Iridoids from Stachys byzantina K. Koch (Lamb's Ears) and Stachys germanica L. (Downy Woundwort)

The family Lamiaceae consists of approximately 200 genera of 3500 species. The woundwort (Stachys L.) genus consists of 300 species. This is the third-largest relationship group of Labiate plants. It grows everywhere in the world with the exception of Australia, New Zealand and the Arctic regions. The number of species is particularly high in the Mediterranean region, Eastern Europe, Cape Province and Chile. Ten species live in Central Europe. The flowers of these annual or perennial herbs are light purple, dark pink, yellow or white. Some species grow in Hungary, too. S. officinalis L. is found in Europe, so in Hungary as well. S. alpina L. likes shady places, and it is found in fresh hornbeam-beech forests. S. germanica L. also grows in Hungary and Slovakia, and it is quite frequent in dry grasslands and pastures. S. byzantina L. is found as an ornamental plant, and S. grandiflora Stev. ex Willd. and S. macrantha K. Koch are found in botanical gardens. S. sylvatica L. can be found in hilly and mountainous zones along shrubs and forest paths, in moist, leafy forests, groves, scrubs and by forest springs. It lives on moist and wet clay and adobe soils which are rich in nutrients and have a neutral pH. S. palustris L. is widespread in the greater part of Europe, and it is common in Hungary, especially along marshes and bogs. S. recta L. is frequent on dry, stony grasses and steppe slopes. S. annua L. is found in most of Southern and Central Europe; it is native to Northern Europe; in Hungary, it is an ordinary plant. It can be found in ploughlands and stubble fields, mainly on hard soils (Tomou et al., 2020; Tutin et al., 1972).

Some members of the Stachys genus (extracts or their content material) have significant antibacterial, antifungal and antiphlogistic effects, and they can also be useful in anoxia, hepatitis and nephritis. It is proved by literature data that Stachys species have long been used in folk medicine for the treatment of genital tumours and cancerous ulcers (Skaltsa et al., 1999, 2001; Tomou et al., 2020).

Stachys species belong to the Lamioideae subfamily, and thus, they contain volatile oils in traces, but they have a great number of other secondary metabolic products, e.g. iridoids. As for their structure, their iridoids usually have 9 C atoms, with an OH group on C5 or C6. They typically contain a methyl or acetyl group on C8, giving C8 a quaternary character. For the most part, these iridoids cannot be detected in UV light, and therefore, a developing reagent is needed to make them visible. Their structure is relatively simple; at the same time, they are very sensitive to acids and enzymes, the presence of which leads to the decomposition of the compound (El-Naggar & Beal, 1980; Kobzar, 1986; Derkach et al., 1987; Jeker et al., 1989; Calis et al., 1991; Boros & Stermitz, 1990, 1991; Isamukhamedova & Pulatova, 1992; Kartev et al., 1994; Miyase et al., 1990; Munoz & Pena, 2001).

The aim of this study was the isolation and identification of iridoids from the aerial parts of Stachys byzantina (lamb's ears) and Stachys germanica (downy woundwort).

Stachys germanica was collected at the Medicinal Plant Garden of the Faculty of Pharmacy, Comenius University in Bratislava. Stachys byzantina was gathered at Hungarian biotope and the Botanical Garden, Vácrátót, Hungary. Voucher specimens were deposited in the Institute of Pharmacognosy, University of Szeged, Hungary. The aerial parts of both species were collected at the flowering time, in June. The samples were conserved at −20°C until processing.

Solvents of analytical purity were purchased by Reanal (Budapest, Hungary), those of HPLC purity by Merck (Darmstadt, Germany).

Aerial parts of both Stachys species (S. byzantina and S. germanica) contained iridoids. Comparing the main iridoid component in the samples obtained from methanolic extract of drugs, harpagide was found in both Stachys samples. In addition to the iridoids in S. byzantina, ajugoside, aucubin and harpagide were identified by TLC (see Fig. 1), while those later two compounds were isolated and determined by RPHPLC and NMR method aucubin and harpagide (see Tables 1 and 2). Harpagide and harpagoside (see Fig. 1) were detected in S. germanica. We could develop a method which can be used for the identification and detection of the iridoids of Stachys species.

Figure 1

In 1945, the Swedish botanist Erdtman subdivided this taxonomic family into two major subfamilies: the Lamioideae and the Nepetoideae. The Lamioideae subfamily is characterized by tricolpate and binucleate pollen, albuminous seeds, spatulate embryos, the presence of iridoid glycosides, lower content of essential oils and rosmarinic acid but higher content of phenylpropanoid glycosides, whereas the Nepetoideae have hexacolpate, trinucleate pollen, exalbuminous seeds, investing embryos, the presence of volatile terpenoids, mainly monoterpenes and high content of essential oil and rosmarinic acid. Both subfamilies contain caffeic acid and its derivatives (Erdtman, 1945; Cantino & Sanders, 1992).

The genus Stachys belongs to the subfamily Lamioideae. Iridoids are a class of secondary metabolites found in this genus. The first investigation of the iridoid content of in three species dates back to the beginning of the 1970s, when Adema detected iridoid glycoside in S. palustris and when Ukrainian researchers determined harpagide and 8-O-acetylharpagide in 17 Stachys species (Adema, 1968; Zinchenko, 1972). The main iridoid components in the Stachys species studied by Gritsenko et al. in 1977 were harpagide and 8-O-acetylharpagide. The presence of aucubin was also detected in some Stachys species (Gritsenko et al., 1977). Ajugol, ajugoside, harpagide and 8-O-acetylharpagide were detected in S. atherocalyx, S. inflate and S. iberica (Komissarenko et al., 1976, 1979). In 1980, the research team started an examination concerning 20 different Stachys species, which supplemented the previous results: beside harpagide and acetylharpagide as the main components, reptoside and diacetyl-reptoside were also found (Pakaln et al., 1980). In 1984, Lenherrr et al. performed the RP-HPLC analysis of 10 species belonging to the S. recta group, and melittoside, harpagide, acetylharpagide, ajugoside and ajugol were identified (Lenherrr, 1984). The data relating to the iridoids isolated until 1980 are summarized by El-Naggar & Beal (1980). El-Naggar's summary was followed by the work by Boros & Stermitz, which contains the summary of iridoids isolated between 1980 and 1990 (Boros & Stermitz, 1990, 1992). Russian researchers continued to investigate the chemical components of the Stachys species growing in Russia (Isamukhamedova & Pulakova, 1992; Kartev et al., 1994). Japanese researchers also conducted research into the iridoids Stachys species (Miyase et al., 1990). Monomelittoside, melittoside, 8-O-acetylharpagide, harpagide, ajugol, catalpol, 7-O-acetyl-8-epi-loganic acid, aucubin and 5-allosyloxyaucubin were isolated by research made by Montenegrian, Greek, Hungarian and Italian scientists (Háznagy-Radnai et al., 2005, 2006, 2007; Munoz & Pena 2001; Kostos et al., 2001; Meremeti et al., 2004; Serrilli et al., 2005).

The isolated iridoid components of Stachys species found in Hungary are included in Table 3. Háznagy-Radnai et al., 2006, 2007 two iridoids, as harpagide and 8-O-acetylharpagide isolated in S. byzantina and S. germanica. Ajugoside and aucubin in both species right now.

The genus Stachys L. is one of the largest genera of the family Lamiaceae and subfamily Lamioideae containing iridoids. Iridoids (aucubin, harpagide, ajugoside and harpagoside) were isolated and identified by combination of different chromatographic methods (NP-TLC, CPC and RP-HPLC) from S. byzantina and S. germanica. The structures were established by one- and two-dimensional NMR and mass spectrometry. Stachys species may also be used as a potential source of iridoids.