbaceous perennial plant belonging to the Scrophulariaceae family. It presents a woody stalk at the base, which turns into a branched and herbaceous one in the upper part. Moreover, it is characterised by some variety for what concerns the shape and the posture of the leaves, both according to its place of growth. It is present in the euro-Mediterranean habitats and in Italy it can be found mostly everywhere on the territory till a height of 1500 m a.s.l. (Pignatti 1982). In the past, it has been widely used in the folkloristic medicine as an antiseptic and cicatrising agent for sheep and cows before it was evidenced its light toxicity due to its chemical constituents of iridoids and saponines (Berdini et al. 1991;Nimis et al. 2013). In fact, the name of the genus evidences the relationship with ABSTRACT A re-examination of Scrophularia canina L. confirmed the presence of iridoid glucosides considered as chemotaxonomic markers for the Scrophulariaceae family, like aucubin, harpagide and 8-Oacetylharpagide, besides the further presence of 8-epiloganic acid, which is, indeed, considered the biogenetic precursor of iridoids normally found in Scrophulariaceae, and was recognised here for the first time in the studied species. Also verbascoside and (E)-phytol were evidenced for the first time in S. canina. The former compound is an almost ubiquitous glycosidic phenyl-ethanoid, which attains systematic importance when in co-occurrence with iridoids, and its taxonomical implications were discussed. The latter compound, even though it is omnipresent, is interestingly endowed with several biological activities, which may give an additional reason for the traditional uses of this plant. the external treatment of scrophula and scabies (Tatli et al. 2015). Nevertheless, in Calabria (Southern Italy) it has been also used as antimalarial and later as larvae killer and growth regulator, because of the presence of other secondary metabolites like phenyl-propanoids, terpenoids and flavonoids as well as iridoids themselves (Germinara et al. 2011).
In the literature, there are only a couple of previous studies on this species and, in particular, for what concerns the polar fraction, there is only one underlining the presence of a ciclopentanoic monoterpenic lactone and some glycosidic iridoids, like harpagide, aucubin, 8-O-acetylharpagide and 10-O-β-glycosilaucubin (Berdini et al. 1991). With the idea of including also the less polar fraction, we have then decided to continue the study of this species, started in our own laboratory in the past (Berdini et al. 1991).

Results and discussion
The study on the polar fraction of S. canina led us to the isolation and the identification of six compounds in total, namely (E)-phytol (1), verbascoside (2), aucubin (3), harpagide (4), 8-O-acetylharpagide (5) and 8-epiloganic acid (6) (Figure 1). These compounds belong to three different classes of natural compounds, which are unsaturated diterpenic alcohols for compound (1), phenyl-propanoids for compound (2), and iridoid glucosides for compounds from (3) to (6). Actually, compounds (3), (4) and (5) have already been found in our previous study on the same species (Berdini et al. 1991) and also in other species of the genus, such as S. dentata Royle ex Benth. (Zhang et al. 2014) and S. ningpoensis Hemsl. (Ji et al. 2014), thus confirming the molecular pattern already reported. These results are also in accordance with the current systematic collocation of this species, since compounds 3, 4, and 5 are considered chemosystematic markers for the Scrophulariaceae. In fact, this family has been recently subjected to a deep reorganisation and several genera previously comprised in Scrophulariaceae have been moved into other families (Plantaginaceae, Orobanchaceae, Phrymaceae, Linderniaceae and Calceolariaceae) (APG II 2003), but not the genus Scrophularia.
On the other side, compounds 1, 2 and 6 were isolated for the first time in this species. Phytol (1) is a very common compound, present, in part, in the chlorophyll structure. It is an almost ubiquitous compound, which may be responsible, synergistically with the other constituents, of the bioactivity since it is endowed with a wide range of biological activities i.e. antimicrobial, cytotoxic, antitumorous, antimutagenic, anti-teratogenic, antibiotic-chemotherapeutic, antidiabetic, lipid lowering, antispasmodic, anticonvulsant, antinociceptive, antioxidant, antiradical, anti-inflammatory and anxiolytic (Pejin, Kartali, et al. 2014;Pejin, Kojic, et al. 2014;Pejin, Savic, et al. 2014;Islam et al. 2015;Pejin et al. 2015). The presence of (E)-phytol may give an additional rationale for the traditional antiparasitic uses of this plant.

Experimental
See Supplementary materials.

Conclusions
With this work we have confirmed the results obtained in previous studies on the polar fraction of S. canina, in particular about some specific components such as aucubin (3), 8-O-acetylharpagide (4) and harpagide (5), considered taxonomic markers for this genus; secondly, we have showed the presence of other compounds never evidenced before such as verbascoside (2), 8-epiloganic acid (6) and (E)-phytol (1) The presence of compound (2) achieves a chemotaxonomic importance, because it was found contemporaneously with glycosidic iridoids (Jensen 1992). In fact, the co-occurrence of glycosidic phenyl-propanoids and iridoid glucosides has been reported in botanical entities comprised in the subclass of Asteridae Venditti, Serrilli, di Cecco, et al. 2013). Moreover, from a biogenetic standpoint, it is worth underlining the isolation of compound (6) because 8-epiloganic acid is considered the natural precursor of all the series of glycosidic iridoids, which are typical of the Scrophulariaceae family and, in general, of the Lamiales order (Jensen 1991). Moreover, the presence of (E)-phytol may give a rationale for the traditional uses of this plant.