Localization of phenolic compounds in the fruits of Silybum marianum characterized by different silymarin chemotype and altered colour.

Silybum marianum (L.) Gaertn (Asteraceae) is a valuable medicinal plant utilized for silymarin production. However, only fragmentary and contradictory information about silymarin localization within S. marianum fruit are available. In this work, a twofold research approach was adopted in order to investigate the distribution and quantification of silymarin and of other phenolic compounds within the different fruit regions (pericarp, seed integument, cotyledon). Two S. marianum wild accessions with contrasting silymarin chemotype (A and B) and a mutant line (C) with an altered fruit colour were analysed. Fruits of Cynara cardunculus were studied as a reference. Firstly, the fruit morpho-anatomy was reviewed by means of light microscopy digital imaging and, secondly, a comprehensive histolocalization of the different classes of polyphenols within the fruit was carried out. The experimental evidences confirmed that silymarin, and its precursor taxifolin, are only accumulated in the seed integuments. The dark colour of fully-ripened fruits is due to the accumulation of condensed tannins in the pericarp subepidermal cell layer. On the contrary, the studied mutant line shows reduced condensed tannin content that probably result from impairment at the level of flavonoid biosynthetic pathway. Condensed tannins content is comparatively low in S. marianum fruits and very low in the identified mutant line. This could represent an advantage for the possible employment of S. marianum fruits and of silymarin extraction by-products in the feed and food sector.

Secretory structures and essential oil composition in Stachys officinalis (L.) Trevisan subsp. officinalis (Lamiaceae) from Italy.

The secretory structures and the volatile fraction of Stachys officinalis (L.) Trevisan subsp. officinalis (Lamiaceae) from Italy were studied for the first time. Peltate and small capitate trichomes were observed on the whole plant (leaves and inflorescences). In the peltate trichomes, an unusual polyphenols content was evidenced by the histochemical methods. The volatile fraction was obtained by a solvent extract from the distillation water of leaves and inflorescences and analysed by GC-MS. Forty-four constituents for leaves, representing 94.1% of the total volatiles, and 57 compounds for flowers, accounting for 90.1% of the total volatiles, were identified. (E)-caryophyllene (20.1%), (E)-nerolidol (14.3%), caryophyllene oxide (6.1%) and γ-cadinene (5.7%) were recognised as the main constituents for the leaf volatile fraction, while caryophyllene oxide (16.5%), (E)-nerolidol (15.4%), humulene epoxide II (9.2%) and α-pinene (7.0%) were the main compounds for the flower volatile fraction.

Phytochemical Analysis, Biological Activity, and Secretory Structures of Stachys annua (L.) L. subsp. annua (Lamiaceae) from Central Italy.

Stachys annua subsp. annua, well-known in central Italy as 'stregona annuale', is an annual, small, slightly-scented herb, commonly found in fields and uncultivated areas in almost all regions of Italy. In folk medicine, its aerial parts were used as anti-catarrhal, febrifuge, tonic, and vulnerary. In the present work, the chemical composition of the flowering aerial parts was studied. The hydrodistilled volatile oil, analysed by GC/MS, showed sesquiterpenoids as the major fraction (42.5%); phytol (9.8%), germacrene D (9.2%), and spathulenol (8.5%) were the most abundant constituents. The volatile oil was assayed for antioxidant and cytotoxic activity by DPPH, ABTS, FRAP, and MTT methods. The cytotoxicity results against HCT116, A375, and MDA-MB 231 human tumor cell lines were significant, with IC50 values of 23.5, 37.2, and 41.5 µg/ml, respectively, whereas the antioxidant power was negligible. The EtOH extract was composed mainly of three glycosidic flavonoids, namely 7-{[2-O-(6-O-acetyl-ß-D-allopyranosyl)-ß-D-glucopyranosyl]oxy}-5,8-dihydroxy-2-(4-methoxyphenyl)-4H-1-benzopyran-4-one (1), 7-{[6-O-acetyl-2-O-(6-O-acetyl-ß-D-allopyranosyl)-ß-D-glucopyranosyl]oxy}-2-(3,4-dihydroxyphenyl)-5,8-dihydroxy-4H-1-benzopyran-4-one (2), and 7-{[6-O-acetyl-2-O-(ß-D-allopyranosyl)-ß-D-glucopyranosyl]oxy}-2-(3-hydroxy-4-methoxyphenyl)-5,8-dihydroxy-4H-1-benzopyran-4-one (3). On the contrary, iridoids, considered chemotaxonomic markers of the genus Stachys, were absent in this species. Finally, the morphological and histochemical survey showed that glandular trichomes were composed of two main types, i.e. peltate type A and capitate types B and C giving positive response for both lipids and polyphenols.

Characterization of secondary metabolites, biological activity and glandular trichomes of Stachys tymphaea Hausskn. from the Monti Sibillini National Park (Central Apennines, Italy).

Stachys tymphaea (Lamiaceae) is a perennial herb growing in forest openings and dry meadows of central and southern Italy. It was investigated for the first time here, determining the content of secondary metabolites, the micromorphology of glandular trichomes, the histochemical localization of secretion, and the biological activity of the volatile oil, namely, the cytotoxic, antioxidant, and antimicrobial properties. The plant showed a peculiar molecular pattern, being rich of biophenolic compounds as flavonoids, phenylethanoid glycosides, and caffeoylquinic acid derivatives, but poor of iridoids, which are known as marker compounds of the genus Stachys. The essential oil was characterized by GC-FID and GC/MS analyses, revealing a high percentage of sesquiterpene hydrocarbons (54.6%), with germacrene D (30.0%) and (E)-ß-farnesene (12.4%) as the most abundant compounds, while other main components were representatives of the diterpenes (19.2%), represented mainly by (E)-phytol (11.9%). This composition supported the taxonomic relationships in the genus Stachys, which comprises oil-poor species producing essential oils rich in hydrocarbons, with germacrene D as one of the predominant components. The micromorphological study revealed three types of glandular hairs, i.e., Type A peltate trichomes, being the primary sites of essential oil biosynthesis, Type B short-stalked trichomes, typical mucopolysaccharide producers, and Type C long capitate trichomes, secreting a complex mixture of both lipophilic and hydrophilic substances, with a major phenolic fraction. Moreover, the MTT assay revealed the potential of the volatile oil to inhibit A375, HCT116, and MDA-MB 231 tumor cells lines (IC50 values of 23.9-34.4 µg/ml).

Phytochemical analysis, biological evaluation and micromorphological study of Stachys alopecuros (L.) Benth. subsp. divulsa (Ten.) Grande endemic to central Apennines, Italy.

Stachys alopecuros subsp. divulsa (Lamiaceae), a perennial herb endemic to central Italy growing on mountain pastures, was investigated for the first time for the content of secondary metabolites, for the micromorphology and histochemistry of glandular trichomes, and for the biological activity of the volatile oil, namely cytotoxic, antioxidant and antimicrobial. The plant showed the molecular pattern of iridoids, among which a new iridoid diglycoside (4'-O-ß-D-galactopyranosyl-teuhircoside) was detected, together with a sterol glucoside and a phenylethanoid glycoside. The essential oil from the flowering aerial parts was characterized by a high proportion of sesquiterpene hydrocarbons (65.1%), with (E)-caryophyllene (33.2%) as the most abundant, while other main components were germacrene D (7.6%), α-humulene (6.4%) and the oxygenated cis-sesquisabinene hydrate (10.2%). Taken together, polar and apolar chemical profiles support the classification of the species within the section Betonica of the genus Stachys. Micromorphological study revealed three types of glandular hairs secreting different classes of compounds, with type A peltate hairs producing the bulk of the essential oil. MTT assay revealed the potential of the volatile oil in inhibiting A375, HCT116 and MDA-MB 231 tumor cells (IC50 values below 20 µg/ml).

Congruence of phytochemical and morphological profiles along an altitudinal gradient in Origanum vulgare ssp. vulgare from Venetian Region (NE Italy).

Plants of Origanum vulgare L. ssp. vulgare from the Veneto region (NE Italy) were selected to study the variability of the essential-oil composition from leaves and inflorescences throughout an elevation gradient. We investigated also the morphology of non-glandular and glandular trichomes, their distribution on the vegetative and reproductive organs, as well as the histochemistry of the secreted products, with special focus on the terpenoidic fraction. Since glandular trichomes are prerequisite for the essential-oil synthesis, the second objective was to establish whether its production is related to glandular hair number, and density. Essential-oil contents decline with increasing altitude, and the micromorphological observations revealed a decrease in trichome density along the same direction. Moreover, GC/MS analysis together with principal component analysis (PCA) showed that the three investigated populations were significantly different in chemical composition. Therefore, an important interpopulation variability for low-, mid-, and high-altitude sites was established, suggesting the likely occurrence of different biotypes associated with altitudinal levels. Hence, the involvement of abiotic factors such as temperature and drought in the chemical polymorphism of O. vulgare associated with elevation is briefly discussed.

The role of secreting structures position on the leaf volatile organic compounds of Hypericum androsaemum.

Hypericum androsaemum L. presents typical translucent, essential oil producing glands, which are distributed on the leaf along both margins (margin glands) and on the lamina (lamina glands). The gland secretion was studied by histochemical and chemical analysis; the gland content was sampled directly from the secretory glands, and the volatile organic compounds (VOC) of the margin and lamina glands were separately analyzed. The lipophilic fraction of the lamina glands had as main components: (E)-2-hexenal (15.5%), hexadecanoic acid (14.7%), beta-caryophyllene (11.2%), germacrene B (11.0%) and gamma-himachalene (9.8%). The lipophilic fraction of the margin glands had as its main components: beta-pinene (22.0%), limonene (17.6%), (E)-beta-ocimene (6.1%), methyl linoleate (5.7%), terpinolene (5.4 %), (E)-2-hexenal (4.9%) and alpha-pinene (4.1%).