Achillea moschata Wulfen: From Ethnobotany to Phytochemistry, Morphology, and Biological Activity

A multidisciplinary investigation on Achillea moschata Wulfen (Asteraceae) is outlined herein. This work, part of the European Interreg Italy–Switzerland B-ICE project, originated from an ethnobotanical survey performed in Chiesa in Valmalenco (Sondrio, Lombardy, Northern Italy) in 2019–2021 which highlighted this species’ relevance of use in folk medicine to treat gastrointestinal diseases. In addition, this contribution included analyses of the: (a) phytochemical profile of the aqueous and methanolic extracts of the dried flower heads using LC-MS/MS; (b) morpho-anatomy and histochemistry of the vegetative and reproductive organs through Light, Fluorescence, and Scanning Electron Microscopy; (c) biological activity of the aqueous extract concerning the antioxidant and anti-inflammatory potential through cell-based in vitro models. A total of 31 compounds (5 phenolic acids, 13 flavonols, and 13 flavones) were detected, 28 of which included in both extracts. Covering and secreting trichomes were observed: the biseriate 10-celled glandular trichomes prevailing on the inflorescences represented the main sites of synthesis of the polyphenols and flavonoids detected in the extracts, along with volatile terpenoids. Finally, significant antioxidant and anti-inflammatory activities of the aqueous extract were documented, even at very low concentrations; for the first time, the in vitro tests allowed us to formulate hypotheses about the mechanism of action. This work brings an element of novelty due to the faithful reproduction of the traditional aqueous preparation and the combination of phytochemical and micromorphological research approaches.


Introduction
Achillea moschata Wulfen (Asteraceae) is a suffruticose chamaephyte. It is endemic to the Alpine region and grows between 1400 m and over 3000 m a.s.l. on cliffs, stony ground, and moraines, exclusively on silica. It is known by various common and vernacular names, including erba iva, taneda, daneda, and aneda. The decoction of its flower heads, which represents the drug, is traditionally used to treat digestive tract disorders [1]. The balsamic time coincides with anthesis (July-August) [1].
This study is derived from an ethnobotanical survey related to the European Interreg Italy-Switzerland B-ICE project. The main purpose of this project is to create a management ground, and moraines, exclusively on silica. It is known by various common and vernacular names, including erba iva, taneda, daneda, and aneda. The decoction of its flower heads, which represents the drug, is traditionally used to treat digestive tract disorders [1]. The balsamic time coincides with anthesis (July-August) [1].
This study is derived from an ethnobotanical survey related to the European Interreg Italy-Switzerland B-ICE project. The main purpose of this project is to create a management model useful in dealing with emerging issues in Valmalenco (Sondrio, Lombardy, Northern Italy) due to climate change. Glacial retreat and diminished snowfall are adversely affecting tourism in the area. Therefore, the B-ICE management model includes strategies aimed at looking for new tourism avenues, such as the revitalization of traditional practices linked to medicinal and food wild plants [1]. Specifically, our work focuses on Chiesa in Valmalenco (Figure 1), which is the main village of the Valley. It has about 2400 inhabitants, is located at approximately 1000 m a.s.l., and is enclosed by the Bernina Alps. Previous ethnobotanical works that mentioned A. moschata referred to the Alpine and Pre-alpine regions, with special focus on the Italian territory [1][2][3][4][5][6]. Common aspects regarding the traditions of use emerged: from the employed plant parts (flower heads, flowered aerial parts, and flowered epigeal organs) to the most popular preparations (decoctions/infusions and digestive liqueurs). Folk uses in the Alpine and Pre-alpine regions mainly referred to a digestive, antispasmodic, and carminative action. They also Previous ethnobotanical works that mentioned A. moschata referred to the Alpine and Pre-alpine regions, with special focus on the Italian territory [1][2][3][4][5][6]. Common aspects regarding the traditions of use emerged: from the employed plant parts (flower heads, flowered aerial parts, and flowered epigeal organs) to the most popular preparations (decoctions/infusions and digestive liqueurs). Folk uses in the Alpine and Pre-alpine regions mainly referred to a digestive, antispasmodic, and carminative action. They also mentioned antiseptic, emollient, anti-inflammatory, and painkiller properties, as well as hypotensive characteristics [1][2][3][4][5][6].
The characterization of the aqueous extract proposed in the literature deserves further investigation, with morphological studies on the target species aimed at investigating the secreting structures responsible for productivity in secondary metabolites lacking. Indeed, previous micromorphological contributions focused on European and Western Asian congeneric species [16][17][18][19][20][21]. Regarding the biological activity, A. moschata extracts were generally screened for their radical scavenger properties with spectrophotometric assays such as the DPPH test [11,12]. Moreover, studies never reported the aqueous extract, which is significant, being the traditional decoction used in folk medicine in the Alpine and Pre-alpine regions. Only recently, Vitalini et al. [9] tested several extracts, including the aqueous one, for their anti-inflammatory activity, evaluating the modulation of interleukins 8 and 1β on CaCo-2 cells. Concerning congeneric species, the antioxidant potential of different extracts was evaluated as radical scavenger activity, but no in vitro studies were reported to date in the literature [10,[22][23][24].
We started our work with a (1) pilot field ethnobotanical research. Its primary focus was the collection and preservation of primary data obtained directly from actual users, concerning A. moschata-based remedies and traditional practices. We then characterized the species by combining complementary research approaches. Specifically, we (2) examined the phytochemical profile of the aqueous and methanolic extracts of the dried flower heads, obtained according to the referred traditional practices, from samples at anthesis gathered at the study area; (3) described the micromorphology of the vegetative and reproductive organs, with a special focus on the drug and secretory structures responsible for the production of secondary metabolites; and (4) evaluated the antioxidant and antiinflammatory activities of the aqueous extract using two different cell models.

Ethnobotanical Research
A total of 125 informants were interviewed, aged between 18 and 96 years old. They provided information on 148 plant species (Table S1), 74 of which were used therapeutically in humans (Table S2). Among these 74, the most cited were Arnica montana L. (16.7% of the total citations), A. moschata (8.9%), Malva sylvestris L. (7.8), Pinus mugo Turra (6.5%), and Hypericum perforatum L. (6.3%). Their use was deeply rooted in the malenca daily life. All ailments retrieved from the informants were tabulated in 18 categories of pathology [1]. Among these, musculoskeletal system disorders and traumas (27.0% of the total citations), respiratory tract infections (18.6%), digestive tract disorders (16.1%), and skin disease and traumas (8.4%) were the most cited. Focusing on the treatment of digestive tract discomforts, 28 species were employed and most of the malenca traditional knowledge in this field of application was concentrated on the use of a single plant species: A. moschata. In fact, of the total 239 citations for this category, 112 referred to this autochthonous plant, immediately followed by Matricaria chamomilla L. (n = 19), Gentiana lutea L. (n = 18), and Artemisia genipi Weber ex Stechm. (n = 13), which clearly represented a steep decline. For the treatment of these disorders, all of the informants were consistent in using the flower heads (rarely described as aerial parts) in the form of decoction to aid digestion, as a gastric antispasmodic, for abdominal pain, and stomachache. Within the eight ethnobotanical works conducted in the Alpine and Pre-alpine regions in the past 15 years [1,2,4,5,[25][26][27][28], seven documented the traditional use of A. moschata. Further confirmation on the same traditional use of the species was obtained by five of those seven works [1,2,4,5,27]. Furthermore, they described the use of decoctions/infusions of the flowered epigeal organs as a digestive, antispasmodic, and carminative agents. On the other hand, Cornara et al. [26] documented the use of compresses for eye inflammation, while Bruschi et al. [28] simply reported the category DSD (Digestive System Disorders), without mentioning specific pathologies.

Phytochemical Investigation
Regarding the phytochemical investigation, the qualitative profile of the aqueous and methanolic extract of the dried flowered aerial parts of A. moschata was evaluated. One of the elements of novelty offered by this work was the faithful reproduction of the traditional aqueous preparation, which was directly documented in the form of decoction through the ethnobotanical survey conducted in the study area. Another original aspect of this study concerned the characterization of the polyphenolic content of the traditional preparation, with the aim to better understand its potential in terms of bioactive compounds. Furthermore, this analysis was enriched with the characterization of the methanolic extract obtained by extraction with solvents of increasing polarity. This, in turn, facilitated a comparison with the literature data currently available about the target species, which mainly refer to this type of extract [10][11][12]15]. Figure 2 shows the Total Ion Currents (TICs) of the two extracts analyzed in negative ion mode, showing that all the main components of the mixture were identified. Table 1. LC-ESI-(+/−)-MS identification of the constituents of the two extracts of Achillea moschata. The peak numbers refer to those reported in Figure 2. * Compounds found only in the methanolic extract.  alpine regions in the past 15 years [1,2,4,5,[25][26][27][28], seven documented the traditional use of A. moschata. Further confirmation on the same traditional use of the species was obtained by five of those seven works [1,2,4,5,27]. Furthermore, they described the use of decoctions/infusions of the flowered epigeal organs as a digestive, antispasmodic, and carminative agents. On the other hand, Cornara et al. [26] documented the use of compresses for eye inflammation, while Bruschi et al. [28] simply reported the category DSD (Digestive System Disorders), without mentioning specific pathologies.

Phytochemical Investigation
Regarding the phytochemical investigation, the qualitative profile of the aqueous and methanolic extract of the dried flowered aerial parts of A. moschata was evaluated. One of the elements of novelty offered by this work was the faithful reproduction of the traditional aqueous preparation, which was directly documented in the form of decoction through the ethnobotanical survey conducted in the study area. Another original aspect of this study concerned the characterization of the polyphenolic content of the traditional preparation, with the aim to better understand its potential in terms of bioactive compounds. Furthermore, this analysis was enriched with the characterization of the methanolic extract obtained by extraction with solvents of increasing polarity. This, in turn, facilitated a comparison with the literature data currently available about the target species, which mainly refer to this type of extract [10][11][12]15]. Figure 2 shows the Total Ion Currents (TICs) of the two extracts analyzed in negative ion mode, showing that all the main components of the mixture were identified.   Table 1. Table 1 reports the name of the components characterized by their accurate mass, isotopic and fragmentation pattern (where available), both in negative and positive ion mode, and listed based on their elution order.
Regarding the phytochemical profiling of the phenolic content of A. moschata, the literature proposed only five works. Two of these highlighted the quali-quantitative characterization of the methanolic extract [11,12]; one proposed the quali-quantitative analysis of the acetone/water extract, later solubilized in methanol/water [15]; one focused on the quantification of the total polyphenolic content of the methanolic fraction [10]; and, finally, a more recent work, proposed a quantitative comparison between the methanolic extract and the aqueous preparation, without presenting any chromatogram and fragmentation pattern of the listed compounds [9].
These phytochemical data deepened the field work conducted by our research group on the traditional uses of A. moschata in the very distribution area of the species. This aspect further distinguishes our survey from previous works, which based their phytochemical investigation on ethnobotanical data obtained in the scientific literature [9,11,12,15], or sometimes analyzing commercial forms of the herbal drug, instead of spontaneous ones [15].

Botanical Description
Achillea moschata presents creeping woody stems from which ascending sterile shoots and erect flowering branches start ( Figure 3). The latter are sub-glabrous or with short hairs. The leaves are alternate, deeply pinnate with a lanceolate-spatulate outline and 7-10 segments on each side ( Figure 3). The basal leaves are pedicellate, while the cauline ones are sessile, toothed, and progressively linear. The leaves are strongly aromatic, hairless, or with scattered hairs [30]. The flowers are grouped in small flower heads (capitula) with a diameter of 2-6 mm, in turn gathered in loose corymbs, with each composed of about 10 flower heads ( Figure 3). The peduncles of the flower heads are cylindrical, striated, sub-glabrous, or with scattered hairs [31]. The capitula are composed of both ray (6-9) and disk (10)(11)(12)(13)(14)(15) florets. The receptacle is conic or convex, enclosed by various layers of imbricate involucral bracts. The bracts are obovate-oblong, sub-glabrous [31], with narrow transparent membranous margin, yellowish-to-brownish, and whole or notched only towards the apex with shallow denticles. The disk florets vary in number, from 10 up to 15 per head and are hermaphrodite. Each floret is subtended by an interfloral scale called "palea". The corolla tube is yellow with the top split in 5 apical laciniae overhanging the tube. The stamens are 5 with free filaments and connate anthers, welded in a sleeve surrounding the stylus with deeply bifid stigma; the carpels are 2 forming a unilocular inferior ovary. The ray florets are 6-9 per head, pistillate, fertile, 3-4 mm long. The corollas are white, with a tubular lower portion surmounted by a ribbon-like extension, the ligule, ending with sub-round denticles, with scattered golden glands on the lower side [30]. The stigma is 2-lobed, with a papillose surface.
Molecules 2022, 27, x FOR PEER REVIEW 7 of 23 cylindrical, striated, sub-glabrous, or with scattered hairs [31]. The capitula are composed of both ray (6-9) and disk (10)(11)(12)(13)(14)(15) florets. The receptacle is conic or convex, enclosed by various layers of imbricate involucral bracts. The bracts are obovate-oblong, sub-glabrous [31], with narrow transparent membranous margin, yellowish-to-brownish, and whole or notched only towards the apex with shallow denticles. The disk florets vary in number, from 10 up to 15 per head and are hermaphrodite. Each floret is subtended by an interfloral scale called "palea". The corolla tube is yellow with the top split in 5 apical laciniae overhanging the tube. The stamens are 5 with free filaments and connate anthers, welded in a sleeve surrounding the stylus with deeply bifid stigma; the carpels are 2 forming a unilocular inferior ovary. The ray florets are 6-9 per head, pistillate, fertile, 3-4 mm long. The corollas are white, with a tubular lower portion surmounted by a ribbonlike extension, the ligule, ending with sub-round denticles, with scattered golden glands on the lower side [30]. The stigma is 2-lobed, with a papillose surface.

Indumentum
The indumentum of the examined organs in all the replicates showed a general consistency both in terms of trichome morphotypes and distribution pattern (Figures 4-7). Both covering and glandular hairs were observed ( Figure 4). The formers were simple, multicellular, uniseriate, and distinguished in two subtypes: short and 5-7 celled with isodiametric cells of uniform diameter from the base to the apex, so as to appear digitiform ( Figure 4a); long and filamentous with a pointed apex which may be straight or slightly twisted (Figure 4b). Although the cell number could not be defined based on LM and SEM analyses, the literature data indicated the occurrence of 5-celled filamentous hairs in some congeneric species [15,17]. The basal cells of both subtypes were usually protruding from the epidermal surface of the organs bearing them, and sporadically appeared swollen. The short digitiform hairs were distributed on flowering stems and on both leaf sides (Figure 5a-f), while the long filamentous ones were on the leaf abaxial sides, on the abaxial side of the involucral bracts, and on peduncles of the capitulum (Figures 5c and 6a-d). The

Indumentum
The indumentum of the examined organs in all the replicates showed a general consistency both in terms of trichome morphotypes and distribution pattern (Figures 4-7). Both covering and glandular hairs were observed ( Figure 4). The formers were simple, multicellular, uniseriate, and distinguished in two subtypes: short and 5-7 celled with isodiametric cells of uniform diameter from the base to the apex, so as to appear digitiform ( Figure 4a); long and filamentous with a pointed apex which may be straight or slightly twisted (Figure 4b). Although the cell number could not be defined based on LM and SEM analyses, the literature data indicated the occurrence of 5-celled filamentous hairs in some congeneric species [15,17]. The basal cells of both subtypes were usually protruding from the epidermal surface of the organs bearing them, and sporadically appeared swollen. The short digitiform hairs were distributed on flowering stems and on both leaf sides (Figure 5a-f), while the long filamentous ones were on the leaf abaxial sides, on the abaxial side of the involucral bracts, and on peduncles of the capitulum (Figures 5c and 6a-d). The glandular trichomes belonged to a single morphotype (Figure 4b-g) that was a 10-celled biseriate, composed of two rows of five cells each (Figure 4c,f,g). There are two basal cells, two stalk cells, and six secretory cells, as already described in some congeneric species [19,20]. At full growth, the mature glands presented a peculiar bilobed, heart-like shape, resulting from the expansion of the cuticle covering the apical cells. In fact, during development, the subcuticular space at the apex grew into a sac-like structure, although it was unclear whether the sac embraced four or six apical cells (Figure 4f,g). These hairs may be sunken or may protrude to varying degrees from the epidermal surface because of the different elongation of the basal cells (Figure 4d-g).
They appeared sunken on the flowering stems, leaves, involucral bracts, and interfloral scale of the disk florets (Figures 5a-f and 6a-d,g-j), but protruded from the epidermis of the corolla of both disk and ray florets and on their ovaries (Figures 6k-m and 7a-f). On the abaxial surfaces of involucral bracts, trichomes presented a peculiar distribution pattern: filamentous glands and trichomes were arranged in two rows in troughs along either side of the leaf midrib (Figure 6b-d). Most of the secretion was discharged externally following the rupture of the cuticle, along a predetermined line of weakness, visible on the apical cuticular layer (Figures 4e and 5e-f). The cuticular envelope appeared broken to several degrees in many of the mature glands. Fractured cuticles also revealed the flocculent appearance of the secreted materials within the subcuticular chamber (Figure 4e).     No previous micromorphological contribution was found on the target species, while the literature data referred to works focused on European and Western Asian congeneric species. Secretory canals and both covering and glandular trichomes were described on both vegetative and reproductive organs by light and electron microscopy [16][17][18][19][20][21]. The canals were never thoroughly investigated and the literature reported only generic references [16,18], whereas information regarding glandular trichomes was more detailed. We did not detect secretory canals in the examined samples, while the biseriate, 10-celled glandular trichomes that resulted were consistent with those already known for Achillea genus [16][17][18][19][20][21]. Finally, the morphological features of the covering hairs, both the short digitiform and the long filamentous, were invariably uniform to the literature data, regardless of their localization on the different plant parts [16][17][18][19]21].

Histochemistry
The secretion products of the biseriate glandular trichomes exhibited positive responses both to lipophilic tests, such as Fluoral Yellow-088 and Nadi reagent (Figure 8a,b), and to the FeCl 3 test (Figure 8c,d), specific to polyphenols, and to the AlCl 3 test (Figure 8e) and Naturstoff reagent A specific to flavonoids. They showed weak coloration or negative responses to the hydrophilic dyes Alcian Blue (Figure 8f) and Ruthenium Red. Concerning internal tissues, the histochemical observations turned out to be uneven, since the results were generally negative for all the employed dyes, with sporadic weak positive responses only for polyphenols. Only one previous histochemical investigation was performed on a congeneric species, A. millefolium, and the production of monoterpenes was ascertained [17]. The secretory products are released externally after cuticle rupture: the volatile terpenoids emitted in the air are responsible for the typical aroma of A. moschata, while the polyphenolic/flavonoidic components presumably cover the epidermis constituting epicuticular exudates. Considering the overall micromorphological results, we exclude the presence of internal secretory structures, as well as the massive accumulation at tissue level of the polyphenols and flavonoids detected in the aqueous and methanolic extracts. The main sites of synthesis, accumulation, and release of such substances, along with volatile terpenoids, were represented by the biseriate glandular trichomes, distributed on the whole plant epidermal surfaces, being particularly abundant on the inflorescences (involucral bracts, interfloral scales, corollas, and ovaries of both disk and ray florets). No previous micromorphological contribution was found on the target species, while the literature data referred to works focused on European and Western Asian congeneric species. Secretory canals and both covering and glandular trichomes were described on both vegetative and reproductive organs by light and electron microscopy [16][17][18][19][20][21]. The canals were never thoroughly investigated and the literature reported only generic epidermis constituting epicuticular exudates. Considering the overall micromorphological results, we exclude the presence of internal secretory structures, as well as the massive accumulation at tissue level of the polyphenols and flavonoids detected in the aqueous and methanolic extracts. The main sites of synthesis, accumulation, and release of such substances, along with volatile terpenoids, were represented by the biseriate glandular trichomes, distributed on the whole plant epidermal surfaces, being particularly abundant on the inflorescences (involucral bracts, interfloral scales, corollas, and ovaries of both disk and ray florets).  These observations were consistent with previous contributions on congeneric species. Some authors have evaluated the variability regarding the density of glandular trichomes on different organs (flowering stems, leaves, and florets) at different stages of development (pre-anthesis and anthesis). It was documented that the overall density increased with the advancing of the phenological stages, from the pre-anthesis phase to complete anthesis. Furthermore, the highest density rates were found for florets, followed by leaves and stems [16,18]. Therefore, the studies on trichome density confirmed that the local population collects the drug at the exact balsamic time.
In addition, based on these results, it is reasonable to hypothesize that the compounds characterizing the aqueous extract are stored at the level of these epidermal appendages.

Biological Activity
The aqueous extract was evaluated for its antioxidant and anti-inflammatory potential in two different cell lines. Regarding the antioxidant activity, we tested the ability of the extract to activate the response of NRF2, a transcription factor associated with antioxidant enzymes, and which plays a master role in redox homeostasis in the cells. Data reported in Figure 9A show significant increase of NRF2 activation compared to the control. The activity is already significant at 10 µg/mL, while at the highest tested dose, 100 µg/mL, the increment is even higher than the one registered with BDX (Bardoxolone), a synthetic NRF2 activator used as a positive control. Concerning the anti-inflammatory activity, Figure 9B highlights some remarkable results. Indeed, the extract was able to significantly decrease the inflammatory response due to the stimulation of TNFα already at 1 µg/mL, while at 100 µg/mL, the response was comparable to control values. Cell viability was assessed and found to be comparable to the control values in both cell lines for all the tested concentrations (data not shown).
100 μg/mL, the increment is even higher than the one registered with BDX (Bardoxolone), a synthetic NRF2 activator used as a positive control. Concerning the anti-inflammatory activity, Figure 9B highlights some remarkable results. Indeed, the extract was able to significantly decrease the inflammatory response due to the stimulation of TNFα already at 1 μg/mL, while at 100 μg/mL, the response was comparable to control values. Cell viability was assessed and found to be comparable to the control values in both cell lines for all the tested concentrations (data not shown).  The statistical significance difference of each concentration in respect to the control was analyzed by one-way ANOVA followed by the Bonferroni post-test. * p < 0.05, **** p < 0.0001, ns = not significant.
Notably, it was the first time that these biological activities were reported in vitro for the aqueous extract of A. moschata. The antioxidant activity of A. moschata was previously reported using the DPPH assay, demonstrating, in in vitro conditions, a direct radical scavenging activity of the polyphenolic constituents of the species [9]. However, it is now well established that in in vivo conditions, the scavenging of radicals by polyphenols, and in general by antioxidants, cannot occur due to kinetic constraints (with the exception of vitamin E as a scavenger of lipid peroxyl radicals) [32]. Hence, the in vivo antioxidant defense of small antioxidants, such as polyphenols, is not due to a direct radical scavenging mechanism but rather to the activation of the NRF2 (NF-E2-related factor 2) signaling pathway which upregulates the enzymatic removal of the precursors of radical species, such as hydroperoxides, in two-electron redox reactions. We demonstrated herein the ability of polyphenols from A. moschata to activate the NRF2 pathway, a mechanism which could result in an in vivo antioxidant defense mechanism.
For the anti-inflammatory activity, an in vitro study was conducted on the aqueous extract by Vitalini et al. [9] on CaCo-2 cells focusing on the modulation of the release of interleukins. However, the novelty of our work regarding the study of the anti-inflammatory mechanism, which once activated, led to an anti-inflammatory response of which the release of inflammatory interleukins is one of the effects. The activation of NFKB is only partially related to the transcription of interleukins; it is responsible for numerous other mediators [33]. A strict crosstalk between inflammation and oxidative stress and between the two transcriptional factors involved, NFKB and NRF2, is now well established. When inflammation occurs as a primary disorder, it induces oxidative stress as a secondary disorder, which can further enhance inflammation. On the other hand, when oxidative stress is induced, inflammation develops as a secondary disorder and further enhances oxidative stress [34,35]. Based on this mechanism, the anti-inflammatory activity of the aqueous extract could be related not only to its action on the NFKB pathway, but also to the activation of NRF2. Because of the striking importance these two pathways have in the regulation of anti-inflammatory and antioxidant processes, it is notable that the aqueous extract is able to significantly work even at low concentrations on both processes.
Regarding the previous studies on the potential biological activity of A. moschata extracts [9,11,12,15], the phenolic profile was generally investigated concerning antioxidant and anti-bacterial properties. Once again, the tested activities rarely considered the traditional uses, or they did not describe them properly [9,15]. Moreover, even in such cases as these, the tested extracts deviated from the traditional remedies documented in the considered ethnobotanical studies.
With reference to the composition of the hot aqueous extract, which faithfully reproduces the decoction traditionally used and which was firstly characterized in this work, we proposed hereafter some considerations on the available literature data. Regarding the biological activity, we performed a survey with special focus on the gastrointestinal tract. The scientific literature documented an interesting in vitro anti-inflammatory ability ascribable to some of the main compounds found in the aqueous and methanolic extracts. As for the mechanisms, a few studies conducted on apigenin 7-glucoside (15) and chlorogenic acid (2) recorded important antioxidant and radical scavenging activities. The inhibition of the expression and translocation of Nuclear factor-kappa B (NF-κB) and of the production of pro-inflammatory cytokines, such as Tumor necrosis factor-α (TNF-α) and Interleukin-8 (IL-8), was also detected [36][37][38][39][40][41]. Apigenin 7-glucoside was also considered as a potential candidate against gastric cancer progression [42], due to the positive modulation of intracellular Reactive Oxygen Species (ROS) production. Moreover, some authors attributed to chlorogenic acid the healing properties of Taraxacum spp. on gastrointestinal disorders such as dyspepsia and gastritis [43]. The inhibition of the nuclear translocation of NF-κB, with the subsequent dwindling of COX-2 gene expression and of Prostaglandin E2 (PGE2) production, could be the mechanism underlying the anti-inflammatory activity of dicaffeoylquinic acids (13). In addition, another mechanism can be considered, namely, the inhibition of the expression of Inducible Nitric Oxide Synthase (iNOS) and the production of Nitric Oxide (NO) [44,45]. Karbab et al. [46] ascribed to isorhamnetin 3-O-beta-glucoside, which is one of the potential compounds for peak number 16, anti-inflammatory properties related to the inhibition of protein denaturation. Little has been reported on compound number 22, apigenin 7-malonyl-glucoside, which characterizes the aqueous extract. Han et al. [47] showed that it is one of the potential NF-κB inhibitor activity components of Flos Chrysanthemi, while Martínez-Vázquez et al. [48] described a sedative effect of the Dracocephalum moldavica L. aqueous extract with a high content of apigenin-and luteolin 7-malonyl-glucosides. Malonyl-glucosides can either be hydrolyzed in the gastrointestinal tract up to their relative glucosides [49], or absorbed in their intact form [50], thus exerting their activity in both conditions.

Ethnobotanical Research-Interview Management and Data Archiving
From 2019 to 2021, the ethnobotanical fieldwork was conducted as reported in Bottoni et al. [1]. Open and semi-structured interviews were conducted through a questionnaire, consisting of an "Informant Sheet" and a "Species Sheet" (the summary of the questionnaire is reported in Table S3). The former was used to document the personal data of the informant and feedback of the interview, while the latter consisted of a seven-column table arranged to fix common and vernacular name of the cited species, field of use, detailed use, preparation form, administration form, part of the plant, and other information. Each "Informant Sheet" was matched to the corresponding "Species Sheet" through an alphanumeric identification code, and subsequently filed in chronological order. For each species, information was organized according to the field of use and, eventually, in more detailed categories. For example, the therapeutic field was divided into several categories, with one for every anatomical apparatus (digestive tract disorders, musculoskeletal system disorders and traumas, etc.). All data obtained from the interviews were filed in a database in the form of an Excel spreadsheet (Microsoft, Redmond, WA, USA) where each row represented a citation, defined as a single use reported for a single species by a single informant. The overall data referring to the therapeutic use of the plants for the treatment of human diseases were processed using pivot tables, basing the analysis mainly on the number of citations.

Plant Material
For the phytochemical and micromorfological investigations, flower heads of Achillea moschata were gathered in July 2019 in Alpe Mastabbia, at 2200 m a.s.l. (Chiesa in Valmalenco, Sondrio, Lombardy, Northern Italy). The collection site is characterized by a siliceous substrate and by niches with accumulations of raw humus, at windy and exposed ridges. Voucher specimens were labelled with the codes GBG118/GBG119 and deposited in the Herbarium of the Ghirardi Botanical Garden of the University of Milan (DISFARM, Toscolano Maderno, Brescia, Italy). Prof. G. Fico and Prof. C. Giuliani identified the species according to Pignatti et al. [30].

Preparation of the Decoction
The decoction was reproduced as documented during the ethnobotanical fieldwork, following the dosages and indications provided by the informants. To prepare the volume of a cup of tea (150-200 mL), 5-6 small bundles of 10-12 flower heads were each placed in a small pot with water. Once it boiled, it was turned off, left to rest for 3-4 min, and then filtered. Traditionally, at this point, the product was taken orally when lukewarm, with or without the addition of sugar, due to its strong bitter taste. For the laboratory research path, the decoction, once filtered, was freeze-dried. Vials (Schott, G) were filled with 10 mL of the product, semi-stoppered, and placed on the shelves of an Alpha 1-4 LSC plus freeze dryer (Martin Christ, G). Product temperature was monitored by a single wire thin thermocouple. First, samples were frozen at the rate of 0.5 K min −1 to a minimum shelf temperature of −20 • C that was held for 6 h to assure complete freezing. In the primary drying, samples were kept at 10 • C and 0.2 mbar for 24 h. Afterwards, the shelf temperature was increased to 25 • C at the rate of 0.1 K min −1 to initiate the secondary drying. The desorption phase was performed over a 6 h period.

Preparation of the Methanolic Extract
Flower heads of Achillea moschata were dried at room temperature, before 45 g of the sample were extracted through three sequential extractions with increasing polarity solvents: n-hexane, dichloromethane, and methanol. Maceration was performed at room temperature and continued until the drug was exhausted for about 1 month for each solvent used for a total of 3 months. The methanolic extract was then stored at 4 • C until its characterization.

Chemicals
Formic acid and LC-MS grade solvents were purchased from Merck KGaA, Darmstadt, Germany. LC-grade H 2 O (18 MΩ cm) was prepared with a Milli-Q H 2 O purification system (Millipore, Bedford, MA, USA).

LC-MS Conditions
The extracts were dissolved in water (aqueous extract) and methanol (methanolic extract) to obtain a concentration of 4 mg/mL, then diluted in H 2 O/HCOOH, 100/0.1% v/v (mobile phase A) at the final concentration of 1 mg/mL for the analysis. The chromatographic separation was performed on a reversed-phase Agilent Zorbax SB-C18 column (150 × 2.1 mm, i.d. 3.5 µm, CPS Analitica, Milan, Italy) by an UltiMate 3000 system (Dionex) with a multistep program (80 min) of mobile phase A (H 2 O/HCOOH, 100/0.1, % v/v) and B (CH 3 CN/HCOOH, 100/0.1, % v/v) after the injection of 20 µL of each sample. An LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) was used as a detector working in negative and positive ion mode, as described by Baron et al. [51]. Xcalibur 4.0 (Thermo Fisher Scientific, Milan, Italy) and Chromeleon Xpress 6.80 (Thermo Fisher Scientific, Milan, Italy) were used for instrument control and spectra analysis. A targeted data analysis was undertaken on a database built searching the literature for known Achillea compounds [9][10][11][12]14,15,29,37]: the identification was conducted by using the accurate mass and the isotopic and fragmentation patterns.

Botanical Line Drawings
Original botanical line drawings depicting the whole plant, along with details of the vegetative and reproductive organs, were realized. Special attention was paid to diacritic features described in Pignatti et al. [30]. Both macrographs of living specimens at the study area and properly prepared exsiccata were used for the realization of the botanical illustrations. They were drawn in continuous graphite tone and then retraced with a felt-tip fine-point pen.

Microscopy
The micromorphological investigation under Light Microscopy (LM), Fluorescence Microcopy (FM), and Scanning Electron Microscopy (SEM) was performed on flowering stems, leaves, and flower heads collected from at least two different individuals at each of the three collection sites, concurrently with the sampling for the phytochemical investigation. At least five replicates for each of the investigated plant parts were examined to evaluate the variability in the morpho-anatomical features, as well as the type, distribution, and histochemistry of the secretory structures.

Light Microscopy (LM) and Fluorescence Microscopy (FM)
The micromorphological investigation under LM and FM was performed on both fresh material and fixed samples included in historesin (Technovit ® 7100). For the fresh material, sections of thickness ranging from 30 to 50 µm were obtained using a vibratome or a cryostat. Samples were also fixed in F.A.A. solution (Formaldehyde:Acetic Acid:Ethanol 70% = 5:5:90) for 10 days at 4 • C. Subsequently, fixed samples were washed in 70% ethanol for 12 h and then dehydrated progressively in ascending ethanol series, up to absolute. Pre-inclusion was then performed first with ethanol and historesin in 1:1 ratio for one night, then with a 1:2 ratio for 2 h, and in pure historesin for 3 h. Finally, the inclusion was performed in a polypropylene capsule with the addition of a hardener with a ratio of 1:15 of basic resin [52]. The historesin samples were cut in 2 µm sections by means of an ultramicrotome. The following dyes were employed [52]: Fluoral Yellow-88 for total lipids; Nile Red for neutral lipids; Nadi reagent for terpenes; Alcian Blue for mucopolysaccharides; Ruthenium Red for pectins; Ferric Trichloride for polyphenols; Aluminum Trichloride and Naturstoff reagent A for flavonoids. Observations were made with a Leitz DM-RB Fluo (Oberkochen, Germany) optical microscope equipped with a Nikon digital camera.

Scanning Electron Microscopy (SEM)
For SEM observations, small segments of the examined samples were F.A.A.-fixed for 7 days, dehydrated with ascending ethanol series up to absolute, critical-point dried, mounted on aluminum stubs, and carbon gold-coated [53]. Observations were performed under a Zeiss ® EVO MA15 SEM (Oberkochen, Germany) operating at 10 kV at the Interdepartmental Center for Electron Microscopy and Microanalysis Services (M.E.M.A.) of the University of Florence (Florence, Italy).

Antioxidant Activity
The aqueous extract was evaluated for its ability to modulate the antioxidant response pathway by monitoring the luciferase activity, strictly correlated with NRF2 activation as reported by Ferrario et al. [54] with minor changes. Experiments were performed using NRF2/ARE Responsive Luciferase Reporter HEK293 stable cell line (Signosis, Santa Clara, CA, USA) in Dulbecco's modified Eagle medium (DMEM; Lonza, Verviers, Belgium) supplemented with 10% fetal bovine serum (FBS; Gibco, Gaithersburg, MD, USA), 1% Peni-cillin/Streptomycin (Lonza), and 50 µg/mL of G418 sulfate solution (Promega Corporation, Madison, WI, USA). HEK293 cells were treated with different concentrations (1, 10, 50, and 100 µg/mL) of extract for 18 h after seeding in a white 96-well plate (BRANDplates ® , cell grade) at 10,000 cells/well. Subsequently, to avoid any interference on the reading of luciferase activity, media were removed and 50 µL/well of PBS was added. ONE-Glo™ Luciferase Assay Substrate (purchased from Promega Corporation, Madison, WI, USA) (50 µL/well) was directly added to the wells, followed by a luciferase measurement performed using a luminometer (Wallac Victor2 1420, Perkin-Elmer™ Life Science, Monza, Italy). Experiments were performed with biological and technical replicates, with results shown as mean ± SD compared to untreated control cells. Statistical analysis was performed using one-way ANOVA with Bonferroni's multiple comparisons test (p < 0.05 was considered significant). The cell viability was assessed with MTT assay on HEK293 cells treated with all the concentrations of the extract tested for the antioxidant activity.

Anti-Inflammatory Activity
The in vitro anti-inflammatory activity of the aqueous extract was evaluated using a cell model that has been previously described [55]. Briefly, R3/1-Nf-κB cells were seeded (5000 cells/well) in a white 96-well plate (BRANDplates ® , cell grade). Cells were pretreated with different concentrations of the extracts (1, 10, 50, and 100 µg/mL) for 18 h in complete medium (DMEM 10% FBS, 1% L-glutamine, 1% Penicillin/Streptomycin). This process was followed by a 6 h stimulation with 10 ng/mL TNFα. To avoid components' interference with the reading of the luciferase assay, cells were washed once with 100 µL of warm PBS before 50 µL of DMEM was added. Subsequently, 50 µL ONE-Glo™ Luciferase Assay Substrate (purchased from Promega Corporation, Madison, WI, USA) was directly added to the wells, followed by a luciferase measurement performed using a luminometer (Wallac Victor2 1420, Perkin-Elmer™ Life Science, Monza, Italy). Experiments were performed with biological and technical replicates and the results are shown as mean ± SD compared to untreated control cells. Statistical analysis was performed using one-way ANOVA with Bonferroni's multiple comparisons test (p < 0.05 was considered significant). The cell viability for all the concentrations tested in the anti-inflammatory assay was verified by MTT assay on R3/1-Nf-κB cells.

Conclusions
The novelty of our study was the combination of complementary research approaches: from ethnobotany to phytochemistry and morphology, up to biological activity. The ethnobotanical research conducted for the first time at the study area highlighted the traditional value of the target species. The phytochemical investigation of the aqueous extract obtained following the traditional procedure revealed a richer composition than previously described in the literature. The micromorphological investigation allowed for the first time an accurate description of flowers and inflorescences that represent the herbal drug. Moreover, the applications of histochemical dyes not only confirmed the production of phenolic derivatives, but also allowed for the enrichment of the literature data concerning the sites of synthesis and release of terpenic substances. Finally, the biological activity tests represented an important validation of the traditional use of the aqueous extract, being active at very low concentrations.
With regards to future research perspectives, the importance emerged of investigating the composition of the plant exudates, with the aim of confirming the histochemical outcomes. Indeed, this evidence allowed us to hypothesize that the accumulation of the active compounds occur at the level of the epidermal appendages. Finally, these results are consistent with the traditional use of the whole dried inflorescences, directly immersed in water without being chopped or pounded.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules27238318/s1. Table S1: Plant species traditionally used in Chiesa in Valmalenco (Sondrio, Lombardy, Northern Italy). Species are reported in decreasing order of citations. Table S2: Plant species used in the therapeutic field in Chiesa in Valmalenco (Sondrio, Lombardy, Northern Italy). Species are reported in decreasing order of citations. Table  S3: Summary of the questionnaire, in both the Italian and English languages, proposed during the interviews to the local community in Chiesa in Valmalenco (Sondrio, Lombardy, Northern Italy).