Traditional, Therapeutic Uses and Phytochemistry of Terrestrial European Orchids and Implications for Conservation

The Orchidaceae family accounts for about 28,000 species, and most of them are mentioned in the folk medicine of nations around the world. The use of terrestrial orchids in European and Mediterranean regions has been reported since ancient times, but little information is available on their medicinal properties, as well as on their phytochemicals and biological activities. However, plant collection for human use is still listed as one of the main threats for terrestrial orchids, alongside other menacing factors such as wrong habitat management and disturbance to symbionts, such as pollinators and mycorrhizal fungi. Therefore, the primary aim of this review was to resume and discuss available information regarding the past and current popular uses of European orchids. We then grouped phytochemical data to evaluate the presence of bioactive compounds of pharmacological relevance, and we discussed whether these could support the therapeutic employment of the different organs. Finally, we briefly debated the sustainability of orchid utilizations, considering the different threatening factors and conservation actions including plant propagation methods.


Introduction
Family Orchidaceae, with approximately 28,000 species distributed worldwide except in the poles and deserts, is considered one of the most fascinating and diverse group of plants among angiosperms [1,2]. In fact, orchids show a wide variety of life forms, habitat preferences, reproductive strategies, sizes, colours, and scents, characteristics that have placed them at the centre of the attention of many researchers and passionate people [2][3][4]. Despite their evolutionary success, orchids are among the most endangered plants in the world [2,5], mainly because of their strict dependency on interactions with pollinators and mycorrhizal fungi for spreading and persistence, which leads to species being negatively affected by climate change, use of pesticides, anthropogenic pressure, human harvesting, etc. According to recent estimates, hundreds of species are threatened, with terrestrial orchids particularly represented in the IUCN Red list [6][7][8]. All members of Orchidaceae have therefore been included in Appendix II or higher of the Convention on International Trade in Endangered Species, CITES. Orchids are indeed highly represented in the commerce, being traded legally or not for their ornamental value, or as source of components for cosmeceuticals and medicine, or as food [9,10]. Orchids are also known in the folk tradition of many nations around the world [11][12][13]. Since ancient times, orchids have been used as nourishment and have also been employed in medicinal preparations. The first descriptions of orchids and their therapeutic utilizations have been found in China since 2800 B.C. [14], 58 are tuberous and four are rhizomatous. Overall, the hypogean apparatus was the most cited portion. Harvesting of tubers for Salep (see below) was considered separately from the collection for alimentary/medicinal home consumption, as well as the ritual use.
Multiple uses were found for 40 species. For 47 taxa, the hypogean portion was cited for Salep production (75.80% of the total investigated species), while in 41 cases it was consumed as medicinal food (66.13%); the tuber was utilized in rituals only in five cases (8.06%). Other organs were less cited: leaves/flowering stem were used as medicinal in eight cases (12.90%), and in rituals in five cases (8.06%). In 16 cases, the flowering stem was used as decorative (25.80%), while in four cases the whole plant was harvested and moved to kitchen gardens as ornamental (6.45%).
The complete list of all the ethnobotanical information that we found by examining original papers, books and reviews is provided in Table 1. Part of the source bibliographic material that we collected was still untranslated, being available only in the authors' original language (especially for Italy). Salep obtained from tubers used as medicinal food against cold, inflammation, cough and gastro-intestinal problems or as tonic, medicine, mind-developer, aphrodisiac (Bulgaria [26], Serbia [32], Turkey [33][34][35], and Greece [36]); in Turkey, tubers are used in decoction for strengthening and against wounds, abscess, inflammation, digestive diseases, and to improve circulatory system [34] and mental wellness [37]; Salep is used as food in Turkey [38,39].
LC (Europe). National protection in Belgium, France, Luxembourg. Regionally EX in Czech Republic, Estonia, Netherlands; CR in Germany; EN in Belarus, Switzerland; VU in Croatia and France; NT in Hungary. Regional protection in Italy [40].
Habitat conversion; extensive drainage, agricultural use of the habitat including fertilization and plant collection.
Anacamptis laxiflora (Lam.) R.M. Bateman, Pridgeon and M.W. Chase S; T Tuber used as medicinal food (Serbia [41,42], Turkey [43]); tuber's mucilages used as astringent, expectorant, for anti-diarrhetic properties and for the treatment of bronchitis and convalescence (Southern Europe [44]). Tuber is used to produce Salep in Serbia [32] and Bulgaria [26]. Used in decoction for the treatment of respiratory disorders in Bulgaria, where the subspecies elegans is used to obtain Salep [26]. In Turkey the tuber is sold as source of Salep [35,38,39,58]. The flowering stem is cut and kept in vessels as ornamental in Turkey [59].
LC (Global), LC (Mediterranean). Protected in some regions of France. Regional protection in Italy [60].
Drainage, infilling, agriculture pressure on wetland habitats, intensive collection. The tuber is used to produce Salep and against cough or as aphrodisiac (Turkey [25], Bulgaria [26], and Greece [36]). The flowering stem was collected and used as ornament in houses (Sardinia, Italy [53] and Turkey [25]); in Sicily (Italy), the flowering stem was employed in spells and evil eye [61].
LC (Europe). Protected in Belgium, Luxembourg, in some regions of France. VU in Bulgaria.
Animal grazing, agricultural and urbanisation pressures on habitat, fire, and deforestation.
LC (Europe). Protected in Belgium, Luxembourg, in some regions of France, Czech Republic, Slovakia. The Maltese variety A. urvilleana is listed in the Annex II of Habitat Directive; VU in Bulgaria.
Habitats (garrigue and steppe) in decline, often due to its classification as wasteland and to the spreading of invasive species (ruderals and aliens); urbanisation, quarrying, generic land use.
Cypripedium calceolus L. R; RZ Powdered roots and rhizome in sugar water were said to act as sedative, promote sleep, and reduce pain (Europe [11]). A tea prepared from roots is used to treat jangling nerves and headaches (European Russia [68]).
NT (Europe), LC (Global). Present in the Italian Red list (LC). [69,70] Collection. Habitat decline due to inadequate forest management and to the use of pesticides/herbicides. Overgrazing or grazing abandonment, which can give rise to competition with other plant species, are both threating factors.
High probability of hybridization.

Dactylorhiza euxina
(Nevski) Czerep T; L Tuber used in infusion in Turkey [73]: used to cure cough, inflammation and skin affections including boils; used as aphrodisiac, tonic. Leaves crushed and used to cure wounds (Turkey [74]).
NT (Global) [75] Biological resource use. Tuber as source of Salep, which is used as medicinal food and to treat cold, flu, rheumatism, and as a body warmer in Turkey [35,58,64,65,76].
VU (Europe) Listed as Endangered in the national Red List of Cyprus [77] Ecological range is narrow and exposed to changes in hydrology by groundwater extraction.
Other threats: competition with other species, hybridization and recreational activities, tourism development, urbanization, trampling, and plant collection. Tuber as food (Northern Italy [52]); tuber used to produce Salep and taken as tonic, aphrodisiac (Bulgaria [26]).
LC (Europe). National protection in Belgium and Luxembourg, regional protection in France; CR in Bulgaria Luxembourg; EN in Croatia, Germany; VU in France; NT in Hungary, Switzerland; LC in Norway, United Kingdom. Regional protection in Italy [78] Drainage, overgrazing, agriculture and urbanisation pressure, tourism, intensive recreation; high probability of hybridization.

FS
Employed as decoration in houses in Nuoro province, Sardinia (Italy [53] [79] Habitat (wetlands) in decline due to soil drainage, tourism, overgrazing, and reservoir construction; collection; high tendency to hybridization.
LC (Europe). Protected in Belgium and Luxembourg. Regional protection in Italy [83].
LC (Global and Europe).
Regional protection in Italy [94]. In Turkey, tuber is prepared in infusion to treat skin problems such as wounds, abscess, inflammation, and as tonic [73,95].
Over-grazing in some regions of the Caucasus. This species was cited by Rumphius in his Herbarium Amboinense, where he reported that the roots and rhizome were used in Europe to treat insanity and against rheumatisms [11]. The species is cited for the treatment of gout in European folklore [13]. In Sardinia, leaves were used to cure wounds [97]. Used as aphrodisiac (Mediterranean Europe [85]).
Habitat in decline due to the coniferization or clearance of woodlands, use of soil, overgrazing; urbanisation, tourism, recreational activities.

S; T; FS; UN
Decoction of tuber for treatment of lung diseases and strengthening of sexual potency (Bosnia-Herzegovina [49]). Tuber used as source of Salep or as medicine (Serbia [41,42]); flowering stem was collected for its perfume and used as ornamental [99]. In Abruzzo (Italy), tubers were employed in rituals to reunite or separate couples [82]. The species was cited among medicinal plants of the Carpathian area in the herbal of Stefan Falimirz (~1550 A.C) (Central Europe [56]). LC (Europe).
Habitat conversion and decline due to ploughing, agriculture, abandonment of grazing, overgrazing, urbanisation, tourism. Flowers are used to product cheese (Aosta Valley) and as flavouring in sweets (Lombardy) [102]; flowers are used against fever and respiratory diseases; liquoristic use is reported; aphrodisiac and ritual use (Aosta Valley, Italy [103,104]. In Piedmont, (North-Western Italy), as ritual, decoction of tubers was drunk in secret to reunite or separate couples, while flowers were used as ornament for S. Martino in Moena and Predappio (North-Eastern Italy) [82].   [109,110]); tuber used as tonic, aphrodisiac, for coughs and gastritis in Spain [111]. Tuber was prepared in infusion with milk or plain in Turkey [112]; flowering stem is used as decorative [25]; whole plant is collected as ornamental in Italy (personal observations M.B., J.C.). LC (Europe), LC (Mediterranean). Regional protection in Italy; LC in France [113,114].  [52]); tuber as source of Salep (Serbia [32], Bulgaria [26], and Turkey [33,38] Neottia ovata (L.) Bluff and Fingerh S; RZ; UN A tincture of rhizome was used for improving skin tone and for the treatment of gastrointestinal problems (Spain [117]); the species was cited in Gerard's Herbal in 1597 for the cure of green wounds, bursting and ruptures [13] and in the Sussex list of wound cures (Great Britain [118]; rhizome was used to produce Salep in the Highlands (Great Britain [118] [82,108]; in this region it was also cited as anti-inflammatory gastrointestinal, against childhood diarrhoea, cystitis, and nephritis [120]. The tuber is used for Salep production in Turkey [84]. This species was cited as medicinal by Turner in 1568, while Langham in 1579 reported as antipyretic, anti-consumption and for anti-diarrhoeal effects [85]; flowering stem is harvested as ornamental in Genoa (Italy (personal observation M.B. and L.C.)).

Cav. S
Used to prepare Salep against cold, flu, and as body warmers, psychedelics, pleasure and medicinal tea (Turkey [63]); in Villarino de los Aires (Spain) the whole plant is collected for its shape, or as a souvenir, or as a talisman, while in previous years it was hunted and given by boys to the girls they were interested in [128].
LC (Europe). LC in France; total protection in some regions of Italy [129].

Ophrys speculum
Link. W Whole plant was harvested for ornamental purposes in Genoa (Italy [99]).
LC (Europe). Total protection in some regions of Italy; VU in France's Red List [130].
Urbanisation, construction work, residential buildings; plant collection.

Mill. S; T; FS
Used for Salep (Turkey [38,131]). This species was cited as medicinal by Turner in 1568, while Langham in 1579 reported it as antipyretic, anti-consumption and for anti-diarrhoeal effects [85]. Flowering stem of O. sphegodes subsp. atrata was used as decoration in Sardinia (Italy [53]).
LC (Europe). Total protection in some regions of Italy [132].
Plant collection; risk of hybridization with Orchis quadripunctata on Cyprus and Crete. Tourism, land use changes, deforestation, expansion of villages and infrastructures.

S; T
Tuber as source of Salep (Turkey [139] and Greece [36]); tuber used as medicinal food for the treatment of respiratory diseases (Sardinia, Italy [97,140]).
LC (Europe). National protection in Belgium, Luxembourg, United Kingdom; regional protection in France and Italy [141].
Competition with other plants and shrubs; overgrazing; ploughing; infrastructures; tourism; inadequate timing of road verges clearing, use of pesticides and herbicides; herbivory.
LC (Europe). National protection in Belgium, Luxembourg; regional protection in France and Italy [146].
LC (Europe). EN in Bulgaria; National protection in Belgium, Luxembourg, United Kingdom; regional protection in France and Italy [147].
Pressure on species' habitat: use of fertilisers, overgrazing, drainage, ploughing, infrastructures, tourism, herbivory. Habitat conversion due to the abandonment of rural activities, which causes the rising of competing species and shrub development. Tuber as source of Salep and used to treat cough or as aphrodisiac (Bulgaria [26] and Serbia [32]).
Changes in habitat due to the abandonment of the practice of coppicing and to the decrease in the amount of light available. Drought, late frost, animals digging and herbivory; urbanisation and infrastructures; tourism, deforestation, plant collection.
LC (Europe). CR in Bulgaria; listed in the Appendix I of the Berna Convention [149].
Changes in woodland composition leading to decreases in the amount of light; urbanisation; infrastructures; tourism; plant collection.

Orchis punctulata
Steven ex Lindl. S; T Salep obtained from powdered tubers is mixed with milk and used as food in Turkey [35]. Infusion of tuber is employed to cure cold, flu, and as body warmers, psychedelics, pleasure and medicinal tea in Turkey [63].
VU (Europe). Included in the Red Data Book of Ukraine and Greece [150].
This species is very rare, and its habitat is menaced by several anthropogenic threats like fires, grazing, deforestation, tourism, urbanisation, buildings/infrastructure, and plant collection especially in unprotected sites.
LC (Europe). National protection in Belgium, Luxembourg; regional protection in Italy and France [152].
Tuber as medicinal in Serbia [41,42]. Tubers used in infusion to treat cold, flu, and as body warmers, psychedelics, pleasure and medicinal tea in Turkey [63]. This species was cited as medicinal by Turner in 1568, while Langham in 1579 reported as antipyretic, anti-consumption and for anti-diarrhoeal effects [85].
LC (Europe). National protection in Belgium, Luxembourg; regional protection in Italy and France [153].
Harvesting of tuber for Salep production; horticulture; animal digging, herbivory; overgrazing; habitat in decline for changes in woodland management, reforestation, ploughing, agriculture, use of fertilisers; urbanisation, tourism, infrastructures. LC in France; regional protection in Italy [154].

Orchis
Damage to populations due to annual winter sport activities and tourism; collection for decorative purpose and for Salep production; animal digging and herbivory.  [49]), cold, flu, body warmers; tuber used for Salep (Turkey [13]); tuber as medicinal food in Serbia [41,42]. In North-Eastern Italy, leaves were used against rheumatism and as antineuralgic, by putting swell dry leaves in water-vinegar and by applying swollen leaves to the affected area [155].

Current Uses
We considered as current the uses reported in relative ethnobotanical investigations carried out and published after the year 2012 (past ten years). We found that the folk uses continue today for 54 species. All the tuberous orchids cited for Salep are currently harvested, while the consumption of tuber as food/medicinal ingredient other than Salep continues for 23 species. In these cases, the highest number of citations was found for Turkey, Bulgaria, Greece, and Serbia. Only Dactylorhiza euxina in Turkey and Gymnadenia rhellicani in Italy are harvested today at the above-ground level for medicinal purposes. We found that 10 species are still mentioned for ornamental aims: in these cases, cited countries were mainly Italy and Turkey. Finally, ritual uses are no longer reported. A graphical comparison between current and past utilizations of the different portions is shown in Figure 1.

Orchids and Salep
The mixture called Salep, for which the majority of our studied species were mentioned, is the most famous folk preparation that includes orchids. Given its ethnic importance and the associated concerns it creates for orchid conservation, much of the literature has already been dedicated to this product. For thousands of years, as reported for other species from Indian region and Middle East, terrestrial orchids have been harvested to ground their tubers and produce Salep powder [13,[24][25][26][27]32,66,139,[162][163][164]. Salep is also the name attributed to dried orchid tubers. This preparation originated in the Middle East in ancient times but had returned to be famous in the Europe of Renaissance after the publication of Gerard's Herbal in 1633 [13]. We found 46 tuberous and one rhizomatous species mentioned for Salep, but Tamer et al. [162] stated that 90 different taxa are overall harvested for this aim: tubers belong mainly to genera Orchis, Anacamptis, and Ophrys, but also Dactylorhiza, Neotinea, Himantoglossum, Serapias, and Platanthera are cited. As highlighted in Table 1 and as confirmed by various authors, the consumption of Salep is not only intended for alimentary use, but it is highly considered for the treatment of coughs, fever, diarrhoea, intestinal problems, asthenia, and to increase appetite and sexual desire. The substance is served as the homonymous hot drink Salep or it is an ingredient for sweets, being especially employed in the derivate ice-cream called "Salepi dondurma" in Turkey and "Kainaki" in Greece [24,32,139,162,165].

Uses of Orchid Below-Ground Portions
In Europe and especially in the Mediterranean Area, orchid tubers are not only harvested for Salep preparation, but they are mentioned for home consumption as medicine

Orchids and Salep
The mixture called Salep, for which the majority of our studied species were mentioned, is the most famous folk preparation that includes orchids. Given its ethnic importance and the associated concerns it creates for orchid conservation, much of the literature has already been dedicated to this product. For thousands of years, as reported for other species from Indian region and Middle East, terrestrial orchids have been harvested to ground their tubers and produce Salep powder [13,[24][25][26][27]32,66,139,[162][163][164]. Salep is also the name attributed to dried orchid tubers. This preparation originated in the Middle East in ancient times but had returned to be famous in the Europe of Renaissance after the publication of Gerard's Herbal in 1633 [13]. We found 46 tuberous and one rhizomatous species mentioned for Salep, but Tamer et al. [162] stated that 90 different taxa are overall harvested for this aim: tubers belong mainly to genera Orchis, Anacamptis, and Ophrys, but also Dactylorhiza, Neotinea, Himantoglossum, Serapias, and Platanthera are cited. As highlighted in Table 1 and as confirmed by various authors, the consumption of Salep is not only intended for alimentary use, but it is highly considered for the treatment of coughs, fever, diarrhoea, intestinal problems, asthenia, and to increase appetite and sexual desire. The substance is served as the homonymous hot drink Salep or it is an ingredient for sweets, being especially employed in the derivate ice-cream called "Salepi dondurma" in Turkey and "Kainaki" in Greece [24,32,139,162,165].

Uses of Orchid Below-Ground Portions
In Europe and especially in the Mediterranean Area, orchid tubers are not only harvested for Salep preparation, but they are mentioned for home consumption as medicine or food. During the Renaissance, Mattioli [51] had already indicated tubers from A. morio as source of nourishment for the inhabitants of Trentino and Ladinia Dolomitica (Northern Italy). Orchids were reported to be eaten in several Italian regions to recover some nutrients, especially during wars and famine. Mattirolo [52], in this context, listed eight orchid species (see Table 1) in his book Phytoalimurgia Pedemontana (1918), with which he aimed to help rural Italian people to survive the food crisis caused by the First World War. The large tubers of H. robertianum (Figure 2a) were roasted and eaten in Sicily [109,110]. Similar uses for this plant, together with O. apifera, N. ustulata, and other unspecified Ophrys and Dactylorhiza species, were reported for Sardinia [82,108] where tubers were consumed in the traditional preparation "Kasùgottu" (literally, "cooked cheese"). For this region, Ballero and Fresu [166] indicated tubers from Orchis s.l. for the cure of childhood diarrhoea. For Italy, Guarrera [82] reported alimentary and medicinal use for Dactylorhiza, Ophrys, and Orchis. Tubers of O. anthropophora were reported as food source and to cure phthisis, pectoral ailments, and hemoptysis [97,140]. In Northern Europe, Fousch (1549), and then Turner (1568), Langham (1579), and Parkinson (1640), indicated orchid tubers as medicinal resource to treat skin and gastrointestinal problems, and as anti-pyretic, anti-septic, anti-consumption, anti-diarrhoea, and to enhance sexual activity [14].  (1640), indicated orchid tubers as medicinal resource to treat skin and gastrointestinal problems, and as anti-pyretic, antiseptic, anti-consumption, anti-diarrhoea, and to enhance sexual activity [14]. In South-Eastern Europe (Turkey, Serbia, Bulgaria, Albania, South Kosovo and the Balkans, Hungary) tubers are still mentioned for medicinal purposes. Orchid species are prepared in decoction/infusion and taken as panacea to treat several conditions, such as cough, cold, inflammation, infections/abscess, wounds, boils and skin diseases, respiratory and gastrointestinal disorders; furthermore, uses as tonic, for mental wellness and aphrodisiac have been reported. Similarly, hypogean portions of rhizomatous species were harvested as herbal remedies: Cypripedium calceolus, Epipactis helleborine, Neottia ovata and Spiranthes spiralis were employed to obtain sedative and neuroactive effects, or to treat rheumatisms, skin and mucosal problems, or as aphrodisiac [11,13,117].
Ritual uses of orchid tubers have also been cited, always referring to the sexual or emotional sphere. Decorticated tubers from Orchis sp. pl. were reported to be aphrodisiac in the folk tradition of Friuli-Venezia-Giulia (Italy) and were employed to prepare "love decoctions" [167]. In Great Britain, a similar use was indicated for taxa that according to successive botanical investigations could be possibly identified with O. mascula, D. purpurella (T. Stephenson and T.A. Stephenson) Soó, or D. maculata [118]. In Trentino and Ladinia Dolomitica (Italy), male farmers had to look for orchids with palmate tubers (Figure 2b) and the girls those with testicular ones to eat them as a mutual demonstration of sexual sympathy [50]. In Northern Piedmont (Italy) a decoction with Nigritella's tubers (syn = Gymnadenia sp.) was secretly administered to the members of a couple: if during cooking the tubers remained close together it meant concord, vice versa discord. In Abruzzo (Central Italy), the tubers of G. conopsea or O. purpurea were used in ceremonies to reunite or separate couples [82,151]; for the same reason, D. maculata was collected in In South-Eastern Europe (Turkey, Serbia, Bulgaria, Albania, South Kosovo and the Balkans, Hungary) tubers are still mentioned for medicinal purposes. Orchid species are prepared in decoction/infusion and taken as panacea to treat several conditions, such as cough, cold, inflammation, infections/abscess, wounds, boils and skin diseases, respiratory and gastrointestinal disorders; furthermore, uses as tonic, for mental wellness and aphrodisiac have been reported. Similarly, hypogean portions of rhizomatous species were harvested as herbal remedies: Cypripedium calceolus, Epipactis helleborine, Neottia ovata and Spiranthes spiralis were employed to obtain sedative and neuroactive effects, or to treat rheumatisms, skin and mucosal problems, or as aphrodisiac [11,13,117].
Ritual uses of orchid tubers have also been cited, always referring to the sexual or emotional sphere. Decorticated tubers from Orchis sp. pl. were reported to be aphrodisiac in the folk tradition of Friuli-Venezia-Giulia (Italy) and were employed to prepare "love decoctions" [167]. In Great Britain, a similar use was indicated for taxa that according to successive botanical investigations could be possibly identified with O. mascula, D. purpurella (T. Stephenson and T.A. Stephenson) Soó, or D. maculata [118]. In Trentino and Ladinia Dolomitica (Italy), male farmers had to look for orchids with palmate tubers (Figure 2b) and the girls those with testicular ones to eat them as a mutual demonstration of sexual sympathy [50]. In Northern Piedmont (Italy) a decoction with Nigritella's tubers (syn = Gymnadenia sp.) was secretly administered to the members of a couple: if during cooking the tubers remained close together it meant concord, vice versa discord. In Abruzzo (Central Italy), the tubers of G. conopsea or O. purpurea were used in ceremonies to reunite or separate couples [82,151]; for the same reason, D. maculata was collected in Sardinia [82].

Uses of Orchid Aerial Parts or Whole Plant
In the European traditional knowledge, while tubers are widely mentioned, few reports on the utilization of other portions of orchids are available. Both food and medicinal, ritual, and ornamental uses are known. In Turkey, a culinary recipe that mentions the use of leaves of O. mascula subsp. mascula cooked with onion and eggs was found ( [66] and references therein). In Sardinia (Italy), leaves of E. helleborine were used for the treatment of wounds [97], while those of D. euxina are still cited for this purpose in Turkey [74]. In North-Eastern Italy, dry leaves of Platanthera bifolia were cooked in water-vinegar and then directly applied on the skin for the cure of rheumatism and as anti-neuralgic [155]; in Dorset (UK), aerial parts of P. chlorantha were included in an ointment to be applied on ulcers [118]. Leaves and flowers of D. sambucina were harvested in Liguria (Praglia; Genoa, Italy) to prepare a decoction against coughs [92]. In the 1577, Mattioli [51] reported for Northern Italy the use of flowers from genus Gymnadenia (G. nigra, G. rhellicani and related, G. odoratissima, G. conopsea) in infusion to produce digestive decoctions or distillates. For alpine regions of Northern Italy, the good-smelling inflorescence of G. rhellicani is one of the most mentioned: it is reported in various culinary recipes including liquors, as a herbal remedy for cold and respiratory diseases, or for increasing sexual desire [82,[101][102][103][104]. Furthermore, shepherds from Aosta Valley had stated that if the cows ate too many G. rhellicani flowers, the fontina cheese became bitter [104]. Concerning other uses of orchid epigean portions, the inflorescence of D. maculata was considered psychoactive and was used by the sorcerer "Marendìn" to hypnotize women in Northern Italy during the XVII century [80]. The flowering stem of Anacamptis papilionacea was employed in spells and evil eye in Sicily, Italy [61]. In Liguria (Vara Valley-Italy), in early May ("au primmu su de Mazzu [ . . . ]") children were rubbed with the inflorescence of O. mascula and other orchids to protect them from the bites of snakes and other animals [145]. In Aosta Valley, Italy, flowers of G. rhellicani were gifted by boys to girls they were interested in [104]; a similar approach was reported for Ophrys scolopax in Spain by González et al. [128]. Several species are mentioned for being collected as ornamentals at the stem level or as entire plants because of their beauty and/or scents: both in Italy and Turkey, this practice was particularly widespread. Among the reports, many different Ophrys are cited, i.e., O. tenthredinifera and O. sphegodes (see Table 1), together with O. argolica in Turkey [25] that is listed as Vulnerable, and O. speculum in Liguria [99], nowadays rarely observed in this region. The harvest of the rare D. insularis in Sardinia [53] also deserves a special mention.
Finally, skincare products industrially made with extracts from flowers of Orchis mascula, D. maculata, or A. morio are commercially available [9].

Phytochemical Information and Pharmacology of European Orchids
We found phytochemical information for 88 orchids diffused in European territories, of which 85 were selected based on the presence of known bioactive compounds; details on infraspecific taxa were also included. Table 2 provides a partial database of literature reporting compounds detected, and relative evaluations of biological properties, if present. As highlighted in the table, most of the available information consists in metadata collected from articles written for very different aims, namely works on chemical ecology for the evaluation of plant-pollinator interactions, or phytochemical analyses on the presence of specific classes of compounds compared in different taxa, even in a phylogenetic key (i.e., Strack et al. [168]). From a phytochemical point of view, the flowers/inflorescence are therefore the portions on which more information is reported, with more than 70 species being investigated; the most studied are O. mascula s.l., A. coriophora s.l., G. conopsea, H. robertianum, Ophrys sphegodes complex, and P. bifolia. Specific groups of chemical components of the leaves were found for 33 species, especially thanks to the work of Williams [169], van Damne and colleagues [170,171], and Balzarini [172]; the most cited orchids are E. atrorubens (Hoffm.) Besser, E. helleborine, N. ovata, and A. papilionacea. Phytochemical details for the hypogean apparatus (tuber/rhizome) are available for a total of 17 species, especially deriving from investigations on phytoalexin compounds and their production in response to external stimuli (see below). Many authors focused on the biochemicals and biological properties of extracts from tuber of D. viridis (L.) R.M. Bateman, Pridgeon and M.W. Chase, and G. conopsea (two orchids presenting a wide global distribution); the latter taxon has been already the subject of a review [16] (these results were not included in the table). These two species in particular (and a few others as well, i.e., Goodyera repens (L.) R.Br., Orchis mascula), being also present in the folk traditions and pharmacopoeia of other extra-EU countries, have therefore been studied by non-European working groups. Finally, starch [173], ash, sugar, sucrose [13], and glucomannan [38,84] were detected in orchid tubers.   [174] Saturated hydrocarbons (heneicosane, nonadecane, tricosane, pentacosane, heptacosane); unsaturated hydrocarbons (9-pentacosene 9-heptacosene; 9-tricosene; 1-hexadecene); aldehydes (nonanal, phenylacetaldehyde, anisaldehyde); alcohols (2,5-dimethoxybenzyl alcohol) and terpenes (thymol and α-copaene).

Inflorescence
EOs isolated by steam distillation from A. coriophora subsp. fragrans
High content in flavonoids, carotenoids; chlorophyll a-b and reduced glutathione.

Inflorescence
EOs isolated by steam distillation

Leaves from plants belonging to serpentine dumps
Ethanol extract Phytochemical screening by In vitro assays and total protein determination Non-enzymatic antioxidants play a role in the adaptive potential in plants belonging to polluted sites [203] Epipactis helleborine (L.) Crantz.

Inflorescence
EOs isolated by steam distillation
p-hydroxybenzylalcohol and orchinol are produced when the plant is challenged with Rhizoctonia repens.

Flowers
Methanol extract TLC, HPLC, and thin-layer electrophoresis [168] Stem, leaves and inflorescence Ethanol extracts No inhibition of the growth and biofilm formation of methicillin-resistant Staphylococcus aureus was observed [220] Coumarin; p-cresol; pentacosane; hexadecanoic acid; p-vinyl-phenol and other less abundant compounds.

Flowers
Floral scent HS-SPME-GC-MS analysis [236] Phenolic compounds and flavonoids detected in higher quantity in individuals from disturbed habitats in respect to those from natural sites.
Different amounts of the same compounds reported for P. bifolia (see above).

Bioactive Compounds, Tissue Distribution and Main Biological Properties
Among secondary metabolites, polyphenols and derivatives are largely studied, and the various polyphenol subgroups have been frequently reported in the investigated species. These subgroups consist in compounds such as flavonoids (including flavanones, anthocyanins, flavonols) and phenolic acids (including caffeic acid or chlorogenic acid). Among the health-promoting activities of polyphenols, antioxidant, cytotoxic, anti- Inflorescence DHA-GC-MS analysis [239] Serapias cordigera L.

Bioactive Compounds, Tissue Distribution and Main Biological Properties
Among secondary metabolites, polyphenols and derivatives are largely studied, and the various polyphenol subgroups have been frequently reported in the investigated species. These subgroups consist in compounds such as flavonoids (including flavanones, anthocyanins, flavonols) and phenolic acids (including caffeic acid or chlorogenic acid). Among the health-promoting activities of polyphenols, antioxidant, cytotoxic, anti-inflammatory, antihypertensive, skin-preserving, and anti-diabetic properties have been evaluated by both In vitro and In vivo assays [240].
The extensive investigation performed by Strack et al. [168] and Uphoff [177] on the anthocyanin content and relative patterns of abundance allowed to obtain information for many European orchids. Interestingly, the relative content of identified pigments (chrysanthemin, cyanin, seranin, orchicyanin I, ophrysanin, serapianin, orchicyanin II, mecocyanin, and epipactin), and unidentified ones from this water-soluble class of flavonoids was found to be highly variable but genus specific. According to these authors, Arditti and Fisch [241] and Uphoff [242], the mixture of acylated and non-acylated anthocyanins underlies the great variability of orchid flower colours, with orchicyanin I recognized as one of the key compounds responsible for intensive flower pigmentation. With about 600 identified compounds, anthocyanins have strong antioxidant properties and a validated defensive role for plants against biotic or abiotic stressors. These pigments are also active in delaying organ senescence, therefore contributing to the prolongation of tissue survival and increasing reproductive success. Patterns of anthocyanins also have a well-documented role in pollinator attraction [243,244]; i.e., in the case of Ophrys species, chrysanthemin and ophrysanin have been traced to the darker pigmentation of the labellum [168], which is typically one of the traits helping the flower in mimicking the female of the insect by which it is pollinated. Intriguingly, Vignolini and colleagues [218] found that the appearance of the speculum in O. speculum depends not only on the morphology of the surface cell layer, but also on the concentrated localization of cyanidin pigments. Colourful pigmentation (which is based on anthocyanins) has also been linked to the increased attention of people in regards of plants [4].
In orchid species, compounds with a recognized role as phytoalexins have been found (see Table 2; [13,16,178,179,194,227,[245][246][247]). These secondary metabolites are a supergroup of compounds (such as flavonoids, terpenoids, coumarins, stilbenoids/phenanthrenes and derivatives, glycosteroids and alkaloids). They are categorized as phytoalexins if their production starts in response to microbial attacks [248], playing a key role in the resistance against groups of microorganisms. For instance, they are known for exerting antibacterial [17] and fungistatic activity ( [13] and references therein, [227] and references therein). Among phenanthrenes and derivatives recognized as phytoalexins, hircinol, militarine, loroglossol, and orchinol are the most common molecules recorded [13,249]. Studies on orchid phytoalexins have been conducted, especially on the hypogean portions (i.e., Orchis mascula, O. militaris, H. robertianum). However, these compounds, such as loroglossin in A. papilionacea [179], were also reported in flowers and leaves. Phytoalexins such as loroglossol and hircinol recently re-isolated from the tuber of H. robertianum by Badalamenti et al. [17], showed In vitro antioxidant and immuno-stimulatory effects, together with anti-microbic and anti-cancer activities. 4-hydroxybenzyl alcohol (or p-hydroxybenzyl alcohol) is another well-known molecule that has been detected in different orchid tissues and species (see Table 2), for example, in the tuber of A. coriophora ( [13] and references therein) or in the flowers of D. maculata [184]. This compound has interesting biological activities as assessed by both In vitro and In vivo tests, such as: effects on the central nervous system (sedative, hypnotic, sleep-promotion properties [250], neuroprotective and anti-Parkinson activity [251], antioxidant, antimicrobial, and skin preserving properties, including anti-tyrosinase activity [13,252]).
Alkaloids were found in different portions of several orchids, including leaves and flowers ( Table 2). These molecules, in addition to being in some cases recognized as phytoalexins [248], are well-known for their activity on the animal nervous system. Alkaloids were detected in species like C. longifolia, Goodyera repens and E. helleborine [13,182,204]. In this latter orchid, oxycodone and other morphinan/indole derivatives were also found in the flower nectar by Jakubska et al. [205], which proposed that these molecules could be at the basis of the sluggish effect and the disorientation of visiting insects.
Phytochemicals detected in the flowers/inflorescence (whose presence has been mainly collected from anthecology articles) belong to several classes: apart from polar compounds/less volatile ones like polyphenols [176,184,253], saturated and unsaturated hydrocarbons, fatty acids and derivatives, and ketones are frequently found and proposed to contribute to pollinator attraction or herbivory avoidance ( [175] and references therein); the same role has been hypothesized for other classes such as aldehydes, alcohols, esters, coumarins, or terpenes, which are well-known as fragrant compounds. Among these, coumarin and terpenes are important metabolites that possess various physiological, ecological, and therapeutic functions. For example, orchid-derived compounds belonging to diterpenoids, sesquiterpenoids, and triterpenoids have shown interesting antiviral activities, including anti-SARS-CoV-2 properties due to the inhibitory competition on 3CL viral protease. Other compounds such as eugenol and methyl-eugenol are recognized as spicy and have several biological properties, among which are anaesthetic and hepatotoxic [13].

Causes of Biochemical Variations
Some species have been chemically analysed in several studies, but the guilds of compounds were found to be very variable (see Table 2). This may depend on several aspects, including the extraction methods and the analytical tools chosen [20,254,255]. For instance, more polar compounds such as polyphenols are less volatiles and therefore the use of protic solvents (such as methanol, diethyl ether or water-ethanol) results in substantially different chemical characterizations in respect to dynamic headspace sorption methods (i.e., [176,185,192]). Individual situations also contribute to changing metabolic processes and thus varying phytochemical profiles. It is known that the concentration of antioxidants such as polyphenols or carotenoids can change depending on plant physiological/phenological status ( [256] and references therein). For example, Maleva et al. [237] found that leaves from plants belonging to disturbed habitats showed an increased content of flavonoids. The sources of variability in orchid floral colour and scent have been already reviewed by Dormont et al. [257]. Concerning the content of anthocyanins in particular, changes have been recorded in plants collected in different populations [168] or in individuals facing nutrient deficiency ( [243] and references therein). Differences in alkaloid content between plants from diverse countries have been noted ( [13] and references therein). Chemical patterns can also differ due to random genetic drift [233]; some species were found to be phylogenetically similar but chemically distant [258], showing significant differences even between subspecies. Ayasse et al. [259] demonstrated that odour variation between plants of Ophrys sphegodes favoured cross pollination; furthermore, Schiestl and Ayasse [260] observed that pollinated flowers increased the production of the repellent farnesyl hexanoate. In support to these reports, Dormont et al. [261] confirmed that factors such as habitat characteristics, flower age, pollination, circadian rhythm, herbivory, and inflorescence morphology are responsible for chemical variations in Orchis mascula. Finally, as previously mentioned, the production of phytoalexins increases in tissues under microbial colonization. In this context, it is still to define whether the content of these secondary metabolites is dependent on the plant, or on hosts such as mycorrhizal fungi [13,262,263]. The relative abundance of the biochemical components should therefore be considered rather as a "snapshot" of the species' phytochemistry in response to a given environmental (physiological or ecological) situation.

Validation of Traditional Uses
When comparing the data on portions used as herbal remedies and the available scientific literature on phytochemistry/biological activities, only a few direct matches can be found. In the following cases, however, only a partial explanation that usually stops at In vitro evaluations is available.
Dactylorhiza romana subsp. georgica: Tuber was cited for the cure of cough. Kotiloglu et al. [190] and Bozkir et al. [191] recently analysed different extracts, including ethanol extracts from both dried and fresh tuber. Polyphenols and flavonoids were detected. Among constituents, p-hydroxybenzoic acid, kaempferol, rosmarinic acid, and caffeic acid were present. In both these studies, extracts demonstrated antioxidant and antimicrobial activities.
Himantoglossum robertianum: The tuber was prepared in infusion as medicinal tea and was cited for the cure of coughs. Badalamenti et al. [17], as mentioned above, worked on two phenanthrenes, loroglossol and hircinol, isolated from the tuber. The compounds exhibited In vitro antioxidant and immune-stimulatory activity, increasing the activity of superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST) in polymorphonuclear leukocytes (PMN); they also have antimicrobial properties as demonstrated by tests with Escherichia coli and Staphylococcus aureus; they have also anti-proliferative effect on gastric tumour cell lines by induction of apoptotic effect. However, the importance of other components of the plant complex in the entire traditional preparation cannot be excluded.
As mentioned above, glucomannan, ash, mucilage, water, and starch have been detected in orchid hypogean portions, therefore justifying their alimentary consumption, and indirectly, that of Salep. However, in this case, some authors in the XIX century already argued that its nutritive potential was overestimated ( [13] and references therein). No scientific evidence has been found demonstrating the claimed beneficial and aphrodisiac activities of Salep or constitutive portions of the species employed. Some of the secondary metabolites from orchid tubers (i.e., polyphenols/flavonoids) have well-documented biological properties ( [240] and references therein) but have no confirmed role in Salep. It should also be remembered that orchid tubers are strongly processed before pulverisation [264] and that spices such as cinnamon or ginger are added as flavours [13].
Some of the traditional uses cited can be at least partially explained by the available literature on phytochemicals. Compounds such as p-hydroxybenzyl alcohol (see above) can underline uses of tubers as anti-inflammatory or for the treatment of skin/gastrointestinal problems (i.e., A. coriophora or A.morio); the same can be hypothesized for antimicrobial molecules such as orchinol, loroglossol, militarine, or orchinol found in different species (i.e., Orchis spp., Anacamptis spp., Himantoglossum spp., Dactylorhiza spp., Gymnadenia spp., N. ovata). The presence of p-hydroxybenzyl alcohol, known as neuroactive and sedative, could be also at the basis of the alleged psychoactive properties of A. pyramidalis tuber or of D. maculata flowers (see Table 1). The tuber of D. osmanica was used to cure coughs, inflammation, ulcers, and skin boils: Kiziltas et al. [188], while investigating the tuber extract to evaluate other biological properties, found that fumaric acid, p-coumaric acid, rosmarinic acid, and vanillic acid were present. A contribution of these compounds to the beneficial effect of D. osmanica's tuber use cannot be excluded. Leaves and flowers of D. sambucina were prepared in infusion and cited for cough treatment: these portions were chemically investigated by Pagani [176], who listed health-promoting compounds such as coumarin, quercetin derivatives, or chlorogenic acid. In this species, water-soluble antioxidant anthocyanins such as cyanin, seranin, ophrysanin, orchicyanin II were recognized by Strack et al. [168]; quoted antibacterial terpenoids such as caryophyllene were found in the floral scent [192]. Leaves of E. helleborine, used to cure wounds, showed the presence of the antioxidant, antimicrobial, and wound healer quercetin [169]. Furthermore, alkaloids were detected in this species by Lüning [204].
Finally, leaves/aerial parts of both P. bifolia and P. chlorantha were employed as herbal remedies, to cure rheumatism, neuralgias, and skin ulcers, respectively. In the case of P. bifolia, well-known flavonoids like quercetin and kaempferol were detected in these tissues by Williams [169]; the presence of phenolic compounds, although variable between polluted and unpolluted sites, was confirmed by Maleva et al. [237].
Finally, there are species whose selected portions have been analysed phytochemically and for testing biological activities not reported in the ethnobotanical literature.
Dactylorhiza romana subsp. georgica: Antioxidant and antimicrobial activities of the tuber from this species have been already cited above. Kotiloglu et al. [190] and Bozkir et al. [191] also observed antidiabetic properties by evaluating α-amylase and α-glucosidase inhibitory activity during In vitro assays.
Dactylorhiza osmanica: This species and its phytochemicals have been already mentioned above. Kiziltas et al. [188] also found that both tuber and flowering stem extracts have anti-Alzheimer and anti-diabetes properties, as evaluated by In vitro enzymatic assays (inhibitory activity against acetylcholinesterase (AChE), α-glycosidase, and α-amylase).
Epipactis helleborine and N. ovata: The rhizomes of these species were cited as medicinal, but information on compounds and biological properties is available only for the leaves. Mannose-specific lectins obtained from homogenate plant material were tested on MT-4, HEL, HeLa, and MDCK cell lines and showed several antiviral activities against HIV-1, HIV-2, CMV, RSV, and influenza A [170][171][172].

Conservation Concerns and Action
According to Wraith and Pickering [7], biological use, which includes illegal collection of plants from the wild for medicine, food, and trade [265], is the most common threat for orchids globally, and particularly for terrestrial species. This was observed also for European species, since 12 out of the 16 species examined with threats were affected by biological use. Tuber collection, indeed, is still practiced, but for ornamental and private gardening uses in Italy and other developed European countries, with collectors often unaware of the identity and protection levels for orchid species ( [24][25][26][27]; personal observations by M.B. and J.C). As mentioned above, in Europe, thanks to the Habitat Directive and the Natura2000 network [266], but also to regional and national level laws, the collection of threatened species is regulated or forbidden, and it is often mandatory to ask special permission from the local authorities for collecting plants or any portion of them.
As recently reviewed by Masters et al. [267], there are many Salep patents including both industrial and medicinal products, which are easily found even with a quick search on e-commerce platforms like eBay. As highlighted in Table 1, this traditional preparation is still very popular in Turkey, Greece, the Balkans, and in neighbouring Iran as well, increasing the risk of extinction for several species [24][25][26][27]139,165]. Indeed, it has been reported that thousands of individuals are harvested every year [81,165,268]. The practice is now forbidden in many Mediterranean countries and the product is proposed mainly with guar-gum (Cyamopsis tetragonoloba (L.) Taub.) as a substitute for tubers (or only as a starch pudding with vanilla flavour); other alternatives consist in Salep made with cereal starch or synthetic carboxymethyl cellulose [38,165]. However, illegal harvesting still occurs in Turkey, Greece, and Iran [13,25,163,165]. Ghorbani et al. [163,164] listed non-disruptive collection as a compromise to meet Salep request, with the possibility of introducing local bans in the case of substantial population decline. However, levels of sustainable harvesting are still under scientific debate, especially because there is, in general, an increasing demand of Salep made by the rising middle-class among developing countries, with a major request of exportation to Western nations (with Germany leading) that has been linked to illegal collection [27,81,163,164,269]. Kreziou et al. [24] noticed that there had been a change in the utilization of Dactylorhiza species from previous reports, and they hypothesized that this was caused by the increased rarity of the more desired Anacamptis and Ophrys following overharvesting.
It has been reported that in the late 1800s, harvesting was performed by selecting tubers by size, with small tubers being replanted to promote regrowth; in support to this, Sumpter et al. [270] provided experimental evidence of the regenerative capacity of old orchid tubers, while Caliskan et al. [131] found that O. sphegodes harvested at early flowering stage was able to develop new tubers. However, Kreziou et al. [24] recorded neither dispersal of young tubers nor replanting of old tubers in their study in North Greece.
In vitro propagation has been proposed in extra-EU countries as potential alternative to overcollection [271][272][273][274][275][276]. However, orchids used for Salep are generally difficult to propagate and cultivate on a commercial scale for their tubers. Currently, only a few smallscale cultivation trials for orchid tubers exist, therefore they are only available commercially via wild harvesting [24,165].
A few companies have started to propagate European orchids for ornamental purposes such as Albiflora (https://albiflora.be/index.php, accessed by 14 December 2022, Phytesia (http://www.phytesia.com/en/, accessed by 14 December 2022), or Bewdley Orchids (https://www.bewdleyorchids.com/, accessed by 14 December 2022). However, apart for the latter which specifies that local native orchids are propagated with the aim to help dwindling populations, other companies do not indicate the provenience of seed material. This sets potential problems of genetic pollution, increasing hybridization with local orchid species ( [277] and references therein), and also the introduction of allochthonous microorganisms used in the potting mix, in particular bacteria and fungi, which could compete with the endophytes found in native populations, i.e., [278][279][280]. Given that recent studies seem to suggest that orchids can act as reservoir of orchid mycorrhizal fungi [281], conservation efforts would not only be pivotal for orchid populations persistence but also for the hosted microorganisms, which deserve more scientific attention as potential sources of active signalling molecules of ecological, agronomical, and industrial interest [263].
With the aim of helping local harvesters and resource managers to apply the best choices in terms of sustainability, very recently Ticktin et al. [282] reviewed existing data on the harvest pressure on orchids and consequent effects on their ecology and demography. These authors finally constructed a dichotomous key based on 12 characteristics, which can be used to determine in a local context whether and how a population could be subjected to a sustainable harvest. Characteristics include natural distribution and local abundance, habitat management, the existence of methods for the species cultivation, harvest type, and demand; for the development of the key, authors also considered characteristics of five Salep species (A. morio, A. pyramidalis, D. sambucina, Orchis italica, O. mascula).

Concluding Remarks
In conclusion, in this review we grouped citations of 62 orchids in the European folk tradition, and we examined the available information on phytochemicals and pharmacological activities for 85 species. As expected, the harvesting of tubers for Salep was highly mentioned and is still currently practiced: we confirm that there is no scientific evidence including pharmacological trials on humans that can justify the claimed aphrodisiac and healthy effects. Orchid tubers were found to contain interesting bioactive compounds that could at least partially explain the home consumption for medicinal purposes; however, also in this case, pharmacological confirmations are still lacking. Furthermore, it should be emphasised that tubers are heavily processed upstream of the final Salep powder, and that in chemical characterizations only compounds such as glucomannan, starch and ash were recognized. Claimed therapeutic effects of Salep should therefore be rather linked to mind suggestion and to the symbolic meaning that has been attributed to the testicle-like appearance of tubers for centuries. Currently, no efficient use of plant propagation methods has been found to fully supply Salep request: therefore, further studies are needed to better evaluate costs, benefits, and risks of an alternative production.
It should be mentioned that, as reported by Kreziou et al. [24], many Salep consumers were not aware that the product included material from threatened species. Conservation measures including dissemination/education programs aimed at raising public awareness on issues of nature protection are important to be considered and have already proved to be strategically useful (i.e., see [128,282]; www.globalorchidtrade.wixsite.com, accessed by 14 December 2022; www.lifeorchids.eu, accessed by 14 December 2022).
Compounds with demonstrated high therapeutic values have also been found in other orchid tissues less mentioned in the folk tradition: although even in these cases pharmacological trials need to be performed, leaves and flowers showed bioactive phytochemicals such as quercetin, kaempferol, chlorogenic acid, coumarins, phenanthrenes, alkaloids, and anthocyanins. Considering this, the possibility of deepening the study on orchid phytochemistry without exerting excessive pressure on individuals by harvesting, and possibly by obtaining material from In vitro micropropagation, may suggest ideas for new research and applications.

Conflicts of Interest:
The authors declare no conflict of interest.