Leaves micromorphology, chemical profile, and bioactivity of in vitro-propagated Nepeta cyrenaica (Lamiaceae).

INTRODUCTION: The endemic species Nepeta cyrenaica Quézel & Zaffran, native to northeastern Libya, is valued as an important honey-bearing plant. OBJECTIVES: This study was aimed to examine the micromorphology, phytochemistry, and bioactivity of in vitro-propagated N. cyrenaica for the first time. MATERIALS AND METHODS: The leaf indumentum was examined using light and scanning electron microscopy and further characterised for histochemistry. The chemical composition of essential oil (EO) was performed using GC-MS analysis, while dichloromethane (DCM), methanol (ME), ethanol (ET), and aqueous (AQ) extracts were analysed using qualitative and quantitative LC/MS analyses. The antioxidant activities of EO and extracts were assessed using three parallel assays, while enzyme-inhibiting effects were evaluated against four enzymes. RESULTS: The leaves bear various types of glandular trichomes, with lipophilic secretion predominating. The main EO component of EO was 1,8-cineole. A considerable number of phenolics and iridoids were tentatively identified in the ME extract. Quantitative LC/MS analysis confirmed that ferulic acid, rosmarinic acid, and epigallocatechin gallate were present in the highest amount in the extracts, in which three iridoids were also quantified. Although the ME extract contained the highest amount of polyphenolics and iridoids, the DCM extract showed the best overall biological potential. Additionally, EO exerted the strongest acetylcholinesterase and tyrosinase inhibition. CONCLUSION: This study demonstrated that the endemic N. cyrenaica can be efficiently grown under in vitro conditions, where it develops various glandular trichomes that are thought to secrete and/or accumulate bioactive compounds with valuable medicinal potential.

A Study of Phytochemistry, Genoprotective Activity, and Antitumor Effects of Extracts of the Selected Lamiaceae Species.

This study was designed to evaluate the genoprotective, antigenotoxic, as well as antitumor potential of methanolic, ethanolic, and aqueous extracts of Melissa officinalis, Mentha × piperita, Ocimum basilicum, Rosmarinus officinalis, Salvia officinalis, and Satureja montana (Lamiaceae), in different model systems. The polyphenols in these extracts were quantified both spectrophotometrically and using HPLC-DAD technique, while DPPH assay was used to assess the antioxidant activity. The genoprotective potential was tested on pUC19 Escherichia coli XL1-blue, and the antigenotoxicity on Salmonella typhimurium TA1535/pSK1002 and human lung fibroblasts, while the antitumor activity was assessed on colorectal cancer cells. Rosmarinic acid, quercetin, rutin, and luteolin-7-O-glucoside were among the identified compounds. Methanolic extracts had the best DPPH-scavenging and SOS-inducing activities, while ethanolic extracts exhibited the highest antigenotoxicity. Additionally, all extracts exhibited genoprotective potential on plasmid DNA. The antitumor effect was mediated by modulation of reactive oxygen species (ROS), nitric oxide (NO) production, and exhibition of genotoxic effects on tumor cells, especially with O. basilicum ethanolic extract. Generally, the investigated extracts were able to provide antioxidant protection for the acellular, prokaryotic, and normal human DNA, while also modulating the production of ROS and NO in tumor cells, leading to genotoxicity toward these cells and their decrease in proliferation.

Bis-Bibenzyls from the Liverwort Pellia endiviifolia and Their Biological Activity.

Based on previous investigations where bis-bibenzyls isolated from liverworts showed various biological activities (cytotoxic, antimicrobial, and antiviral), we investigated their cytotoxic activity in several human cancer cell lines. From the methylene-chloride/methanol extract of the liverwort Pellia endiviifolia, three bis-bibenzyls of the perrottetin type were isolated, namely perrottetin E, 10'-hydroxyperrottetin E, and 10,10'-dihydroxyperrottetin E. The last two were found for the first time in this species. Their structures were resolved using 1D and 2D NMR, as well as by comparison with data in the literature. Cytotoxic activity of the isolated compounds was tested on three human leukemia cell lines, HL-60 (acute promyelocytic leukemia cells), U-937 (acute monocytic leukemia cells), and K-562 (human chronic myelogenous leukemia cells), as well as on human embryonal teratocarcinoma cell line (NT2/D1) and human glioblastoma cell lines A-172 and U-251, and compared to the previously isolated bis-bibenzyls (perrottetins) of similar structure. The isolated compounds exhibited modest activity against leukemia cells and significant activity against NT2/D1 and A-172. Overall, the most active cytotoxic compounds in this investigation were perrottetin E (1), isolated in this work from Pellia endiviifolia, and perrottetin F phenanthrene derivative (7), previously isolated from Lunularia cruciata and added for a comparison of their cytotoxic activity.

Essential Oil Composition and Micromorphological Traits of Satureja montana L., S. subspicata Bartel ex Vis., and S. kitaibelii Wierzb. Ex Heuff. Plant Organs.

The essential oil (EO) composition of Satureja plants is highly variable. Recent studies suggest that there is an even difference in the EO composition from different plant organs within the same plant. This study aims to examine the chemical profile of EOs and the micromorphological characteristics of different organs of three Satureja species. The relationship between the number of glandular trichomes and EOs profile and relative yield is also investigated. Individuals from five populations were visualized using a scanning electron microscope, while EOs of leaves, calyces, corollas, and whole aerial parts were isolated using simultaneous distillation and extraction and analyzed using gas chromatography/mass spectrometry. Three types of glandular trichomes were detected. Peltate trichomes were present on all plant organs of studied species, while two types of capitate trichomes show different organ and species preferences. The EOs profiles differed across the plant parts, but showed a species specific composition. Univariate and multivariate statistics were used to show a correlation between the peltate trichomes and EO yield, and chemophenetic significance of EO profiles.

A comprehensive assessment of the chemical composition, antioxidant, genoprotective and antigenotoxic activities of Lamiaceae species using different experimental models in vitro.

This research was aimed to assess the potential of Glechoma hederacea, Hyssopus officinalis, Lavandula angustifolia, Leonurus cardiaca, Marrubium vulgare and Sideritis scardica (Lamiaceae) methanolic, ethanolic and aqueous extracts against the damaging effects of oxidative stress using different experimental models. The chemical characterization was done spectrophotometrically by quantifying total phenolics, phenolic acids, flavonoids and flavonols in the extracts, as well as by employing HPLC-DAD technique. Moreover, DPPH assay was used to assess the extracts' radical scavenging potential. Genoprotective properties of the extracts were evaluated using plasmid pUC19 Escherichia coli XL1-Blue, whereas their antigenotoxic potential was determined using Salmonella typhimurium TA1535/pSK1002 and normal human lung fibroblasts. All of the extracts showed antioxidant activity in DPPH assay. Furthermore, the results have shown that aqueous extracts provided the best protection for plasmid DNA, while alcoholic extracts most effectively contributed to the preservation of prokaryotic DNA. Additionally, each of the tested samples significantly protected the eukaryotic cells against genomic damages. Finally, despite not showing exceptional results in DPPH assay, S. scardica extracts are regarded as the most favorable in maintaining the integrity of DNA, which might be due to high quantities of phenolics such as quercetin (up to 17.95 mg g-1), naringin (up to 5.07 mg g-1) and luteolin-7-O-glucoside (up to 3.54 mg g-1). Overall, this comprehensive concept highlights the ability of these Lamiaceae species to safeguard the DNA from reactive oxygen species, to curtail the inflicted damage and also improve the efficiency of the DNA repair mechanisms, while emphasizing the importance of polyphenols as their active principles.

Lamiaceae in the treatment of cardiovascular diseases.

Lamiaceae (Labiatae) are an important group of medicinal plants, which have been used for treating heart disease in traditional medicine for centuries. These mainly aromatic plants are used as essential oils, extracts or isolated components (polyphenols, phenolic compounds, terpenes, iridoids etc.). Some Labiatae species (more than 30, such as cornmint, lavender, patchouli, rosemary etc.) are famous for their use in essential oil production worldwide. In this review, cardioprotective effects of Lamiaceae and their active secondary metabolites, as well as mechanism of action against cardiovascular diseases (hypertension, angina pectoris, hyperlipidemia, thromboembolism, coronary heart disease, heart failure, venous insufficiency, arrhythmia) will be discussed. Use of Labiatae as food or food additives (such as spices) may prevent risk of cardiovascular diseases, diabetes and cancer. This approach is also described as a part of the article. Studies on developing new, effective and safe natural products from Lamiaceae (rich source of flavonoids and other active compounds) are promising and may offer prevention and treatment for patients with coronary disease and other related diseases.

The Healing Effects of Spices in Chronic Diseases.

Spices are not only just herbs used in culinary for improving the taste of dishes, they are also sources of a numerous bioactive compounds significantly beneficial for health. They have been used since ancient times because of their antimicrobial, anti-inflammatory and carminative properties. Several scientific studies have suggested their protective role against chronic diseases. In fact, their active compounds may help in arthritis, neurodegenerative disorders (Alzheimer's, Parkinson, Huntington's disease, amyotrophic lateral sclerosis, etc.), diabetes, sore muscles, gastrointestinal problems and many more. In the present study, possible roles of spices and their active components, in chronic diseases (cancer, arthritis, cardiovascular diseases, etc.) along with their mechanism of action have been reviewed.

Juniperus phoenicea var. turbinata (Guss.) Parl. Leaf Essential Oil Variability in the Balkans.

In the present work, the leaf essential oil from 97 individuals of Juniperus phoenicea var. turbinata (Guss.) Parl. from the Balkan Peninsula was analyzed. The essential oil was dominated by monoterpene hydrocarbons (45.5 - 71.8%), of which α-pinene was the most abundant in almost all of the samples (38.2 - 55.8%). Several other monoterpenes and sesquiterpenes were also present in relatively high abundances in samples such as myrcene, δ-3-carene, ß-phellandrene, α-terpinyl acetate, (E)-caryophyllene and germacrene D. Multivariate statistical analysis suggested the existence of three possible chemotypes based on the abundance of the four components. Even though the intrapopulation variability was high, discriminant analysis (DA) was able to separate populations. DA showed high separation between western and eastern populations but also grouped geographically closer populations along the west Balkan shoreline. The potential influence of the climate on the composition of the essential oil was also studied.

Short communication: Cheese supplemented with Thymus algeriensis oil, a potential natural food preservative.

The essential oil of Thymus algeriensis was analyzed as a potential preservative in soft cheese. We developed a novel method to test the preserving properties of essential oil in soft cheese. Contamination incidence of Penicillium aurantiogriseum was absent after 30 d of storage at 4°C with 25 µL of essential oil added. The antimicrobial activity was tested against 8 bacteria and 8 fungi. Thymus algeriensis oil showed inhibitory activity against tested bacteria at 0.03 to 0.09 mg/mL, and bactericidal activity was achieved at 0.05 to 0.15 mg/mL. For antifungal activity, minimum inhibitory concentrations ranged between 0.01 and 0.04 mg/mL and minimum fungicidal concentrations between 0.01 and 0.04 mg/mL. Furthermore, the oil was also screened for antiradical activity using the 1,1-diphenyl-2-picrylhydrazyl assay. The results showed that the oil was active and achieved half-maximal inhibitory activity at 0.132 mg/mL. We used gas chromatography, gas chromatography-mass spectrometry, and nuclear magnetic resonance spectrometry to investigate the volatile compounds from the oil. Carvacrol was identified as the main compound in the oil, represented by 80.9% of the total constituents, followed by p-cymene (7.7%).

Research on Chemical Composition and Biological Properties Including Antiquorum Sensing Activity of Angelica pancicii Vandas Aerial Parts and Roots.

The essential oil, different extracts, and isolated compounds of Angelica pancicii Vandas (Apiaceae) were investigated for the first time. The GC-FID and GC-MS analyses revealed sesquiterpenoids as the main constituents of A. pancicii essential oil of aerial parts with bornyl acetate (8.08%), n-octanol (5.82%), kessane (4.26%), and ß-selinene (4.26%) as the main constituents. Analysis of methanol extracts, using an HPLC-DAD/ESI-ToF-MS system, showed a total of 52 compounds in the aerial parts and 53 in the roots, indicating coumarins as the main constituents. In addition, new chromone (1) and six known furanocoumarins (2-7) were isolated from the roots and structurally elucidated by combined spectroscopic methods. The aerial part extracts exhibited higher polyphenolic contents and antioxidant activity evaluated by three radical scavenging assays. Using a microwell dilution method, the strongest antibacterial activity profiles were determined for ethanol and methanol root extracts (minimum bactericidal concentrations (MBCs) = 0.25-3.00 mg/mL), which were comparable to the activity of streptomycin (MBCs = 0.34-1.24 mg/mL), while the strongest antibacterial compound of A. pancicii was oxypeucedanin hydrate (MBCs = 0.50-8.00 mg/mL). Antifungal potential was in moderate extent, and the highest activity was obtained for root methanol extract (minimum fungicidal concentrations (MFCs) = 4.00-14.00 mg/mL). Tested sub-minimum inhibitory concentrations (subMICs) of the extracts and isolated compounds inhibited selected Pseudomonas aeruginosa PAO1 virulence determinants. The most reduced growth of P. aeruginosa colony was in the presence of isolated oxypeucedanin. Ethanol (17.36-46.98%) and methanol (34.54-52.43%) root extracts showed higher anti-biofilm activity compared to streptomycin (49.40-88.36%) and ampicillin (56.46-92.16%).

Heracleum orphanidis: chemical characterisation, and comparative evaluation of antioxidant and antimicrobial activities with specific interest in the influence on Pseudomonas aeruginosa PAO1.

GC-FID and GC-MS were used to determine the chemical composition of the essential oil of the aerial parts of Heracleum orphanidis Boiss. Also, the HPLC-DAD/ESI-ToF-MS profile of the methanol extracts of aerial parts and roots was determined. The main components of the essential oil were n-octanol, octyl hexanoate and n-octyl acetate, while coumarins were the most prevalent compounds in methanol extracts. An evaluation of antioxidant activity showed that the methanol and aqueous extracts of the aerial parts had the highest potential. In terms of antimicrobial activity determined by microdilution assays, the essential oil and methanol extract of the roots showed the greatest effectiveness. The colonies of Pseudomonas aeruginosa PAO1 treated with H. orphanidis samples produced less toxic pyocyanin, showed lower twitching and flagella mobility and biofilm formatting was reduced. The analyses in this study showed the considerable biological potential of H. orphanidis considering free radicals and various pathogenic strains, including the wild type of P. aeruginosa.

Essential Oil Composition of Centaurea atropurpurea and Centaurea orientalis Inflorescences from the Central Balkans - Ecological Significance and Taxonomic Implications.

The essential oil composition of Centaurea atropurpurea and Centaurea orientalis flowering heads (capitula) from Central Balkans have been determined by GC-FID and GC/MS analyses. In total, 121 compounds were identified, representing on average 97.7% of the oil composition. In all samples, sesquiterpenes were most abundant group, representing 53.9 - 74.0% of the total oil. Sesquiterpene hydrocarbons dominated in all studied populations of C. orientalis and C. atropurpurea, except C. atropurpurea f. flava in which essential oil was characterized with high level of oxygenated sesquiterpenes. The dominant components differed between species, and also between typical C. atropurpurea and C. atropurpurea f. flava. The most abundant compounds of essential oil of C. orientalis were germacrene D and α-cadinol. In C. atropuruprea, germacrene D and ß-caryophyllene were the most abundant, while caryophyllene oxide and ß-caryophyllene were dominant in C. atropurpurea f. flava oil. Taxonomical and ecological implications are further discussed.

Micromeria thymifolia Essential Oil Suppresses Quorum-sensing Signaling in Pseudomonas aeruginosa.

The chemical composition, antimicrobial and antiquorum sensing activity of the essential oil of Micromeria thymifolia (Scop.) Fritsch were investigated. Limonene, piperitone epoxide and piperitenone epoxide were found as the main constituents using a gas chromatography-mass spectrometry technique. In vitro antimicrobial activity of the oil was tested against six bacterial and seven fungal strains and high antimicrobial potential was noticed. Minimum inhibitory concentration varied from 0.031 mg/mL to 0.5 mg/mL for bacterial and 0.062 mg/mL to 0.5 mg/mL for fungal strains. The antiquorum properties of the essentidl oil were evaluated on Pseudomonas aeruginosa PAO 1. The oil was tested at subMIC concentrations for anti-quorum sensing activity. The analyses on quorum-sensing functions have been carried out by evaluating twitching and swarming of bacterial cultures and the total amount of pyocyanin production produced by P. aeruginosa. This study showed that M thymifolia essential oil exhibited antiquorum sensing activity and may be used as an antipathogenic drug.

Essential-oil variability of Juniperus deltoides R.P.Adams along the east Adriatic coast - how many chemotypes are there?

The composition of the essential oils isolated from twigs of ten Juniperus deltoides R.P. Adams populations from the east Adriatic coast was determined by GC-FID and GC/MS analyses. Altogether, 169 compounds were identified, representing 95.6-98.4% of the total oil composition. The oils were dominated by monoterpenes (average content of 61.6%), which are characteristic oil components of species of the Juniperus section. Two monoterpenes, α-pinene and limonene, were the dominant constituents, comprising on average 46.78% of the essential oils. Statistical methods were deployed to determine the diversity of the terpene classes and the common terpenes between the investigated populations. These statistical analyses revealed the existence of three chemotypes within all populations, i.e., a α-pinene, limonene, and limonene/α-pinene type.

Essential-oil composition of the needles collected from natural populations of Macedonian pine (Pinus peuce Griseb.) from the Scardo-Pindic mountain system.

The needle-terpene profiles of two natural Pinus peuce populations from the Scardo-Pindic mountain system (Mt. Osljak and Mt. Pelister) were analyzed. Among the 90 detected compounds, 87 were identified. The dominant constituents were α-pinene (45.5%), germacrene D (11.1%), ß-pinene (10.8%), and camphene (10.3%). The following eight additional components were found to be present in medium-to-high amounts (0.5-10%): bornyl acetate (5.0%), ß-phellandrene (3.4%), ß-caryophyllene (2.9%), ß-myrcene (0.9%), germacrene D-4-ol (0.9%), tricyclene (0.7%), (E)-hex-2-enal (0.7%), and bicyclogermacrene (0.6%). Although the general needle-terpene profiles of the populations from Mt. Osljak and Mt. Pelister were found to be similar to those of the populations from Zeletin, Sjekirica, and Mokra Gora (Dinaric Alps), principle component analysis (PCA) of eight terpenes (α-pinene, ß-myrcene, α-terpinolene, bornyl acetate, α-terpinyl acetate, ß-caryophyllene, trans-ß-farnesene, and germacrene D) in 139 tree samples suggested a divergence between the two population groups, i.e., the samples from the Scardo-Pindic mountain system and those from the Dinaric Alps. Genetic analysis of the ß-pinene content demonstrated a partial divergence between the two geographical groups. The profiles of both population groups differed from those published for populations from the Balkan-Rhodope mountains system (literature results), which were characterized by high contents of bornyl acetate and citronellol (Greek populations) or δ-car-3-ene (Bulgarian populations).

Satureja horvatii essential oil: in vitro antimicrobial and antiradical properties and in situ control of Listeria monocytogenes in pork meat.

The dominant compounds in Satureja horvatii oil were p-cymene (33.14%), thymol (26.11%) and thymol methyl ether (15.08%). The minimum inhibitory concentration (MIC) varied from 0.03 to 0.57 mg/mL for bacteria, and from 0.56 to 2.23 mg/mL for yeast strains, while minimum bactericidal/yeast-cidal concentration (MBC/MYC) varied from 0.07 to 1.15 mg/mL and 1.11 to 5.57 mg/mL for bacteria and yeasts, respectively. The antiradical potential of the essential oil was evaluated using hydroxyl radical (•OH) generated in Fenton reaction. The meat preserving potential of essential oil from Satureja horvatii was investigated against L. monocytogenes. Essential oil successfully inhibited development of L. monocytogenes in pork meat. Sensorial evaluation on flavor and color of meat was performed. The color and flavor of meat treated with essential oil improved after 4 days of storage. S. horvatii essential oil can act as a potent inhibitor of food spoiling microorganisms, in meat products and also can be a useful source of natural antioxidants.

Essential oil composition of Cachrys cristata--a rare and endangered species in the flora of Serbia.

Cachrys cristata DC. (Apiaceace) is a rare and critically endangered species in the flora of Serbia. Essential oil isolated by hydrodistillation from the aerial parts and fruits of this species was analyzed using combined GC and GC/MS. The oil from the aerial parts was rich in sesquiterpenes (45.7%) and oxygenated sesquiterpenes (32.9%), while the fruit oil consisted of a higher percentage of sesquiterpenes (48.3%) and oxygenated sesquiterpenes (36.7%). The dominant constituents in the oil from aerial parts were phytol (13.1%) and germacrene D (12.9%), while in the fruit oil suberosin (19.7%) and germacrene D (12.3%) were the most abundant.

Composition and antimicrobial activity of Seseli globiferum essential oil.

The essential oil from aerial parts of Seseli globiferum Vis. obtained by hydrodistillation with Clevenger-type apparatus was analyzed by GC-MS. Twenty-eight compounds were identified, representing 99.4% of the total oil. The main components of the oil were sabinene (38.0%), alpha-pinene (21.2%) and beta-phellandrene (13.5%). The microbial growth inhibitory properties of the isolated essential oil were determined using the broth microdilution method against seven bacterial species: Salmonella typhimurium (ATCC 13311), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Enterobacter cloacae (clinical isolates), Bacillus cereus (clinical isolates), Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 12228), Micrococcus flavus (ATCC 10240) and three fungal species: Aspergillus niger (ATCC 6275), Aspergillus versicolor (ATCC 11730), Trichoderma viride (IAM 5061) and Penicillium funiculosum (ATCC 36839). The essential oil showed activity against bacteria P. aeruginosa, followed by M flavus, L. monocytigenes and E. coli, and all investigated fungal species.

Essential oil variability in natural populations of Picea omorika, a rare European conifer.

This study is the first report on the composition and variability of essential oil in the relic, endemic, and vulnerable tree species Serbian spruce, Picea omorika, in its natural populations. In the needles of 108 trees of four natural populations, 49 components of essential oils were identified. The main compounds were bornyl acetate (29.2%), camphene (18.7%), and alpha-pinene (12.9%). Fourteen additional components had the contents of up to 0.5%: alpha-cadinol (6.1%), limonene (5.8%), santene (3.5%), (E)-hex-2-enal (2.9%), T-cadinol (2.9%), delta-cadinene (2.3%), tricyclene (2.1%), myrcene (1.6%), beta-pinene (1.2%), borneol (0.9%), germacrene D (0.9%), alpha-muurolene (0.6%), and two unidentified compounds. Population IV from Milesevka Canyon had a much higher content of bornyl acetate (42.9%). Populations I-III from Mt. Tara were more abundant in sesquiterpenes (up to 18.2%). The content of bornyl acetate, the multi-variation analyses according to seven selected components, especially the cluster analysis and genetic analysis of alpha-cadinol, which suggested the monogenic type of heredity, showed a clear differentiation of the two geographic areas, the similarity of populations I-III from the area of Mt. Tara, and the separation of the population IV from Milesevka Canyon.