Teucrium fruticans L., a Multi-Scale Study: From Trichomes to Essential Oil.

This work represents the first multi-scale study on Teucrium fruticans L. cultivated at the Ghirardi Botanic Garden (Lombardy, Northern Italy), combining a micromorphological and a phytochemical survey on the plant's aerial parts. Micromorphological investigations, performed by Light Microscopy, Fluorescence Microscopy and Scanning Electron Microscopy, highlighted the presence of five trichomes morphotypes, distinguished by a different distribution pattern: peltates, short-stalked and ball-like medium-stalked capitates, ubiquitous on the whole plant, medium-stalked and long-stalked capitates, exclusive to the floral whorls. Both peltates and medium-stalked capitates were recognized as the main terpene production sites. Phytochemical characterization focused on the essential oils (EOs), obtained by Clevenger-type hydrodistillation in February and April 2022 and characterized by Gas Chromatography-Mass Spectrometry (GC/MS), which resulted mainly formed by sesquiterpene hydrocarbons. The February EO profile was characterized by ß-caryophyllene (28.30 %) and germacrene D (19.16 %) as main compounds, while in April ß-myrcene was detected at high percentage (13.77 %), in addition to the previous two components (15.72 % and 11.55 %, respectively). Literature data, dealing with the biological activities of the main oil constituents, highlighted an anti-microbial, anti-inflammatory, and anti-tumor potential, due to the high content in sesquiterpenes and, particularly, of ß-caryophyllene and germacrene D.

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.

Ca' Granda, Hortus simplicium: Restoring an Ancient Medicinal Garden of XV-XIX Century in Milan (Italy).

This work is based on the study of 150 majolica vases dated back to the mid XVII century that once preserved medicinal remedies prepared in the ancient Pharmacy annexed to the Ospedale Maggiore Ca' Granda in Milan (Lombardy, Italy). The Hortus simplicium was created in 1641 as a source of plant-based ingredients for those remedies. The main objective of the present work is to lay the knowledge base for the restoration of the ancient Garden for educational and informative purposes. Therefore, the following complementary phases were carried out: (i) the analysis of the inscriptions on the jars, along with the survey on historical medical texts, allowing for the positive identification of the plant ingredients of the remedies and their ancient use as medicines; (ii) the bibliographic research in modern pharmacological literature in order to validate or refute the historical uses; (iii) the realization of the checklist of plants potentially present in cultivation at the ancient Garden, concurrently with the comparison with the results of a previous in situ archaeobotanical study concerning pollen grains. For the species selection, considerations were made also regarding drug amounts in the remedies and pedoclimatic conditions of the study area. Out of the 150 vases, 108 contained plant-based remedies, corresponding to 148 taxa. The remedies mainly treated gastrointestinal and respiratory disorders. At least one of the medicinal uses was validated in scientific literature for 112 out of the 148 examined species. Finally, a checklist of 40 taxa, presumably hosted in the Hortus simplicium, was assembled.

Lavandula dentata from Italy: Analysis of Trichomes and Volatiles.

This study presented a micromorphological and phytochemical survey on Lavandula dentata L. cultivated at the Ghirardi Botanic Garden (Toscolano Maderno, BS, Italy). The morphological investigation revealed the presence of peltate, short- and medium-stalked capitate trichomes. The histochemical survey showed terpene production by peltates and medium-stalked capitates, hydrophilic secretions by short-stalked capitates. The phytochemical survey was developed on leaf and flower volatile organic compounds (VOCs) and on the essential oil (EO) from the flowering aerial parts. The VOC profiles represented an element of novelty and were dominated by oxygenated monoterpenes, among which 1,8-cineole and ß-pinene were the most abundant (77.40 %, 7.11 % leaves; 81.08 %, 10.46 % flowers). The EO of L. dentata was dominated by oxygenated monoterpenes with a high percentage of 1,8-cineole (69.08 %), followed by ß-pinene, trans-pinocarveol and myrtenal. Evaluations about the ecological role, the potential biological activity and the sensory attributes were proposed, based on literature contributions.

Using Medicinal Plants in Valmalenco (Italian Alps): From Tradition to Scientific Approaches.

This ethnobotanical survey was carried out in Caspoggio (Valmalenco, SO, Italy) with the purpose of investigating the traditional uses of medicinal plants. Moreover, a bibliographic research meant to validate or refute the uses, focusing on the potentially responsible compounds, was performed. Fifty-nine species, attributable to 30 families (Asteraceae, Pinaceae, Malvaceae, and Lamiaceae the most cited), were mentioned. Arnica montana, anti-inflammatory for traumas and musculoskeletal pains; Pinus mugo, expectorant; Malva sylvestris, anti-inflammatory and soothing; Achillea moschata, digestive. The compounds, responsible for the therapeutic activities, are often polyphenols and terpenoids: helenanin in A. montana, α-pinene, δ-3-carene, and limonene in P. mugo, gossypin and malvin in M. sylvestris, luteolin and apigenin in A. moschata. Scientific evidence for at least one of the traditional activities described was found for 50 species but only in 26 out of 196 works consulted, it is possible to make a comparison between investigated extracts and traditional preparations. This study is thus a stimulus to new phytochemical investigations, mimicking as much as possible the traditional preparations. This work is part of the European Interreg Italy-Switzerland B-ICE project, aimed at creating a management model for the ongoing climate change and searching for new sources of territory valorization as attractions for tourists.

Beyond the border: comparative ethnobotany in Valmalenco (SO, Italy) and Valposchiavo (Canton of Grisons, Switzerland).

BACKGROUND: The ethnobotanical analysis of two bordering areas allows for the in-depth understanding of the dynamics of Local Ecological Knowledge, which mirrors the naturalistic, historical, and sociopolitical features of each area. As part of the Interreg Italy-Switzerland B-ICE&Heritage and GEMME projects, this work is an ethnobotanical comparative study of two neighboring Alpine territories: Valmalenco (Italy) and Valposchiavo (Switzerland). METHODS: A total of 471 informants were interviewed on different fields of use (medicinal, food, veterinary, etc.). All data were organized in Excel™ spreadsheets. Informant Consensus Factor was calculated for the pathologies reported. Jaccard's similarity indices were calculated to compare the Valmalenco and Valposchiavo areas. Subsequently, another comparison between Valmalenco/Valposchiavo and Italian/Swiss Alpine neighboring areas was carried out. RESULTS: The number of taxa for Valmalenco was 227 (77 families) and 226 in Valposchiavo (65). Out of the 10 most cited species, 7 were mentioned in both. Arnica montana L. was the most cited in Valmalenco, and Sambucus nigra L. in Valposchiavo. The 5 most cited families were the same. Regarding the medicinal and food fields, the similarity indices were fairly low (0.31 and 0.34 for the species; 0.22 and 0.31 for the uses). Concerning the comparison with Italian and Swiss Alps, similarity values were slightly higher with Italy (Valmalenco food species: 0.38 with Italy and 0.26 with Switzerland, medicinal: 0.26 IT and 0.14 SW; Valposchiavo food species: 0.36 with IT and 0.26 with SW, medicinal: 0.21 IT and 0.14 SW). CONCLUSION: Although Valmalenco and Valposchiavo partly share natural environment, language, history, and culture, they had low similarity indices. They both seemed to have more similarities with Italy than Switzerland, maintaining low values with the surrounding territories too. They showed a common core of Local Ecological Knowledge with several divergent branches possibly due to pivotal historical happenings, as well as more modern external influences.

An Integrative Approach to Selected Species of Tanacetum L. (Asteraceae): Insights into Morphology and Phytochemistry.

In this work, we studied Tanacetum vulgare, Tanacetum parthenium, and Tanacetum corymbosum (Asteraceae) cultivated at the Ghirardi Botanic Garden (Toscolano Maderno, Brescia, Northern Italy) of the University of Milan. An integrative research approach was adopted: microscopic and histochemical, with special focus on the secretory structures responsible for the productivity of secondary metabolites; phytochemical, with the analysis of the essential oil (EO) profiles from the air-dried, flowered aerial parts collected in June 2021; bio-ecological, with emphasis, based on literature data, on the ecology and biological activity of the main EO components. In all three species, two basic trichome morphotypes (flagellar non-glandular and biseriate glandular) occurred with different distribution patterns. The glandular ones produced terpenes, along with flavonoids. A high level of chemical variability in the EO compositions emerged, specifically for qualitative data. T. vulgare profile was more complex and heterogeneous than those obtained from T. parthenium and T. corymbosum, with camphor as the predominant compound, followed by farnesol and α-santalone, respectively. Finally, the obtained scientific findings were made available to the visitors of the botanic garden through new dissemination labeling that highlights the "invisible", microscopic features of the plants, from an Open Science perspective ("Botanic Garden, factories of molecules…work in progress"-Lombardy Region Project Lr. 25/2016, year 2021).

From Primary Data to Ethnopharmacological Investigations on Achillea erba-rotta subsp. moschata (Wulfen) I.Richardson as a Remedy against Gastric Ailments in Valmalenco (Italy).

(1) Background: Within the framework of the European Interreg Italy-Switzerland B-ICE & Heritage project (2018-2022), this study originated from a three-year ethnobotanical survey in Valmalenco (Sondrio, Italy). Following a preliminary work published by our group, this research further explored the folk therapeutic use of Achillea erba-rotta subsp. moschata (Wulfen) I.Richardson (Asteraceae) for dyspepsia disorders, specifically its anti-inflammatory potential at a gastrointestinal level. (2) Methods: Semi-structured interviews were performed. The bitter taste was investigated through molecular docking software (PLANTS, GOLD), while the anti-inflammatory activity of the hydroethanolic extract, infusion, and decoction was evaluated based on the release of IL-8 and IL-6 after treatment with TNFα or Helicobacter pylori. The minimum inhibitory concentration and bacterial adhesion on the gastric epithelium were evaluated. (3) Results: In total, 401 respondents were interviewed. Molecular docking highlighted di-caffeoylquinic acids as the main compounds responsible for the interaction with bitter taste receptors. The moderate inhibition of IL-6 and IL-8 release was recorded, while, in the co-culture with H. pylori, stronger anti-inflammatory potential was expressed (29-45 µg/mL). The concentration-dependent inhibition of H. pylori growth was recorded (MIC = 100 µg/mL), with a significant anti-adhesive effect. (4) Conclusions: Confirming the folk tradition, the study emphasizes the species' potentiality for dyspepsia disorders. Future studies are needed to identify the components mostly responsible for the biological effects.

Ecotoxicological evaluation of an aqueous phytoextract of Melia azedarach L.

Melia azedarach L. is a Meliaceae that has shown important insecticidal activities. However, few researchers have extensively studied the toxicology of aqueous extracts of M. azedarach (MAE). Therefore, the main objective of this study was to characterize the phyto-eco-toxicological profile of MAE. First, a botanical and phytochemical characterization of MAE was performed using a histological, and metabolomic multi-analytical approach. Second, the toxicological effects on pollinating insects (Apis mellifera ligustica) and soil collembola (Folsomia candida) were evaluated. In addition, acute toxicity was evaluated in zebrafish (Danio rerio) to assess effects on aquatic fauna, and toxicity was determined in human neuroblastoma (SH-SY5Y) and fibroblast (FB-21) cell models. Finally, phytotoxic effects on germination of Cucumis sativus L., Brassica rapa L. and Sorghum vulgare L. were considered. Metabolomic analyses revealed the presence of not only limonoids but also numerous alkaloids, flavonoids and terpenoids in MAE. Histological analyses allowed us to better localize the areas of leaf deposition of the identified secondary metabolites. Regarding the ecotoxicological data, no significant toxicity was observed in bees and collembola at all doses tested. In contrast, severe cardiac abnormalities were observed in zebrafish embryos at concentrations as low as 25 µg/mL. In addition, MAE showed toxicity at 1.6 µg/mL and 6.25 µg/mL in FB-21 and SH-SY5Y cells, respectively. Finally, MAE inhibited seed germination with inhibitory concentrations starting from 5.50 µg/mL in B. rapa, 20 µg/mL in S. vulgare, and 31 µg/mL in C. sativus. Although M. azedarach extracts are considered valuable natural insecticides, their ecological impact cannot be underestimated. Even the use of an environmentally friendly solvent (an aqueous solution), for the first time, is not without side effects. Therefore, the data collected in this study show the importance of evaluating the dosages, modes of administration and production methods of M. azedarach phytoextracts in agricultural settings.

Alpine Diet in Valmalenco (Lombardy, Italy): Nutritional Features of Spontaneous Plants and Traditional Dishes.

BACKGROUND: Along the Alps, the Alpine diet is considered to be one of the most common nutritional models. Next to traditional animal-based products, spontaneous plants of the territory are collected and eaten. AIM: The aim of this study is to evaluate the nutritional features of selected autochthonous plants of the territory and the typical recipe of green gnocchi. METHODS: The analyses of proximate composition, carotenoid, total phenol, and mineral contents in raw and cooked plant samples and the chemical composition and in vitro starch digestibility in green and control gnocchi were performed. RESULTS: Except for Aruncus dioicus, all the wild plants contained high levels of carotenoids (15-20 mg/100 g FW), mainly as xanthophylls. Rumex acetosa showed the highest levels of total phenols (554 mg GAE/100 g FW), and Urtica dioica can be considered to be a good dietary source of iron, calcium, and magnesium (4.9, 410, and 72 mg/100 g FW). Cooking significantly decreased the potassium and magnesium contents in all wild species, and total phenols and carotenoids in Aruncus dioicus, Blitum bonus-henricus, and Silene vulgaris (p < 0.05). The slowly digestible fraction of starch (%SDS/available starch), which is inversely correlated to insulin demand, was significantly increased in green gnocchi compared to matched control gnocchi (p < 0.05). CONCLUSIONS: Traditional consumption of spontaneous plants in the Alpine regions might increase the dietary intakes of several bioactive substances and contribute to cover the nutritional needs of micronutrients.

Remnants from the Past: From an 18th Century Manuscript to 21st Century Ethnobotany in Valle Imagna (Bergamo, Italy).

BACKGROUND: This project originated from the study of an 18th century manuscript found in Valle Imagna (Bergamo, Italy) which contains 200 plant-based medicinal remedies. A first comparison with published books concerning 20th century folk medicine in the Valley led to the designing of an ethnobotanical investigation, aimed at making a thorough comparison between past and current phytotherapy knowledge in this territory. METHODS: The field investigation was conducted through semi-structured interviews. All data collected was entered in a database and subsequently processed. A diachronic comparison between the field results, the manuscript, and a 20th century book was then performed. RESULTS: A total of 109 interviews were conducted and the use of 103 medicinal plants, belonging to 46 families, was noted. A decrease in number of plant taxa and uses was observed over time, with only 42 taxa and 34 uses reported in the manuscript being currently known by the people of the valley. A thorough comparison with the remedies in the manuscript highlighted similar recipes for 12 species. Specifically, the use of agrimony in Valle Imagna for the treatment of deep wounds calls back to an ancient remedy against leg ulcers based on this species. CONCLUSIONS: The preliminary results of this study allow us to outline the partial passage through time fragments of ancient plant-based remedies once used in the investigated area.

Botanic Garden as a Factory of Molecules: Myrtus communis L. subsp. communis as a Case Study.

A novel perception of botanic gardens as complex "factories of molecules" (Lombardy Region Project-Lr. 25/2016, year 2021), that mediate plant-environment interactions, and are the basis of their utility for humans, is presented. The core-topic is the medicinal plant heritage of the Ghirardi Botanic Garden (Toscolano Maderno, Brescia, Italy) of the University of Milan. In this work, we studied Myrtus communis L. subsp. communis (Myrtaceae) at multiple scale levels: macro- and micromorphological, with special emphasis on the secretory structures responsible for the production of secondary metabolites; phytochemical, with the analysis of the essential oil (EO) composition from leaves (fresh, dried, stored at -20 °C and at -80 °C) and fruits over two consecutive years (2018 and 2019); bio-ecological, with a focus, based on literature data, on the ecology and biological activity of the main EO components. The occurrence of secretory cavities producing terpenes, along with flavonoids, was proven. A high level of chemical variability across the obtained EO profiles emerged, especially that concerning quantitative data. However, regardless of the different conservation procedures, the examined plant part, or the phenological stage, we detected the presence of three ubiquitous compounds: α-pinene, 1,8-cineole, and linalool. The overall results will serve to enrich the Ghirardi Botanic Garden with novel labeling showing accurate and updated scientific information in an Open science perspective.

Sub-Tissue Localization of Phytochemicals in Cinnamomum camphora (L.) J. Presl. Growing in Northern Italy.

In the present paper, we focused our attention on Cinnamomum camphora (L.) J. Presl. (Lauraceae), studied at three levels: (i) micromorphological, with the analysis of the secretory structures and a novel in-depth histochemical characterization of the secreted compounds; (ii) phytochemical, with the characterization of the essential oils from young stems, fruits, and leaves, subjected to different conservation procedures (fresh, dried, stored at -20 °C, stored at -80 °C) and collected in two different years; (iii) bioactive, consisting of a study of the potential antibacterial activity of the essential oils. The micromorphological investigation proved the presence of secretory cells characterized by a multi-layered wall in the young stems and leaves. They resulted in two different types: mucilage cells producing muco-polysaccharides and oil cells with an exclusive terpene production. The phytochemical investigations showed a predominance of monoterpenes over sesquiterpene derivatives; among them, the main components retrieved in all samples were 1,8-cineole followed by α-terpineol and sabinene. Conservation procedures seem to only influence the amounts of specific components, i.e., 1,8-cineole and α-terpineol, while analyses on each plant part revealed the presence of some peculiar secondary constituents for each of them. Finally, the evaluation of the antibacterial activity of the essential oil showed a promising activity against various microorganisms, as Listeria monocytogenes, Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa. In conclusion, we combined a micromorphological and phytochemical approach of the study on different plant parts of C. camphora, linking the occurrence of secretory cells to the production of essential oils. We compared, for the first time, the composition of essential oils derived from different plant matrices conserved with different procedures, allowing us to highlight a relation between the conservation technique and the main components of the profiles. Moreover, the preliminary antibacterial studies evidenced the potential activity of the essential oils against various microorganisms potentially dangerous for plants and humans.