separations Article Phytochemical and Biological Traits of Endemic Betonica bulgarica (Lamiaceae) Tsvetelina Mladenova 1, Plamen Stoyanov 1,2 , Krasimir Todorov 1, Delyana Davcheva 3,4, Gergana Kirova 4,5, Tanya Deneva 3,4, Donika Gyuzeleva 1, Rumen Mladenov 1,2 and Anelia Bivolarska 6,* 1 Department of Botany and Methods of Biology Teaching, Faculty of Biology, University of Plovdiv “Paisii Hilendarski”, 24 Tzar Assen Street, 4000 Plovdiv, Bulgaria; [email protected] (T.M.); [email protected] (R.M.); [email protected] (P.S.); [email protected] (K.T.); [email protected] (D.G.) 2 Department of Bioorganic Chemistry, Faculty of Pharmacy, Medical University of Plovdiv, 15A Vasil Aprilov Boulevard, 4000 Plovdiv, Bulgaria 3 Department of Clinical Laboratory, Faculty of Pharmacy, Medical University of Plovdiv, 15A Vasil Aprilov Boulevard, 4000 Plovdiv, Bulgaria; [email protected] (D.D.); [email protected] (T.D.) 4 Division of Pharmaceutical Innovations for Personalized Medicine, Research Institute at Medical University of Plovdiv, 15A Vasil Aprilov Boulevard, 4000 Plovdiv, Bulgaria 5 Department of Chemical Sciences, Faculty of Pharmacy, Medical University of Plovdiv, 15A Vasil Aprilov Boulevard, 4000 Plovdiv, Bulgaria; [email protected] 6 Department of Medical Biochemistry, Faculty of Pharmacy, Medical University of Plovdiv, 15A Vasil Aprilov Boulevard, 4000 Plovdiv, Bulgaria * Correspondence: [email protected]; Tel.: +359-887611786 Abstract: Betonica bulgarica is an endemic species distributed in Bulgaria. The chemical composition of the essential oil analysed by GC–MS (Gas chromatography–mass spectrometry) and the content of trace elements analysed by ICP–MS (Inductively coupled plasma mass spectrometry) were deter- Citation: Mladenova, T.; Stoyanov, P.; mined. Additionally, a study on the types and distribution of trichomes was done using a microscope Todorov, K.; Davcheva, D.; Kirova, G.; with a camera. The essential oil was characterized using a high concentration of sesquiterpene Deneva, T.; Gyuzeleva, D.; Mladenov, hydrocarbons, whose major compounds are β-caryophyllene (17.4%), germacrene D (9.9%), and R.; Bivolarska, A. Phytochemical and β-bourbonene (6.7%). The contents of manganese (177.2 µg/g) and strontium (156.8 µg/g) were Biological Traits of Endemic Betonica highest among the investigated micronutrients. Two types of trichomes were identified on the bulgarica (Lamiaceae). Separations adaxial and abaxial epidermises of the leaves of B. bulgarica—covering and glandular. Peltate stacked 2021, 8, 11. https://doi.org/10.3390/ glandular trichomes with a four-celled head of type B were observed on the leaf surface. separations8020011 Keywords: Betonica bulgarica; ICP–MS (Inductively coupled plasma mass spectrometry); GC–MS (Gas Received: 7 January 2021 chromatography–mass spectrometry); multielement analysis; trace elements; trichomes; endemic; Accepted: 20 January 2021 essential oil; lamiaceae Published: 23 January 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in 1. Introduction published maps and institutional affil- iations. Endemic plants make up 12.8% of the representatives of Bulgarian flora and have specificity and certain genetic peculiarities [1]. They are some of the most sensitive and vulnerable natural ecosystems in the country [2]. The genus Betonica L. is separate from the genus Stachys L., with four species [3]. The taxonomic independence of two of them—Betonica officinalis and Betonica bulgarica—is Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. currently controversial. This article is an open access article In Bulgaria, B. officinalis can be found throughout the country at an altitude of up distributed under the terms and to 1400 m in grassy and shrubby areas [4]. B. officinalis is used in folk medicine for the conditions of the Creative Commons treatment of nervous exhaustion, gallstones, stomach acids, high blood pressure, migraine, Attribution (CC BY) license (https:// and neuralgia, as an ointment for cuts and wounds as well as against sweating [5,6]. creativecommons.org/licenses/by/ The Bulgarian endemic B. bulgarica Degen et Neiˇc.(Stachys bulgarica Hayek), described 4.0/). by the Hungarian botanist A. V. Degen and the Bulgarian botanist I. Neichev in 1906 on Separations 2021, 8, 11. https://doi.org/10.3390/separations8020011 https://www.mdpi.com/journal/separations Separations 2021, 8, x FOR PEER REVIEW 2 of 8 Separations 2021, 8, 11 2 of 8 The Bulgarian endemic B. bulgarica Degen et Neič. (Stachys bulgarica Hayek), de- thescribed basis by of the materials Hungarian collected botani inst A. the V. Central Degen Staraand the Planina Bulgarian Mountains, botanist I. grows Neichev outdoors in in stony1906 on and the sandy basis soilsof materials in oak collected and beech in foreststhe Central of the Stara Stara Planina Planina Mountains, Mountains grows (Central, East)outdoors and in the stony Thracian and sandy Lowlands soils in oak [7– and9]. beechB. bulgarica forests ofis the an Stara endemic Planina plant Mountains protected by the(Central, Biological East) and Diversity the Thracian Act [10 Lowlands] and included [7–9]. B. inbulgarica the Red is an Data endemic Book ofplant Bulgaria protectedunder the categoryby the Biological “endangered” Diversity [ 9Act]. Three [10] and flavonoids included in have the Red been Data found Book inof Bulgaria significant under amounts the in B.category bulgarica “endangered”—rutin, quercetin, [9]. Three and flavonoids hispidulin—associated have been found in with significant the antioxidant amounts in activity B. of thebulgarica plant—rutin, [11]. quercetin, and hispidulin—associated with the antioxidant activity of the plantIn [11]. recent years, the Bulgarian populations of B. bulgarica have been studied by Grozeva et al.In [12 recent–14]. Alongyears, withthe Bu morphological,lgarian populations palinomorphological, of B. bulgarica have and been ecological studied studies, by the authorsGrozeva alsoet al. draw [12–14]. attention Along towith the morphological, epidermis covering palinomorphological, the leaves, stems, and and ecological flower parts ofstudies, the plants the authors as the mainalso draw structure attention responsible to the epidermis for the accumulationcovering the leaves, of essential stems, and oils. flowerAlthough parts of the the plants healing as the properties main struct ofure the responsible species of for the the Lamiaceae accumulation family of essen- are likely tial oils. due to their essential oils and flavonoids, in recent years, there has also been increasing Although the healing properties of the species of the Lamiaceae family are likely due interest in certain trace elements [15], whose presence could have a synergistic effect with to their essential oils and flavonoids, in recent years, there has also been increasing interest thesein certain components trace elements [16]. Most[15], whose trace elementspresence could are essential have a synergistic for higher effect plants, with mainly these in the compositioncomponents [16]. of metalloenzymes Most trace elements and are metalloproteins. essential for higher They plants, are also mainly involved in the in com- a number ofposition redox of processes, metalloenzymes processes and of metalloproteins. photosynthesis, They breathing, are also involved expression in a and number regulation of of genes,redox proteinprocesses, synthesis, processes and of protectivephotosynthesis mechanisms, breathing, of plantsexpression [17]. and So far,regulation there have of been nogenes, data protein on the synthesis, presence and of trace protec elementstive mechanisms in B. bulgarica of plantsspecies. [17]. So far, there have been no dataThe on purpose the presence of this of trace study elements was to in complement B. bulgarica species. the available information about the endemicThe speciespurpose B.of bulgaricathis studywith was regardto comple to thement anatomic the available morphological information structures about the of the epidermis,endemic species the essential B. bulgarica oil with composition, regard to andthe anatomic the microelement morphological composition structures of of the the species fromepidermis, the plant the essential habitat, oil which composition, we discovered and the inmicroelement the Bulgarka composition Nature Park. of the species from the plant habitat, which we discovered in the Bulgarka Nature Park. 2. Results and Discussion 2.1.2. Results Biological and Traits Discussion 2.1. BiologicalAnalysing Traits the leafy and stem surfaces of B. bulgarica, two types of trichomes were identified:Analysing covering the leafy trichomes and stem andsurfaces glandular of B. bulgarica trichomes., two types The resultsof trichomes obtained were are in accordanceidentified: covering with the trichomes claims ofand Metcalfe glandular and trichomes. Chalk [The18], results Maleci obtained Bini and are Giuliani in ac- [19], andcordance Mladenova with the et claims al. [20 of] regardingMetcalfe and the Chalk types [18], of trichomes Maleci Bini in and the Giuliani representatives [19], and of the LamiaceaeMladenova family.et al. [20] regarding the types of trichomes in the representatives of the Lami- aceaeIn family. this study, two types of trichomes were identified on the upper and lower epi- In this study, two types of trichomes were identified on the upper and lower epider- dermises of the leaves of B. bulgarica-covering and glandular. Covering