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.     

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)‐β‐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).     

Glandular trichomes and essential oil constituents ofCalamintha menthifolia (Lamiaceae)

Plant material ofCalamintha menthifolia was collected from two populations grown in the Vikos-Aoos area (NW. Greece). The structure and ontogeny of the glandular trichomes and the chemical composition of the secreted essential oil were studied. The leaves ofC. menthifolia have numerous glandular trichomes of three morphological distinct types: 1) glandular scales with a unicellular foot and stalk, and a 12-celled head, 2) glandular hairs with a unicellular foot and stalk, and a unicellular pear-shaped head and 3) glandular hairs with a unicellular foot, a bicellular stalk and a unicellular elongated head. Qualitative and quantitative GC-MS analyses of the essential oils revealed piperitone oxide as the main constituent in both populations. In the light of these results the relationships beweenC. menthifolia and other members of theSatureja group as well as its taxonomy are discussed.     

Phytomorphological and Essential‐Oil Characterization in situ and ex situ of Wild Biotypes of Oregano Collected in the Campania Region (Southern Italy)

Oregano is an aromatic species of great interest, which spreads spontaneously over the Mediterranean area, but its genetic resources are not yet adequately developed. Moreover, the results of studies of wild strains of different origin – although quite numerous – are not always comparable, and, therefore, the influence of the environment and genotype on the variability cannot be distinguished. Hence, the plant morphology and the essential‐oil composition of three wild, white‐flowering biotypes of oregano collected in the Campania region (southern Italy) were characterized, and the effects of genotype and environment were assessed by comparison in situ and ex situ. This allowed deducing that the biotypes belonged to two different subspecies, i.e., Origanum vulgare ssp. virens and O. vulgare ssp. viridulum. The essential‐oil yield was higher for the biotype belonging to ssp. virens, and it was significantly correlated with the glandular and stomatal density. The chemical composition of the oils obtained by hydrodistillation was found to be influenced by the genotype and the conditions of plant growth.     

冬凌草腺毛的形态学及组织化学研究

利用光学显微镜对药用植物冬凌草地上部分腺毛的形态、分布和组织化学进行了研究。结果表明:(1)冬凌草的叶表皮有3种形态显著不同的毛,即非腺毛、盾状腺毛和头状腺毛;盾状腺毛和头状腺毛均具1个基细胞、1个柄细胞和头部;成熟的盾状腺毛的头部一般由4个分泌细胞组成,而头状腺毛头部由2个分泌细胞组成。(2)组织化学鉴定结果显示:2种腺毛中均含有黄酮类成分,盾状腺毛中还含有单萜、倍半萜等萜类成分;冬凌草甲素可能只存在于盾状腺毛中,但需要更直接的证据证明。研究认为,高密度的盾状腺毛可以作为筛选冬凌草高甲素含量品种的一项重要依据。     

Phytochemical analysis of the labdanum-poor Cistus creticus subsp. eriocephalus (Viv.) Greuter et Burdet growing in central Italy

Phenolic constituents and essential oil from the aerial parts of Cistus creticus subsp. eriocephalus (Viv.) Greuter et Burdet growing in central Italy were analysed by HPLC-MSⁿ and GC–MS, respectively. Furthermore, six constituents were isolated by semipreparative HPLC from the methanol extract and their structures were determined on the basis of 1D and 2D NMR measurements as well as MS spectra. Isolated compounds were one new natural product, i.e. the shikimic acid ester 3,5-diihydroxy-4-(O-β-d-glucopyranosyl)-cyclohex-1-en-1-(O-β-d-glucopyranosyl)-ester (27), and six flavonoid glycosides, namely quercetin-3-O-β-D glucopyranoside (16), quercetin-3-O-rhamnoside (17), tricetin-4′-O-β-D glucopyranoside (24), tricetin-4′-O-β-D rutinoside (21), 3′-methoxy-quercetin-3-O-(3-β-Dglucopyranosyl-2-rhamnopyranosil-4-glucopyranosyl-2-rhamnopyranosil)-glucoside (25) and 3′,4′dimethoxyquercetin-3-O-rhamnopyranoside (26). GC–MS analysis of the essential oil highlighted the occurrence of aliphatic compounds, mainly fatty acids, whereas labdane-type compounds were very scant. Our results showed that C. creticus subsp. eriocephalus has a different chemical profile with respect to the other subspecies due to the lack of labdane derivatives. On the other hand, this subspecies contains several phenolic constituents like ellagitannins, gallotannins and flavonoids, some of which can be of chemotaxonomic value.     

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‐β‐D ‐allopyranosyl)‐β‐D ‐glucopyranosyl]oxy}‐5,8‐dihydroxy‐2‐(4‐methoxyphenyl)‐4H‐1‐benzopyran‐4‐one ( 1 ), 7‐{[6‐O‐acetyl‐2‐O‐(6‐O‐acetyl‐β‐D ‐allopyranosyl)‐β‐D ‐glucopyranosyl]oxy}‐2‐(3,4‐dihydroxyphenyl)‐5,8‐dihydroxy‐4H‐1‐benzopyran‐4‐one ( 2 ), and 7‐{[6‐O‐acetyl‐2‐O‐(β‐D ‐allopyranosyl)‐β‐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.     

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.     

Chemical composition and antimicrobial activity of the essential oil of endemic Dalmatian black pine (Pinus nigra ssp. dalmatica)

The chemical composition and the antimicrobial activity of the essential oil isolated from the needles of endemic Dalmatian black pine (Pinus nigra ssp. dalmatica) from Croatia were investigated. The chemical composition of the essential oil was determined by GC and GC/MS analyses, and the main compounds identified were α-pinene, β-pinene, germacrene D, and β-caryophyllene. Disc-diffusion and broth-microdilution assays were used for the in vitro antimicrobial screening. The Dalmatian black pine essential oil exhibited a great potential of antibacterial activity against Gram-positive bacteria (MIC=0.03-0.50% (v/v)) and a less pronounced activity against Gram-negative bacteria (MIC=0.12-3.2% (v/v)). The volatile compounds also inhibited the growth of all fungi tested, including yeast.     

Leaf essential oil composition of three species of Myrcianthes from Monteverde, Costa Rica

To examine the chemical diversity of essential oils from Myrcianthes species (Myrtaceae) as well as potential chemotaxonomic relationships between them, the leaf essential oils of M. fragrans (Sw.) McVaugh, M. rhopaloides (Kunth) McVaugh, and an undescribed species, Myrcianthes 'black fruit', from Monteverde, Costa Rica, were isolated by hydrodistillation and analyzed by GC/MS. The most abundant components of the essential oil of M. fragrans were 1,3,5-trimethoxybenzene (15.7%), (Z)-hex-3-en-1-ol (10.0%), alpha-cadinol (10.4%), eudesma-4(15),7-dien-1beta-ol (9.0%), caryophyllene oxide (7.8%), and spathulenol (7.5%). The leaf oils of two different samples of Myrcianthes rhopaloides were quantitatively different with one sample composed mostly of linalool (17.7%), alpha-cadinol (14.4%), spathulenol (11.1%), tau-cadinol (9.6%), and 1-epicubenol (6.9%), and the other was made up largely of (E)-hex-2-enal (46.1%), 1,8-cineole (12.5%), linalool (9.1%), alpha-cadinol (6.7%), and alpha-terpineol (4.4%). The major components in the leaf essential oil of Myrcianthes 'black fruit' were 1,8-cineole (38.3%), alpha-terpineol (21.2%), heptan-2-ol (15.5%), terpinen-4-ol (4.2%), and beta-pinene (3.8%). The leaf oil compositions of Myrcianthes in this study are very different from leaf oils from other members of Myrcianthes reported in the literature. A cluster analysis reveals large chemical variation not only between members of the genus, but also between samples of the same species.     

Chemical composition and antimicrobial activity of the essential oil of Teucrium ramosissimum (Lamiaceae)

The phytochemical composition of the essential oil of Teucrium ramosissimum (aerial parts), harvested in a mountainous region of Tunisia, was analyzed. A total of 68 compounds, accounting for 99.44% of the essential oil, were identified by GC and GC/MS. The major compounds were beta-eudesmol (61; 44.52%), caryophyllene oxide (56; 9.35%), alpha-thujene (1; 5.51%), sabinene (4; 4.71%), and T-cadinol (59; 3.9%). The essential oil, which is being used in Tunisian folk medicine against infectious diseases, was tested for its antimicrobial properties against five different bacteria, and found to have weak to moderate activity, with minimal-inhibitory-concentration (MIC) and minimal-bactericidal-concentration (MBC) values in the range 0.24-0.36 and 1.3-2.9 mg/ml, resp.     

Seasonal variation of the chemical constituents of the essential oil of Santolina etrusca from Italy

The seasonal variation of the essential oil, in terms of both yield and composition, of a population of Santolina etrusca (Asteraceae) is reported in detail for different aerial parts, including sterile branches, fertile branches, and capitula. The yields generally increased from November to June, and then decreased in August and September. The main constituents of the sterile branches were 1,8-cineole and viridiflorol. The oil from fertile branches was characterized by myrcene as principal compound, whereas the essential oil obtained from the capitula contained germacrene D as the main constituent. Several compounds, as identified by GC and GC/MS, were found to prevail during the cold season.     

Chemical composition and antibacterial activity of the essential oil of Drimys granadensis L.f. leaves from Colombia

Drimys granadensis L.f., a native plant from Colombia, has been included in the Vademecum de Plantas Medicinales de Colombia by the Instituto de Investigación de Recursos Biológicos Alexander von Humboldt, due to its widespread use in the folk medicine for the treatment of gastrointestinal ailments. The chemical composition of the essential oil of Drimys granadensis obtained by hydrodistillation of the leaves was analyzed by GC and GC/MS analyses. A total of 85 components were identified, with the major compounds germacrene D (1, 14.7%), sclarene (9.5%), a-cadinol (7.3%), longiborneol acetate (2, 6.3%), drimenol (4.2%), (Z)-β-ocimene (3, 4.2%), a-pinene (4, 3.2%), and β-elemene (5, 2.7%). The essential oil was also tested against eight bacteria using the Kirby-Bauer disk-diffusion method. Most of the Gram-positive bacteria tested were susceptible to the D. granadensis essential oil, while the Gram-negative bacteria tested were not.     

Anatomical studies of the secretory structures: Glandular trichomes and ducts, in Grindelia pulchella Dunal (Astereae, Asteraceae)

The ultrastructure of the glandular trichomes and secretory ducts of Grindelia pulchella was studied. Plastids, mitochondria and endoplasmic reticulum are involved in the secretory process of both, trichomes and ducts. A special tissue with “transfer cells” is associated with the duct epithelial cells. The secretion is produced in the transfer cells and then is transferred to the duct epithelial cells where it accumulates in the vacuoles. The occurrence of cavities within the cell walls of the trichome cells and duct epithelial cells is described. The secretion is accumulated between the cell wall and the cuticle of these cells. When the cuticle is broken the secretion is released. We conclude that granulocrine secretion operates in this species.     

Chemical composition, seasonal variation, and biosynthetic considerations of essential oils from Baccharis microdonta and B. elaeagnoides (Asteraceae)

The chemical composition and seasonal variation throughout one year of the essential oils from leaves of Baccharis microdonta and B. elaeagnoides, collected in Campos do Jordão, SP, were investigated. The composition of the latter species has been described for the first time. By GC (RI) and GC/MS analysis, 43 compounds were identified, and a predominance of oxygenated sesquiterpene derivatives was found in both species. The main components of the B. microdonta oils were elemol ( 31 ; 11.7–30.6%), spathulenol ( 34 ; 4.7–9.1%), β‐caryophyllene ( 19 ; 3.7–6.2%), and germacrene D ( 24 ; 2.9–12.2%), and those of the B. elaeagnoides oils were 34 (10.1–21.5%), viridiflorol ( 35 ; 3.6–18.4%), 24 (0.9–13.8%), and 19 (3.5–9.4%). The identified compounds were grouped according to their respective C‐skeletons, and the percentages of occurrence of the C‐skeletons in the essential oils of leaves collected in the four seasons allowed identifying the preferential accumulation of different types of C‐skeletons as well as the seasonal variation of the biosynthetic routes over the studied period.     

Composition of the essential oils in various organs at different developmental stages of Ammi visnaga (L.) Lam. from Tunisia

The composition of the essential oils isolated by hydrodistillation from various organs at different development stages of Ammi visnaga (L.) Lam. growing in Tunisia was determined by GC/MS analysis. In particular, the oil profiles of the leaves, stems, flower buds, roots, umbels, and fruits have been examined during the whole life cycle. The oil from the flowering aerial parts was characterized by a high content of isoamyl 2-methylbutanoate. After flowering and during desiccation and fructification, the umbels and fruits expressed a high content of linalool. The oils, extracted from the roots collected in the vegetatif, buds floral, and floral stages, were rich in monoterpene aldehydes, oxygenated monoterpenes, and monoterpene hydrocarbons. The highest level of non-terpene hydrocarbons was found at the flower-bud stage, represented by 61.3% of nonane. Among the monoterpenes, sabinene (12.5%) and β-pinene (8.5%) were identified in the flower buds.     

Salvia verbenaca L. essential oil: Variation of yield and composition according to collection site and phenophase

The effect of the collection sites and phenophase on yield and chemical composition of Salvia verbenaca essential oils was evaluated. The essential oil constituents were assessed by gas chromatography (GC) and gas chromatography–mass spectrometry (GC–MS). The highest essential oil yields were observed for samples of the higher semi-arid bioclimate and at the flowering period. Eighty-five volatile constituents were identified and their percentages varied significantly (p < 0.05) depending on the collection site and the phenological stage. According to the plants origin, essential oils were dominated by monoterpene hydrocarbons, oxygenated monoterpenes and sesquiterpene hydrocarbons. The monoterpene hydrocarbons (31.9%) predominate at the flowering stage whereas oxygenated sesquiterpenes (27.5%) at the early fruiting stage. The sesquiterpene hydrocarbons (28.2%) was the most represented chemical class at late fruiting. On the basis of GC-MS data, the major identified volatile constituents were viridiflorol (3.4–17.7%), α-pinene (0.7–15.9%), β-caryophyllene (1.0–15.3%) and p-cymene (1.3–14.2%). S. verbenaca contains a diversity of bioactive constituents which shows large variations as affected by the collection sites and phenophase.     

Chemical composition and antimicrobial activity of the essential oils of Anthospermum emirnense and Anthospermum perrieri (Rubiaceae)

The essential oils of Anthospermum emirnense Baker and Anthospermum perrieri Homolle ex Puff, obtained by hydrodistillation in 0.03 and 0.02% yield, respectively, were analyzed by GC/MS. In both cases, the major constituents consisted of sesquiterpene hydrocarbons and oxygenated sesquiterpenes. The two species showed an important qualitative similarity, with 40 compounds common to A. emirnense and A. perrieri, including β-elemene, trans-β-caryophyllene, caryophyllene oxide, and τ-cadinol, which were major components in both cases. When tested for antimicrobial activity, both essential oils showed similar profiles and exhibited interesting minimal-inhibitory-concentration (MIC) values towards Bacillus subtilis, Chryseobacterium indologenes, Flavimonas oryzihabitans, and Yersinia enterocolitica.     

Effects of variety and growth year on the essential oil properties of lavender (Lavandula angustifolia Mill.) and lavandin (Lavandula x intermedia Emeric ex Loisel.)

Essential oil (EO) properties of six L. angustifolia (LA) and two L. x intermedia (LI) cultivars were evaluated during summer harvest periods of 2017 and 2018, The cultivars were grown in Dörgicse, Hungary. According to our results, the essential oil (EO) content was highly dependent on the varieties in both experimental years (2017: p < 0.0001 and 2018: p < 0.0001). In 2017, the EO content values of LI accessions were all higher than those of the LA cultivars. However, in 2018, the mean EO content value of ‘Budakalászi’ (LA) (8.2 mL/100g) exceeded that of ‘Grosso’ (LI) (7.7 mL/100g). The EO of ‘Mailette’ (LA) and ‘Budakalászi’ (LA) contained the highest linalool proportions (52.9% and 47.0%) while ‘Beate’ (LA) showed the lowest (18.1%); and this latter variety was richest in linalyl-acetate (58.9%). LI varieties produced lower linalyl acetate and total ester ratios in comparison to the LA cultivars. Concerning EO composition, only the UK varieties ‘Hidcote Blue’ (LA) and ‘Munstead’ (LA) met the requirements of the Ph. Eur.This study provided evidence that the effect of growth year on the EO content was significant only in two varieties out of the eight. However, in the more rainy year 2018, the accumulation of linalool and linalyl acetate was significantly affected in several cases, where linalool proportions decreased and linalyl acetate ratios increased.