Introduction

There is an increasing public concern about the harmful effects of chemicals used in conventional agriculture and food production on human health leading to an increasing demand for more natural and healthier food. This habit encourages researchers to investigate about organic grown plants, especially wild species as new source for food or food additives and/or ingredients1, 2.

The integration of wild species into crop fields, particularly in organic farms, is of great importance to support the biodiversity and stability of agro-ecology systems and to enhance farmers’ livelihood3. This kind of wild species is useful for several reasons, such as the use of their extracts as source of natural herbicidal compounds4, used in the food sector, and to increase the plant and animal biodiversity. Some of the wild species recently “domesticated” are aromatic and medicinal plants, which showed economical and agronomical potentials greater than the parental cultivated species5, 6.

Lagoecia cuminoides L. (Apiaceae), described by Linnaeus7, is a Mediterranean-Turanian element, though disjointed being reported as native in Bulgaria, Greece, Crete with Karpathos, East Aegean islands, Cyprus, Israel and Palestine (Fig. 1a), Jordan, Lebanon, Syria, Libya, Portugal, Spain8, Albania9, 10, North Macedonia11, Crimea12, Iran13, 14, and Iraq15, 16. The presence of L. cuminoides was considered doubtful in Italy as it had not been reported for a long time from 1925 to 2018 (Fig. 1b), while it is considered an “alien” species, with status as casual, in France, Germany and Norway17. In Italy it was probably cultivated since the eighteenth century18, 19, for sure growing wild in southern Italy20,21,22, where it was discovered in Apulia, in the province of Taranto at Leucaspide, by D. Profeta, who described its small fruits and its cumin like taste23, and confirmed for the same region by other botanists22, 24, but after 1912 nobody else recorded it neither in Leucaspide nor in other places of Apulia.

Figure 1
figure 1

Herbarium samples: (a) Jerusalem (Palestine) (BI 25616); (b) Statte (Taranto—Italy) (BI 43986).

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Thanks to Lattanzi25, this taxon was discovered on 2018 in two sites in the municipality of Statte (Taranto province, Apulia), by the Working Group for floristic investigation of the Italian Botanical Society (SBI), and later by other botanists26. These Italian reports, together with those of the other Mediterranean countries, allowed to update the distribution map of the species (Fig. 2).

Figure 2
figure 2

Geographic distribution of Lagoecia cuminoides L. in the native countries (updated) and study area (created with CoreIDRAW version 12.0.0.458, https://www.coreldraw.com/).

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L. cuminoides was preserved and cultivated as an aromatic and medicinal plant in various Italian botanical gardens, and Mattioli27 gave also information on its use for the treatment of various diseases.

Metabolomics studies on this species and investigation about the functional properties of its extracts, such as antioxidant and antimicrobial activities, have been carried out mainly in Turkey and Iran14, 28, whereas no studies exist describing the ecology and the metabolomics of this species in Italy.

The seeds of L. cuminoides are used as a cumin substitute, giving to food spicy and aromatic characteristics29. It is used importantly in curries as an ingredient, and often is used as a flavour for cakes, bread and biscuits, where it has also a beneficial effect by improving the digestion process30, 31. When fully ripe the seeds are harvested and then dried and stored in jars14. The aerial part of the plant is frequently used to prepare an infusion for treating gastrointestinal diseases32. An essential oil from the seeds is used as a food flavouring33.

The main objective of the present work is to investigate the ecological context in which L. cuminoides grows in Italy, assess its conservation status in Italy, identify and characterize the metabolic pattern of the plant extract, evaluate their biological activity and finally its potential use in the food sector to establish a link with organic farms.

History, taxonomy, nomenclature and morphology

The name Lagoecia (from the Greek lagṓs oikos: hare’s home) alludes to the inflorescences of the species that resemble the bed made by the hare for her cubs34. The name cuminoides (from the Greek εἷδος eídos: similar to cumin) linked only to the fragrance of the small seeds which looks like that of Cuminum cyminum L. (cumin), native species of Afghanistan, Iran and Iraq35, that has been in use as a spice for thousands of years. C. cyminum itself is sometimes confused with caraway (Carum carvi L.), another spice of the same family (Apiaceae), and often in many European countries it is not clearly distinguished from these latter two species. In addition, Slavic and Uralic languages refer to cumin as "Roman caraway" or "spice caraway". Finally, Bunium persicum (Boiss.) B.Fedtsch., Bunium bulbocastanum L. and the unrelated Nigella sativa L. are sometimes called "black cumin", with the latter belonging to a different family (Ranunculaceae).

Taxonomy

The taxonomy of Lagoecia L. is controversial. The genus was described by Linnaeus7, under Monogynia (ovary with only one carpel) and not Digynia (ovary with two carpels) in which many of Apiaceae species were placed, due to reduction to only one carpel in Lagoecia genus36. Several botanists suggest a different history from the other Apiaceae. Drude37 and Wolff38 recognized the tribe Lagoecieae with three genera: Lagoecia, Petagnia Guss., and Arctopus L. Calestani39 divided Umbelliferae (= Apiaceae) into four subdivisions with the Lagoecineae that included the single tribe Lagoecieae. Cerceau-Larrival40 considered Lagoecia a monotypic tribe in Endressioideae. Later, Valiejo-Roman36 based on genetic studies (sequencing of nuclear ribosomal DNA) showed the affinity of Lagoecia with Crithmum L., Trachyspermum Link, Scaligeria DC., Bunium L., Elaeosticta Fenzl, Pyramidoptera Boiss. and Oedibasis Koso-Pol., with an outside position from all other Saniculoideae. Finally, Doğru-Koca et al.41 from a phylogenetic point of view suggested that Froriepia K.Koch should be the sister genus of Lagoecia cuminoides, and although both genera belong to the same tribe (Piramidoptereae), they are morphologically very different from each other. Even if the collocation of the genus Lagoecia is complex, from a morpho-taxonomic point of view Lagoecia should be considered very close to Petagnia, having in common a specific character: the abortion of an ovarian lodge21 and the consequent maturation of a single achene.

Chromosome number. Lagoecia cuminoides: 2n = 1642

Nomenclature

Apiales Nakai (1930)

Apiaceae Lindl. (1836)

Lagoecia L. (1753)7

Lagoecia cuminoides L. (1753)7

Synonyms

Cuminoides obliqua Moench in Methodus: 94 (1794); Cuminum cuminodes (L.) Kuntze in Revis. Gen. Pl. 1: 266 (1891).

Morphology

L. cuminoides is an annual herb, 10–30 cm high if it grows in desired moist soil (Fig. 3). Basal leaves with ovate and dentate segments, those cauline with segments deeply divided into short, lanceolate and aristate lobes. Umbels compound, subspherical, and feathery 0.5–1.5 cm in diameter, dense, globose; rays numerous. Bracts and sepals like leaves. Bracteoles 4, 2-pinnatisect, with setaceous lobes. Sepals pinnatisect, with lobes setaceous and sometimes 2–3-fid. White petals extended into two linear horns. Style 1. Fruit 2 mm, cylindrical-curved, covered with short, brittle, clavate hairs that look-like a pappus43, 44.

Figure 3
figure 3

L. cuminoides. Habit (a), flowers (b), and leaf (c) at Statte (Taranto), 13 May 2023. Pictures by E.V. Perrino.

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Materials and methods

The in-field surveys on the only two known Italian populations (Fig. 1) were conducted in 2022, from April to July. During the surveys some specimens were collected and used for laboratory analysis accordingly to the methods described in the following paragraphs.

Ecological characterization

Ecological characterization was performed through the phytosociological method of the Zurich–Montpellier school45 with information on physiographic data, as relevé identification code, geographic coordinates in WGS84, altitude (m a.s.l.), aspect, slope, relevé area (m2), stoniness, rockiness, cover total, average height of herbaceous layer (cm), number of individuals in the population (estimate), and habitat of Directive 92/43/ECC (https://environment.ec.europa.eu/). For the identification of taxa, Flora Europaea43 and Flora d’Italia44 were used; for nomenclature Bartolucci et al.46 and Galasso et al.47 were followed and for syntaxonomic nomenclature Mucina et al.48 was consulted. The collected plant material was stored at the Herbarium Horti Botanici Barensis of the University of Bari (BI) (3 March 2022, E.V. Perrino).

Assessment of the conservation status in Italy

The conservation status of the species in Italy was assessed according to the IUCN protocol49. The area of occupancy (AOO) was calculated with a 2 × 2 km cell grid, while the extent of occurrence (EOO) was calculated as convex hull.

Metabolites extraction

Plant samples collected during botanical surveys were air dried at room temperature and in the dark. All aerial parts were used (leaves, stem and flowers). The drying phase was considered accomplished when a constant weight was reached. After the drying phase, the plant material was grinded using a coffee grinder for 15 s. The obtained powder was weighted in amber glass bottles and added of hot water or hydroalcoholic solution using 1:30 w/vol ratio. The water decoction was performed using distilled water heated at 90 °C and 15 min time infusion. Then the extracts were cooled down and filtered using 0.45 µm cellulose recycled filters and stored at − 20 °C until the analysis. For hydroalcoholic extraction, dried vegetable material was added of 70% vol/vol ethanol, and, after mixing, the bottle was kept in the dark for 3 days and mixed every 12 h. The extract was then filtered using 0.45 µm cellulose recycled filters and stored at − 20 °C until the analysis.

Total polyphenol content

Total polyphenols were determined by spectrophotometric Folin assay according to Wrolstad et al.50. In plastic cuvettes 4 mL capacity, were placed 1.58 mL of water HPLC grade, 20 µL of extract and 100 µL of Folin reagent. The cuvettes were covered with parafilm and mixed and left for 5 min timed, then added of 300 µL of freshly prepared Na2CO3 20% and mixed. After 90 min the absorbance was read at 765 nm against a blank made at the same way of sample but using clean extraction solution (water or hydroalcoholic solution) instead of sample extract. Calibration was done using gallic acid standard in the range 10–800 mg/L. Results were expressed as mg of gallic acid equivalent on 100 g of plant material dry weight or per mL of extract.

Total antioxidant activity

Total antioxidant activity was determined using the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assay calibrated with Trolox. The ABTS radical was obtained by mixing 10 mL of ABTS 7 mM with an equal volume of persulphate 4.95 mM. The mixture was left at room temperature in the dark for 12 h, then stored refrigerated for a maxim of 7 days. Using the stock solution of ABTS radical, a dilution 1:25 was prepared, obtaining an absorbance at λ 730 nm of 0.7 units. The calibration range was 25–800 nmol/mL of Trolox equivalent. The assay was carried out as follow. In plastic cuvettes of 1 cm of optical length, 980 µL of ABTS diluted radical solution were placed and then 20 µL of sample or standard added. The cuvettes were closed using parafilm, mixed and left for 25 min timed. A blank was also prepared in the same manner but using the extraction solution instead of sample. After 25 min the absorbance was read at 730 nm against a cuvette with water. The difference between the sample and the blank containing only the extraction solution was determined and used in the calculation and expressed as µmol/kg of Trolox Equivalent Antioxidant Capacity (TEAC).

Non-volatiles metabolites identification by liquid chromatography coupled with mass spectrometry (LC/MS)

Three microliters of the extract were injected in the UHPLC Ultimate 3000 system (Dionex Thermo Fisher Scientific) equipped with LPG-3400RS pump, WPS-3000 autosampler, TCC-3000 column oven, and a Photodiode Array Detector PDA 3000. Chromatographic separation was obtained by the column Zorbax Eclipse XDB C18, 10 cm of length, 2.1 mm of internal diameter, 1.8 µm of particles size (Agilent) using a binary gradient with formic acid 0.1% in water (solvent A), methanol/acetonitrile/formic acid (50/50/0.1 vol). The solvent B gradient program was 5% initial, isocratic for 1 min, increased to 28% in 4 min, to 55% in 20 min, to 90% in 2 min, isocratic for 3 min, equilibration to the initial conditions for 5 min. The column temperature was set at constant temperature of 30 °C, and the mobile phase flow rate at 0.25 mL/min. The identification of compounds was performed by using a TSQ Quantum™ Access MAX Triple Quadrupole Mass Spectrometer equipped with a HESI interface. The MS conditions were capillary temperature 330 °C; source heater temperature 280 °C; nebulizer gas N2; sheath gas flow 35 psi; auxiliary gas flow 10 arbitrary units; capillary voltage − 2.8 kV. Data were acquired in negative ionization mode using a data-dependent method. The data-dependent settings were: Full scan from 250 to 850 m/z, activation level 500 counts, isolation width 1 Da, default charge state 2, collision induced dissociation energy (CID) 35 eV, collision gas pressure 1.5 mTorr of Argon bip. All data were acquired and processed using Xcalibur v.2 (Thermo Fischer Scientific). The identification of compounds was achieved by comparing λmax, [M-H]- and MS/MS fragmentation patterns with literature data51. Quantitative data were estimated by comparing the area of syringic acid to the area of each compound in the UV chromatogram at 280 nm. Syringic acid was used as internal standard, and was added to the sample before the injection as methanolic solution at 70 µg/mL final concentration.

Solid phase micro extraction (SPME) followed by gas chromatography mass spectrometry GC/MS analysis of volatiles metabolites

The extraction of volatiles compounds was obtained using the solid phase micro extraction technique with a three-phase fiber, Divinylbenzene/Carboxen/Polydimethylsiloxane (DVB/CAR/PDMS) 50/30 µm, 1 cm length (Supelco). Samples (10 mg of flowers and leaves or 100 µL of extracts) were placed in 20 mL dedicated SPME vials, then equilibrated at 50 °C per 2 min, and finally the fiber was exposed in the headspace of vials for 5 min for volatiles absorption. The volatiles were desorbed by exposing the fiber in the injector port of the GC system heated at 230 °C. All the process of equilibration, extraction and injection was performed by robotic autosampler Combi-PAL tx. The Gas Chromatography coupled with Mass Spectrometry (GC–MS) was composed of a Clarus 680 GC equipped with an Elite-5 MS fused silica capillary column (30 m × 0.25 mm and 0.25 μm film thickness) and interfaced with a single quadrupole mass spectrometer Clarus SQ8C (Perkin Elmer). Mass spectra of target compounds were obtained by electron impact ionization system with standardized ionization energy of 70 eV. Helium 5.5 was used as a carrier gas at a constant flow rate of 1 mL/min. The injection was performed in splitless (closed split valve for 1 min) at 230 °C. The oven temperature was programmed from 50 °C to 110 °C at 3 °C/min, then raised to 230 °C at 5 °C/min, hold at the final temperature for 3 min. Transfer line and source temperatures were set at 250 °C. Data were collected in full scan mode in the range 33 − 300 m/z. Qualitative results include compound identification and area percentage of related peak in the total ions chromatogram. Compounds identification was performed by both Retention Indexes (RI) and mass spectra (MS) search in NIST and Wiley databases and bibliography52. The linear retention index of each identified compounds was calculated according to Van Den Dool and Kratz53. The reference standard for linear retention index calculation was the alkane standard mix C8-C20 (Supelco), it was injected using a 1:100 split ration and analysed with the upper mentioned chromatographic and mass spectrometric conditions.

Results

Vegetation and ecological characterization

The context in which L. cuminoides grows, in relation to its limited national and regional distribution, seems to be linked to microenvironments with low anthropic input. They are transitional environments between the natural habitats with mosaic of scrublands and annual meadows and cultivated fields with extensively management. The phytosociological survey showed that this taxon, in relation to its peculiar ecology, is one of the characteristic species of Stipion retortae O. de Bolòs 1957 (Syn.: Stipion capensis O. de Bolòs 1957) alliance, that encloses the plant communities of Western Mediterranean ephemeral winter pastures on loamy soils and over calcareous substrates48.

The vegetation was surveyed in two localities in the municipality of Statte, Province of Taranto (southern Italy) (relevés codes: 17-05-22-01 and 22-05-22-01). The sites have the same altitude (272 m a.s.l.), exposition (SW), slope (2°) and rockiness (2%), while the stoniness at Pineta di San Giovanni (PSG) site (30%) exceed of 10% that of the Gravina di Mazzaracchio (GM) (20%). The soil is always silty-loam, characterized by poor total carbonate, and low phosphorus availability but rich in organic carbon and total nitrogen. Remarkably, a significant higher number of individuals in the L. cuminoides population in the GM site was found, in addition to an increase of total coverage (Table 1).

Table 1 Phytosociological data related to the two sampling areas.
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Assessment of the conservation status in Italy

According to the IUCN protocol49, the only criterion B was used for the assessment of the conservation status of L. cuminoides in Italy. With an AOO less than 10 Km2, an EOO less than 100 Km2, a single location (sensu IUCN), and a continuing decline projected in area of occupancy, extent and quality of habitat, number of mature individuals, the species is Critically Endangered in Italy: CR B1ab(ii,iii,v) + 2ab(ii,iii,v).

Antioxidant activity and total polyphenol content

Total polyphenols and antioxidant activity assays were performed on the ethanolic extract and in the water infusion extract. Results were calculated in mg per g of dry weight of plant material and shown in Table 2. The 2-ways ANOVA revealed no significant difference in total polyphenol content by comparing the sampling areas or the type of extraction. Nevertheless, the ethanol extracts of this taxon had two times higher antioxidant activity than the water infusion extract (p < 0.01). Comparing the two areas of collection, considering both ethanol and water extracts, samples collected in the second area (GM) reported a higher value of antioxidant activity.

Table 2 Antioxidant activity in µmol/g and total polyphenol in mg/g on dry weight of the extracts obtained from L. cuminoides samples collected in the two target areas (PSG = Pineta di San Giovanni; GM = Gravina di Mazzaracchio).
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As reported in Table 2, there was a significant interaction between the extraction method and the sampling area. This significance was illustrated in Fig. 4. In fact, the significant difference between the two areas could be highlighted only by the water infusion method.

Figure 4
figure 4

Results of the total antioxidant activity (TEAC) of L. cuminoides comparing the type of extraction and the site of collection (with pattern = PSG, white = GM).

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It could be hypothesized that, in GM samples there were a higher concentration of some polar constituents with anti-scavenging activity than in the PSG samples, and that these compounds could be glycated flavonoids.

Metabolites identification and quantification by chromatography techniques coupled to mass spectrometry (LC/MS–MS and GC/MS)

Non-volatiles metabolites present in the extracts obtained by water infusion or hydroalcoholic extraction were identified and tentatively quantified by ultra-high performance liquid chromatography coupled with triple quadrupoles detectors. Results show the presence of 12 compounds, ten of them were tentatively identified using literature data, whereas two unknown compounds were only described for their molecular mass, UV absorption and MS2 spectra (Table 3).

Table 3 Identification data obtained by LC/MS–MS of metabolites extracted from L. cuminoides samples.
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Eight compounds were identified as quercetin derivate linked with one or more molecules of sugar and or organic acid, accordingly to Abas et al.51. In this case, the identification was based on the presence in MS2 spectrum of the characteristic signal at 301 m/z and the peak absorbance in the UV at 250 and 350 nm. The firs eluted compound was the caffeoylquinic acid, also known as chlorogenic acid, whereas the last ones are luteolin and the two unknowns.

In Table 4 the semiquantitative data of identified compounds are listed, comparing the hydroalcoholic and the hot water infusion extracts. The first five compounds, which are more hydrophilic were more concentrated in the water extract than in the ethanolic one. The opposite occurred for the last compounds, which are more hydrophobic.

Table 4 Semiquantitative data obtained by LC/MS–MS, expressed in µg/mL, of metabolites identified in the L. cuminoides samples extracts.
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A further analytical determination was performed to identify and compare the volatiles fraction of metabolites present in plant material and in the water and ethanolic extracts by SPME-GC/MS. The aim of this analysis is to evaluate the L. cuminoides as potential source of volatiles compounds with interesting biological activities in the plant aerial part and their fate during the preparation of a water or alcoholic extract for human consumption. The choice to use the SPME method of extraction instead of the classical hydrodistillation can be clarified taking into account the following considerations. First of all, L. cuminoides is a very rare species in the investigated area, it is a small plant and collecting all individuals found did not give a sufficient quantity for hydrodistillation nor to obtain a minimum quantity of essential oil to be quantified or analysed by GC/MS. Furthermore, the hydrodistillation produce an extract containing only part of the volatile compounds present in the matrix. The SPME is itself selective in relation to the extraction condition and the stationary phase used, but it is extremely more sensible then the hydrodistillation, and the chose to use a triple phase fibre gave us the possibility to have an exhaustive extraction, as much as possible, of the different chemical class of volatiles compounds.

Thirty-five volatiles’ compounds were identified in the head space of L. cuminoides aerial part of plant samples (Table 5). As expected, the elution order was monoterpens, oxygenated monoterpenes, sesquiterpene, and diterpenes. The most represented chemical group was that of sesquiterpenes, with 14 identified compounds. Not considering the quinone form of thymol, seven compounds were identified for both monoterpenes and oxygenated monoterpene groups. Four alcohols were detected, three C6 and one C8, and three of them had a double bond. Finally, one ester (3-octylacetate) and one diterpene (geranyl-p-cymene) were also detected.

Table 5 Results of volatiles metabolites identified in the plant and in the ethanol and water extracts of L. cuminoides by SPME-GC/MS.
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All identified compounds, apart from α-Chamigrene whose retention index was not found in bibliography, had a retention index close to the reference value plus or minus 6, validating in this way the identification process. Results in Table 5 show an important difference among the volatiles profile of plant and extracts. There is a considerable reduction of the number of volatile compounds present in the extracts in respect to the raw material. In fact, 10 compounds were identified in the head-space of hydroalcoholic extract. Considering the percentage of each chemical group, sesquiterpenes is the more affected by the extraction process, passing from 14 compounds and 58% of total area in the plat, to only 2 compounds and 2.63% of total area in the hydroalcoholic extract. Furthermore, sesquiterpenes are not detected at all in the water extract.

Discussion

Environmental and vegetation issue

The two studied sites have similar pedo-climatic characteristics, in fact they are geographically close one to each other and share climatic condition, altitude, vegetation cover, stoniness, rockiness and other vegetational features. The microenvironments in which L. cuminoides was detected, can be referred to the same annual meadows of Stipion retortae O. de Bolòs 1957 (syn.: Stipion capensis Br.-Bl. et O. de Bolòs ex Izco 1974) (Fig. 5). However, the vegetational context needs to be further investigated to define the syntaxon at the association level, including surveys in the other Mediterranean countries where it is already reported. From a conservation and management point of view the community aspects of this alliance fall within the priority habitat of the directive 92/43/EEC “Pseudo-steppe with grasses and annuals of the Thero-Brachypodietea” (code 6220*, the symbol * means that it is a priority habitat for the purposes of Directive 92/43/EEC), as the technical report in Subtype 3 (Brachypodietalia distachyi) includes many annual aspects, like Stipion capensis which is synonymous of Stipion retortae54.

Figure 5
figure 5

Plant communities on survey days. Pineta di San Giovanni (PSG) with L. cuminoides in bloom (a) (15 May 2023) and Gravina di Mazzaracchio (GM) (b) (20 May 2022). Pictures by E.V. Perrino.

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The greatest threats to this habitat are the abandonment of traditional activities being integrated into so called cultural landscapes, never constituting the potential vegetation of their area. The abandonment of those activities thus triggers the reactivation of natural succession and therefore the substitution of those communities by others. When grazing disappears from these plant communities, the first effect is an increase in the cover of perennial species and decrease in biological diversity, and the consequence is the encroachment of woody vegetation as a result of the re-activation of natural succession. This situation results not only in the reduction in biodiversity but also in a dramatic increase in the risk of wildfire55,56,57. For these reasons the livestock is being increasingly used for creating and maintaining firebreaks in many Mediterranean countries58, 59. The abandonment of traditional activities usually results also in the disappearance of traditional infrastructures which might be important as observed in the first site with the dry-stone walls (Fig. 5a).

Another threat is the agricultural transformation taking away natural areas with results in an instantaneous disappearance of these plant communities. Especially due to their semi-pioneer character, it should be necessary a management, especially of grazing, to perpetuate them. Therefore, as a rule, traditional extensive management schemes should be considered as the desired conservation management model. The active management should be sheep or goat livestock, better if sheep, with a minimum impact of livestock on vegetation. The stocking rates must be never exceeding the capacity of 0.1 livestock unit ha−1 year−154. The grazing system can be continuous with peaks in spring and sometimes autumn, depending on the time of onset of the autumn rain. Browse from shrub or forest formations and agricultural sub-products (e.g., stubble) are complementary sources of food for livestock. The traditional infrastructures, as dry-stone walls, play a key role in landscapes where these habitats, where they are usually of a high value for wildlife, and for the endangered plant species, as observed for L. cuminoides. Their conservation or restoration is therefore an advisable measure for this habitat type.

The environmental data collected and especially the differences in phenological stage between the two sites, with individuals in PSG in the flowering stage, those in GM with fully mature seeds, explain the slight differences in chemical composition and antioxidant activity in the Italian population of this taxon. Unfortunately, no scientific contribution gave useful elements on the aspects of environmental interaction and phenological stages on this taxon.

Conservation status in Italy

L. cuminoides is a widespread species ranging from Portugal to Iran. However, in Italy it is very rare, with only two known sites, very close to each other, in Apulia region. Given to its restricted distribution and to the projected decline related to the detected threats, as the abandonment of pastoral traditional activities, the species is Critically endangered in Italy. Therefore, in situ and ex situ conservation actions should be implemented for the conservation of this rare and threatened species in Italy.

Interestingly, other species with a wide distribution range and very rare in Italy occur in Apulia, and they are also threatened with extinction in Italy60,61,62.

Antioxidant activity and total polyphenol content

Ethanol and hot water infusion, as techniques for nutraceuticals compounds extraction, were chosen to investigate the potential use of this plant as infusion (like Matricaria chamomilla) or as ingredient in herbal mixture for liqueur production. In the supplementary materials (Table 1S) results of antioxidant activity and total polyphenols were reported in different units to make easier the comparison with already published results related to already known and used herbal species for these purposes. Considering the most commonly used plants worldwide to make hot water infusion preparation, we can consider Matricaria chamomilla, Taraxacum officinale (L.) W.W.Weber ex F.H.Wigg. and Melissa officinalis L. as references. Ivanova et al.63 investigated the polyphenols content and the antioxidant activity of the hot water infusion obtained using 26 Bulgarian plant species. The authors reported value range for total polyphenols (TP) 40–1700 µM (quercetin equivalent) and a total antioxidant activity ranging from 0.1 to 7 mM TEAC. The concentration of total polyphenols observed in the infusion of L. cuminoides expressed in quercetin equivalent ranged between 412 and 840 µM (Table 1S), whereas the antioxidant capacity was between 0.4 and 0.8 mM TEAC. These values are very close to that observed and reported for M. chamomilla and Taraxacum officinale. Similar conclusions could be deduced considering Jiménez-Zamora et al.64 who reported the antioxidant capacity and the total polyphenols content of 36 plant species commonly used for infusion. It was surprising to observe that the antioxidant activity of L. cuminoides (~ 1900 µmol/100 g as mean value) on dry weight has the same magnitude range of the green tea infusion (400–2000 µmol/100 g). Considering the results obtained on the hydroalcoholic extract, L. cuminoides infusion showed a TEAC of 34 µmol/g and a TP of about 6 mg of gallic acid equivalent (GAE) per gram dry weight65. Georgieva and Mihaylova66 reported a mean TEAC and TP for M. chamomilla ethanol extract of 0.45 µmol/g and 4.5 mg/g dw respectively. Finally, Issa-Issa et al.67 studied the TEAC and TP values of the ethanol extract of 15 plant species commonly used in the production of a Spanish liqueur, reporting 827 mg/L TEAC and 228 mg/L GAE for M. chamomilla. The values obtained in the present paper expressed in the same units of concentration were 285 mg/L TEAC and 225 mg/L GAE. All that considered, L. cuminoides showed interesting properties both if used as infusion or as source of antioxidants in the production of liqueurs.

LC/MS–MS and GC/MS analysis

Since the TP and TEAC assays revealed interesting results on the extracts of L. cuminoides and values close to that of other species with well-known beneficial effects for human consumption, a deeply characterization of the obtained extracts was performed using the most advanced systems of LC/MS–MS for non-volatiles compounds, and the GC/MS for the volatiles ones.

As already mentioned in the results section, ten compounds were identified in the hydroalcoholic and in the water infusion extracts of L. cuminoides and most of them are glycated derivatives of quercetin flavonoid. Lin and Harnly68 studied the active substances extracted from M. chamomilla flowers during the hot water infusion preparation followed by LC–MS analysis. These authors identified 30 compounds among caffoyl quinic derivatives and glycated flavonoids. It is noteworthy that most of these later compounds have a common aglycone which is apigenin. In the extract of L. cuminoides both caffeoyl quinic isomers and glycated flavonoids were identified. These later showed a common aglycone which was quercetin. In both species lutein was also identified but for L. cuminoides only in the hydroalcoholic extract. There are many scientific reviews about the biological importance of quercetin and its derivates, among all these activities it should be highlighted the antioxidant, anticancer and antidiabetic effects on human health69, 70. By searching for the mean content of quercetin glucoside in the “phenols database”71 it was noted that quercetin 3 glucoside was found in a concentration range between 0.1 and more than 10 mg/100 mL considering wine and tea infusion among the different food sources of this flavonoid. Considering the quantitative data of the presented results obtained by LC–MS, the concentration of quercetin 3 glucoside in the water infusion or in the hydroalcoholic extract was 0.9 and 1.5 mg/100 mL respectively, within the upper mentioned range. Another important compound found in the extracts was the caffeoyl quinic acid. This compound is also known for its biological activity72 and the concentrations found (0.8 and 1.4 mg/100 mL in hydroalcoholic and in the water infusion extract respectively) are higher than that experienced in the black tea (0.3 mg/100 mL).

In our knowledge, only two papers discuss about the volatiles organic compounds of L. cuminoides. Bahmanzadagan et al.73 investigated the volatiles of L. cuminoides found in the south of Iran by hydrodistillation and head space analysis. These authors identified 35 volatiles, and among these, thymol, γ-terpinene and p-cymene were the most abundant. Baser and Tümen28 reported the volatiles composition of L. cuminoides collected in three different areas of Turkey and extracted by hydrodistillation. Also, these authors found thymol, γ-terpinene and p-cymene as the most abundant compounds. Apart from the differences in the percentages, probably related to the different techniques of extraction, both paper results are in agreement with the results of the present paper, highlighting a common biochemical pathway.

Considering the volatiles compounds identified in the plant material, β-farnesene, thymol, γ-terpinene and p-cymene were the most abundant. β-farnesene belongs to the sesquiterpene class. It is naturally produced by many plant species and by aphids as an alarm pheromone74. More recently, it is also studied as a potential anti-inflammatory modulator of human neutrophils75. Thymol, the second compound in percentage in Italian population of L. cuminoides, is a monoterpene widespread in many vegetable oils of wild plants76, especially in oil of Thymus sp. pl. Thymol is known as disinfectant in traditional medicine77, and antifungal78. It has also application in food packaging to prevent food spoilage during storage and increase the shelf life79. γ-Terpinene is another monoterpene present with a good frequency in this taxon, the third after thymol. The percentage found for γ-Terpinene (14.7%) in the analysed samples of L. cuminoides collected in the present work is close to the percentage value observed in the Iranian population of this species (15.6%)73, and in the Turkish populations (15.8%)28. The antibacterial and antifungal activity of γ-terpinene were documented by Yoshitomi et al.80, and Tahvilian et al.81 respectively. Finally, p-Cymene is a monoterpene found in over 100 plant species used for medicine and food purposes82. It shows a range of biological activities including antioxidant, anti-inflammatory, antinociceptive, anxiolytic, anticancer and antimicrobial effects83, 84. Considering the fate of these compounds during the preparation of the extracts, it worth to be mentioned the presence of all these four compounds in the headspace of the ethanolic extract but with an important increase of the relative percentage of γ-terpinene and p-cymene in respect to the value found in the plant material. Only two compounds, thymol and terpinene-4-ol, were detected in the hot water infusion, showing the low efficiency of water in extracting volatile terpenoids from vegetable sources.

Conclusions

The present research on Lagoecia cuminoides allowed to: 1) characterize the vegetation of this very rare taxon, at least in Italy, with identification of the alliance (Stipion retortae) that falls within the priority habitat of the directive 92/43 EEC “Pseudo-steppe with grasses and annuals of the Thero-Brachypodietea” (code 6220*). This is a very important aspect for conservation purposes, suggesting to define its level of threat according to the IUCN guidelines; 2) assess the conservation status of L. cuminoides in Italy according to the IUCN guidelines; 3) deepen and clarify its metabolites with modern methodologies compared to the few previous works on this species; 4) evaluate the potential biological activity of the extracts obtainable by hot water infusion or hydroalcoholic extraction as preparation techniques for human consumption (decoction or liqueurs respectively). The results revealed a richness of L. cuminoides in beneficial compounds for human health (antioxidant, anti-inflammatory, antinociceptive, anxiolytic, and anticancer active compounds). It would be appropriate a collaboration with chemists and botanists in the other Mediterranean countries in which this species grows, to better define its phenotype and biological diversity in relation to the environmental factors, as well as evaluating its use in specific environmental restoration programs at Mediterranean level, as in REACT4MED (https://react4med.eu/) and EcoplantMed (http://www.ecoplantmed.eu/project/) projects.

The valorisation of L. cuminoides and its potential use in food and agronomical sectors could play an important role in any future development programs and strategies that aim to enhance the territory and foster the resilience of communities and natural habitats, especially in marginal areas, not suitable for agricultural purposes but extremely important for biodiversity safeguard.