Claudio Frezza

Iridoids and phenylethanoid from Pedicularis kerneri Dalla Torre growing in Dolomites, Italy

In this study, we report the first phytochemical analysis of polar fraction of Pedicularis kerneri Dalla Torre growing in Dolomites, Italy. Several iridoid glucosides were isolated, namely aucubin (1), monomelittoside (2), plantarenaloside (3), euphroside (4), mussaenosidic acid (5) and 8-epiloganic acid (6), showing a composition in accordance with previous study on this genus. The studied samples, collected from Dolomites, presented a chemotype already recognised in species from North America, characterised by euphroside (4) and aucubin (1) as main components, but the main character was the presence of monomelittoside (2) never reported in this genus. The identification of verbascoside (7), leucosceptoside A (9) and echinacoside (10) complete the systematic framing of this species since is ascertained the co-occurrence of phenylethanoid glycosides with iridoids in Lamiales species.

Polar Constituents and Biological Activity of the Berry-Like Fruits from Hypericum androsaemum L.

Hypericum androsaemum, also known as Tutsan, is a small evergreen shrub common in the Mediterranean basin where it is traditionally used as diuretic and hepatoprotective herbal drug. This plant possesses the peculiarity to produce fleshy and berry-like fruits that ripen from red to shiny black. In the present work, the chemical constituents of methanolic extracts and infusions of red and black fruits were analyzed by HPLC, and correlated with their antioxidant properties which were evaluated by the DPPH, β-Carotene/linoleic acid, and hypochlorous acid tests. In addition, the red pigment of the fruit was isolated by column chromatography and structurally elucidated by NMR. Results showed that H. androsaemum fruits contain high amounts of shikimic and chlorogenic acids, while their color was given by a tetraoxygenated-type xanthone, reported for the first time in Hypericum species. The red berries infusion gave the highest content of total phenolic compounds, DPPH, and hypochlorous acid scavenging activity, and β-carotene bleaching. Cytotoxicity of the berries extracts on three human tumor cell lines (malignant melanoma, breast adenocarcinoma, and colon carcinoma) was evaluated by MTT assay, and relevant inhibition on colon carcinoma cells (IC50 value of 8.4 μg/mL) was found. Finally, the effects of red berries extract on the immune system were evaluated by peripheral blood mononuclear cell (PBMC) proliferation assay that revealed a strong stimulation on lymphocytes at low doses (0.4–6 μg/mL).

Phytochemistry, micromorphology and bioactivities of Ajuga chamaepitys (L.) Schreb. (Lamiaceae, Ajugoideae): Two new harpagide derivatives and an unusual iridoid glycosides pattern.

Ajuga chamaepitys (L.) Schreb, well-known as Camaepitium or Ground Pine, is an annual herb typical of the Mediterranean area accounting several uses in the traditional medicine. In this work we have, analyzed the plant iridoid fraction together with the essential oil composition and study of the plant indumentum. Finally, we assayed the polar extracts and essential oil obtained from the aerial parts for antioxidant activity and cytotoxicity on tumor cells. The analysis of the monoterpene glycosides allowed us to isolate from roots and aerial parts and to structurally elucidate by NMR and MS the following compounds: ajugoside (1), reptoside (2), 8-O-acetylharpagide (3), harpagide (4), 5-O-β-d-glucopyranosyl-harpagide (5), asperulosidic acid (6), deacetyl asperulosidic acid (7) and 5-O-β-d-glucopyranosyl-8-O-acetylharpagide (8), among which 5 and 8 were two new natural products. Chemotaxomic relevance of these constituents was discussed. The chemical analysis of A. chamaepitys essential oil by GC-FID and GC-MS showed ethyl linoleate (13.7%), germacrene D (13.4%), kaurene (8.4%), β-pinene (6.8%), and (E)-phytol (5.3%) as the major volatile components. The micromorphological and histochemical study showed that iridoids and essential oil are mainly produced in the type III capitates and peltate trichomes of leaves and flowers. Biological evaluations of A. chamaepitys polar extracts and essential oil showed that the former were more potent as radical scavengers than the latter. MTT assay revealed that essential oil and ethanolic extracts were moderately cytotoxic on tumor cells with IC50 of 36.88 and 59.24μg/mL on MDA-MB 231 cell line, respectively, and IC50 of 60.48 and 64.12μg/mL on HCT116, respectively.

Secondary metabolites from Scrophularia canina L.

Abstract A re-examination of Scrophularia canina L. confirmed the presence of iridoid glucosides considered as chemotaxonomic markers for the Scrophulariaceae family, like aucubin, harpagide and 8-O-acetylharpagide, besides the further presence of 8-epiloganic acid, which is, indeed, considered the biogenetic precursor of iridoids normally found in Scrophulariaceae, and was recognised here for the first time in the studied species. Also verbascoside and (E)-phytol were evidenced for the first time in S. canina. The former compound is an almost ubiquitous glycosidic phenyl-ethanoid, which attains systematic importance when in co-occurrence with iridoids, and its taxonomical implications were discussed. The latter compound, even though it is omnipresent, is interestingly endowed with several biological activities, which may give an additional reason for the traditional uses of this plant.

Secoiridoids and other chemotaxonomically relevant compounds in Pedicularis: phytochemical analysis and comparison of Pedicularis rostratocapitata Crantz and Pedicularis verticillata L. from Dolomites

Abstract We compared the respective metabolite patterns of two Pedicularis species from Dolomites. Seven phenylethanoid glycosides, i.e., verbascoside (1), echinacoside (2), angoroside A (3), cistantubuloside B1 (4), wiedemannioside C (5), campneoside II (11) and cistantubuloside C1 (12), together with several iridoid glucosides as aucubin (6), euphroside (7), monomelittoside (8), mussaenosidic acid (9) and 8-epiloganic acid (13) were identified. Pedicularis verticillata showed also the presence of greatly unexpected secoiridoids, ligustroside (14) and excelside B (15), very rare compounds in Lamiales. Both PhGs and iridoids are considered of taxonomical relevance in the Asteridae and their occurrence in Pedicularis was discussed. In particular, the exclusive presence of several compounds such as 8-epiloganic acid (13), campneoside II (11), cistantubuloside C1 (12), ligustroside (14) and excelside B (15) in Pedicularis rostratocapitata, and angoroside A (3), cistantubuloside B1 (4) and wiedemannioside C (5) in P. verticillata could be considered specific markers for the two botanical entities.

Polar constituents, protection against reactive oxygen species, and nutritional value of Chinese artichoke (Stachys affinis Bunge)

In the present work, we studied the chemical composition of Chinese artichoke (S. affinis tubers) by analyzing its polar constituents and its macro- and micro- nutrients. A total of nine compounds were isolated from the tuber ethanolic extract and structurally elucidated by Nuclear Magnetic Resonance (NMR) spectroscopy and mass spectrometry (MS). The marker compounds identified were oligosaccharide stachyose and the organic acid, succinic acid, as well as phenylethanoid and iridoid glycosides. The macronutrient profile was dominated by carbohydrates (36.9% dw), whereas potassium (2.36%) was the most abundant micro-nutrient. The tuber ethanolic extract was able to efficiently protect human cells (Caco-2, SHSY-5Y and K562) against t-BHP-induced oxidative damage.

Iridoids and phenylethanoid glycosides from the aerial parts of Ajuga tenorei, an endemic Italian species

Abstract We report the first analysis in absolute, and in particular, concerning the phytochemical pattern, about an endemic Italian species, Ajuga tenorei C. Presl. The analysis, performed by means of techniques such as Column Chromatography and NMR spectroscopy and Mass spectrometry, led to the isolation and the identification of five compounds namely verbascoside (1), echinacoside (2), ajugoside (3), harpagide (4) and 8-O-acetylharpagide (5). The presence of these compounds is important from both chemotaxonomic and ethno-pharmacological point of view. For what concerns the first point is confirmed the correct botanical classification of the species. The isolated compounds are also known to exert peculiar pharmacological activities and their presence may give a rationale to the historical medicinal properties associated to the Ajuga genus in general, since these plants have a long traditional use in many parts of the world. Such fact might suggest the use of also this species in this sense.

Unusual molecular pattern in Ajugoideae subfamily: the case of Ajuga genevensis L. from Dolomites

Abstract We analysed the ethanolic extract from Ajuga genevensis L. (Lamiaceae) growing in Dolomites, part of Italian Alps. Three new compounds for this species were identified: rosmarinic acid (1), oleanolic acid (2) and maslinic acid (3), representative of two different classes of chemical compounds (phenylpropanoids and pentacyclic triterpenes). A. genevensis resulted to be a valuable source of these compounds endowed with interesting biological activities (i.e. antioxidant, neuroprotective, anti-inflammatory, antiproliferative). The recognition of compounds (1), (2) and (3) may also confirm the ethnomedicinal uses of this plant. From a chemotaxonomical point of view, it is worth noting that iridoids were not evidenced in this accession. Iridoids are considered chemotaxonomic marker in Lamiales, and, in contrast with a previous study on this species, the presence of aucubin was not confirmed. In addition, the presence of large amounts of rosmarinic acid (1) was unexpected for a species that does not belong to subfamily Nepetoideae.

Secondary metabolites, glandular trichomes and biological activity of Sideritis montana L. subsp. montana from central Italy

Sideritis montana subsp. montana is a small annual herb occurring in countries bordering the Mediterranean and Balkan regions. The secondary metabolism of this plant has not been fully explored so far. The aim of the present study was to understand the complex mixture of secondary metabolites and the type of secretory structures. The polar constituents were isolated by column chromatography from the ethanolic extract, and their structure was elucidated by NMR and MS. The essential oil was isolated by hydrodistillation and analysed by GC/MS. The plant indumentum was studied by light and scanning electron microscopy. To complete the work, the essential oil antioxidant activity and cytotoxicity on tumor cells were evaluated by DPPH, ABTS, FRAP, and MTT methods. Four different classes of secondary metabolites were isolated, namely flavonoids, caffeoylquinic derivatives, glycosidic hydroquinones and iridoids. The essential oil was mainly characterized by sesquiterpenene hydrocarbons. Peltate and long‐capitate hairs were the main sites where terpenes and polar constituents are produced. The secondary metabolites found in S. montana subsp. montana are of chemotaxonomic interest, some of them being typical of the genus Sideritis. The trichomes types observed partially differ from those described in other members of the genus Sideritis. The essential oil showed noteworthy inhibition on tumor cells.

Iridoids of Chemotaxonomy Relevance, a New Antirrhinoside Ester and Other Constituents from Kickxia spuria subsp. integrifolia (Brot.) R.Fern.

In this work, the secondary metabolite content of the EtOH extract of Kickxia spuria subsp. integrifolia (Brot.) R.Fern. is reported. Fourteen compounds were isolated and identified by means of column chromatography and NMR and MS instrumental techniques, respectively. Among the identified compounds, the chemotaxonomic markers of the species were evidenced, whereas others were reported for the first time in the genus. Among these, a new antirrhinoside derivative (12) was recognized. The iridoid content showed a molecular pattern very similar to those reported for other taxa comprised in the Antirrhinae tribe of Plantaginaceae, thus providing an additional evidence that supports the current botanical classification of the Kickxia genus. Anyway, most of the recognized components are able to exert important pharmacological properties which might suggest the possible employment of also this species in traditional medicine just like it happens for some other species of the Kickxia genus.