Dragoljub L. Miladinović

Investigation of the chemical composition–antibacterial activity relationship of essential oils by chemometric methods

The antibacterial effects of Thymus vulgaris (Lamiaceae), Lavandula angustifolia (Lamiaceae), and Calamintha nepeta (Lamiaceae) Savi subsp. nepeta var. subisodonda (Borb.) Hayek essential oils on five different bacteria were estimated. Laboratory control strain and clinical isolates from different pathogenic media were researched by broth microdilution method, with an emphasis on a chemical composition–antibacterial activity relationship. The main constituents of thyme oil were thymol (59.95%) and p-cymene (18.34%). Linalool acetate (38.23%) and β-linalool (35.01%) were main compounds in lavender oil. C. nepeta essential oil was characterized by a high percentage of piperitone oxide (59.07%) and limonene (9.05%). Essential oils have been found to have antimicrobial activity against all tested microorganisms. Classification and comparison of essential oils on the basis of their chemical composition and antibacterial activity were made by utilization of appropriate chemometric methods. The chemical principal component analysis (PCA) and hierachical cluster analysis (HCA) separated essential oils into two groups and two sub-groups. Thyme essential oil forms separate chemical HCA group and exhibits highest antibacterial activity, similar to tetracycline. Essential oils of lavender and C. nepeta in the same chemical HCA group were classified in different groups, within antibacterial PCA and HCA analyses. Lavender oil exhibits higher antibacterial ability in comparison with C. nepeta essential oil, probably based on the concept of synergistic activity of essential oil components.

An In Vitro Synergistic Interaction of Combinations of Thymus glabrescens Essential Oil and Its Main Constituents with Chloramphenicol

The chemical composition and antibacterial activity of Thymus glabrescens Willd. (Lamiaceae) essential oil were examined, as well as the association between it and chloramphenicol. The antibacterial activities of geraniol and thymol, the main constituents of T. glabrescens oil, individually and in combination with chloramphenicol, were also determined. The interactions of the essential oil, geraniol, and thymol with chloramphenicol toward five selected strains were evaluated using the microdilution checkerboard assay in combination with chemometric methods. Oxygenated monoterpenes were the most abundant compound class in the oil, with geraniol (22.33%) as the major compound. The essential oil exhibited in vitro antibacterial activity against all tested bacterial strains, but the activities were lower than those of the standard antibiotic and thymol. A combination of  T. glabrescens oil and chloramphenicol produced a strong synergistic interaction (FIC indices in the range 0.21–0.87) and a substantial reduction of the MIC value of chloramphenicol, thus minimizing its adverse side effects. The combinations geraniol-chloramphenicol and thymol-chloramphenicol produced synergistic interaction to a greater extent, compared with essential oil-chloramphenicol association, which may indicate that the activity of the thyme oil could be attributed to the presence of significant concentrations of geraniol and thymol.

Antibacterial activity chemical composition relationship of the essential oils from cultivated plants from Serbia

The antibacterial effects of essential oils from Serbian cultivated plants, Thymus vulgaris L. (Lamiace) and Lavandula angustifolia L. (Lamiace) on different bacteria were investigated, with an emphasis on an antibacterial activity-chemical composition relationship. Essential oil was obtained from airdried aerial parts of the plants by hydrodistillation for 3 h using a Clevenger-type apparatus. The essential oil analyses were performed simultaneously by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) systems. The main constituents of thyme oil were thymol (59.95%) and p-cymene (18.34%). Linalyl acetate (38.23%) and linalool (35.01%) were main compounds in lavender oil. The antibacterial activity of the essential oils samples was tested towards 5 different bacteria: laboratory control strain obtained from the American Type Culture Collection and clinical isolates from different pathogenic media. Gram negative bacteria were represented by Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 43895 and Salmonella enteretidis ATCC 9027 while researched Gram positive strains were Bacillus cereus ATCC 8739 and Staphylococcus aureus ATCC 25923. A broth microdilution method was used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Essential oils from thyme have been found to have antimicrobial activity against all microorganisms tested, with a range of MIC values from 0.025 to 0.10 l/ml and MBC values from 0.05 to 0.78 l/ml. Lavender oils demonstrated MIC values from 0.025 to 0.20 l/ml and MBC values from 0.05 and 0.78 l/ml. Reference antibiotic tetracycline was active in concentrations between 0.025 and 0.05 l/ml. The Gram-positive bacteria were more sensitive to the essential oil of thyme, while Gram-negative bacteria were more sensitive to the essential oil of lavender. Essential oils from thyme and lavender may be used at low concentrations for prevention and treatment of infective diseases in animals and humans caused by pathogenic bacterial species.

VOLATILE CONSTITUENTS OF Euphrasia stricta

The genus Euphrasia is fairly large with 450 species distributed in both the hemispheres in Europe, Asia, Northern parts of America, mountains of Indonesia to New Zealand, and South America [1]. The Serbian flora recognizes eight species of Euphrasia genus [2]. Euphrasia stricta J. P. Wolff ex J. F. Lehm. (eyebright) has been a part of the traditional folk medicine for centuries and is used for the treatment of various eye problems such as cataracts, conjunctivitis, and red, inflamed, irritated, and sore eyes [3–5]. The literature data on its constituents and their action is limited. The principal compounds in the aerial part are iridoids, phenolic acids, and phenylpropane, and flavonoid glycosides [6–8]. To the best of our knowledge, there is no report found in the literature on the chemical composition of Euphrasia stricta volatile constituents. Aerial parts of Euphrasia stricta were collected during the flowering period, in August 2011, from natural populations of Bojana s Waters, Suva Planina Mountain, Serbia. A voucher specimen, with the accession number 16517, is deposited at the Herbarium of the Department of Botany, Faculty of Biology, University of Belgrade – Herbarium Code BEOU. Oil was obtained from air-dried aerial parts of the plant in 0.02% (w/w) yield by hydrodistillation for 4 h using a Clevenger-type apparatus. The oil analyses were performed simultaneously by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) systems. The GC analysis of the oil was carried out on an HP-5890 II gas chromatograph equipped with a split-splitless injector on an HP-5MS capillary column (30 m 0.25 mm, 0.25 m film thickness) with helium as the carrier gas (1 mL/min) and flame ionization detector (FID). Operating conditions: injector temperature 250 C and interface temperature 280 C, temperature program from 50 C (3 min) to 250 C at a rate of 3 C/min. GC/MS analyses were performed on an Agilent Technologies apparatus, Model GS 6890N, at 70 eV coupled with a mass selective detector MSD 5975C under the same gas-chromatograph conditions. The identification of compounds was based on a comparison of Kovats retention indexes using AMDIS software (ver. 2.64) in combination with a chemometric multivariate resolution method and selective ion analysis (SIA) [9]. The identity of the compounds was confirmed by comparing their data with those from the literature [10], and their mass spectra with the Wiley 275 and NIST/NBS libraries. Relative retention indexes (RRI) were obtained by co-injection a C9–C28 standard mixture of aliphatic hydrocarbons. The results of GC and GC/MS analyses of the investigated oil are summarized in Table 1. Forty-seven compounds were identified, representing 84.0% of the total oil. Fatty acids and their derivatives were found in large quantities. Palmitic acid was detected as the major component, constituting 20.3% of the oil. Other significant compounds were -palmitolactone (11.4%), ethyl linolate (7.6%), 9,12,15-octadecatrien-1-ol, (Z,Z,Z) (6.1%) and phytol (5.2%), while other constituents were in less than 5%.

In vitro interactions of Peucedanum officinale essential oil with antibiotics

The chemical composition and antibacterial activity of Peucedanum officinale L. (Apiaceae) essential oil were examined, as well as the association between it and antibiotics: tetracycline, streptomycin and chloramphenicol. The interactions of the essential oil with antibiotics were evaluated using the microdilution checkerboard assay. Monoterpene hydrocarbons, with α-phellandrene as the dominant constituent, were the most abundant compound class of the essential oil of P. officinale. The researched essential oil exhibited slight antibacterial activity against the tested bacterial strains in vitro. On the contrary, essential oil of P. officinale possesses a great synergistic potential with chloramphenicol and tetracycline. Their combinations reduced the minimum effective dose of the antibiotic and, consequently, minimised its adverse side effects. In addition, investigated interactions are especially successful against Gram-negative bacteria, the pharmacological treatment of which is very difficult nowadays.

Chemical Composition of the Essential Oil of Geum Coccineum

The genus Geum with ca. 60 species in the family Rosaceae is primarily found in temperate or montane regions of Europe, Asia, North and South America, Africa, and New Zealand [1]. Geum coccineum is representative of the subendemic taxa and native to the Balkan region and Asia Minor [2]. Geum species are used in traditional medicine and exhibit antiviral, anti-inflammatory, anticoagulant, antidizziness (Meniere s syndrome), angiogenesis, and myogenesis activities [3–6]. Phytochemical studies on the Geum species have revealed the occurrence of many secondary metabolites, such as terpenoids, flavonoids, tannins, and phenylpropanoids [7–10]. To the best of our knowledge, there is no report in the literature on the chemical composition of the essential oil of Geum coccineum Sibth. et Sm. Aerial parts of Geum coccineum were collected in June 2013 from natural populations of Jablanica Mountain, Macedonia (41 13 44.33 N, 20 31 41.97 E, Elev. 1810 m). The plant material was authenticated by one of the authors (V. N. Randjelovic). A voucher specimen, with the accession number 16732, is deposited at the Herbarium of the Department of Botany, Faculty of Biology, University of Belgrade – Herbarium Code BEOU. Oil was obtained from air-dried aerial parts of the plant with 0.01% (w/w) yield by hydrodistillation for 4 h using a Clevenger-type apparatus. The oil analyses were performed simultaneously by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) systems. The GC analysis of the oils was carried out on a GC HP-5890 II apparatus equipped with a split-splitless injector, an HP-5MS capillary column (30 m 0.25 mm, 0.25 m film thickness) with helium as the carrier gas (1 mL/min), and a FID. Operating conditions: injector temperature 250 C and interface temperature 280 C, temperature program from 50 C (3 min) to 250 C at a rate of 3 C/min; volume injected, 1 L of the oil in ether (0.5%). GC/MS analyses, under the same gas-chromatograph conditions, were performed on an Agilent Technologies apparatus, Model GS 6890N, coupled with an MSD 5975C mass selective detector. The MS operating parameters were as follows: ionization potential 70 eV; ion source temperature 250 C; quadrapole 150 C, solvent delay 3.0 min, mass range 50–550 amu, and Em voltage 1435 V. Identification of the compounds was based on comparison of Kovats retention indexes (applying calibrated automated mass spectral deconvolution and identification system software (AMDIS ver. 2.64) in combination with selective ion analysis (SIA) resolution method [11]), comparison with the spectral data from the available literature [12], and comparison of their mass spectra with those from Wiley 275 and NIST/NBS libraries using various search engines. Relative retention indexes (RRI) were obtained by co-injection with an aliphatic hydrocarbons C7–C40 standard mixture. The results of GC and GC/MS analyses of the investigated oil are summarized in Table 1. Thirty-three compounds were identified, representing 97.4% of the total oil. The major component was phytol constituting 24.3% of the oil. Other significant compounds were myrtenal (13.4%), cis-myrtanol (9.6%), tricosane (9.2%) and palmitic acid (6.1%), while other constituents were present in less than 5%. The essential oil of the aerial part of Geum iranicum Khatamaz had palmitic acid (10.6%) and linoleic acid (9.6%) as the main constituents [13]. The major compounds in the oil of the roots and rhizomes of Geum kokanicum were eugenol (80.9%) and myrtenol (5.2%) [14].

Essential Oil of Euphrasia tatarica

The genus Euphrasia has a worldwide distribution, in Europe, Asia, Northern parts of America, mountains of Indonesia to New Zealand and South America [1]. This genus is represented by eight species in the flora of Serbia [2]. Euphrasia species are used in folk medicine for centuries, as a traditional remedy for the treatment of various eye problems, and also for coughs and hoarseness [3–5]. The literature data on Euphrasia tatarica Fisch. constituents and their mechanism of action are limited. The principal compounds in the aerial part are lipophilic and hydrophilic compounds. The dominant components of the the lipophilic complex are alkanes, -sitosterol, squalene, and fatty acids, while the most common hydrophilic compounds are flavonoids [6–8]. To the best of our knowledge, there is no report in the literature on the chemical composition of Euphrasia tatarica essential oil. Aerial parts of Euphrasia tatarica were collected during the flowering period, in July 2014, from natural populations of Basarski Kamen, Vidlic Mountain, Serbia. A voucher specimen, with the accession number 17140, is deposited at the Herbarium of the Department of Botany, Faculty of Biology, University of Belgrade – Herbarium Code BEOU. Oil was obtained from air-dried aerial parts of the plant in 0.01% (w/w) yield by hydrodistillation for 4 h using a Clevenger-type apparatus. The oil analyses were performed simultaneously by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) systems. The GC analysis of the oils was carried out on a GC HP-5890 II apparatus, equipped with the split-splitless injector, HP-5MS capillary column (30 m 0.25 mm, 0.25 m film thickness) with helium as the carrier gas (1 mL/min) and FID. Operating conditions: injector temperature 250 C and interface temperature 280 C, temperature program from 50 C (3 min) to 250 C at a rate of 3 C/min. Volume injected, 1 L of the oil in ether (0.5%). GC-MS analyses, under the same gas-chromatograph conditions, were performed on an Agilent Technologies apparatus, Model GS 6890N, coupled with a mass selective detector MSD 5975C. The MS operating parameters were as follows: ionization potential 70 eV; ion source temperature 250 C; quadrupole 150 C, solvent delay 3.0 min, mass range 50–550 amu, and Em voltage 1435 V. Identification of the compounds was based on comparison of Kovats retention indexes (applying calibrated automated mass spectral deconvolution and identification system software (AMDIS ver. 2.64) in combination with selective ion analysis (SIA) resolution method [9]), comparison with the spectral data from the available literature [10], and comparison of their mass spectra to those from Wiley 275 and NIST/NBS libraries using various search engines. Relative retention indexes (RRI) were obtained by co-injection with an aliphatic hydrocarbons C7–C40 standard mixture. The results of GC and GC-MS analyses of the investigated oil are summarized in Table 1. Seventy-nine compounds were identified, representing 86.1% of the total oil. Palmitic acid was detected as the major component, constituting 15.7% of the oil. Other significant compounds were 1-octen-3-ol (9.1%), hexahydrofarnesyl acetone (5.8%), and phytol (4.8%), while other constituents were in less than 3%. Recent GC-MS analysis of Euphrasia rostkoviana Hayne essential oil revealed more than 70 constituents, with palmitic acid (18.5%) as the main component [11]. However, there is no other compound present in significant amount that would indicate the relatedness of these two Euphrasia species.

Chemical Composition of the Essential Oil of Geum rhodopeum

were collected in June 2010 at Prestojceva mahala (Cemernik Mountain-42 44 45.43N, 22 18 40.01 E, Elev. 1339 m). The plant material was authenticated by one of the authors (V. N. Randjelovic). A voucherspecimen, with the accession number 16680, is deposited at the Herbarium of the Department of Botany, Faculty of Biology,University of Belgrade – Herbarium Code BEOU.Oil was obtained from air-dried aerial parts of the plant with 0.01% (w/w) yield by hydrodistillation for 4 h using aClevenger-type apparatus. The oil analyses were performed simultaneously by gas chromatography (GC) and gaschromatography-mass spectrometry (GC-MS) systems. The GC analysis of the oils was carried out on a GC HP-5890 IIapparatus equipped with a split-splitless injector, an HP-5MS capillary column (30 m 0.25 mm, 0.25 m film thickness) withhelium as the carrier gas (1 mL/min), and FID. Operating conditions: injector temperature 250 C and interface temperature280 C, temperature program from 50 C (3 min) to 250 C at a rate of 3 C/min. Volume injected, 1 L of the oil in ether (0.5%).GC/MS analyses, under the same gas-chromatograph conditions, were performed on an Agilent Technologies apparatus,Model GS 6890N coupled with a mass selective detector MSD 5975C. The MS operating parameters were as follows: ionizationpotential 70 eV; ion source temperature 250 C; quadrapole 150 C, solvent delay 3.0 min, mass range 50–550 amu, Em voltage1435 V.Identification of the compounds was based on comparison of Kovats retention indexes (applying calibrated automatedmass spectral deconvolution and identification system software (AMDIS ver. 2.64) in combination with selective ion analysis(SIA) resolution method [11]), comparison with the spectral data from the available literature [12], and comparison of theirmass spectra to those from Wiley 275 and NIST/NBS libraries using various search engines. Relative retention indexes (RRI)were obtained by co-injection with an aliphatic C

Antioxidative responses to seasonal changes and chemiluminescence assay of Astragalus onobrychis leaves extract

AbstractThe aim of this study was to research the seasonal changes of antioxidant enzyme activity and total antioxidant capacity in leaves of Astragalus onobrychis L. subsp. chlorocarpus (Griseb.) S. Kozuharov et D.K. Pavlova. Leaves of A. onobrychis were collected during the different stages of growth and analyzed for antioxidant enzyme activity: superoxide dismutase, catalase, guaiacol peroxidase, glutathione peroxidase. Quantities of malonyldialdehyde, superoxide radicals, and hydroxyl radicals were measured as well as the content of soluble proteins. Furthermore, total antioxidant capacity was determined by the inhibition of chemiluminescence activity of blood phagocytes by leaf extracts. Stages of vegetation significantly affected the accumulation of superoxide radicals, but there were no significant differences in hydroxyl radical quantity and lipid peroxidation levels during vegetation. Soluble proteins vary greatly between different stages of growth. Seasonal changes were found to have an effect on enzymatic activities. During the spring season, guaiacol peroxidase showed the highest levels. Catalase and glutathione peroxidase increased their activities in summer, while, during the autumn season, superoxide dismutase showed maximum activity. On the basis of chemiluminescence assay, it can be concluded that leaf extract of A. onobrychis possesses a significant antioxidant capacity thus protecting plants during environmental stress.