The chemical composition of the aerial parts essential oil of S. spreitzenhoferi Heldr. (Lamiaceae) growing in Kythira island (Greece)

Abstract Stachys is a large genus of economically important plants belonging to Lamiaceae family that includes about 300 species as annual or perennial herbs. Several species of this genus are extensively used in various traditional medicines. In the present study the chemical composition of the essential oil from aerial parts of Stachys spreitzenhoferi Heldr., a very rare plant, belonging to Section Candidae, endemic of South Greece and collected in the island of Kythira, was analysed by GC-MS. No one reports have been previously published on this species. The result showed the presence of large quantity of diterpenoids with manoyl oxide (22.1%), as the most abundant component. Other metabolites present in high quantity were trans-nerolidol (18.5%), β-caryophyllene (11.0%) and germacrene D (8.1%). Chemotaxonomic considerations with respect all the other oils of Stachys taxa, belonging to the same section studied so far, were carried out. Graphical Abstract


Introduction
The genus Stachys L., belonging to Lamiaceae family, is composed by more than 300 species, distributed in temperate and tropical regions of Mediterranean, Asia, America and southern Africa. It has been divided in two subgenera Betonica L. and Stachys L. The subgenus Stachys includes 19 sections, while the subgenus Betonica comprises 2 sections (Bhattacharjee 1980).
The genus name derived from the Greek word «stachys (rs avt1)», that it is related to the type of the inflorescence which is characterised as 'spike of corn' and resembles to the inflorescences of the species of genus Triticum L. (Gramineae). Many taxa of the genus are largely used in traditional medicine of several countries to treat a broad array of disorders and diseases, including stress, skin inflammations, stomach disorders and genital tumors. The traditional uses, phytochemistry and bioactivity of this genus have been reviewed (Tundis et al. 2014;Tomou et al. 2020) as well as the occurrence of diterpene metabolites both in essential oils and non-volatile extracts Bruno 2009, 2011).
S. spreitzenhoferi Heldr. var. spreitzenhoferi is a very rare perennial semi-shrubby species, which forms tufts like 'cushions', endemic to Kythira and SE Laconia, in Elafonisos and the Maleas peninsula (Greece). Its hairy shoots are 5-20 cm high. The leaves are hairy with serrated margins, gray-green on the upper surface and whitish on the lower. The flowers come out in groups of 4-6, one on top of the other, at the edge of the shoots. The petals are white and form a tube ending in 2 lips, the lower with dark purple-red shapes and the upper dark purple. It grows on rock crevices, walls, in limestone and other sedimentary rocks, at altitudes of 5-50 m (Greek flora 2021). A different variety of this species, S. spreitzenhoferi Heldr. var. virella (D. Perss.) Krestovsk, growing on the mainland, can be distinguished from var. spreitzenhoferi by sparsely pubescent and green leaves, instead of white or greyish-green. According to Krestovskaya (2017) both taxa belong to section Candidae R. Bhattacharjee of the Stachys genus that includes also S. candida Bory et Chaub, S. chrysantha Boiss. et Heldr, S. saxicola Coss. et Balansa and S. iva Griseb, the last one being assigned to subsection Stenophyllae Krestovsk (Table S1).
Previous phytochemical investigations on the non-volatile metabolites of these species indicate the occurrence of flavonoids, phenolic derivatives, phenylethanoid glycosides and iridoids, etc in S. candida (Michailidou 2018;Skaltsa et al. 2020;Michailidou et al. 2021), S. chrysantha (Skaltsa et al. 2020) and S. iva (Lazarevi cet al. 2010; Pritsas et al. 2021). Other investigations have been carried on the essential oils of Stachys taxa belonging to section Candidae (Table S2) and their composition will be discussed below. On the other hands no one paper has been published on S. spreitzenhoferi Heldr.
Studies about the chemotaxonomy of these plants could be crucial to help and facilitate a clear partition of the genus and species and, also, provide important information about the effects of the geographic isolation of populations, related to the processes of speciation and differentiation, as well as the specific variability in geographical areas with different edaphic and weather conditions.
The analysis carried out in the present work concern to plant samples picked up on the cliffs of Avlemonas in the island of Kythira, South Greece.
Consequently, in the frame of our on-going researches on Mediterranean plant essential oil ( Bruno et al. 2019;Ilardi et al. 2020;Badalamenti et al. 2021;D'Agostino et al. 2021;Gagliano Candela et al. 2021) and on chemotaxonomic differences (Catinella et al. 2021;Bancheva et al. 2021aBancheva et al. , 2021b we decided to investigate the composition of the essential oil of S. spreitzenhoferi Heldr., collected in Kythira.

Results and discussion
Hydrodistillation of S. spreitzenhoferi Heldr. aerial parts gave a pale-yellow oil (Ss). Overall, twenty-two compounds were identified, representing 97.7% of total components, listed in Table S3 according to their retention indices on a DB-Wax column and classified into four classes based on their chemical structures. Diterpenoids formed the main class, representing 36.9% of the oil, with manoyl oxide (22.1%) as the most abundant component. In the same class, abietatriene (6.8%) was also present in good amounts. Oxygenated sesquiterpenes was the second most abundant class (30.5%), with trans-nerolidol (18.5%) as the main component of this class. Sesquiterpene hydrocarbons occurred in similar amount (28.7%) being b-caryophyllene (11.0%) and germacrene D (8.1%), the principal constituents of the class. On the other hands monoterpenes were totally absent.
Comparison with the essential oils of other taxa belonging to section Candidae of genus Stachys (Table S2) allowed us to carry out some interesting comments. In fact, manoyl oxide, the main compound of Ss, was present, although in lesser amounts, in S. candida (12.1%) (Skaltsa et al. 1999) and S. chrysantha (1.0%) (Skaltsa et al. 1999), both collected in Greece, but absent in S. horvaticii Micevski and S. iva from North Macedonia (Radulovi c et al. 2007;Kremer et al. 2016). It is noteworthy that these two last species contain good amount of monoterpenoids (17.0-5.7%), poorly represented in S. candida, S. chrysantha and S. spreitzenhoferi Heldr. On the other hands, b-caryophyllene, the main sesquiterpene hydrocarbon of Ss, was present, and in comparable amount (9.6-6.3%), in all the other taxa of this section (Table S2). Finally, it is worthy of mention that trans-nerolidol, the second most abundant metabolite in Ss, is practically absent in all the other taxa.
According to Tundis et al. (2014) Stachys species, on the basis of the composition of their essential oils, have been divided in three groups: a) species mainly characterised by monoterpene hydrocarbons; b) species that contain mainly oxygenated monoterpenes and c) species that contain similar amounts of hydrocarbon and oxygenated sesquiterpenes.

Conclusions
The comparison of the chemical compositions of the essential oils shows that in many cases they can be used as chemotaxonomic markers to support taxonomic decisions related to the establishment of separate taxa. In our study, the GC analysis of the S. spreitzenhoferi Heldr. EO showed that this is rich in diterpenoids, representing 36.9% of the oil, with manoyl oxide (22.1%) as the most abundant component. An aspect that emerges from the chemotaxonomic analysis of the main compounds present in the various essential oils of Stachys taxa belonging section Candidae (Table S2), is the distinction of the taxa into two different groups: the first one includes species devoid in monoterpenenes and with good amount of diterpenoids (S. spreitzenhoferi Heldr., S. candida and S. chrysantha); in contrast, the second one (S. horvaticii and S. iva) groups the species that contain monoterpenes but that are very poor or devoid of diterpenoids.

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

Funding
This work was supported by grant from MIUR-ITALY PRIN 2017 (Project N. 2017A95NCJ).