Effects of Different Methods of Isolation on Volatile Composition of Artemisia annua L.

In order to determine influence of extraction method on volatile oil composition of Artemisia annua L., steam distillation, hydrodistillation, organic solvent extraction, and headspace sampling have been applied. The relative abundance of volatile compounds from the odorous aerial parts of A. annua, obtained by different extraction techniques, was analyzed by GC-MS. Exactly fifty constituents were identified. The leaf and flower essential oil yield ranged from 0.9 to 2.3% (v/w). Oxygenated monoterpenes were predominant in all samples ranged from 42.6% for steam-distilled fraction of petroleum ether extract to 70.6% for headspace of plant material. Essential oils isolated by steam distillation and hydrodistillation indicate that A. annua belongs to artemisia ketone chemotype with its relative content of 30.2% and 28.3%, respectively. The principal constituent in headspace sample of plant material was also artemisia ketone (46.4%), while headspace of petroleum ether extract had camphene (25.6%) as the major compound. The results prove the combined approaches to be powerful for the analysis of complex herbal samples.

In vitro and in vivo antitumour effects of phenylboronic acid against mouse mammary adenocarcinoma 4T1 and squamous carcinoma SCCVII cells.

The cytotoxic activity of phenylboroxine acid was evaluated in vitro on mouse mammary adenocarcinoma 4T1, mouse squamous cell carcinoma SCCVII, hamster lung fibroblast V79 and mouse dermal fibroblasts L929 cell lines. The cytotoxic effects were dose dependent for all tested tumour and non-tumour cell lines. Under in vivo conditions, three application routes of phenylboronic acid were studied: intra-peritoneal (i.p.), intra-tumour (i.t.) and per-oral. After tumour transplantation in syngeneic mice, phenylboronic acid was shown to slow the growth of both tumour cell lines (4T1 and SCCVII) compared with the control. The inhibitory effects were pronounced during the application of phenylboronic acid. For both tested tumour cell lines, the most prominent antitumour effect was obtained by intraperitoneal administration, followed significantly by oral administration.

Advantages of an Electrochemical Method Compared to the Spectrophotometric Kinetic Study of Peroxidase Inhibition by Boroxine Derivative.

In this study, boroxine derivative (K2[B3O3F4OH]) was tested as an inhibitor of horseradish peroxidase (HRP) by spectrophotometric and electrochemical methods. The activity of horseradish peroxidase was first studied under steady-state kinetic conditions by a spectrophotometric method which required the use of guaiacol as a second substrate to measure guaiacol peroxidation. The results of this method have shown that, by changing the concentration of guaiacol as the literature suggests, a different type of inhibition is observed than when changing the concentration of hydrogen peroxide as the substrate. This suggests that guaiacol interferes with the reaction in some way. The electrochemical method involves direct electron transfer of HRP immobilized in Nafion nanocomposite films on a glassy carbon (GC) electrode, creating a sensor with an electro-catalytic response to the reduction of hydrogen peroxide. The electrochemical method simplifies kinetic assays by removing the requirement of reducing substrates.

Sea fennel (Crithmum maritimum L.): phytochemical profile, antioxidative, cholinesterase inhibitory and vasodilatory activity.

Sea fennel, a rediscovered star of the coastal cuisine, has been investigated for its phytochemical profile and biological potential. Sea fennel flowers, stems and leaves were analyzed for essential oils (EOs) isolated by hydrodistillation, as well as non-volatiles obtained by ethanolic extraction. Limonene were found to be a dominant compound in EOs and ethanolic extracts; ranging from 57.5-74.2 % and 0.7-8.1 mg/g dry plant material, respectively. In addition total phenolic content was determined for ethanolic extracts. All samples and their main phytochemicals were tested for various methods. EO and extract obtained from flowers were tested for vasodilatory activity on rat aortic rings. Antioxidant activity of EOs was extremely low in comparison to extracts, on the contrary to cholinesterase inhibition where EOs showed better activity than extracts. Flower extract and chlorogenic acid showed stronger vasodilators in comparison to EO and limonene. The obtained results point out the potential impact of the dominant compounds from EO and extract on the biological properties of the sea fennel.

Impact of calcium ion on cytotoxic effect of the boroxine derivative, K2[B3O3F4OH].

The effect of Ca2+ ions on the cytotoxic ability of boron heterocyclic compound dipotassium-trioxohydroxytetrafluorotriborate (K2[B3O3F4OH]), on in vitro tumor cells (mammary adenocarcinoma 4T1, melanoma B16F10 and squamous cell carcinoma SCCVII) and non-tumoral fibroblast cells (mouse dermal L929 and hamster lung V79) was examined. At small concentrations of Ca2+ ions (0.42 mM), K2[B3O3F4OH] (3.85 mM) has a very strong cytotoxic effect on all cancer cells tested (89.1, 85.6 and 84.6%) and significantly less effect on normal cells (19.5 and 24.2%), respectively. Applying larger concentrations of Ca2+ ions (9.42-72.42 mM), at the same concentration of K2[B3O3F4OH], no significant cytotoxic effect was detected on cancer cells and normal cells investigated. The selective ability of K2[B3O3F4OH], in the medium with a low concentration of Ca2+ ions has a strong cytotoxic effect on cancer cells and very weak effect in normal cells, opens up the possibility of its application in antitumor therapy.

Effects of dipotassium-trioxohydroxytetrafluorotriborate, K2[B3O3F4OH], on cell viability and gene expression of common human cancer drug targets in a melanoma cell line.

Recently it was found that dipotassium-trioxohydroxytetrafluorotriborate, K2(B3O3F4OH), is a potent and highly specific inhibitor of precancerous cell processes. We conducted gene expression profiling of human melanoma cells before and after treatment with two concentrations (0.1 and 1 mM) of this boron inorganic derivative in order to assess its effects on deregulation of genes associated with tumor pathways. Parallel trypan blue exclusion assay was performed to assess the cytotoxicity effects of this chemical. Treatment with K2(B3O3F4OH) induced a significant decrease of cell viability in melanoma cellline at both tested concentrations. Furthermore, these treatments caused deregulation of more than 30 genes known as common anti-tumor drug targets. IGF-1 and hTERT were found to be significantly downregulated and this result may imply potential use of K2(B3O3F4OH) as an inhibitor or human telomerase and insulin-like growth factor 1, both of which are associated with various tumor pathways.

Dipotassium-trioxohydroxytetrafluorotriborate, K2[B3O3F4OH], is a potent inhibitor of human carbonic anhydrases.

The boron heterocyclic compound dipotassium-trioxohydroxytetrafluorotriborate (K2[B3O3F4OH]) was investigated as inhibitor of the zinc enzyme, carbonic anhydrase (CA, EC 4.2.1.1). Eleven human (h) CA isoforms, hCA I-IV, VA, VI, VII, IX and XII-XIV, were included in the investigations. The anion, similar to tetraborate or phenylboronic acid, inhibited most of them. hCA III was not inhibited by K2[B3O3F4OH], whereas hCA VA, hCA VI, hCA IX and hCA XIII were inhibited in the submillimolar range, with KIs of 0.31-0.63 mM. hCA I and II (cytosolic, widespread isoforms), hCA IV (membrane-bound isoform), hCA XII (tumor-associated, transmembrane) and hCA XIV (transmembrane) were much more effectively inhibited by this anion, with inhibition constants ranging from 25 to 93 µM. hCA VII, a cytosolic enzyme present in the brain and associated to oxidative stress, was very effectively inhibited by K2[B3O3F4OH], with a KI of 8.0 µM. We propose that K2[B3O3F4OH] binds to the metal ion from the enzyme active site, coordinating to the Zn(II) ion monodentately through its B-OH functionality. We hypothesize that some of the beneficial antitumor effects reported for K2[B3O3F4OH] may be due to the inhibition of CAs present in skin tumors.

In vitro and in vivo antitumor activity of the halogenated boroxine dipotassium-trioxohydroxytetrafluorotriborate (K2[B3O3F4OH]).

Dipotassium-trioxohydroxytetrafluorotriborate K2[B3O3F4OH] was listed as a promising new therapeutic for cancer diseases. For in vitro and in vivo investigation of its antitumor effects 4T1 mammary adenocarcinoma, B16F10 melanoma and squamous cell carcinoma SCCVII were used. The detailed in vitro investigation undoubtedly showed that K2[B3O3F4OH] affects the growth of cancer cells. The proliferation of cells depends on the concentration so that aqueous solution of K2[B3O3F4OH], the concentrations of 10(-4) M and less, does not affect cell growth, but the concentrations of 10(-3) M or more, significantly slows cells growth. B16F10 and SCCVII cells show higher sensitivity to the cytotoxic effects of K2[B3O3F4OH] compared to 4T1 cells. Under in vivo conditions, K2[B3O3F4OH] slows the growth of all three tumors tested compared to the control, and the inhibitory effect was most pronounced during the application of the substance. There is almost no difference if K2[B3O3F4OH] was applied intraperitoneally, intratumor, peroral or as ointment. Addition of 5-FU did not further increase the antitumor efficacy of K2[B3O3F4OH].

A study of the inhibition of catalase by dipotassium trioxohydroxytetrafluorotriborate K2[B3O3F4OH].

In the development of boronic acid-based enzyme inhibitors as potential pharmaceutical drugs, dipotassium trioxohydroxytetrafluorotriborate K2[B3O3F4OH] was listed as a promising new therapeutic for treatment of these diseases. The catalase-mediated conversion of hydrogen peroxide, in the presence and absence of K2[B3O3F4OH] was studied. The kinetics conformed to the Michaelis-Menten model. Lineweaver-Burk plots were linear and plotted the family of straight lines intersected on the abscissa indicating non-competitive inhibition of the catalase. It appears that in the absence of inhibitor, catalase operates the best at conditions around pH 7.1 and in the presence of K2[B3O3F4OH] the optimum is around pH 6.2. The uncatalyzed reaction of hydrogen peroxide decomposition generally has a value of activation energy of 75 kJ mole(-1), whereas catalase, in the absence of inhibitor, lowers the value to 11.2 kJ mole(-1), while in the presence 69 mmoles L(-1) of K2[B3O3F4OH] it was 37.8 kJ mole(-1).

Campanula portenschlagiana ROEM. et SCHULT.: chemical and antimicrobial activities.

The phytochemical profile and the antimicrobial effects of the volatile oil and the aqueous extract of Campanula portenschlagiana, a wild growing plant endemic to Croatia, were described. In the volatile oil, 53 compounds were identified by GC-FID and GC/MS analyses. Diterpene alcohols constituted the major compound class with labda-13(16),14-dien-8-ol as the main compound. The aqueous extract was characterized by the total phenolic content. The antimicrobial potential of the volatile oil and the aqueous extract was evaluated against a diverse range of microorganisms comprising food-spoilage and food-borne pathogens. The volatile oil exhibited interesting and promising antimicrobial effects against the tested species, which were generally more pronounced against Gram-negative bacteria. In addition, the inhibitory effect of this volatile oil was also evaluated against eleven extended-spectrum ß-lactamase (ESBL)-producing isolates. The results suggest that the C. portenschlagiana volatile oil might be used as antimicrobial agent against ESBL-producing isolates and Gram-negative bacteria.

Chemical composition of Hypericum richeri subsp. grisebachii essential oil from Croatia.

The aerial parts of Hypericum richeri Vill. subsp. grisebachii (Boiss.) Nyman were collected from two different locations in Croatia and subjected to hydrodistillation. GC/FID and GC/MS analysis of the isolated essential oils revealed 64 compounds representing 94.7% and 98.2% of the total oils. Predominant constituents in both samples were: germacrene D (10.90%; 6.0%), bicyclogermacrene (4.7%; 3.5%), alpha-pinene (6.8%; 6.9%), beta-pinene (8.1%; 5.1%), decanoic acid (4.5%; 6.8%), beta-caryophyllene (3.3%; 7.5%), delta-cadinene (7.0%; 4.4%), spathulenol (6.0%; 9.5%) and tetracosane (3.1%; 5.8%). Comparison of both samples revealed similarity in the chemical composition with minor fluctuations of constituent percentages. The chemical profile of Croatian oils was in general similar to those reported for other geographic areas regarding major mono- and sesquiterpene constituents. However, spathulenol, delta-cadinene and bicyclogermacrene were more abundant in Croatian oils. The presence of decanoic acid (4.5%; 6.8%) in Croatian oils was the major difference between acids and fatty acids derivatives. Higher abundance of alkanes (particularly tetracosane and docosane) was also noticed.

Phytochemical profiles of volatile constituents from Centaurea ragusina leaves and flowers and their antimicrobial effects.

The volatile oils obtained from the leaves and flowers of the Croatian endemic plant Centaurea ragusina L. were evaluated for antimicrobial activity. The chemical compositions, determined by GC and GC-MS, were complex, with sesquiterpenes the most abundant class of compound. Nonterpene hydrocarbons were also identified in high quantities, while acids and ketones were found in high quantities only in the leaf volatile oil. Aldehydes, alcohols, esters and other compounds were identified in lower quantities. Antimicrobial effects of the volatile samples were investigated by using the disc diffusion and microdilution assays. They were found to inhibit a wide range of bacteria and fungi, causing in vitro growth inhibition at a concentration of 250 microg/mL. The volatile oils displayed great antibacterial potential with MIC values ranging from 16 to 128 microg/mL against the Gram-positive bacteria Bacillus cereus, Clostridium perfringens, Enterococcus faecalis and Staphylococcus aureus, and lesser activity against the Gram-negative bacteria Cronobacter sakazakii, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa, as well as fungal strains (MIC = 32-128 microg/mL).

Screening for acetylcholinesterase inhibition and antioxidant activity of selected plants from Croatia.

The methanol, ethyl acetate and chloroform extracts of selected Croatian plants were tested for their acetylcholinesterase (AChE) inhibition and antioxidant activity. Assessment of AChE inhibition was carried out using microplate reader at 1 mg mL⁻¹. Antioxidant capacities were determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging test and ferric reducing/antioxidant power assay (FRAP). Total phenol content (TPC) of extracts were determined using Folin-Ciocalteu colorimetric method. Out of 48 extracts, only methanolic extract of the Salix alba L. cortex exerted modest activity towards AChE, reaching 50.80% inhibition at concentration of 1 mg mL⁻¹. All the other samples tested had activity below 20%. The same extract performed the best antioxidative activity using DPPH and FRAP method, too. In essence, among all extracts used in the screening, methanolic extracts showed the best antioxidative activity as well as highest TPC.

Chemical characterization and genetic relationships among Ocimum basilicum L. cultivars.

Twenty-seven Ocimum basilicum cultivars were subjected to a chemical characterization of essential oil components by gas chromatography/mass spectrometry (GC/MS) and a genetic characterization using the amplified fragment-length polymorphism (AFLP) technique. Since the same 27 accessions had previously been classified into six morphotypes, these analyses allowed us to make detailed comparisons of chemistry, genetics, and morphology. The chemical composition and morphology of the studied cultivars appeared to have a strong genetic component. The AFLP analysis revealed a distinction between the green and purple morphotypes. The green morphotypes predominantly utilized the terpene biosynthetic pathway, while most purple morphotypes primarily utilized the phenylpropene biosynthetic pathway. The GC/MS analysis led to identification of 87 volatiles. Among the 27 cultivars, five chemotypes were identified. A detailed characterization of the essential oil constituents indicated the existence of both specific combinations of compounds and 'private' compounds with the potential to be used in many aspects of human life. The established relationship between a genetic profile, chemical composition, and morphology represents an important step in future breeding programs and in the cultivation of this species.

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, ß-pinene, germacrene D, and ß-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.

Comparison of chemical composition and free radical scavenging ability of glycosidically bound and free volatiles from Bosnian pine (Pinus heldreichii Christ. var. leucodermis).

The results obtained show that Bosnian pine is rich in glycosidically bound volatile compounds with strong free radical scavenging properties. Since volatiles can be released from nonvolatile glycoside precursors, these compounds can be considered as a hidden potential source of antioxidant substances and may contribute to the total free radical scavenging ability of Bosnian pine.

The effects of essential oils and aqueous tea infusions of oregano (Origanum vulgare L. spp. hirtum), thyme (Thymus vulgaris L.) and wild thyme (Thymus serpyllum L.) on the copper-induced oxidation of human low-density lipoproteins.

In this study, the antioxidative capacity effect of essential oils and aqueous tea infusions obtained from oregano, thyme and wild thyme on the oxidation susceptibility of low-density lipoproteins (LDL) has been studied. The results indicate a dose-dependent protective effect of the tested essential oils and aqueous tea infusions on the copper-induced LDL oxidation. The protective effect of essential oils is assigned to the presence of phenolic monoterpenes, thymol and carvacrol, which are identified as the dominant compounds in these essential oils. The strong protective effect of aqueous tea infusions is proposed to be the consequence of large amounts of polyphenols, namely rosmarinic acid and flavonoids (quercetin, eriocitrin, luteolin-7-O-glucoside, apigenin-7-O-glucoside, luteolin, apigenin), with the most pronounced effect in the case of oregano. These findings may have implications for the effect of these compounds on LDL in vivo.

In vitro acetylcholinesterase inhibitory properties of thymol, carvacrol and their derivatives thymoquinone and thymohydroquinone.

The aim of this study was to examine in vitro the inhibitory activity exerted by the main constituents of essential oil obtained from the aromatic plant Thymus vulgaris L. on acetylcholinesterase (AChE). The total essential oil and selected compounds, specifically linalool and thymol, carvacrol and their derivatives thymoquinone and thymohydroquinone, were tested for AChE inhibition. Thymohydroquinone exhibited the strongest AChE inhibitory effect over the range of concentrations. The AChE inhibitory potential decreased in the following order: thymohydroquinone > carvacrol > thymoquinone > essential oil > thymol > linalool. It is interesting that the AChE inhibitory effect exerted by carvacrol was 10 times stronger than that exerted by its isomer thymol, although thymol and carvacrol have a very similar structure.

Chemical composition and antioxidant test of free and glycosidically bound volatile compounds of savory (Satureja montana L. subsp. montana) from Croatia.

The evaluation of antioxidant power of glycosidically bound volatile aglycones from savory shows that these compounds possess antioxidative activity comparable to that of the essential oil, but less effective compared to the well-known antioxidant butylated hydroxytoluene (BHT) and alpha-tocopherol. Thus, since the glycosides are able to release their aglycones by enzymatic hydrolysis, they could be considered as possible precursors of antioxidant substances in savory.

Chemical composition and in vitro evaluation of antioxidant effect of free volatile compounds from Satureja montana L.

As a part of an investigation of natural antioxidants from Dalmatian aromatic plants, in this paper we report a study of the antioxidant activity related to the chemical composition of savory free volatile compounds. Twenty-one compounds were identified in the essential oil without fractionation, representing 97.4% of the total oil. The major compound was phenolic monoterpene thymol (45.2%). Other important compounds were monoterpenic hydrocarbons p-cymene (6.4%) a nd gamma-terpinene (5.9%)and oxygen-containing compounds carvacrol methyl ether (5.8%), thymol methyl ether (5.1%), carvacrol (5.3%), geraniol (5.0%) and borneol (3.9%). The evaluation of antioxidant power was performed in vitro by the beta-carotene bleaching and thiobarbituric acid (TBA) methods. As determined with both methods, the total savory essential oil as well as different fractions or pure constituents containing hydroxyl group exhibited relatively strong antioxidant effect. The hydrocarbons, when isolated as CH fraction, showed the poorest effectiveness in spite the fact that this fraction contained gamma-terpinene, alpha-terpinene, p-cymene and terpinolene which previously were identified as potential antioxidants.