Ethnobotanical and ethnomedicinal research into medicinal plants in the Mt Stara Planina region (south-eastern Serbia, Western Balkans)

Background Ethnobotanical research in Southeast Europe—one of the most important European hotspots for biocultural diversity—is significant for the acquisition of Traditional Ecological Knowledge related to plants as well as for encouraging the development of local environments. The current ethnobotanical research was conducted in the region of Mt Stara Planina (south-eastern Serbia), which is characterised by rich phytodiversity with a large number of endemic and relict plant species. The aim of the study was to document the diversity of uses of medicinal plants and of traditional knowledge on their therapeutic uses. Methods Ethnobotanical data was collected through both open and semi-structured interviews with locals. Fifty-one inhabitants were interviewed (26 men and 25 women), aged 30–91, and data was analysed by means of use reports, citation frequency, use values (UV), and the informant consensus factor (ICF). Results The study identified 136 vascular medicinal plant taxa and one lichen species belonging to 53 families and 116 genera. Lamiaceae (19), Rosaceae (18), and Asteraceae (17) had the highest species diversity. The plant parts most commonly used to make a variety of herbal preparations were the aerial parts (54 citations), leaves (35 citations), fruits (20 citations), flowers (18 citations), and roots (16 citations), while the most common forms of preparation were teas (60.78%), consumption of fresh tubers, leaves, roots, and fructus (6.86%), compresses (5.88%), juices (5.39%), decoctions (3.92%), ‘travarica’ brandy (3.92%), and syrups (2.45%). Of the recorded species, 102 were administered orally, 17 topically, and 18 both orally and topically. The plants with a maximum use value (UV = 1) were Allium sativum, Allium ursinum, Gentiana asclepiadea, Gentiana cruciata, Gentiana lutea, Hypericum perforatum, Thymus serpyllum and Urtica dioica. The highest ICF value (ICF = 0.95) was recorded in the categories of Skin and Blood, Blood Forming Organs, and Immune Mechanism. Conclusions This study shows that medicinal plants in the research area are an extremely important natural resource for the local population as they are an important component of their health culture and provide a better standard of living.


Background
Europe has a long history of the transmission of medical knowledge despite its geographical, cultural, and linguistic barriers [1].Rural regions in some parts of Europe, such as the mountainous regions of the Balkan Peninsula, are unique hotspots of biological and cultural diversity.As such they are considered highly significant for the conducting of studies with a human ecological focus, thus allowing cross-cultural comparisons of traditional ecological knowledge concerning medicinal plants [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16].Ethnobotanical studies are mainly conducted with the indigenous peoples in local communities where traditional knowledge is scarcely documented.This is mainly due to the way the information is passed down, mostly orally, from generation to generation.Also, an alarming rate of decline in traditional medical knowledge in the region of Southeast Europe is a result of major political turmoil and economic changes that have greatly affected lifestyles, eating habits, and relationships with nature, as well as the transfer of traditional knowledge on health and local medical practices [17].For this reason, the role of traditional knowledge, indigenous communities, and ethnobotanists in achieving sustainable development goals has to be recognised as a matter of urgency [18].It is of the utmost importance that plant biodiversity be preserved so as to provide future structural diversity and important medical components for the sustainable development of human populations worldwide.Well-planned bioprospecting coupled with non-destructive commercialisation could help in the conservation of biodiversity, ultimately benefiting humankind in the long run [19].
Among indigenous people, particularly in the rural regions of the Balkan Peninsula including Serbia, there is a strong belief in the power of medicinal plants, which is linked to traditions originating over the past centuries.The sound knowledge of ethnobotany in these areas is also a result of the specific geographical position, great biological diversity, and ethnic and cultural differences.
In regard to this, we conducted ethnobotanical research focused on the use of medicinal plants in the Mt Stara Planina region, which is located in south-eastern Serbia, in the Western Balkans.This area is characterised by high diversity of plant and animal species and their communities, as well as of geomorphological, geological, hydrological, and hydrogeological phenomena.The traditional way of life is still found here and it abounds in cultural heritage [20].It has also been designated as internationally significant for plants (important plant areas-IPA), diurnal butterflies (primary butterfly areas-PBA), and birds (internationally important bird areas-IBA) and is on the list of Serbian geo-heritage sites (International Association for the Conservation of Geological Heritage-ProGEO) [21].
Knowledge of the diversity and use of medicinal plants, especially in rural areas like the Mt Stara Planina region, enables the development of modern ecologically organised human activities (ecological management, sustainable development, ecotourism, ecological engineering), which improves the living conditions of the local inhabitants and contributes to maintaining the ecological balance in nature.Historically, the Balkan region has provided plant material for the West European market, especially in the last few centuries.Most locally harvested medicinal plants, in dry or raw form, are widely used in local health care in many households and they are at the heart of many economic initiatives and programmes dedicated to rural development [17].The increased volume for trade and increased demand for certain types of medicinal plants has resulted in the need for sustainable management of these natural resources based on biodiversity conservation strategies [22].In this regard, previous or current ethnobotanical studies in Southeast Europe, where the study area is located, are of vital importance for stimulating local development and investigating the dynamics of Traditional Ecological Knowledge (TEK), which relates to plants as an important natural resource in one of the most crucial European hotspots for biocultural diversity [9].Wild medicinal plants are an important commercial product (in the form of raw or dried plant material), but they are primarily a natural resource that should be used wisely and rationally.Therefore, the objectives of this study were: (1) to identify plant species traditionally used for the treatment and prevention of various health conditions and diseases among the local population, (2) to distinguish plant species with a maximum use value (UV), (3) to determine consensus levels among the informants by using the informant consensus factor (ICF), (4) to report on new species data and new usage data not previously recorded for Serbia and the Balkans, and (5) to discuss the significance of these medicinal plants for the herbal market, as foodstuffs, and for ecotourism.
These studies can contribute to the general fund of traditional ethnobotanical and ethnomedical knowledge in the Western Balkans and beyond and to the promotion of the practical importance of the use of medicinal plants and their conservation.This type of research can contribute to the conservation of biodiversity and the sustainable use of medicinal plants in order to develop the economy of the local population.

Study area
Stara Planina Mountains or the Balkan Mountains are a mountain range mainly located in Bulgaria with a much smaller part in south-eastern Serbia.It is part of the Carpathian-Balkan mountain arc and extends from the Black Sea in Bulgaria in the east to Vrška Čuka (Zaječar, Serbia) in the west, with a total length of 530 km.A small section of this range is located in the eastern part of Serbia, lying in the municipalities of Zaječar, Knjaževac, Pirot, and Dimitrovgrad, and as a morphological unit it is bound by the valleys of the Beli Timok, Trgoviški Timok, and Visočica rivers and the state border to the east [23] (Fig. 1).The highest point of the Stara Planina mountain range in the region of south-eastern Serbia, where our research was conducted, is the peak of Midžor (2169 m a.s.l.), while the lowest is at the exit of the Prlitski Potok valley (132 m a.s.l.).In terms of climate, Mt Stara Planina is a subregion of Eastern Serbia, which is impacted primarily by the Vlach-Pontic Basin, but also by the Pannonian and, to a much lesser extent, the Aegean Basins.The prevailing climate at the base of the mountains is continental [24].The climatic elements of Mt Stara Planina exhibit significant spatial variability in terms of their mean values due to the high altitude gradient (up to 2000 m), the direction the main mountain range extends in, and its relief, which sees frequent and large changes in the gradient and exposure of mountain slopes and valley sides.Average temperatures decrease and humidity increases with increase in altitude.The coldest month of the year is January with an average temperature of 0.5 °C, while the hottest month is July with an average temperature of 22 °C.Average annual precipitation is 960.5 mm with the highest levels in May and the lowest in September [25].
The relief of this area is fundamentally tectonic in origin (morphostructures formed by the effects of tectonic movements) and is shaped by eluvial, diluvial, proluvial, colluvial, fluvial, and karst processes [26].The existence of different types of soils on Mt Stara Planina is the result of the specificity of the geological substrate and the prevailing climatic conditions, with rankers, rendzinas, vertisols, cambisols, and recent soils present, while wetlands have developed in places with high groundwater levels [26,27].Moreover, the hydrography of this area is complex and features aquifers, springs, streams, and mountain rivers.The spring with the coldest water (4 °C) in Serbia is found below the peak known as 'Vražja glava' [28].
Due to the exceptional diversity of the flora and fauna, as well as its geomorphological, geological, hydrological, and hydrogeological features, a 142,219.64ha area of the Mt Stara Planina region was proclaimed a Nature Park in 1997.Later, in 2009, a Decree was adopted at the national level, designating the 'Stara Planina' Nature Park a protected natural resource of exceptional importance, i.e.Category 1 status (Official Gazette of the Republic of Serbia, no.23/09).
The population of the study area belongs to a particular variety of ethnic group with ancient Slavic origins known as the 'Shopi' or 'Torlaks' .In terms of language, this group has managed to preserve its own dialect-the Timok-Luznica dialect with elements of Serbian, Bulgarian, and Turkish [29,30]-while the official language of the region is Serbian.According to the 2011 census, inhabitants in the study area identify as Serbs, Bulgarians, and Roma.Bulgarians were in the majority in the villages of Senokos, Vlkovija, Gornji Krivodol, and Donji Krivodol, while Serbs predominated in the other villages (Table 1).In terms of religion, all the interviewees were Orthodox Christians.
Ethnobotanical data on the knowledge of plants and their use for medicinal purposes was collected using semi-structured and structured face-to-face interviews, conducted in Serbian, with inhabitants of the research area on several trips to this region between 2020 and 2022.The main criteria for selecting the interview participants were: the age of the respondents (those chosen were over 50 because it was assumed that they had 'life' experience and had at some point used a plant to treat an ailment), the participation of respondents of both sexes, and their residing permanently in the villages where we conducted the research.We interviewed 26 men and 25 women (n = 51), aged 30-91.The majority of the interviewees were aged between 51 and 70 (50.98%),while only two (3.92%) were older than 91 (Table 2).For all the respondents, we recorded their gender, age, level of education, and occupation.The majority had completed secondary education (54.90%), while traditional knowledge on the use of medicinal plants in the study In Serbia, there are no special rules or regulations pertaining to conducting ethnobotanical research.Before beginning each interview, the purpose, methodology, and nature of the research were explained to all participants, after which voluntary oral consent was obtained from all informants.Moreover, all of the respondents gave their oral consent for their photographs to be taken during the interview and published.Each informant had the chance to end the interview at any time.After the interviews, all the data was deposited at the Department of Ecology of the Institute for Biological Research 'Siniša Stanković' , a National Institute of the Republic of Serbia.Ethnobotanical and ethnomedical research was conducted in accordance with the International Society of Ethnobiology (ISE) Code of Ethics [32].All principles of the Code of Ethics were respected and there were no harmful consequences for the local community.Furthermore, all the recommended standards for conducting ethnobotanical research according to Weckerle et al. [33].
The focus of the interview was the local population's traditional knowledge on the use of wild plants to treat various health problems (Fig. 2).One questionnaire was completed for each plant species mentioned as being used by the respondents.Identification of plants was performed in the field or from herbarium specimens using literature sources [34][35][36].The correct taxonomy and nomenclature of the vascular plants was also checked referring to World Flora Online (WFO) (www.world flora online.org), while for Cetraria islandica (L.) Ach.(Iceland lichen) the LIAS names web interface was used [37],   The International Classification of Primary Care (ICPC-2) of the World Health Organization (WHO) was used for the categorisation of all the diseases and ailments treated [38,39].In this study, the diseases and health problems which were mentioned by the respondents were classified into 16 categories: (1) Blood, Blood Forming Organs, and Immune Mechanism, (2) Respiratory, (3) General and Unspecified, (4) Digestive, (5) Cardiovascular, (6) Endocrine/Metabolic and Nutritional, (7) Ear, (8) Eye, (9) Psychological, (10) Urological, (11) Female Genital, (12) Male Genital, (13) Pregnancy, Childbearing, Family Planning, (14) Skin, (15) Musculoskeletal, and ( 16) Neurological.
MS Excel (2016) was used for data entry and summary.As part of the quantitative data analysis, the number of citations for each of the mentioned health problems was calculated as well as the informant consensus factor (ICF) as an index used to test the homogeneity of the knowledge shared by the informants and the UV of the plants which the respondents mentioned.
The informant consensus factor is determined using the formula: ICF = Nur − Nt/Nur − 1, where Nur refers to the total number of reports (number of mentions) on use for each disease category and Nt indicates the number of species used in that category [40].Its values vary between 0 and 1.
The use value of species is a quantitative method that shows the relative importance of locally known species.It is calculated according to the formula UV = Ui/N, where Ui is the number of reports of use mentioned by each informant (i) and N is the total number of surveyed informants for a given plant species [40].

Diversity of medicinal plants
Results of ethnobotanical research in the Mt Stara Planina region showed that 137 species classified into 51 families and 116 genera had practical applications in the ethnomedicine of the local population.Most species were Angiospermae (96.35%) and the total proportion of gymnosperms, pteridophytes, and lichen was 3.65% (Table 3).In terms of plant habit, 10 families, 17 genera, and 20 species were trees, 4 families, 6 genera, and 8 species were shrubs, 36 families, 92 genera, and 108 species were herbs, and 1 family, 1 genus, and 1 species was lichen (Tables 3 and 4).The richest families in terms of species were Lamiaceae (19), Rosaceae (18), and Asteraceae (17), which accounted for 39.42% of the total number of species.Twenty-nine families were represented by one species and 10 families by 2 species, while 12 families contained ≥ 3 plant species (Table 4, Fig. 3).The genera with the highest number of species were Allium, Gentiana, Mentha, and Tilia (three species each) (Table 4).
In terms of location, 122 (89.05%) medicinal species were only found to be growing in the wild.Eight (5.84%) species were only cultivated and 7 (5.11%)species were found both in the wild and cultivated (most often in gardens) (Table 4).
According to the 'Regulations on the Proclamation and Protection of Protected Wild Plants, Animals, and Fungi' that apply in Serbia, 55 of the total number of recorded plant species are protected, while 3 (Agrimonia eupatoria L., Lycopodium clavatum L., and Ruta graveolens L.) are strictly protected [41].The research showed that populations of Gentiana lutea L. were scarce due to excessive use of the rhizome, which was widely used by the local population to prepare a herb brandy known as 'lincura' , a highly valued medicinal product.

Preparation and use of medicinal plants
In addition to botanical information, Table 4 also contains information relating to the use of the plants in ethnomedicine: plant part/product, form of preparation, mode of application, diseases treated/number of respondents (ICPC-2), number of informants, and UV index.

Use value (UV)
Use value is an essential tool for identifying highly valuable medicinal plants for potential further detailed pharmacological research.There is a strong correlation between use value and use reports for a certain plant.The high usage of reported medicinal plants implies a strong association and the reliance of local populations on the surrounding flora, particularly for the treatment of various diseases [42].In this study, the use value ranged from 0.019 to 1 (Table 4).The highest use value (UV = 1) was reported for Alium sativum L., Allium ursinum L., Gentiana asclepiadea L., Gentiana cruciata L., Gentiana lutea L., Hypericum perforatum L., Thymus serpyllum L., and Urtica dioica L. (UV = 1) (Fig. 6; Table 4).Allium sativum (garlic) and A. ursinum (wild garlic) were mainly used against high blood pressure and for health maintenance/preventive healthcare.Moreover, ethnobotanical research in other regions of Southeast Europe confirms the long-lasting, continuous tradition of using these plants in traditional medicine [2-6, 9, 11, 15, 43-45].In addition, pharmacological research has shown that preparations made from Allium ursinum reduce hypertension and inhibit angiotensin-converting enzyme in vivo when tested on rats with spontaneous hypertension [46].Similarly, Reuter established that the activity of A. ursinum as an angiotensin-converting-enzyme (ACE) inhibitor was considerably greater than that of A. sativum [47].
Species of the genus Gentiana (G.asclepiadea, G. cruciata, and G. lutea) were the most popular plant species among the population of the study area.The aerial parts of G. cruciata and G. asclepiadea were used in the preparation of 'travarica' herb brandy to treat stomach diseases and cancer, while the root of G. lutea was used to prepare 'gentian brandy' (lincura) to treat stomach problems and to improve digestion.Because of its healing properties and its wide variety of uses, the inhabitants of the Mt Stara Planina region considered G. cruciata to be a panacea (for nervousness, diabetes, killing parasites, liver disease, cholelithiasis, treating the prostate, etc.).The use of G. asclepiadea was similar: to treat malignancy, liver disease and jaundice, etc.An analysis of ethnobotanical literature showed that G. cruciata, G. asclepiadea, and G. lutea have similar uses in other mountainous regions in the Western Balkans [5, 8-10, 13, 14, 48, 49].Phytochemical and biological research has shown that G. lutea contains physiologically important compounds that include iridoids, bitter constituents (gentiopicroside, amarogentine, swertiamarin, and sweroside), xanthones (gentisin, isogentisin), triterpenoid derivatives, and essential oils Fig. 5 Form of preparation of medicinal plants [50].Secoiridoid glycosides that are most common in G. lutea contribute most to the anti-inflammatory, antitumour, hepatoprotective, wound healing, antifungal, antibacterial, and antioxidant activities of this species [51].Also, xanthones and xanthone glycosides from G. lutea have been found to be potent inhibitors of monoamine oxidase in vitro [52].In similar studies, it was found that methanolic extracts of the aerial part and root of G. asclepiadea exhibited hepatoprotective properties, while HPLC analysis revealed that the extracts were rich in gentiopicrin [53].Experimental research also found that G. cruciata extracts significantly reduced DNA damage in liver cells caused by carbon tetrachloride.The antioxidant and antigenotoxic activities of this species confirm its therapeutic benefits and justify its use in traditional medicine [54].Furthermore, phytochemical research into the dry extract of G. cruciata has shown the presence of tannins and polyphenols, which have antibacterial and antifungal properties.The native extract is highly active against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Candida albicans [55].
In the study area, all the respondents mentioned the use of the species Hypericum perforatum, which they readily pick for their own needs but also to sell.The aerial part of this plant was used to prepare teas and oil, which were applied orally and topically.For the local population, its most important use was in the treatment of skin problems (burns, lacerations, skin injuries, etc.) and it is interesting to note that the medicinal properties of H. perforatum were known even in Hippocrates' time, when it was farmed for its anti-inflammatory and wound healing properties [56].Recent ethnobotanical studies conducted in other rural areas of the Balkan Peninsula have shown that H. perforatum is frequently used in ethnomedicine and is a well-known and favourite plant of the local population [2, 5, 6, 9, 13-15, 48, 57-59].
Thymus serpyllum was one of the 'most significant' medicinal plants, both ethnomedically and economically, for the current inhabitants of the Mt Stara Planina region.In the study area, the aerial part of this plant was mainly used for its antibacterial properties, to relieve cold symptoms, to treat respiratory diseases, influenza, and skin complaints, to regulate the menstrual cycle, to relieve heartburn, and as a sedative.In addition, numerous ethnobotanical studies have shown that this species has similar uses in the treatment of respiratory and gastrointestinal problems in other parts of the world, including the Western Balkans, where the study area lies [2,5,10,13,14,48,[59][60][61][62].
In the area of 'Stara Planina' Nature Park, respondents mentioned the species Urtica dioica 103 times, most often to treat iron deficiency anaemia (40 times) and leukaemia (12 times) and for health maintenance/preventive healthcare (11 times).Similarly, this species is widely used in traditional medicine across the world and the health benefits of nettle are numerous: it improves circulation, heart health, and gastrointestinal health, it has antidiabetic properties, boosts the immune system, reduces inflammation, prevents kidney stones, aids in controlling blood sugar levels, helps detoxification, improves women's health, reduces the risk of prostate cancer, strengthens the bones, treats respiratory problems, and aids in pregnancy [63].Phytochemical research has identified a wide range of biological properties that the leaves of this plant possess: antimicrobial, antiviral, antioxidant, anti-inflammatory, antiulcer, hypolipidemic, etc. [64].This is a result of the presence of biologically active compounds: vitamins, amino acids, carotenes, fatty acids, terpenoids, fibres, and phenolic compounds [65].
The species Hypericum perforatum and Urtica dioica occupy an important place in Serbian traditional medicine, as well as in common beliefs.It is thus believed that on St. George's Day (6th May) you should take a bath in water which nettles have been soaked in.This will aid your general health.For St. John's Wort, also known in folk medicine as Virgin Mary grass, there is the belief in the south of Serbia that it got its spots after drops of water fell from the hands of the Virgin Mary onto its leaves [66].
A very popular species among the local population was Sempervivum tectorum L. (UV = 0.980), which was used to treat skin, ear, and cardiovascular problems.It was also cultivated as a decorative plant in yards and was often found on the roofs of houses because of the folk belief that it protects the house and household from lightning.Ethnobotanical studies conducted in the field of the distribution of Sempervivum tectorum show that this species is used for the same or similar health problems elsewhere [2,5,6,8,9,58,[67][68][69].
Twenty-nine respondents (56.9%) from the study area mentioned the species Hylotelephium spectabile (Boreau) H. Ohba, which was grown in gardens, primarily for its medicinal properties (for the treatment of skin diseases), but also as an ornamental plant.By reviewing the ethnobotanical literature, we found that this species is mentioned in a small number of ethnobotanical studies [8,13,[70][71][72], which is why it needs to be examined in more detail from a phytochemical and pharmacological point of view.
A special feature of this region was the use of the plant Sambucus ebulus L., which was cited eight times in the treatment of malignant diseases.In particular, respondents emphasised that one teaspoon of boiled jam should be consumed in the morning, before breakfast.This species is also used in other parts of the Western Balkans, but in the treatment of musculoskeletal disorders [6,73].

Informant consensus factor (ICF)
Analysis of ICF values showed that there was high homogeneity when it came to respondents' statements on the use of medicinal species within certain categories of diseases.The study included 2783 use reports for all taxa, referring to 138 diseases in 16 categories of health disorders (Table 5).
The highest ICF value was recorded in the categories of skin diseases (S; ICF = 0.95) and Blood, Blood Forming Organs, and Immune Mechanism (B; ICF = 0.95).The homogeneity of statements was also high in the following categories of diseases: Musculoskeletal (L; ICF = 0.91), Respiratory (R; ICF = 0.90), General and Unspecified (A; ICF = 0.89), Digestive (D; ICF = 0.88), Cardiovascular (K; ICF = 0.87), Ear (H; ICF = 0.86), Endocrine/Metabolic and Nutritional (T; ICF = 0.85), and Female Genital (X; ICF = 0.85) (Table 5).The high ICF values found in most of the categories of medicinal uses can be explained by the fact that there was high homogeneity of consensus among informants on the therapeutic uses of a set of species and on their efficacy [74].The lowest ICF value was in the neurological category (N; 0.25).
In the skin category, respondents mentioned 18 different types of skin diseases and problems.These were treated using 22 plant species, which were cited a total of 412 times.They were most commonly used in the treatment of skin infections (S76-84 times), skin injuries (S19-68 times), and chronic skin ulcers (S97-63 times).They were applied externally and the respondents most often mentioned Hypericum perforatum (79 times), Plantago lanceolata L., and Plantago major L. (72 times each).
In the Blood, Blood Forming Organs, and Immune Mechanism category, respondents listed five different types of diseases.These were treated using seven plant species, which were cited a total of 113 times and were most often mentioned in the treatment of iron deficiency anaemia (B80-94 times) (Table 2).In this category of health problems, the most commonly mentioned plant species were Urtica dioica (52 times), Cornus mas L. (41 times), and Rubus fruticosus L. (13 times), which were also an integral part of the diet of the inhabitants in the study area.The fruits (Conus mas and Rubus fruticosus) and aerial parts (Urtica dioica) were used and taken orally.
The second highest ICF was found in the musculoskeletal category.Ten types of plants were used in the treatment of bone fractures, joint pain, and rheumatic pain and respondents cited them 103 times.The most important plant for the treatment of musculoskeletal problems was Symphytum officinale L. (cited 84 times).Its root was used to make ointment (the ground root is cooked in milk and left to stand overnight; the dressing is changed every second day over a period of two months) and applied externally in the form of a compress.This plant is highly valued in Serbian ethnomedicine [5,73,75,76].
In their phytochemical and pharmacological research, Sowa et al. determined that Symphytum officinale root extract contains allantoin and phenolic acids (rosmarinic, p-hydroxybenzoic, caffeic, chlorogenic, and p-coumaric acids), which indicates its strong antioxidant potential and a beneficial effect on human skin fibroblasts [77].Also, allantoin is considered to play an important role in the healing of wounds and bones in the case of fractures.It has been claimed that allantoin is the active ingredient in Symphytum officinale and is responsible for triggering cell division and wound healing, as well as promoting conjunctive tissue, bone, and cartilage growth [78].The results of this and similar studies justify the use of S. officinale in ethnomedicine.The third highest ICF was found for Respiratory problems, where respondents cited the use of 44 plant species mentioned a total of 490 times for the treatment of 13 different types of respiratory complaint.Plants were mostly used in the treatment of respiratory system pain (R01-176 times) and coughs (R05-129 times), while Thymus serpyllum was the most commonly mentioned plant (63 times).Also important for the treatment of respiratory complaints in this region were Tilia cordata Mill.(mentioned 39 times), Mentha x piperita L. (mentioned 39 times), and Satureja montana L. (37 times), which, like Thymus serpyllum, were taken orally, in the form of tea.

Novel ethnobotanical reports
Comparative analysis of the obtained results with the results of other ethnobotanical studies conducted in Serbia and the Balkans found that the species Agrimonia eupatoria, Gentiana asclepiadea, Geranium robertianum, Origanum vulgare, Oxalis acetosella, Polygonum aviculare, and Stachys officinalis are mentioned for the first time as being used in the treatment of jaundice.The aerial parts of Urtica dioica and the leaves of Scrophularia nodosa are also mentioned for the first time in the treatment of goitre.Other recent findings have shown that S. nodosa is used in traditional medicine in the Deliblato Sands region (Serbia), but it is the roots and shoots that are used to treat cancer, goitre, rabies, skin ulcers, haemorrhoids, rashes, and eczema, while the seeds are used as an anthelmintic [79].In the south-western part of the Sharr Mountains (North Macedonia), the aerial parts of S. nodosa are topically applied to treat tuberculosis [58], while in the Prokletije region it is used in the treatment of breast cancer [13].The biological screening of S. nodosa extract and its fractions has revealed that this species may be a good source of antimicrobial, antioxidant, and analgesic compounds [80].
Achilea clypeolata is a Balkan endemic species and is highly valued among the population of the study area.It is used in the ethnomedicine of the region to treat respiratory system pain, bleeding/haemorrhages, and symptoms of female genital diseases; this is the first report on this species being used to treat these health issues in the Balkans and Serbia.However, data does already exist on the use of this species in Serbia (Suva planina and Rtanj), but to treat kidney problems, improve appetite, soothe coughs [5], and treat diabetes [15].
Carlina acaulis is used in the Stara Planina region to treat diabetes, gout, and urinary calculus.The ethnomedicinal properties of this species are familiar to the peoples of the Balkans, but in different regions it has different uses.In the region of central Serbia (Kopaonik), it is used to treat skin complaints, ulcers, acne, eczema, and wounds [2], while in Bosnia it is used to treat skin injuries [81], influenza, high temperatures, headaches, urinary tract infections, worm infections, increased diuresis, renal ailments, and inflammation of the throat [57].In Montenegro (Prokletije), C. acaulis roots are used to treat gastritis, dyspepsia, and bile duct disorders, and when applied externally, to treat dermatoses, wounds, and ulcerations [13].In the region of North Macedonia (Sharr Mountains), the flower, root, and seed are used in the treatment of acne and eczema [82].In Bulgaria, Carlina acanthifolia root is used as a diuretic and urogenital anti-inflammatory [83].
Carlina vulgaris is used in the ethnomedicine of Mt Stara Planina to treat haemorrhoids, which is the first such report for this species.Phytochemical research has shown that C. vulgaris, one of the least studied species of the genus, is rich in polyphenols and minerals, especially populations that grow in uncontaminated areas, which contain more chlorogenic acid and exhibit greater antioxidant activity [84].It has also been found to have a protective effect against H2O2-induced oxidative stress in human skin fibroblasts [85] and the antioxidant and cytotoxic potential of C. vulgaris against human colorectal adenocarcinoma has been determined [86].
Nasturtium officinale is mentioned in the treatment of acne, which is also the first such report for the Balkans, and therefore for Serbia, too.However, this species was mentioned as far back as the eleventh century, in 'The Trotula' , the first textbook on aesthetic medicine, written by Trota of Salerno, where it was noted for its anti-ageing properties [87].Chemical analyses of N. officinale have revealed the presence of alkaloids, flavonoids, saponins, terpenoids/steroids, protein, essential and volatile oils, glycosides, tannins, folic acid, vitamins, and elements [88].Also, pharmacological research has determined the hypolipidemic, anti-inflammatory, hepato-renal protective, antidiabetic, antioxidant, anticancer, antimicrobial, dermatological, antigenotoxic, anti-urolithiatic, and antigenotoxic properties of this species [88].
Heracleum sphondilium is mentioned for the treatment of epilepsy, which is a new finding for Serbia and the Balkans.Previous ethnobotanical research conducted in the Suva planina area (Serbia) revealed the use of this species to treat rheumatoid arthritis [5].Data exists that shows the stem and seeds of Heracleum persicum Desf.ex Fischer are used for the treatment of epilepsy in Iranian ethnomedicine.Biological research has shown that H. sphondylium exhibits antimicrobial, cytotoxic, and vasorelaxative activity [89].
In the ethnomedicine of the study area, Filipendula ulmaria (L.) is used in the treatment of uterine fibroids, heartburn, and peptic ulcers, which is also new data for the use of this species.In the region of Timok and the Svrljig Mountains, this plant is used to treat musculoskeletal (arthritis), digestive (liver disease), urological (urination), endocrinal, metabolic, and nutritional (anti-obesity) issues [73], while in the Kopaonik area it is used in the treatment of rheumatism [2].In Bosnia, it is drunk in the form of a tea to treat the common cold [81], while in Montenegro (Prokletije) it is used in the treatment of coughs, bronchitis, fevers, colds, and rheumatism of the joints and muscles [13].Phytochemical research has revealed the presence of several active compounds, mainly phenolic acids, flavonoids, tannins, and terpenoids.Salicylic acid and its derivatives are the most important compounds found in the essential oil and extracts from different parts of the plant.Pharmacological analyses have determined the analgesic, anti-arthritic, antimicrobial, anti-inflammatory, anticancer, antioxidant, anticoagulant, immunomodulatory, gastro-protective, and hepatoprotective activity of F. ulmaria [90,91].
Sambucus ebulus being used to treat malignancy is a new finding for Serbia and the Balkans.Generally speaking, this plant is very well known to the peoples of the Balkans and, according to ethnobotanical data, is widely used.In other parts of Serbia, this species is used to treat arthritis, gout [73], and gallstones [76], as an antirheumatic and diuretic, to cleanse and induce perspiration, for the removal of warts [79], and as an antidote to snake bites (juice from the ground leaf is applied to the bite) [2,10].In the Kosovo region, S. ebulus is used as an anti-rheumatic, for menstrual pains and regulation of the menstrual cycle, urinary inflammations [9], constipation, and respiratory disorders [92].Menković et al. [13] described the use of this species in the Prokletije region (Montenegro) to treat gout, rheumatic complaints, and oedema, while Pieroni et al. [59] note that it is topically applied to treat snake bites in Western Macedonia (Upper Reka Valley, Mount Korab).In Bosnia and Herzegovina, S. ebulus has traditionally been used against pulmonary ailments, coughs, and hoarseness, to treat increased diuresis and kidney and bladder ailments, for restlessness and blood purification, to reduce blood sugar levels, and against rheumatism and fluid retention [6,57,93].Also, ethnobotanical research has shown that in Eastern Albania S. ebulus is applied externally to treat bruises, wounds, and cold sores [94,95].
Phytochemical and pharmacological research has shown that crude methanol extract of S. ebulus leaves has remarkable wound healing properties.This may be the result of the synergistic effect of the constituents present in the methanolic extract.Namely, S. ebulus methanol extract at one per cent concentration has been found to exhibit considerable wound healing activity in both linear and circular excision animal models.This property, which is linked to 'quercetin 3-O-glucoside' as a flavonoid derivative, is supported by a histopathological examination of wound models [96].Experimental research has shown that the extracts of the flowers and leaves of S. ebulus possess anti-inflammatory potential by effectively suppressing the biosynthesis of tumour necrosis factor (TNF-α), interleukin 1-α (IL1-α), and interleukin 1-β (IL1-β) [97] S. ebulus fruit extract also exhibits antiinflammatory properties through significant nitric oxide removal activity (inflammation indicators) [98].Saravi et al. [99] investigated the antitumour properties of S. ebulus and found that ethyl acetate extract of this species exhibits high cytotoxicity against human hepatocarcinoma and human colon carcinoma cell lines.In addition, experimental research has shown that S. ebulus also possesses antioxidant, antimicrobial, wound healing, antigiardial, scolicidal, analgesic, antidepressant, and neuroprotective properties [100].It is clear that S. ebulus has a wide range of therapeutic effects, which is why there is a need for further research to assess its effectiveness and how safe it is.In general, the raw fruits of this plant are considered poisonous, but other parts of the plant can also be toxic if used in excess: they can induce vomiting, especially in children [100].In this regard, respondents who stated that they used this species emphasised the need to limit consumption (it must be measured out, 1 teaspoon in the morning).
Data on the use of Sanicula europaea for health maintenance/preventive health care and treatment of strep throat and gout in the study area is new for the Balkan region.In general, there is not much data on the use of this species in ethnobotanical literature.In the Balkans, in addition to the study area (Mt Stara Planina), it has also been recorded as a ritual plant and a plant that solves 'love disorders' in eastern Serbia (Timok and Svrljig Mountains) and as a plant used to heal wounds and to treat skin problems and bleeding in Montenegro [13] and Bosnia and Herzegovina [62].Pharmacological research has established the in vitro anti-inflammatory activity of S. europaea roots due to the reduced production of IL-8 and E-selectin after stimulation with TNF-α and LPS [101].
Our research also revealed interesting new findings for Serbia and the Balkans for the following species: Tanacetum vulgare (induced abortion), Dioscorea communis (gout), and Consolida regalis (prostatitis).The importance of these new reports on the specific uses of certain plants in Serbia and the Balkans as a whole lies in the chance to find new uses for medicinal plants and to discover new herbal remedies.

Conservation status of medicinal plants
Plants are a very important part of any ecosystem because they form its physical structure and are essential for the functioning of the planet's atmosphere.However, many plant species, especially those intended for a particular purpose, such as being used for medicinal purposes, are directly threatened by over-exploitation and the impact of invasive species, as well as climate change posing an increasing threat [102].For these reasons, plants in the priority conservation class in Serbia are protected by national legislation and some international regulations such as The Convention on Biological Diversity, the Bern Convention (The Convention on the Conservation of European Wildlife and Natural Habitats), CITES (The Convention on International Trade in Endangered Species of Wild Fauna and Flora), and the Habitat Directive (Council Directive 92/43/EEC on the Conservation of Natural Habitats and of Wild Fauna and Flora).This study identified the endemic species Achillea clypeolata Sm. (Balkan endemic), used for medicinal purposes.According to the IUCN list (https:// www.iucnr edlist.org/), 15 plant species are in the category of Least Concern (LC).However, the species Anacamptis morio (L.) R.M. Bateman, Pridgeon & M.W.Chase is in the category of Near Threatened (NT) and also on the CITES list (Annex II), while the Data Deficient (DD) category includes the species Malus sylvestris Mill.The list of the Habitat Directive (HD; Annex V) includes the species Gentiana lutea.The identified medicinal species with their related degree of protection are shown in Table 4.
The biggest threat to protected species in the study area is the impact of the anthropogenic factor.Namely, people picking certain plants in excess, e.g.Gentiana lutea, has contributed to a significant reduction in populations of these species.A key measure that should be regularly undertaken in the study area would be to educate the local population on the importance of medicinal plants.

Medicinal plants as foodstuffs and a valuable resource for the herbal market and ecotourism
The Mt Stara Planina region is a rural one with preserved traditional village-type settlements, human activities, and local agricultural products, which are a recognisable brand and identity of this part of south-eastern Serbia.The region's inhabitants are engaged in livestock farming and traditional agriculture, with their primary task being to provide sufficient food [29].In order to improve both their standard of living and to foster the economic development of this rural area, the local population is also involved in picking forest fruits, mushrooms, and wild medicinal plants, which are then purchased by local traders.
In terms of how plant species are used as food, there are great similarities with neighbouring regions.For instance, the local population in Bulgaria gathers the aboveground parts of plants, e.g.Urtica dioica, Rumex patientia, and Taraxacum officinale, mainly during the spring and uses them as vegetables, while fruits are mostly gathered from woody and shrubby plants from the families Rosaceae, Viburnaceae, Ericaceae, and Vitaceae [103].Nedelcheva et al. [103] highlight that the jams and jellies prepared from cornel (Cornus mas) have an astringent effect, while soup (or other meals) made from U. dioica (nettle) is generally used to strengthen the constitution especially after a long illness, to treat anaemia, and as a natural blood purifier, similar to in the Mt Stara Planina region.Ethnobotanical research conducted in eastern and south-eastern Albania, as well as in nearby villages located in North Macedonia, showed that the most commonly used wild plants included Cornus mas fruits, Urtica dioica and Rumex patientia leaves, Origanum sp. and Sideritis sp.aerial parts, and Prunus cerasifera fruits [104].In south-eastern Romania, several taxa are used as foodstuffs, e.g.Rumex, Malva sylvestris, Sambucus nigra and Urtica dioica, with U. dioica clearly identified as a distinctive feature in the diet of Romanians [105].
Data relating to the use of plants to make alcoholic beverages is interesting: 'travarica' herb brandy (Pyrus piraster), liquors (Artemisia absinthium, Cornus mas, Juglans regia, Prunus avium), and cocktails (Fragaria vesca).Flavoured alcoholic drinks specific to certain regions are deeply rooted in the tradition of herbalism and are an important part of local culinary identity in Eastern Europe including the Balkan countries [106,107].
Our research also found that 65 plant species are of economic significance for the local population in that they are traded (Table 6).This is particularly important given the current and potential contribution of these species to the local economies and their increased value to those harvesting them in the long term.Most often it is the aerial parts (43.59%), fruits (17.95%), and leaves (12.82%) that are harvested.They are bought and sold both fresh and dried (Fig. 9).
Based on the existing renewable resources of Mt Stara Planina, there is great potential to develop an area of significant economic activity, including the gathering and processing of forest berries and fruits, and the harvesting, processing, and final production of medicinal and aromatic plants.Traditionally, on St. John's Day (Ivanjdan, 24th June), there is an organised harvest of medicinal and aromatic plants on Mt Stara Planina.
In Serbia, the harvesting and trade of medicinal plants dates back to 1930, when 1,810 tons of medicinal plants were exported [108].Recent data shows that in the period 2004-2016, 17,807,282.86 kg were exported and a profit of EUR 54,461,300.36 was made [109].There are 110 companies that deal in the purchase and sale of medicinal plants in Serbia [110].There is no precise data for the Mt Stara Planina region, but it is known that there are a number of family-run and local companies that are engaged in harvesting and buying up medicinal      Ecotourism based on medicinal plant resources is becoming increasingly popular, reflecting the growing demand for medicinal plants as a field of alternative medicine.'Stara Planina' Nature Park, declared as such in 1997, represents the main tourism potential of the Pirot municipality.However, ecotourism in this region is still at an early stage, with rural tourism poorly organised and insufficiently linked to other forms of tourism (e.g.hiking, skiing, hunting and fishing tourism, etc.) [111].

Conclusion
The ethnobotanical research conducted in the Mt Stara Planina region is an attempt to update our knowledge on the diverse therapeutic applications of different medicinal plants, which are clearly of great importance in the lives of the local population.The knowledge of the medicinal properties of the plants and how they are used by the local population is a result of the specific geographical location of the study area, high biodiversity, ethnic and cultural differences, and folk traditions that have evolved over the centuries.Traditionally, plants have been used to treat a wide range of health problems.This study has presented data relating to the traditional use of medicinal plants in human medicine (136 plant species and 1 lichen species).Eight plant species had a maximum use value (UV = 1).The highest ICF value was observed in the categories of Skin and Blood, Blood Forming Organs, and Immune Mechanism.Our research, focussing on Serbia and the Balkans, revealed new ways of using certain medicinal plants from the Mt Stara Planina region-Agrimonia eupatoria, Gentiana asclepiadea, Geranium robertianum, Origanum vulgare, Oxalis acetosella, Polygonum aviculare and Stachys officinalis to treat jaundice, Urtica dioica and Scrophularia nodosa to treat goitre, the endemic Achilea clypeolata to treat respiratory system pain, bleeding/haemorrhages, and symptoms of female genital diseases, Carlina acaulis to treat diabetes, gout, and urinary calculus, Carlina vulgaris to treat haemorrhoids, Nasturtium officinale for acne, Heracleum sphondilium for epilepsy, Filipendula ulmaria to treat uterine fibroids, heartburn, and peptic ulcers, Sambucus ebulus to treat malignancy, and Sanicula europaea for health maintenance/preventive health care and the prevention of strep throat and gout.New findings for the following species are also significant: Tanacetum vulgare (inducing abortions), Dioscorea communis (gout), and Consolida regalis (prostatitis).Therefore, the richness of the biodiversity and the unique biocultural heritage of the local people from the Mt Stara Planina region are highly valued.This is reflected by measures that have been undertaken with the formation of the protected 'Stara Planina' Nature Park for biodiversity conservation.However, further efforts in terms of the conservation of human Traditional Ecological Knowledge diversity and cultural heritage are also necessary.This study has documented traditional knowledge on the use of medicinal plants in ethnomedicine, which should be preserved to prevent it from being lost and forgotten.The importance of medicinal plants used as foodstuffs and their significance for the local market and exports as well as ecotourism should not be ignored either.Furthermore, this research proves that the Western Balkans is a unique area, suitable for further, detailed studies on traditional medical ethnobotany and phytotherapy, which is in line with its biological, ethnic, and cultural diversity.

Fig. 2
Fig. 2 Field work a,b Data collection from local informants; c-f Methods of preparation of medicinal plants for further use

Fig. 3 Fig. 4
Fig. 3 Dominant families of medicinal plants in the study area

Fig. 6
Fig. 6 The plants with a maximum use value: a Allium sativum, b Allium ursinum, c Gentiana asclepiadea, d Gentiana cruciata, e Gentiana lutea, f Hypericum perforatum, g Thymus serpyllum, h Urtica dioica (All photographs by Jarić S.) plants and thus have a positive impact on the development of the local community (e.g.'Stara Planina' Association for Medicinal Plants, 'Babin Nos' Association, Stanko Madić-Teas from Stara Planina, The Agricultural Estate of Dragiša Dimitrova from Temska-Medicinal Plants, Mushrooms, and Teas, etc.).The only company authorised by the 'Srbijašume' Public Enterprise to purchase

Fig. 7
Fig. 7 Percentage use of plant parts in the diet of the local population

Fig. 8
Fig. 8 Frequency of medicinal plant preparation forms

Fig. 9
Fig. 9 Medicinal plants, plant parts, and herbal products of commercial importance

Table 2
Demographic features of the local informants in the study area (n = 51) (https:// liasn ames.lias.net/).Voucher specimens were deposited at the herbarium of the Institute for Biological Research 'Siniša Stanković'-National Institute of the Republic of Serbia (IBISS) in Belgrade.

Table 3
Classification of medicinal plants in the study

Table 4
Traditional use of medicinal plants in the Mt Stara Planina region (south-eastern Serbia)

Table 4 (
continued) c Cultivated species, ws Wild species, Ap Aerial part, Bk Bark, Br Branches, Fl Flower, Fr Fruit, Le Leaf, Ne Needles, Re Resin, Rh Rhizome, Ro Root, Se Seeds, Tu Tuber, WhPl Whole plant, E Endemic, IUCN International Union for Conservation of Nature, LC Least Concern, NT Near threatened, DD Data Deficient, HD Habitats Directive Annexes

Table 5
Informant Consensus Factor values for ICPC-2 ailment categories

Table 6
Economically important medicinal plants and medicinal plants as foodstuffs