**Zenon W˛eglarz, Olga Kosakowska \*, Jarosław. L. Przybył, Ewelina Pióro-Jabrucka and Katarzyna B ˛aczek**

Department of Vegetable and Medicinal Plants, Institute of Horticultural Sciences, Warsaw University of Life Sciences–SGGW, 02-787 Warsaw, Poland; zenon\_weglarz@sggw.edu.pl (Z.W.);

jaroslaw\_przybyl@sggw.edu.pl (J.L.P.); ewelina\_pioro\_jabrucka@sggw.edu.pl (E.P.-J.);

katarzyna\_baczek@sggw.edu.pl (K.B.)

**\*** Correspondence: olga\_kosakowska@sggw.edu.pl; Tel.: +48-22-593-2247

Received: 23 October 2020; Accepted: 13 November 2020; Published: 15 November 2020

**Abstract:** The purpose of the study was to determine the differences between two subspecies: *O*. *vulgare* L. subsp. *hirtum* (Link) Ietswaart (Greek oregano) and *O*. *vulgare* L. subsp. *vulgare* (common oregano) growing in cultivation conditions within temperate climate of Central Europe. The characteristic of the subspecies was undertaken in terms of selected morphological parameters and the quality of the raw material. The herb of both subspecies was evaluated on the content and composition of essential oil by hydrodistillation followed by GC-MS and GC-FID (gas chromatography coupled with mass spectrometry and flame ionization detector), the total content of phenolic acids (according to PP 6th ed.) and the content of rosmarinic acid (by HPLC). The sensory evaluation (QDA) was performed, as well. Greek oregano was distinguished by visibly higher number of glandular trichomes on the leaves (up to 4.85 per 1 mm2) followed by higher content of essential oil in the herb (up to 3.36 g <sup>×</sup> 100 g−<sup>1</sup> DW) in comparison to common oregano. Based on the essential oil composition, Greek oregano was classified as mixed carvacrol/γ-terpinene chemotype, while common oregano as mixed sabinyl/cymyl type rich in sesquiterpenes. Greek oregano was also characterized by higher total content of phenolic acids (up to 6.16 g <sup>×</sup> 100 g−<sup>1</sup> DW) and rosmarinic acid (up to 6787.2 mg <sup>×</sup> 100 g−<sup>1</sup> DW) than common oregano. Essential oil content reached the maximum at the beginning of blooming (common oregano) and at the full blooming stage (Greek oregano). In turn, the amount of phenolic acids followed by rosmarinic acid was the highest at the beginning of seed-setting stage, in the case of both subspecies. The differences between subspecies concerning chemical composition (especially essential oil) were reflected in the sensory attributes, where both odor and taste notes were found at higher level for Greek oregano. Results of our work indicate that Greek oregano is well adapted to grow in the temperate zone conditions. Such adaptation was reflected mainly in the satisfied yield and maintaining characters typical for the Mediterranean plant, e.g., a high essential oil content followed by high carvacrol share, traits the most important from practice viewpoint.

**Keywords:** *Origanum* subspecies; morphological traits; glandular trichomes; essential oil composition; rosmarinic acid; sensory evaluation

#### **1. Introduction**

Plants belonging to *Origanum* genus (*Lamiaceae* family) have been known as culinary and medicinal plants since ancient times. This genus contains 49 taxa belonging to 10 sections. Some species, including *Origanum vulgare* L., are rich in essential oil and commonly known as "oregano" [1]. *Origanum vulgare* L., an aromatic, perennial sub-shrub, is widely distributed all over Eurasia and North Africa [2]. The species is regarded to be extremely variable, both in its morphological features and chemical composition. Given its specific biological character and significant economic importance, *O*. *vulgare* has been placed in the List of Priority Species in Europe [3]. According to the widely accepted taxonomy, six subspecies of *O*. *vulgare* have been recognized [2,4]. Among them, *O*. *vulgare* L. subsp. *hirtum* (Link) Ietswaart so-called Greek oregano, endemic to the Mediterranean area, is cultivated almost all over the world and regarded as the most valuable one [5]. Another subspecies important from economic point of view, is *Origanum vulgare* L. subsp. *vulgare* (common oregano). It frequently occurs on the region of Northern and Central Europe and is the only representative of *O*. *vulgare* in Poland [6,7]. The upper, not woody parts of flowering shoots (herb) of both subspecies is commonly used and traded raw material. Besides the range of occurrence, these two subspecies differ in terms of many features, whereas the content and composition of essential oil seems to be the most important, because it determines medicinal properties of the herb and its sensory value [2]. Greek oregano is rich in essential oil (about 5%), while common oregano contains less amount (up to 2%) of this substance. Subspecies create few various chemotypes defined on the basis of the dominant compound in essential oil. Greek oregano accumulates mainly phenolic monoterpenes (thymol and carvacrol) followed by its precursors (*p*-cymene and γ-terpinene). In turn, common oregano is distinguished by less active biosynthesis of "cymyl" compounds in favor of the bicyclic "sabinyl" (i.a. sabinene, *cis*/*trans* sabinene hydrate and its acetates) or acyclic once (i.a., β-ocimene, β-myrcen, linalyl acetate, linalool). This kind of chemotype is often accompanied by high content of sesquiterpenes (i.a. germacrene D, β-caryophyllene and caryophyllene oxide) [1,7–18]. Both *Origanum* subspecies contain also considerable amounts of non-volatile phenolic compounds such as flavonoids and phenolic acids. Rosmarinic acid followed by caffeic, vanillic, *o*-coumaric and protocatechuic acids dominate in common oregano herb [7,19,20]. When given Greek oregano, rosmarinic and lithospermic acids are the present in the highest amounts [13,21,22]. In both subspecies, flavonoids are represented mainly by derivatives of luteoline and apigenine [7,13,20–24]. In relation with such a wide range of biologically active compounds, both *Origanum* subspecies indicate various pharmacological activities, especially antimicrobial, choleretic and antioxidant. Common oregano herb reveals also diuretic and expectorant properties, while Greek oregano—stimulative, carminative, antispasmodic, and anticancer [25,26]. It is worth noting that Greek oregano is listed in the European Pharmacopeia and is recommended as a remedy for gastrointestinal disorders treatment, temporary loss of appetite and to stimulate bile secretion [25–27]. Common oregano, even though not mentioned in European Pharmacopeia, used to be applied in the same way in both modern and folk medicine [28]. Both *Origanum* subspecies are widely used not only in pharmaceutical industry but also as a food preservative and flavoring, cosmetic ingredient, and, most importantly—as a culinary herb [29,30].

Despite abovementioned intraspecific diversity, *O*. *vulgare*is still treated as a collective taxon [31–34]. Moreover, many varieties, landraces, forms, ecotypes, and cultivars are nowadays available for stakeholders, creating possibility of subspecies misleading [35]. This altogether may lead to decrease homogeneity and the quality of raw material. It is especially important, since herbal products standardization requirements are taken into consideration [36].

Up to now, Greek oregano cultivation areas have been located mainly within a warmer climate. Recent studies have showed that this subspecies may be cultivated in temperate zone of Central Europe, as well [13,37,38]. However, in Poland, the cultivation of this subspecies is at its infancy [18]. In turn, common oregano used to be collected in Poland both from natural sites and cultivation [28]. However, the harvest from the wild may result in heterogeneous raw material. Moreover, the number of wild growing common oregano populations has recently significantly decreased what can lead to genetic erosion. Thus, the collection of common oregano herb exclusively from cultivation would provide natural resources protection as well as ensure high quality of raw material [39].

The aim of the study was to determine the differences between Greek oregano and common oregano in cultivation conditions within temperate climate of Central Europe. The characteristic of the subspecies was undertaken in terms of selected morphological parameters and the quality of raw material, reflected in the content of biologically active compounds (the total content and composition of essential oil and phenolic acids) and sensory evaluation.

#### **2. Materials and Methods**

### *2.1. Plant Material*

The experiment was carried out at the experimental field of the Department of Vegetable and Medicinal Plants, Warsaw University of Life Sciences (WULS-SGGW) (5210180 N; 2105234 E), on heavy alluvial soil. Seeds of Greek oregano and common oregano originated from Polish Gene Bank collection (accession numbers: 406735 and 401291, respectively). Seeds were sown in the first week of February (2020) into multi-pots filled with a peat substrate, in a greenhouse. A total of 180 seedlings of each subspecies were randomly selected and planted out into the field in the last week of April. The randomized block design (60 seedlings per plot; in 3 replications) was applied, with a spacing of 40 × 60 cm. The harvest of the herb (upper, not woody parts of shoots) was performed on 1-year old plants, at three stages of plant's development: at the beginning of blooming (fourth week of June), at the full blooming (third week of July) and at the beginning of seed-setting (second week of August). The herb was cut at a height of about 15 cm above ground. The fresh and dry weight of the herb was determined (g per plant). After drying at 35 ◦C, the herb was ground and prepared for chemical analysis. Climatic parameters were recorded (Table 1).


**Table 1.** Climatic parameters in the vegetation season of 2020.
