Iridoids and phenylethanoid glycosides from the aerial parts of Ajuga tenorei, an endemic Italian species

Abstract We report the first analysis in absolute, and in particular, concerning the phytochemical pattern, about an endemic Italian species, Ajuga tenorei C. Presl. The analysis, performed by means of techniques such as Column Chromatography and NMR spectroscopy and Mass spectrometry, led to the isolation and the identification of five compounds namely verbascoside (1), echinacoside (2), ajugoside (3), harpagide (4) and 8-O-acetylharpagide (5). The presence of these compounds is important from both chemotaxonomic and ethno-pharmacological point of view. For what concerns the first point is confirmed the correct botanical classification of the species. The isolated compounds are also known to exert peculiar pharmacological activities and their presence may give a rationale to the historical medicinal properties associated to the Ajuga genus in general, since these plants have a long traditional use in many parts of the world. Such fact might suggest the use of also this species in this sense.


Introduction
Ajuga tenorei C. Presl (synonym of Ajuga acaulis Brocchi), better known with the common name of Tenore's Bugle, is a herbaceous hemicryptophyte perennial plant belonging to the tribe Ajugoideae of the Lamiaceae family.
Its name, 'Ajuga', is possibly derived from the union of the Greek word 'a' (meaning 'not' or 'without') and the latin word 'jugum' (meaning a yoke), reflecting that the flowers of this genus are without the upper lip of the corolla. The specific term 'tenorei' is in honour of its discoverer, Professor Michele Tenore who called it A. decumbens Ten. in 1811 (Tenore 1811(Tenore -1838. Its height can reach up to 10 cm and it's characterised by the fact that it has no stem or this is very short for which it can be easily distinguished from the other species of the genus. Its leaves are all collected in a basal rosette and have an oblanceolate shape and are crenate. The flowers are small and are blue-violet coloured blooming from May to June (Pignatti 1982).
This species is endemic to Italy and grows in stony pastures from 1200 to 2200 m a.s.l. Actually, it can't be found in the central and southern regions of Italy with several exceptions: it is confirmed with absolute certainty only in Latium, Campania, Abruzzo, Molise, Calabria and Sicily, while there are doubts about its presence in Umbria, Marche and Basilicata and moreover, it has been no longer reported in Apulia (Peruzzi et al. 2014).
Several species of the Ajuga genus own an ancient traditional use in folk medicine in some areas of the world and in particular in Africa, where it has been employed as antihypertensive and anti-diabetics (Ziyyat et al. 1997;eddouks et al. 2002) and against intestinal disorders (Bellakhdar 1978), and in China, where it has been employed especially as diuretics (Aliotta & Pollio 1994), instead. Nevertheless, they present many other important pharmaceutical properties such as anti-bacterial (Chen et al. 1997), antifungal (Kariba 2001), insecticide (Wessner et al. 1992), anti-malarial (Kuria et al. 2001), anti-microbial (Cantrell et al. 1999), anti-inflammatory (Marc et al. 2008, antioxidant (Chenni et al. 2007) and anti-tumour (Rodriguez-Hahn et al. 1994).
In literature, there are no previous works on any argument concerning the species A. tenorei and for this reason, we have decided to start this study in order to make the first phytochemical analysis in absolute and even better the first general analysis about it.
Verbascoside (1) is a very common PhG and in fact, it has been already evidenced in several species of the genus (Shimomura et al. 1987), in other entities of Lamiaceae (Venditti et al. 2013c(Venditti et al. , 2013d(Venditti et al. , 2014 as well as in other families presenting a systematic correlation with that one like Scrophulariaceae (Venditti et al. 2015), Orobanchaceae (Serafini et al. 2005;Venditti et al. 2016aVenditti et al. , 2016b and Plantaginaceae (Venditti et al. 2012;Cheriet et al. 2015). From a pharmacological point of view, it owns a pronounced antimicrobial activity, in particular against Staphylococcus aureus (Avila et al. 1999), together with the anti-inflammatory property (Speranza et al. 2010). Verbascoside (1) was found in huge amount in the studied sample and this may explain some of the traditional uses of the plants belonging to the Ajuga genus. On the other side, from a mere chemotaxonomic standpoint, it gains much more importance when it is found in co-occurrence with iridoids like in this case (Jensen 1992).
Echinacoside (2) is a congener of compound (1) presenting an additional glucose residue linked to the first one by interglucosidic bond. Compound (2) is less common in respect of 1 but, anyway, it has been already evidenced in this genus (Göger et al. 2015), in the family i.e. in the Sideritis genus (Stanoeva et al. 2012) and again in systematically near families like Orobanchaceae and especially in the Pedicularis genus (Venditti et al. 2016a(Venditti et al. , 2016b. echinacoside (2) shows also several pharmacological activities, i.e. antimicrobial, in particular against Staphylococcus aureus and Streptococci even if with minor efficacy than verbascoside (1) (Stoll et al. 1950), antioxidant (Xiong et al. 1996;Wu et al. 2007) and anti-inflammatory (Caufin et al. 2014).

Experimental
See Supplementary materials.

Conclusion
In conclusion, the first total phytochemical analysis on the leaves of A. tenorei C. Presl, an Italian endemic species, allowed us to evidence the presence of five compounds namely verbascoside (1), echinacoside (2) (5) belonging to two different classes of chemical compounds i.e. phenyl-ethanoid glycosides for the first two and iridoids for the remaining ones.
According to literature data, all of these compounds are endowed with several interesting and important pharmacological properties, in particular, the anti-microbial, anti-oxidant, anti-inflammatory and anti-tumour ones.
Several species of the Ajuga genus, especially A. reptans L., A. iva (L.) Schreb. and A. decumbens Thunb. have been, and still are, used in the traditional medicine of many areas of the world, for several different purposes in medicinal field.
The discovery in this species of such compounds, not only underlines the correct botanical classification of the species but also by a mere phytochemical point of view, but may also suggest the possibility of a future employment of this species in medicine, too.

Disclosure statement
No potential conflict of interest was reported by the authors.