Ethnomedicinal application of species from genus Thymus in the Pirot County (Southeastern Serbia)

The species from genus Thymus are polymorphous plants from the family Lamiaceae, spread in Serbia in many species, subspecies, varieties, and forms. The aerial parts of species from genus Thymus have a long tradition of being used worldwide. The subject of this study was the research of ethnomedicinal application of thyme in Pirot County (Southeastern Serbia). Ethnomedicinal surveys were conducted among the population in four municipalities: Pirot, Babušnica, Bela Palanka, and Dimitrovgrad. It was noticed that 56.9 % of interviewed persons use the species of genus Thymus in the Pirot County. They are usually used in the county’s folk medicine in the form of herbal tea against colds to relieve dry and spastic cough, especially in bronchitis and asthma, and for sedation.


INTRODUCTION
The species from genus Thymus are perennial herbs or semishrubs of the family Lamiaceae. In Serbia, they are highly polymorphous species, spread in 30 species, and many subspecies, varieties, and forms occurred differing in size, branching, and hairiness of the stems, the colors, the shape, the size of the hair of the leaves, and other morphological characteristics (Diklić, 1974;Jančić et al., 1995). The genus Thymus is very complex from the taxonomical and systematic point of view, demonstrating significant polymorphism in morphological characteristics and the composition of essential oils (Jarić et al., 2015a).
Thymus species have been used since ancient times to treat diseases of the respiratory and digestive systems (Zarzuelo and Crespo, 2002). In ancient Egypt, they were used to make perfumed balms for embalming and medical purposes, and in ancient Greece, according to Dioscorides, thyme was used to treat asthma and loosen congestion in the throat stomach (Jarić et al., 2015a). The pharmacological records of the Chilandar Medical Codex (15 th and 16 th centuries) mention the use of thyme as a remedy against headaches caused by colds, laryngitis, and diseases of digestive organs and as an antitussive (Jarić et al., 2014).
The aerial part of species from genus Thymus have applications not only in folk medicine but also in modern medicine. It has antiseptic, fungicide, expectorant, spasmolytic, carmina-tive, sedative, diuretic, and diaphoretic activities (Aneva et al., 2018). Due to its pharmacological characteristics, the essential oil of species from genus Thymus represents an important natural resource for pharmaceutical industry, beside its antioxidnant and antimicrobial properties (Nikolić et al., 2019). Ilić et al. (2017) have evaluated the antibacterial and streptomycinmodifying activity of Thymus glabrescens essential oil, and its components geraniol, geranyl acetate, and thymol. In this study it was noticed that all substance-streptomycin combinations produced antagonistic interactions, and combinations between geraniol and thymol showed dominant additive effect. Species from genus Thymus are also the sources of natural products for nutritional supplements or functional food components in the dietary industry Jarić et al. (2015a);Nikolić et al. (2019). In Pirot County (Southeastern Serbia), the species from genus Thymus are widespread at meadows and pastures, with collecting period May-September. The following compounds have been previously reported in Thymus spp.: geraniol, geranyl acetate and thymol as the main components of the essential oil, tannins, bitter substance, flavonoids (Marković et al., 2010) . A detailed review of aromatic plants presence in investigated and described plant communities on the Vidlič Mountain in the Pirot County was presented by Marković et al. (2019). They have noticed the following species from the genus Thymus: Thymus striatus Vahl., Thymus glabrescens Willd., Thymus praecox Opiz subp. jankae (Čelak.) Jalas, Thymus pulegioides L. genus Thymus, also known as "babina dušica", "dušičina", "majčina dušica," or "majkina dušica" to the local population of study area. This paper will discuss species' application from the genus Thymus based on a population survey in the studied area: Pirot, Babušnica, Bela Palanka, and Dimitrovgrad. Also, a comparison with other regions in Serbia was performed.

METHODOLOGY
The study area includes four municipalities Pirot, Bela Palanka, Babušnica, and Dimitrovgrad in Southeastern Serbia ( Figure 1). It covers 2761 km 2 (Pirot County GIS, 2019). The climate is temperate continental, with the transitional climatic variations to sub-mountain and mountain climate at altitude over 600 meters (Marković et al., 2010). Lists of medicinal plants in the Pirot County were recorded by Randelović et al. (1991), and Marković et al. (2010), while on the Vidlič Mountain in the study area were reported 60 aromatic plants were recorded by Marković et al. (2019;2009). Information collected in the form of a questionnaire provided data about medicinal plants' knowledge among the local community at Pirot County. Data gathered from the participants were the common name of plants, the disease for which the plant is used, the plant part they use, and preparation forms. Special selection criteria were not carried out in the choice of the informants. Plant species were identified according to Josifović (1970Josifović ( -1986, Jordanov (1963Jordanov ( -1979, and Tutin et al. (1964Tutin et al. ( -1980. The voucher specimens of collected plant material deposited in HMN herbarium are presented in Table 1. The nomenclature of the taxa listed, given at subspecies level, was compiled from database: The EURO+MED PlantBase -the information resource for Euro-Mediterranean plant diversity (Euro+Med, 2006-). The results were systematized using Microsoft Excel and presented in Table 2 with the frequency of forms of application for traditional use, in Table 3 systematized regarding therapeutic groups with number of use reports per indication. Comparison with other regions in Serbia was also done using tabular overview (Table 4).

Quantitative analysis
It was noticed that applications of species from genus Thymus (Thymus spp.) are well known among population of the Pirot County. A total of 359 of 631 interviewed persons i.e. 56.9 % respondents mentioned "babina dušica", dušičina", "majčina dušica" or "majkina dućica" and their medicinal usage (Table 2,3), out of which 301 (83.85 %) of them were of Serbian nationality, 55 (15.32 %) were of Bulgarian nationality, and 3 (0.83 %) were Roma. Among respondents, a total of 180 were male, and 179 were female. In Pirot municipality, Thymus spp. were known to 198 (55.15 %) respondents, in municipality Babušnica 46 (12.81 %), in municipality Bela Palanka 73 (20.34 %) and municipality Dimitrovrgad 42 (11.70 %) interviewed people. The age of respondents who mentioned Thymus spp. was from 16 to 85. The majority of interviewed persons mentioned the use of the aerial part of the Thymus spp. in the form of tea in the treatment of common cold (149 persons, 41.50 %), cough (60 persons, 16.71 %), and for sedation (42 persons, 11.70 %). In the disease prevention, Thymus spp. were mentioned by 19 persons, and the same number of respondents were familiar with its application against stomach diseases, and eight respondents against lung diseases (asthma, bronchitis). In the treatment of sore throat Thymus spp. was mentioned by seven persons, and the same number of people were familiar with their effect on stomach.
Five respondents mentioned application in nutrition as a spice. The same number of respondents were familiar with the usage of Thymus spp. in treating bronchitis and respiratory diseases (each one). The application for improving the immune system was mentioned by three respondents. The same number of persons was familiar with their effect against high temperature and the heart and nerves (sedative effects). Likewise, three people mentioned "majčina duišica" or "dušičina", but did not know its use. Two persons mentioned improving hearing, and the same number of respondents were familiar with its effect against headaches, against high blood pressure, and kidney and bladder diseases. One respondent mentioned usage of "majčina dušica" in the form of tea for rinsing the oral cavity, against thyroid diseases, one as an antibacterial antiseptic, for the treatment of influenza, against menstrual pain, against diabetes, against inflammation, and one was familiar with usage in the filling the pillow against insomnia and for good sleep.
The most common form of usage is the form of tea for oral application. Rarely used forms are extract, dry herba, and form of oil. Extract in alcohol is used against cough, dry herba in the nutrition as a spice, or for filling the pillow against insomnia. The form of oil is used for sedation.

Comparative ethnopharmacological analysis
The results of usage of species from genus Thymus of previous studies in neighboring regions in Serbia are presented in Table  4.  Table 1 Results presented in Table 2 Presented data Furthermore, Matejić et al. (2020) were identified Thymus praecox Opiz for Timok and Svrljig region. However, they mentioned genus level (Thymus spp.) because there are other species from genus Thymus which may be used and have the same effect. The present study results are in accordance with the findings of ,  and Matejić et al. (2020) probably as a result of proximity of investigated areas having in mind geographical locations of the geographical locations of the Svrljig, Timok, Rtanj Mountain, and Pirot County, which is probably caused by the proximity of these investigated areas. The following therapeutical applications of species from genus Thymus in the Pirot County systematized in therapeutic groups, are different and new from the above-mentioned ethnopharmacological studies in Serbia: • Nervous system: epilepsy, filling the pillow against insomnia.
• Inflammation: rinsing of the oral cavity, inflammation in general.
• Cardiovascular system: for the heart, high blood pressure.
• Urogenital system: kidney and bladder diseases.

CONCLUSION
Based on the results of interviews with the local population in the Pirot conducted in this study, it can be concluded that species from genus Thymus commonly known as "babina dušica", "dušičina", majčina dušica", or "majkina dušica" are well known to people living in the rural areas, and they are used for indications from various therapeutic areas. The aerial part of Thymus spp. is frequently used in the form of tea for the treatment of cold, cough, and for sedation, and rarely in disease prevention, against stomach diseases, lung diseases, against sore throat, for the stomach, against respiratory diseases, improvement of the immune system, against high temperature, for the heart, for nerves, improving hearing, against headache, against high blood pressure, for rinsing the oral cavity, against thyroid diseases, as an antibacterial antiseptic, against epilepsy, against influenza, against menstrual pain, against inflammation, against diabetes. Except as tea, the species from genus Thymus are used as an extract against cough, dry herba is used in the nutrition as a spice or for filling the pillow against insomnia, and oil is used for sedation.