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).     

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.     

Chemical Composition and Antimicrobial Activity of the Essential Oil of Stachys officinalis (L.) Trevis. (Lamiaceae)

Characterization by GC‐FID and GC/MS analyses of the Stachys officinalis (L.) Trevis . essential oil obtained by hydrodistillation of the aerial parts allowed the identification of 190 components that represented 97.9% of the total oil content. The main constituents identified were germacrene D (19.9%), β‐caryophyllene (14.1%), and α‐humulene (7.5%). Terpenoids were by far predominant (89.4%), with sesquiterpene hydrocarbons (69.1%) and oxygenated sesquiterpenes (14.8%) being the most abundant compounds detected in the oil. Based on the present and previously published results, multivariate statistical comparison of the chemical composition of the essential oils was performed within the species. Principal component analysis (PCA) and agglomerative hierarchical clustering (AHC) of the data on the volatile profiles of S. officinalis taxa revealed no pronounced differences among the samples originated from the Balkan Peninsula. Additionally, the oil was screened for in vitro antibacterial and antifungal activity using the broth microdilution assay. The oil's best antimicrobial activities were obtained against the mold Aspergillus niger (minimal inhibitory (MIC) and minimal fungicidal (MFC) concentrations of 2.5 and 5.0 mg/ml, resp.) and the yeast Candida albicans (MIC and MFC of 5.0 mg/ml).     

Polar Constituents,Essential Oil and Antioxidant Activity of Marsh Woundwort (Stachys palustris L.)

Stachys palustris, well‐known as marsh woundwort, is a perennial herb growing in wet environments of Europe. Its tubers, leaves and seeds are eaten raw or cooked. Alike other Stachys species, the plant is also used as a traditional remedy. Despite S. palustris has been consumed for centuries, little is known about its chemical constituents. In this work the main secondary metabolites of S. palustris from Hungary and France have been analysed. From the plant ethanolic extracts, ethanoid glucosides, isoscutellarein derivatives, caffeoyl‐quinic acids and iridoids have been isolated and structurally characterized by NMR. The essential oils were analysed by GC/MS and showed (E)‐phytol, fatty acids and carbonylic compounds as the most abundant compounds. The radical scavenging capacity of plant ethanolic extracts, as evaluated by the DPPH assay, was noteworthy, with IC₅₀ values of 92.08 – 105.42 μg/ml.     

Chemical Diversity among the Essential Oils of Wild Populations of Stachys lavandulifolia Vahl (Lamiaceae) from Iran

The variation of the essential‐oil composition among ten wild populations of Stachys lavandulifolia Vahl (Lamiaceae), collected from different geographical regions of Iran, was assessed by GC‐FID and GC/MS analyses, and their intraspecific chemical variability was determined. Altogether, 49 compounds were identified in the oils, and a relatively high variation in their contents was found. The major compounds of the essential oils were myrcene (0.0–26.2%), limonene (0.0–24.5%), germacrene D (4.2–19.3%), bicyclogermacrene (1.6–18.0%), δ‐cadinene (6.5–16.0%), pulegone (0.0–15.1%), (Z)‐hex‐3‐enyl tiglate (0.0–15.1%), (E)‐caryophyllene (0.0–12.9), α‐zingiberene (0.2–12.2%), and spathulenol (1.6–11.1%). For the determination of the chemotypes and the chemical variability, the essential‐oil components were subjected to cluster analysis (CA). The five different chemotypes characterized were Chemotype I (germacrene D/bicyclogermacrene), Chemotype II (germacrene D/spathulenol), Chemotype III (limonene/δ‐cadinene), Chemotype IV (pulegone), and Chemotype V (α‐zingiberene). The high chemical variation among the populations according to their geographical and bioclimatic distribution imposes that conservation strategies of populations should be made appropriately, taking into account these factors. The in situ and ex situ conservation strategies should concern all populations representing the different chemotypes.     

Phytochemical diversity of Origanum vulgare L. subsp. vulgare (Lamiaceae) from Austria

This investigation was aiming at the phytochemical characterisation of Origanum vulgare subsp. vulgare from Austria. To study the qualitative and quantitative composition of essential oil compounds 374 individual plants were analysed via GC. The volatiles of Austrian O. vulgare subsp. vulgare were found to be complex mixtures of 53 mono- and sesquiterpenes. Among the sesquiterpenes β-caryophyllene, germacrene D, E-E-α-farnesene, germacrene D-4-ol and caryophyllene oxide were frequently present in higher amounts. The monoterpenes were mainly made up of sabinyl-compounds [mainly sabinene (up to 48.4%) and cis-sabinene hydrate (up to 57.8%)] and/or cymyl-compounds [mainly p-cymene (up to 49.7%), γ-terpinene (up to 21.5%) and carvacrol (up to 32.9%)] that were accompanied by usually smaller amounts of bornyl-compounds and acyclic compounds. Some exceptional (in O. vulgare rare) chemotypes were detected. The essential oil content of Austrian O. vulgare subsp. vulgare ranged between 0.1 and 1.8%. The content of rosmarinic acid was analysed by HPLC and ranged from 0.6 mg/g dry mass up to 37.2 mg/g dry mass. No arbutin could be detected in the analysed populations.     

Glandular trichomes and essential oil composition of endemic Sideritis italica (Mill.) Greuter et Burdet from central Italy

Sideritis italica (Mill.) Greuter et Burdet belongs to the Lamiaceae family and is endemic to Italy. The glandular trichomes (morphology, distribution, histochemistry, and ultrastructure) of the plant were studied for the first time, along with the chemical composition of the essential oils. Abundant non-glandular hairs and peltate (type A) and capitate (types B, C(1), and C(x)) glandular trichomes were observed both on the vegetative and reproductive organs. The histochemical procedures and the ultrastructural investigation enabled specific location of the main site of essential oil production mainly in type-A peltate hairs. Particular emphasis is given to the release mechanism of the secreted material in all of the types of glands, and the potential taxonomic value of the indumentum in the Lamiaceae family is briefly discussed. Essential oils were hydrodistilled from flowering aerial parts of S. italica, and 136 compounds (112 in flowerheads, 79 in vegetative parts) were identified. The quantitative prevalence of diterpenoids (43.4% in flowerheads and 22.3% in vegetative parts) was the most significant characteristic of the essential oil of S. italica that could be classified as a diterpene-rich essential oil according to the classification of Kirimer.     

Phytochemical Diversity of Murraya koenigii (L.) Spreng. from Western Himalaya

Murraya koenigii (L.) Spreng. (Rutaceae), commonly known as ‘curry leaf tree’, is a popular spice and condiment of India. To explore the diversity of the essential‐oil yield and aroma profile of curry leaf, growing wild in foot and mid hills of north India, 58 populations were collected during spring season. M. koenigii populations were found to grow up to an altitude of 1487 m in north India. Comparative results showed considerable variations in the essential‐oil yield and composition. The essential‐oil yield varied from 0.14 to 0.80% in shade‐dried leaves of different populations of M. koenigii. Analysis of the essential oils by GC and GC/MS, and the subsequent classification by statistical analysis resulted in four clusters with significant variations in their terpenoid composition. Major components of the essential oils of investigated populations were α‐pinene ( 2 ; 4.5–71.5%), sabinene ( 3 ; <0.05–66.1%), (E)‐caryophyllene ( 11 ; 1.6–18.0%), β‐pinene ( 4 ; <0.05–13.6%), terpinen‐4‐ol ( 9 ; 0.0–8.4%), γ‐terpinene ( 8 ; 0.2–7.4%), limonene ( 7 ; 1.1–5.5%), α‐terpinene ( 6 ; 0.0–4.5%), (E)‐nerolidol ( 14 ; 0.0–4.1%), α‐humulene ( 12 ; 0.6–3.5%), α‐thujene ( 1 ; 0.0–2.5%), β‐elemene ( 10 ; 0.2–2.4%), β‐selinene ( 13 ; 0.2–2.3%), and myrcene ( 5 ; 0.5–2.1%). Comparison of the present results with those in earlier reports revealed new chemotypes of M. koenigii in investigated populations from Western Himalaya. The present study documents M. koenigii populations having higher amounts of sabinene ( 3 ; up to 66.1%) for the first time.     

Chemical Composition of Volatiles; Antimicrobial,Antioxidant and Cholinesterase Inhibitory Activity of Chaerophyllum aromaticum L. (Apiaceae) Essential Oils and Extracts

The present study reports the chemical composition of the headspace volatiles (HS) and essential oils obtained from fresh Chaerophyllum aromaticum root and aerial parts in full vegetative phase, as well as biological activities of their essential oils and MeOH extracts. In HS samples, the most dominant components were monoterpene hydrocarbons. On the other hand, the essential oils consisted mainly of sesquiterpenoids, representing 73.4% of the root and 63.4% of the aerial parts essential oil. The results of antibacterial assay showed that the aerial parts essential oil and MeOH extract have no antibacterial activity, while the root essential oil and extract showed some activity. Both of the tested essential oils exhibited anticholinesterase activity (47.65% and 50.88%, respectively); MeOH extract of the root showed only 8.40% inhibition, while aerial part extract acted as an activator of cholinesterase. Regarding the antioxidant activity, extracts were found to be more effective than the essential oils.     

Secondary Metabolites from Pinus mugo Turra subsp. mugo Growing in the Majella National Park (Central Apennines,Italy)

In this study, we examined the composition regarding secondary metabolites of P. mugo Turra ssp. mugo growing in the protected area of Majella National Park, which is the southernmost station of the habitat of this species. Both the nonpolar and polar fractions were considered. In particular, the essential‐oil composition showed a high variety of compounds, and 109 compounds were detected, and 101 were identified, among which abietane‐type compounds have a taxonomic relevance. Abietanes were also isolated from the polar fraction, together with an acylated flavonol and a remarkably high amount of shikimic acid.     

Chemotaxonomic and Micromorphological Traits of Satureja montana L. and S. subspicata Vis. (Lamiaceae)

Satureja montana and S. subspicata are used as spice, pepper substitute, for preparing tea, juice, and as a medicine. Fourteen populations (seven per species) of Satureja montana L. and S. subspicata Vis . growing in Croatia were examined to determine the chemical composition of the essential oil (analyzed by GC‐FID and GC/MS), the content of macroelements (Na, K, Ca, Mg) and trace elements (B, Fe, Cu, Mn, Zn, Al, Pb, Cr, Cd, Ni, Hg, As) analyzed by ICP‐AES, antioxidant compounds (analyzed by UV/VIS spectrophotometer), and the types and distribution of trichomes (analyzed by scanning electron microscopy). The main constituents of the essential oil were carvacrol and thymol in S. montana (all populations belong to one phenol chemotype), while α‐eudesmol, β‐eudesmol, and spathulenol dominated in S. subspicata (three chemotypes could be distinguished). Both species possess considerably higher quantities of Ca and Mg, and moderate concentrations of K and Na, while Hg and As levels were below the limit of quantification. Non‐glandular trichomes, peltate trichomes, and three types of capitate trichomes were observed on leaves, stem, calyx, and corolla.     

Chemical Composition and Biological Studies of the Essential Oil from Aerial Parts of Beta vulgaris subsp. maritima (L.) Arcang. Growing in Tunisia

The chemical composition, antioxidant, cytotoxic, anticholinesterase and anti‐tyrosinase activities of the hydrodistilled essential oil of the aerial parts of Beta vulgaris subsp. maritime (L.) Arcang . from Tunisia have been evaluated. The chemical composition of the oil (yield 0.037% [w/w]), determined by GC‐FID and GC/MS is reported for the first time. Twenty five components, accounting for 98.1% of the total oil have been identified. The oil was characterized by a high proportion of oxygenated sesquiterpenes (39.2%), followed by sesquiterpene hydrocarbons (30.3%) and one apocarotenoids (26.3%). The main compounds were γ‐irone (26.3%), α‐cadinol (12.1%), T‐cadinol (10.6%), bicyclogermacrene (10.4%) and δ‐cadinene (6.0%). The isolated oil was tested for its antioxidant activity using the DPPH^·, ABTS^+·, catalase, and paraoxonase assays and also for its cytotoxic, anticholinesterase, and anti‐tyrosinase activities. The essential oil exhibited high antioxidant activity (IC₅₀ = 0.055 ± 0.006 mg/ml) and important result oncatalase (524.447 ± 2.58 Units/mg protein). Furthermore, it exerted a significant cytotoxic effect against A549 cell line, with IC₅₀ = 42.44 ± 1.40 μg/ml. The results indicate that the essential oil of B. vulgaris subsp. maritima (L.) Arcang . aerial parts may be used in future as an alternative to synthetic antioxidant agents, with potential application in the food and pharmaceutical industries.     

Chemical Composition and Biological Activities of the Essential Oil of Athanasia brownii Hochr. (Asteraceae) Endemic to Madagascar

The essential oil obtained from hydrodistillation of flowering aerial parts of Athanasia brownii (Asteraceae) was studied for its chemical composition by GC/FID and GC/MS, and for biological activity, namely, antioxidant, antimicrobial, and chemopreventive potential, by DPPH (=2,2‐diphenyl‐1‐picrylhydrazyl), ABTS (=2,2′‐azinobis[3‐ethylbenzothioline‐6‐sulfonic acid), and FRAP (=ferric reducing antioxidant power), disk diffusion test, and MTT (=3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) assay, respectively. The oil was characterized by a high content of oxygenated sesquiterpenes (71.2%), with selin‐11‐en‐4α‐ol (24.6%), caryophyllene oxide (8.7%), humulene epoxide II (5.1%), and (E)‐nerolidol (4.9%) as the predominant compounds. The oil showed a moderate activity against streptococci as well as radical‐scavenging potential, while the inhibitory effects against human cancer cells examined such as A375 (malignant melanoma) and HCT 116 (colon carcinoma) were significant, with IC₅₀ values of 19.85 and 29.53 μg/ml, respectively.     

Biochemical Characterization of Helichrysum italicum (Roth) G.Don subsp. italicum (Asteraceae) from Montenegro: Phytochemical Screening,Chemotaxonomy, and Antioxidant Properties

The chemical composition and antioxidant properties of the essential oil and EtOH extract of immortelle (Helichrysum italicum (Roth) G.Don subsp. italicum, Asteraceae) collected in Montenegro were evaluated. The essential oil was characterized by GC/MS analysis, and the content of total phenolics and flavonoids in the EtOH extract was determined using the Folin? Ciocalteu reagent. The free‐radical‐scavenging capacity (RSC) of both the essential oil and the EtOH extract was assessed with the 2,2‐diphenyl‐1‐pycrylhydrazyl (DPPH) method. Moreover, the inhibition of hydroxyl radical (^.OH) generation by the EtOH extract of immortelle was evaluated for the first time here. Neryl acetate (28.2%) and γ‐curcumene (18.8%) were the main compounds in the essential oil, followed by neryl propionate (9.1%) and ar‐curcumene (8.3%). The chemical composition of the oils of the examined and additional 16 selected Helichrysum italicum taxa described in literature were compared using principal component (PCA) and cluster (CA) analyses. The results of the statistical analyses implied the occurrence of at least four different main and three subchemotypes of essential oils. Considering the antioxidant properties, the EtOH extract of immortelle exhibited similar potential as propyl gallate and quercetin, while the essential oil exhibited relatively weak DPPH^.‐scavenging capacity.     

Chemical Variability and Biological Activities of Essential Oils of Micromeria inodora (Desf.) Benth. from Algeria

The chemical composition of the essential oils isolated from the aerial parts of Micromeria inodora (Desf .) Benth . collected in 24 Algerian localities was investigated from the first time using GC‐FID, GC/MS and ¹³C‐NMR. Altogether, 83 components which accounted for 94.7% of the total oil composition were identified. The main compounds were trans‐sesquisabinene hydrate ( 1 ; 20.9%), α‐terpinyl acetate ( 2 ; 19.8%), globulol ( 3 ; 4.9%), caryophyllene oxide ( 4 ; 4.3%), β‐bisabolol ( 5 ; 2.9%) and trans‐7‐epi‐sesquisabinene hydrate ( 6 ; 2.6%). Comparison with the literature highlighted the originality of the Algerian M. inodora oil and indicated that 1 might be used as taxonomical marker. The study of the chemical variability allowed the discrimination of two main clusters confirming that there is a relation between the essential‐oil compositions and the soil nature of the harvest locations. Biological activity of M. inodora essential oil was assessed against fourteen species of microorganisms involved in nosocomial infections using paper disc diffusion and dilution agar assays. The in vitro study demonstrated a good activity against Gram‐positive strains such as Staphylococcus aureus, Bacillus cereus, Bacillus subtilis, and Enterococcus faecalis, and moderate activity against Candida albicans. These results might be useful for the future commercial valorization of M. inodora essential oil as a promising source of natural products with potential against various nosocomial community and toxinic infections.     

Chemical Composition and Biological Activity of the Essential Oil of Origanum vulgare ssp. hirtum from Different Areas in the Southern Apennines (Italy)

The chemical composition of the essential oils of Origanum vulgare ssp. hirtum, growing wild in three different localities in the Southern Apennines, was studied by GC‐FID and GC/MS analyses. In total, 103 compounds were identified. The oils were mainly composed of phenolic compounds and all oils belonged to the chemotype carvacrol/thymol. The three essential oils were evaluated for their in vitro phytotoxic activity by determining their influence on the germination and initial radicle elongation of Sinapis arvensis L., Phalaris canariensis L., Lepidium sativum L., and Raphanus sativus L. The seed germination and radicle growth were affected in various degrees. Moreover, the antifungal activity of the three essential oils was assayed against three species causing pre‐ and postharvest fruit decay (Monilinia laxa, M. fructigena, and M. fructicola). At 1000 ppm, the three oils completely inhibited fungal growth. The hemolytic activity of the oils was assayed and showed no effect on the cell membranes of bovine erythrocytes.     

Chemical Composition and in vitro Biological Activities of Essential Oils from Conchocarpus fontanesianus (A. St.‐Hil.) Kallunki & Pirani (Rutaceae)

This study aimed at assessing the chemical composition of the essential oils from leaves and fruits of Conchocarpus fontanesianus, an endemic Brazilian species of Rutaceae. The plant material was harvested from two regions of the Atlantic rainforest in the State of São Paulo. The volatile compounds in the essential oils were extracted by hydrodistillation (HD), and analyzed by GC/FID and GC/MS, allowing the quantification and identification of 54 components in total, which comprise about 97% of the total oil composition. From the leaves collected in Caraguatatuba and Juréia‐Itatins, the major volatile compounds identified were as follows: spathulenol (22.32% and 16.67%) and α‐cadinol (9.7% and 14.76%). However, β‐myrcene (34.56%), (+)‐epi‐bicyclosesquiphellandrene (8.71%), and bicyclogermacrene (5.80%) were dominant in the fruits collected only in Juréia‐Itatins. The in vitro biological activities were tested to evaluate the cytotoxic, antifungal, and antioxidant potential of essential oils from leaves and fruits.