Phytochemical and biological investigations on Centranthus kellereri (Stoj., Stef. & T. Georgiev) Stoj. & Stef. and C. ruber (L.) DC. and their potential as new medicinal and ornamental plants.

INTRODUCTION: Centranthus kellereri is a Bulgarian endemic plant species, found only in two locations in the world: The Balkans Mountains (Stara Planina), above the town of Vratsa, and The Pirin Mountains, above the town of Bansko, Bulgaria. Being endemic and endangered species precluded any significant research on it. The hypothesis of this study was that the populations of C. kellereri may represent genetically, phytochemically, and morphologically distinct forms and these will differentiate from C. ruber. Furthermore, C. kellereri possibly imperfect embryology may preclude its more widespread distribution under natural conditions. RESULTS: This study revealed the phytochemical profile, antioxidant activity, embryology, surface microstructural morphological traits, and genetic differences between the C. kellereri plants from the only two natural populations and compares them to the ones of the related and better-known plant C. ruber. The essential oil (EO) content in aboveground plant parts and in roots was generally low and the EO composition varied significantly as a function of plant part, year of sampling, location, and species. Methylvaleric acid was a major EO constituent in the C. kellereri EO, ranging between 60.2% and 71.7% of the total EO. The EO included monoterpenes, sequiterpenes, long-chain alkanes and fatty acids. Phytochemical analyses of plant tissue revealed the occurrence of 32 compounds that were tentatively identified as 6 simple phenolics, 18 flavonoids, 1 quinone, 1 lipid, 1 alkaloid, 2 diterpenes, and 3 triterpenes. There were differences in detected compounds between the C. kellereri plants at the two locations and between the roots and shoots in both species. The total phenols and flavonoids varied between the two species but were also dissimilar between the plants from the two populations of C. kelleri. Free radical scavenging activity, measured with ABTS and DPPH in aqueous and methanol extracts, had similar values; however, overall, C. kellereri from Vratsa showed the highest antioxidant activity while C. ruber had the lowest activity. Genetic analyses showed a clear differentiation between C. kellereri and C. ruber, and between the two populations of C. kellereri. Embryological studies revealed the peculiarities of the male and female generative spheres of the two species that were defined as being sexually reproducing. The pollen had high viability; however, the low viability of seeds demonstrated possible high sensitivity of C. kellereri to the environmental conditions, perhaps the main factor modifying and restricting the population sizes. The SEM analyses exposed differences in surface microstructural traits between the species (C. kellereri and C. ruber) but also between the two populations of C. kellereri. The observed dissimilarities in genetic makeup, micromorphological characteristics, and phytochemical composition strongly indicate that the two populations can be classified as distinct subspecies or varieties of C. kellereri; var. pirinensis and var. balkanensis. Further research is needed to introduce C. kellereri into culture and develop it as a high-value specialty crop or ornamental in order to conserve C. kellereri natural populations. C. kellereri may be utilized as a source for phytochemicals of interest and as an ornamental plant like C. ruber; however, it may have a greater environmental plasticity and adaptation as evidenced by its current locations.

Chemical Composition of Essential Oils from Nepeta transcaucasica Grossh. and Nepeta cataria L. Cultivated in Bulgaria and Their Antimicrobial and Antioxidant Activity.

The genus Nepeta, belonging to the family Lamiaceae, includes about 300 species, most of which are used in folk medicine due to their pronounced biological properties. The aim of the present study was to evaluate the agrobiological characteristics of Nepeta transcaucasica (N. transcaucasica) Grossh. and Nepeta cataria (N. cataria) L., cultivated in Bulgaria, and obtain their essential oils and determine their antimicrobial and antioxidant activities. The agrobiological characteristics of the two species growing in Kazanlak were analyzed; therefore, high variability in the population of N. transcaucasica and comparative homogeneity in N. cataria was shown. The species N. transcaucasica contained 0.28% essential oil with main components ß-citronellol (52.05%), eucalyptol (7.34%), ß-citronellal (6.06%), germacrene D (5.45%), (Z)-ß-ocimene (5.14%), and ß-caryophyllene (3.06%). The species N. cataria consisted of 0.19% essential oil with main components ß-citronellol (26.31%), geraniol (15.92%), neral (11.45%), nerol (9.56%), carvacrol (6.04%), and ß-citronellal (5.35%). The antibacterial activity against Gram-positive bacteria Listeria monocytogenes and Staphylococcus aureus and Gram-negative bacteria Escherichia coli (E. coli) and Salmonella enterica subsp. enterica serovar Abony was determined. The essential oils showed antimicrobial activity only against E. coli. The diameters of the inhibition zones were found to be 26 mm for the species N. transcaucasica and 10 mm for the species N. cataria. The antioxidant activity of the two essential oils was also determined by four different methods, DPPH, ABTS, FRAP, and CUPRAC, with the highest values for the ABTS radical, for the species N. transcaucasica (48.72 µM TE/mL), and the species N. cataria (310 µM TE/mL).

Chemical Composition Assessment of Structural Parts (Seeds, Peel, Pulp) of Physalis alkekengi L. Fruits.

In recent years there has been an extensive search for nature-based products with functional potential. All structural parts of Physalis alkekengi (bladder cherry), including fruits, pulp, and less-explored parts, such as seeds and peel, can be considered sources of functional macro- and micronutrients, bioactive compounds, such as vitamins, minerals, polyphenols, and polyunsaturated fatty acids, and dietetic fiber. The chemical composition of all fruit structural parts (seeds, peel, and pulp) of two phenotypes of P. alkekengi were studied. The seeds were found to be a rich source of oil, yielding 14-17%, with abundant amounts of unsaturated fatty acids (over 88%) and tocopherols, or vitamin E (up to 5378 mg/kg dw; dry weight). The predominant fatty acid in the seed oils was linoleic acid, followed by oleic acid. The seeds contained most of the fruit's protein (16-19% dw) and fiber (6-8% dw). The peel oil differed significantly from the seed oil in fatty acid and tocopherol composition. Seed cakes, the waste after oil extraction, contained arginine and aspartic acid as the main amino acids; valine, phenylalanine, threonine, and isoleucine were present in slightly higher amounts than the other essential amino acids. They were also rich in key minerals, such as K, Mg, Fe, and Zn. From the peel and pulp fractions were extracted fruit concretes, aromatic products with specific fragrance profiles, of which volatile compositions (GC-MS) were identified. The major volatiles in peel and pulp concretes were ß-linalool, α-pinene, and γ-terpinene. The results from the investigation substantiated the potential of all the studied fruit structures as new sources of bioactive compounds that could be used as prospective sources in human and animal nutrition, while the aroma-active compounds in the concretes supported the plant's potential in perfumery and cosmetics.

Chemical Compounds, Antitumor and Antimicrobial Activities of Dry Ethanol Extracts from Koelreuteria paniculata Laxm.

Koelreuteria paniculata Laxm. is used in traditional medicine and has various established biological activities, however, the species is considered to be a potentially invasive alien tree species for Bulgarian flora. However, there is still much to be studied about the phytochemical and biological characteristics of the species. The present study aimed to determine the chemical composition of the ethanol extracts of aerial plant parts, by GC-MS analysis, and to thereby evaluate their in vitro antitumor and antibacterial properties. All three extracts were tested against the HT-29 and PC3 tumor cell lines using the MTT assay. Fifty-six components were identified from leaf, flower, and stem bark extracts, and over 10% were the following constituents: pyrogallol, α-terpinyl acetate, neryl acetate, and α-terpinyl isobutanoate. The oxygenated monoterpenes predominated in the extracts, followed by the oxygenated aliphatics and phenylpropanoids. Significant antiproliferative activity on the HT-29 cell line (IC50-21.44 µg/mL and 23.63 µg/mL, respectively) was found for the flower and leaf extracts. Antibacterial activity was established for the following bacteria strains: Bacillus subtilis ATCC 6633, Bacillus cereus NCTC 10320, Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 6027, and Proteus vulgaris ATCC 6380. The stem bark and flower extracts showed better antimicrobial potential. K. paniculata could be considered as a potential source of biologically active substances with antitumor and antibacterial properties.

Essential Oil Composition and Bioactivity of Two Juniper Species from Bulgaria and Slovakia.

Juniperus excelsa M. Bieb and J. sabina L. contain essential oil (EO), while J. sabina also contains podophyllotoxin, which is used as a precursor for anti-cancer drugs. Two studies were conducted. The first assessed the variability in the EO profile and podophyllotoxin concentration of the two junipers, depending on the location and tree gender. The main EO constituents of J. excelsa were α-cedrol, α-limonene and α-pinene, while the constituents in J. sabina were sabinene, terpinen-4-ol, myrtenyl acetate and α-cadinol. The podophyllotoxin yield of 18 J. sabina accessions was 0.07-0.32% (w/w), but this was not found in any of the J. excelsa accessions. The second study assessed the effect of hydrodistillation (Clevenger apparatus) and steam distillation (in a semi-commercial apparatus) on the EO profile and bioactivity. The extraction type did not significantly alter the EO composition. The EO profiles of the two junipers and their accessions were different and may be of interest to the industry utilizing juniper leaf EO. Breeding and selection programs could be developed with the two junipers (protected species) in order to identify chemotypes with (1) a high EO content and desirable composition, and (2) a high concentration of podophyllotoxin in J. sabina. Such chemotypes could be established as agricultural crops for the commercial production of podophyllotoxin and EO.

Chemical Composition and Antimicrobial Activity of Laurus nobilis L. Essential Oils from Bulgaria.

Laurel, Laurus nobilis L. is an evergreen plant belonging to the Lauraceae family, native to Southern Europe and the Mediterranean area. This is the first report on the composition and bioactivity of laurel essential oil (EO) from Bulgaria. The oil yield was 0.78%, 0.80%, and 3.25% in the fruits, twigs, and leaves, respectively. The main constituents in the fruit EO were 1,8-cineole (33.3%), α-terpinyl acetate (10.3%), α-pinene (11.0%), ß-elemene (7.5%), sabinene (6.3%), ß-phellandrene (5.2%), bornyl acetate (4.4%), and camphene (4.3%); those in the twig EO were 1,8-cineole (48.5%), α-terpinyl acetate (13.1%), methyl eugenol (6.6%), ß-linalool (3.8%), ß-pinene (3.4%), sabinene (3.3%) and terpinene-4-ol (3.3%); and the ones in the leaf EO were 1,8-cineole (41.0%), α-terpinyl acetate (14.4%), sabinene (8.8%), methyl eugenole (6.0%), ß-linalool (4.9%), and α-terpineol (3.1%). The antibacterial and antifungal properties of laurel EOs were examined according to the agar well diffusion method. The leaf EO showed antibacterial and antifungal activities against almost all strains of the microorganisms tested, whereas the twig EO was only able to inhibit Staphylococcus aureus. Pseudomonas aeruginosa ATCC 9027 and Escherichia coli ATCC 8739 were the bacterial strains that showed the highest resistance to the laurel EO. The results can benefit the EO industry and biopesticide development.

Chemical composition and biological effects of Artemisia maritima and Artemisia nilagirica essential oils from wild plants of western Himalaya.

Artemisia species possess pharmacological properties that are used for medical purposes worldwide. In this paper, the essential oils from the aerial parts of Artemisia nilagirica and Artemisia maritima from the western Indian Himalaya region are described. The main compounds analyzed by simultaneous GC/MS and GC/FID were camphor and 1,8-cineole from A. maritima, and camphor and artemisia ketone from A. nilagirica. Additionally, the oils were evaluated for their antibacterial, antifungal, mosquito biting deterrent, and larvicidal activities. A. nilagirica essential oil demonstrated nonselective antifungal activity against plant pathogens Colletotrichum acutatum, Colletotrichum fragariae, and Colletotrichum gloeosporioides, whereas A. maritima did not show antifungal activity. Both Artemisia spp. exhibited considerable mosquito biting deterrence, whereas only A. nilagirica showed larvicidal activity against Aedes aegypti. Antibacterial effects assessed by an agar dilution assay demonstrated greater activity of A. maritima essential oil against Staphylococcus aureus and Pseudomonas aeruginosa compared to A. nilagirica.

Chemical composition, olfactory analysis and antibacterial activity of Thymus vulgaris chemotypes geraniol, 4-thujanol/terpinen-4-ol, thymol and linalool cultivated in southern France.

The essential oils of four chemotypes of Thymus vulgaris L. (Lamiaceae) were analyzed for their composition and antibacterial activity to assess their different properties. GC-MS and GC-FID analyses revealed that the essentials oils can be classified into the chemotypes thymol (41.0% thymol), geraniol (26.4% geraniol), linalool (72.5% linalool) and 4-thujanol/terpinen-4-ol (42.2% cis- and 7.3% trans-sabinene hydrate, 6.5 % terpinen-4-ol). The olfactory examination confirmed the explicit differences between these chemotypes. Furthermore, antibacterial activity was investigated against several strains of two Gram-positive (Brochothrix thermosphacta and Staphylococcus aureus) and four Gram-negative food-borne bacteria (Escherichia coli, Salmonella abony, Pseudomonas aeruginosa and P. fragi). All essential oil samples were demonstrated to be highly effective against Gram-positive strains, whereas the impact on Gram-negative microorganisms was significantly smaller, but still considerable. The results obtained indicate that, despite their different properties, the essential oils of selected T. vulgaris chemotypes are potent antimicrobials to be employed as useful additives in food products as well as for therapeutic applications.

Chemical composition, olfactory evaluation and antimicrobial activity of selected essential oils and absolutes from Morocco.

The chemical compositions of selected essential oils from North Africa, especially Morocco, of geranium, wild Moroccan chamomile and rosemary as well as absolutes of rose and geranium were determined using GC/FID and GC/MS. These oils and absolutes were tested concerning their antimicrobial activity against some food spoilage strains obtained from fresh milk and minced meat products, like sausages and pork fillet, in accordance with ISO testing procedures. Gram-positive (Bacillus cereus and Staphylococcus aureus) and Gram-negative (Escherichia coli, Salmonella abony and Pseudomonas aeruginosa) strains were used, as well as the yeast Candida albicans. Using a serial broth dilution method, all samples demonstrated weak antimicrobial activity against the Gram-negative bacteria and the yeast, compared with the activity towards the Gram-positive bacteria.

Chemical composition and antimicrobial activity of cumin oil (Cuminum cyminum, Apiaceae).

Cumin oil samples (Cuminum cyminum L.) from four different geographical origins were analyzed using GC-MS and GC-FID for their qualitative and quantitative composition. The major compounds in all cumin oils were the monoterpenes beta-pinene, p-cymene and gamma-terpinene and the terpenoid aldehydes cuminic aldehyde and the isomeric menthadien carboxaldehydes. All essential oils, and cuminic aldehyde, were tested, using agar diffusion and serial dilution methods, against different Gram-positive and Gram-negative bacteria isolated from different sources of food (pork fillet, minced meat and sausages) and clinical isolates, as well as three different Candida albicans isolates. All cumin oils and cuminic aldehyde exhibited a considerable inhibitory effect against all the organisms tested, except Pseudomonas spp.

Chemical composition and antibacterial activity of selected essential oils and some of their main compounds.

The chemical composition of essential oils of cabreuva (Myrocarpus fastigiatus Allemao, Fabaceae) from Brazil, cedarwood (Juniperus ashei, Cupressaceae) from Texas, Juniper berries (Juniperus communis L., Cupressaceae) and myrrh (Commiphora myrrha (Nees) Engl., Burseraceae) were analyzed using GC/FID and GC/MS. The antimicrobial activity of these essential oils and some of their main compounds were tested against eleven different strains of Gram-positive and Gram-negative bacteria by using agar diffusion and agar serial dilution methods. Animal and plant pathogens, food poisoning and spoilage bacteria were selected. The volatile oils exhibited considerable inhibitory effects against all tested organisms, except Pseudomonas, using both test methods. Higher activity was observed against Gram-positive strains in comparison with Gram-negative bacteria. Cabreuva oil from Brazil showed similar results, but in comparison with the other oils tested, only when higher concentrations of oil were used.

Antimicrobial activities of single aroma compounds.

Commercially available aroma samples were evaluated for their olfactory quality by professional perfumers and tested for their antimicrobial activity. Agar diffusion and agar-dilution were used as test methods and a set of two Gram-positive (Staphylococcus aureus and Enterococcus faecalis) and four Gram-negative bacterial strains (Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris G, Klebsiella pneumoniae and Salmonella abony) and a yeast, Candida albicans, were the test microorganisms. All the investigated compounds were active against Gram-positive bacteria, especially beta-caryophyllene against Enterococcus faecalis (MIC 6 ppm), but only few substances showed activity towards Gram-negative bacteria, except for cinnamic acid, which was active against all (MIC 60 ppm) and Candida albicans, against which cinnamic acid and caryophyllene oxide showed high activity (MIC < 60 ppm).

Chemical composition, olfactory evaluation and antioxidant effects of essential oil from Mentha x piperita.

The chemical composition of the essential oil from peppermint (Mentha x piperita L.) was analyzed by GC/FID and GC-MS. The main constituents were menthol (40.7%) and menthone (23.4%). Further components were (+/-)-menthyl acetate, 1,8-cineole, limonene, beta-pinene and beta-caryophyllene. Peppermint oil possessed antiradical activity with respect to DPPH (diphenyl picryl hydrazyl) and hydroxyl (OH*) radicals, exercising stronger antioxidant impact on the OH* radical. The concentrations required for 50% inhibition of the respective radical (IC50) were 860 microg/mL for DPPH and 0.26 microg/mL for OH*. Peppermint essential oil demonstrated antioxidant activity in a model linoleic acid emulsion system in terms of inhibiting conjugated dienes formation by 52.4% and linoleic acid secondary oxidized products generation by 76.9% (at 0.1% concentration).

Chemical composition, olfactory evaluation and antioxidant effects of essential oil from Mentha canadensis.

The chemical composition of the essential oil from cornmint (Mentha canadensis L.) was analyzed by GC/FID and GC-MS. The main constituents were menthol (41.2%) and menthone (20.4%). It was established that cornmint oil had antiradical activity with respect to the DPPH and hydroxyl (OH*) radicals. The concentrations necessary for 50% neutralization of the respective radicals (IC50) were 365.0 microg/mL for DPPH and 0.3 microg/mL for OH*, which was indicative that the antioxidant activity in terms of OH* was higher than that of quercetin. Cornmint oil chelated the Fe3+ ions present in the solution. The oil demonstrated antioxidant activity in a linoleic acid emulsion model system, where at 0.1% concentration it inhibited the formation of conjugated dienes by 57.1% and the generation of secondary oxidized products of linoleic acid by 76.1%.

Composition, quality control, and antimicrobial activity of the essential oil of long-time stored dill (Anethum graveolens L.) seeds from Bulgaria.

The essential oil of long-time stored seeds of dill (Anethum graveolens L.) from Bulgaria was analyzed by physicochemical methods, gas chromatography (GC), GC-mass spectrometry (MS) (achiral and chiral phases), and olfactometry, and its antimicrobial activity was tested by using different strains of microorganisms. More than 40 constituents of the essential dill oil, obtained from seeds stored for more than 35 years, could be identified as essential volatiles, responsible for the pleasant fresh (D-limonene) and spicy (D-carvone) odor of a high quality. As aroma impact compounds, D-carvone (50.1%) and D-limonene (44.1%) were found. Antimicrobial testings showed high activity of the essential A. graveolens oil against the mold Aspergillus niger and the yeasts Saccharomyces cerevisiae and Candida albicans.