Gas Chromatography-Mass Spectrometry Analysis of Volatile Organic Compounds from Three Endemic Iris Taxa: Headspace Solid-Phase Microextraction vs. Hydrodistillation.

Iris taxa are sources of valuable essential oils obtained from aged rhizomes used by various industries, including pharmacy, cosmetic, perfume, and food industry, in which irones are the most important aroma components. In this study, volatile organic compounds (VOCs) obtained from dried rhizomes of three endemics from Croatia, Iris pseudopallida, I. illyrica, and I. adriatica, were studied. The VOCs were isolated by three different methods: headspace solid-phase microextraction (HS-SPME) using divinylbenzene/carboxene/polydimethylsiloxane (DVB/CAR/PDMS) fiber or polydimethylsiloxane/divinylbenzene (PDMS/DVB) fiber, and hydrodistillation (HD). The samples were analyzed by gas chromatography-mass spectrometry (GC-MS). In five out of six samples, the main compounds detected by HS-SPME were perilla aldehyde, butan-2,3-diol, acetic acid, 2-phenylethanol, benzyl alcohol, hexanal, and nonanal, while 6-methylhept-5-en-2-one, trans-caryophyllene, and ethanol were common for all studied samples. The former VOCs were absent from the oldest, irone-rich I. pseudopallida sample, mainly characterized by cis-α-irone (43.74-45.76%). When using HD, its content was reduced (24.70%), while docosane prevailed (45.79%). HD yielded predominantly fatty acids, including myristic, common for all studied taxa (4.20-97.01%), and linoleic (40.69%) and palmitic (35.48%) as the major VOCs of I. adriatica EO. The performed GC-MS analyses of EOs, in combination with HS-SPME/GC-MS, proved to be useful for gaining a better insight into Iris VOCs.

Effects of epidurally administered dexmedetomidine and dexamethasone on postoperative pain, analgesic requirements, inflammation, and oxidative stress in thoracic surgery.

The aim of this study was to compare the effects of dexmedetomidine and dexamethasone as adjuvants to preoperative epidural administration of local anesthetic (ropivacaine) in thoracic surgery on the postoperative level of pain, use of analgesics, inflammation, and oxidative stress. The study enrolled 42 patients who underwent elective thoracic surgery in a one-year period at the University Hospital Dubrava (Zagreb, Croatia). Based on a computer-generated randomization list the patients were assigned to the dexmedetomidine (n = 18) or dexamethasone (n = 24) group. Postoperatively, patients of dexmedetomidine group reported lower pain (VAS value 1 h post surgery, 3.4 ± 2.7 vs. 5.4 ± 1.8, dexmedetomidine vs. dexamethasone, p < 0.01) and had lower anal-gesic requirements in comparison with dexamethasone group. Thus, dexmedetomidine in comparison with dexamethasone was more efficient in lowering pain and analgesia requirements 24 h after the surgery. On the contrary, dexamethasone had better anti-inflammatory properties (CRP level 24 h post surgery, 131.9 ± 90.7 vs. 26.0 ± 55.2 mg L-1, dexmedetomidine vs. dexamethasone, p < 0.01). Both dexmedetomidine and dexamethasone exhibited antioxidant effects, however, their antioxidant properties should be further explored. The results of this study improve current knowledge of pain control in thoracic surgery.

Globularia alypum L. and Related Species: LC-MS Profiles and Antidiabetic, Antioxidant, Anti-Inflammatory, Antibacterial and Anticancer Potential.

Species from the genus Globularia L. have been used as healing agents for various ailments, with utilization of Globularia alypum L. being most frequently reported. The aim of this study was to evaluate the antidiabetic, antioxidant, anti-inflammatory, antibacterial and anticancer potential of G. alypum and three related species, G. punctata Lapeyr., G. cordifolia L. and G. meridionalis (Podp.) O.Schwarz, in relation to their phytochemical compositions. Globularin and verbascoside were identified using LC-PDA-ESI-MSn as the major metabolites of G. alypum with known biological activities. G. alypum demonstrated the greatest α-glucosidase inhibitory activity and DPPH radical scavenging activity (IC50 = 17.25 µg/mL), while its anti-inflammatory activity was not significantly different from those of related species. All investigated species showed considerable antibacterial activity against methicillin-resistant Staphylococcus aureus in the broth microdilution method (MIC = 1.42-3.79 mg/mL). G. punctata also showed antibacterial activities against Escherichia coli (MIC = 1.42 mg/mL), Bacillus subtilis (MIC = 1.89 mg/mL), B. cereus (MIC = 2.84 mg/mL) and Enterococcus faecalis (MBC = 5.68 mg/mL). G. punctata, G. cordifolia and G. meridionalis showed greater anticancer potential than G. alypum. Obtained results indicate investigated Globularia species could serve as sources of diverse bioactive molecules, with G. punctata having the greatest antibacterial potential.

Comparison of Volatile Organic Compounds of Sideritis romana L. and Sideritis montana L. from Croatia.

A study on the headspace volatile organic compounds (VOCs) profile of native populations of Sideritis romana L. and Sidertis montana L., Lamiaceae, from Croatia is reported herein, to elucidate the phytochemical composition of taxa from this plant genus, well-known for traditional use in countries of the Mediterranean and the Balkan region. Headspace solid-phase microextraction (HS-SPME), using divinylbenzene/carboxene/polydimethylsiloxane (DVB/CAR/PDMS) or polydimethylsiloxane/divinylbenzene (PDMS/DVB) fiber, coupled with gas chromatography-mass spectrometry (GC-MS) was applied to analyze the dried aerial parts of six native populations in total. Furthermore, principal component analysis (PCA) was conducted on the volatile constituents with an average relative percentage ≥1.0% in at least one of the samples. Clear separation between the two species was obtained using both fiber types. The VOCs profile for all investigated populations was characterized by sesquiterpene hydrocarbons, followed by monoterpene hydrocarbons, except for one population of S. romana, in which monoterpene hydrocarbons predominated. To our knowledge, this is the first report on the VOCs composition of natural populations of S. romana and S. montana from Croatia as well as the first reported HS-SPME/GC-MS analysis of S. romana and S. montana worldwide.

Characterization of the CYP3A4 Enzyme Inhibition Potential of Selected Flavonoids.

Acacetin, apigenin, chrysin, and pinocembrin are flavonoid aglycones found in foods such as parsley, honey, celery, and chamomile tea. Flavonoids can act as substrates and inhibitors of the CYP3A4 enzyme, a heme containing enzyme responsible for the metabolism of one third of drugs on the market. The aim of this study was to investigate the inhibitory effect of selected flavonoids on the CYP3A4 enzyme, the kinetics of inhibition, the possible covalent binding of the inhibitor to the enzyme, and whether flavonoids can act as pseudo-irreversible inhibitors. For the determination of inhibition kinetics, nifedipine oxidation was used as a marker reaction. A hemochromopyridine test was used to assess the possible covalent binding to the heme, and incubation with dialysis was used in order to assess the reversibility of the inhibition. All the tested flavonoids inhibited the CYP3A4 enzyme activity. Chrysin was the most potent inhibitor: IC50 = 2.5 ± 0.6 µM, Ki = 2.4 ± 1.0 µM, kinact = 0.07 ± 0.01 min-1, kinact/Ki = 0.03 min-1 µM-1. Chrysin caused the highest reduction of heme (94.5 ± 0.5% residual concentration). None of the tested flavonoids showed pseudo-irreversible inhibition. Although the inactivation of the CYP3A4 enzyme is caused by interaction with heme, inhibitor-heme adducts could not be trapped. These results indicate that flavonoids have the potential to inhibit the CYP3A4 enzyme and interact with other drugs and medications. However, possible food-drug interactions have to be assessed clinically.

Optimization of a fast screening method for the assessment of low molecular weight thiols in human blood and plasma suitable for biomonitoring studies.

An adequate level of low molecular weight thiols (LMW-SH, especially glutathione (GSH)) protects cellular macromolecules against toxic agents, and is used as a sensitive biomarker of exposure to toxic compounds. During sample collection, storage and preparation, non-enzymatic and enzymatic oxidation of LMW-SH can occur leading to analytical inaccuracy. The aim of this study was to optimize a fast and reliable screening method for the determination of LMW-SH, mainly GSH, in blood and plasma samples as well as to investigate the impact of storage conditions on the LMW-SH stability. Based on our results, the described spectrophotometric method allows fast and reliable determination of LMW-SH in blood and plasma samples. Results on incubation of samples at 37 °C imply that synthesis of LMW-SH (probably GSH) as well as dynamic interexchange among various thiols forms can be induced in blood cells in in vitro conditions. Importantly, the level of LMW-SH in blood and plasma stored at -20 °C was constant, indicating that they can be stored at -20 °C for at least 30 days. Therefore, the method is suitable for assessment of LMW-SH in long-term human biomonitoring as well as environmental field studies, especially those involving a large number of samples such as epidemiological studies.

Antithrombotic activity of flavonoids and polyphenols rich plant species.

Cardiovascular diseases represent one of the most notable health problems of the modern civilization. Stroke and heart attack often lead to lethal outcome; essential problem underneath being thrombus formation. Prophylactic approaches include acetylsalicylic acid and clopidogrel therapy on the level of primary hemostasis, i.e., primary clot formation. In the last five years, in the USA, health care expenses related to cardiovascular diseases have increased 50 %, to over 350 billion dollars. Thus, application of plant species and medicinal plants rich in polyphenols in prevention of thrombus formation are of interest. This is supported by the fact that the number of publications on antiaggregatory effect of polyphenols has doubled in the last decade. In this review we focus on antiaggregatory effect of most abundant polyphenols - flavonoids, the effect of plant extracts rich in polyphenols (propolis, species Salvia sp., Calamintha nepeta L., Lavandula angustifolia Mill., Melissa officinalis L, Mentha x piperita L., Ocimum basilicum L., Origanum vulgare L., Rosmarinus officinalis L.) on platelet aggregation, association of chemical composition and antioxidant properties with the observed biological effect, and possible clinical significance of the published results.

Screening of flavonoid aglycons' metabolism mediated by the human liver cytochromes P450.

Biological effects of flavonoids have been extensively studied in the last 80 years. As flavonoids represent a rather large group of compounds, data on metabolic biotransformations of these compounds is relatively limited to those well studied. The objective of this study was to screen the metabolism of 30 selected flavonoid aglycons mediated by the most relevant metabolic enzymes, human liver cytochromes P450. For this purpose, in vitro experiments with human liver microsomes and recombinant enzymes were conducted. To evaluate flavonoid's metabolism and structure of the products, high-performance liquid chromatography coupled with high-resolution mass spectrometry was used. Out of 30 flavonoids, 15 were susceptible to oxidative metabolism mediated by cytochromes P450. Dominant reactions were aromatic hydroxylation and O-demethylation, or a combination of these reactions. The dominant enzyme responsible for the observed metabolic reactions is CYP1A2, whereas other human liver cytochromes P450, namely, CYP2C19, CYP2D6, CYP2E1 and CYP3A4, contribute to flavonoid metabolism to a lesser degree. These results, to some extent, contribute to the understanding of the metabolism of constituents found in antioxidant dietary supplements and their possible interactions with other xenobiotics, i.e., medicinal products.

Actualities in the phytochemical research on selected terpenes.

A short review of our recent research on the essential oil phytochemical composition of Petasites albus (L.) Gaertn. and Petasites hybridus (L.) G. Gaertn., B. Mey. & Scherb. (Asteraceae) as well as on the oils of Globularia cordifolia L., Globularia meridionalis (Podp.) O. Schwarz and Globularia punctata Lapeyr. (Plantaginaceae) is presented. All essential oils contained a variety of oxygenated sesquiterpenes among their major constituents, including a bakkane type sesquiterpene fukinanolid (bakkenolide A). The paper is focused on: i) a short overview of the abundance of major terpenes in the essential oils of Petasites and Globularia species from Croatia; ii) possible biosynthetic pathways of major identified sesquiterpenes; and iii) biological activities (literature data) of major sesquiterpenes from Petasites and Globularia species.

Influence of flavonoids' lipophilicity on platelet aggregation.

Flavonoids are natural polyphenolic compounds present in a wide spectrum of plants that have a beneficial effect on human health. In the context of cardiovascular diseases related to plaque and thrombus formation, flavonoids exhibit an anti-aggregatory effect. Previously, it has been reported that all tested flavonoids exhibit an antiaggregatory effect on platelet aggregation when measured by impedance aggregometry on whole blood, in the test of aggregation induced by adenosine diphosphate (ADP). As not all flavonoids have the same targets within signaling pathways, an assumption of a common non-specific mechanism related to lipophilicity is to be considered. To test this hypothesis, reverse-phase thin layer chromatography was used to assess the lipophilicity of flavonoids; impedance aggregometry was used for testing of platelet aggregation and flow cytometry to monitor the influence of flavonoids on platelet activation. Lipophilicity analysis showed a highly negative correlation of logP and MINaAC for groups of flavones and flavanones. As determined by flow cytometry, the exposition of receptors necessary for the promotion of platelet activation and primary clot formation was diminished, i.e., lowered expression of the activated form of integrin αIIbß3 was observed in the presence of flavanone. Platelet membrane stabilization by flavonoids as a mechanism of antiaggregatory effect has been supported by impedance aggregometry experiments when specific inhibitors of platelet aggregation signaling pathways (U73122, indomethacin, verapamil) were used in the presence of a weak (ADP) and a strong (TRAP-6) agonist of aggregation. While individual flavonoids can have specific targets within aggregation signaling pathways, all flavonoids share a common non-specific mechanism of platelet aggregation inhibition related to their lipophilicity and membrane stabilization that, to some extent, contributes to their antiaggregatory effect.

A modern approach to the treatment of plaque psoriasis.

Psoriasis is a common chronic inflammatory skin disease which affects 0.5-1 % of children and 2-3 % of the adult population. In Croatia, 1.6 % of the population suffer from psoriasis. Distribution of the disease is bimodal, with the first peak at the age of 20-30, and the second at the age of 50-60. The etiopathogenesis of the disease is multifactorial, the key factors being genetic predisposition combined with immunological disorders, environmental factors and skin barrier damage. There are several clinical variants of the disease. The main signalling pathways in psoriasis include TNF-α, IL-23 and IL-17. Topical agents are used for the treatment of the mild form, and the systemic conventional therapy is used for the treatment of moderate to severe forms of the disease. In cases where's no response, or intolerance or contraindications are present, new targeted medications are to be administered. Development in the field of immunogenetics of psoriasis leads to personalized medicine.

Nutrition - facts and myths.

Taking responsibility for your life, among other factors, means also considering what to eat and which nutrition pattern to follow. Everyone needs to think about what they put on the plate and which ingredients should be avoided. Food, as such, will never be a drug or medication, like a painkilling tablet relieving pain in a short amount of time, for example. However, proper nutrition is our ally in the prevention of diseases, maintaining balance in our body and our mind. By following the main principles of a healthy diet, the physiological homeostasis can be managed, as well as faster recovery from disease achieved. This review is aimed at summarizing basic principles of nutrition recommendations and at empowering stakeholders (pharmacists, medical biochemists, physicians) to be able to communicate to their patients and customers healthy and sustainable nutrition choices through the personalized advice.

Phenolic compounds of Iris adriatica and their antimycobacterial effects.

Little is known about the pharmacological activities of Iris adriatica (Iridaceae), a plant endemic to Dalmatia (Croatia). We therefore performed a bioassay-guided fractionation including high-performance counter current chromatography (HPCCC) and antibacterial tests using Mycobacterium smegmatis mc2 155. One obtained fraction was found to be antimycobacterially active with a MIC of 64 mg L-1. Furthermore, fractions were tested for resistance modulatory effects using ethidium bromide as substrate. We were able to identify the pure isoflavonic compounds irigenin and irilone and a fraction containing mainly benzophenone 2,4,6-trihydroxy-4-methoxy-benzophenone, responsible for the resistance-modulatory activity of this plant.

Application of medicinal plants in several dermatovenerological entities.

Treatment of skin conditions with medicinal plants has been an ongoing human activity lasting over thousands of years. The use of specific plant species developed regionally, based on local flora. Commonly used medicinal plants for dermatological complaints are: Phlebodium aureum (L.) J. Sm., Ginkgo biloba L., Rosmarinus officinalis L., Panax ginseng C.A.Mey., Allium cepa L., Aloe vera (L.) Burm.f., Capsicum annuum L., Berbe ris aquifolium Pursh, Camellia sinensis (L.) Kuntze, and Podophyllum peltatum L. The demand for complementary therapeutics is an emerging trend due to the awareness of potential side effects that synthetic drugs might cause. More scientific evidence and better documentation are needed before advising dermatologic patients on herbal medicinal treatment. Standardised extracts and formulations with proven clinical efficacy should be developed for this cause. Here provided review entails the use of herbal medicinal products in the treatment of frequent chronic skin diseases, such as vitiligo, alopecia, psoriasis and genital warts.

The Effect of Flavonoid Aglycones on the CYP1A2, CYP2A6, CYP2C8 and CYP2D6 Enzymes Activity.

Cytochromes P450 are major metabolic enzymes involved in the biotransformation of xenobiotics. The majority of xenobiotics are metabolized in the liver, in which the highest levels of cytochromes P450 are expressed. Flavonoids are natural compounds to which humans are exposed through everyday diet. In the previous study, selected flavonoid aglycones showed inhibition of CYP3A4 enzyme. Thus, the objective of this study was to determine if these flavonoids inhibit metabolic activity of CYP1A2, CYP2A6, CYP2C8, and CYP2D6 enzymes. For this purpose, the O-deethylation reaction of phenacetin was used for monitoring CYP1A2 enzyme activity, coumarin 7-hydroxylation for CYP2A6 enzyme activity, 6-α-hydroxylation of paclitaxel for CYP2C8 enzyme activity, and dextromethorphan O-demethylation for CYP2D6 enzyme activity. The generated metabolites were monitored by high-performance liquid chromatography coupled with diode array detection. Hesperetin, pinocembrin, chrysin, isorhamnetin, and morin inhibited CYP1A2 activity; apigenin, tangeretin, galangin, and isorhamnetin inhibited CYP2A6 activity; and chrysin, chrysin-dimethylether, and galangin inhibited CYP2C8. None of the analyzed flavonoids showed inhibition of CYP2D6. The flavonoids in this study were mainly reversible inhibitors of CYP1A2 and CYP2A6, while the inhibition of CYP2C8 was of mixed type (reversible and irreversible). The most prominent reversible inhibitor of CYP1A2 was chrysin, and this was confirmed by the docking study.

Essential Oil Composition of Different Plant Parts from Croatian Petasites albus (L.) Gaertn. and Petasites hybridus (L.) G.Gaertn., B.Mey. & Scherb. (Asteraceae).

Essential oil compositions of fresh leaves, flower stems and rhizomes obtained by hydrodistillation from different Croatian populations of Petasites albus (L.) Gaertn. and Petasites hybridus (L.) G.Gaertn., B.Mey. & Scherb. (four of each species) were investigated using gas chromatography-flame ionization detection and gas chromatography-mass spectrometry. Altogether, 118 constituents were identified, accounting for 81.19-96.81 % of total oil composition. All essential oils were characterized by oxygenated sesquiterpenes, with distinct compounds recorded for both investigated species. Clear separation between the two species was confirmed by principal component analysis and hierarchical cluster analysis. This is the first study that recorded intraspecific variations of essential oil constituents from P. albus and P. hybridus. Obtained results could contribute to the understanding of their medicinal and nutritional value.

The Inhibitory Effect of Flavonoid Aglycones on the Metabolic Activity of CYP3A4 Enzyme.

Flavonoids are natural compounds that have been extensively studied due to their positive effects on human health. There are over 4000 flavonoids found in higher plants and their beneficial effects have been shown in vitro as well as in vivo. However, data on their pharmacokinetics and influence on metabolic enzymes is scarce. The aim of this study was to focus on possible interactions between the 30 most commonly encountered flavonoid aglycones on the metabolic activity of CYP3A4 enzyme. 6ß-hydroxylation of testosterone was used as marker reaction of CYP3A4 activity. Generated product was determined by HPLC coupled with diode array detector. Metabolism and time dependence, as well as direct inhibition, were tested to determine if inhibition was reversible and/or irreversible. Out of the 30 flavonoids tested, 7 significantly inhibited CYP3A4, most prominent being acacetin that inhibited 95% of enzyme activity at 1 µM concentration. Apigenin showed reversible inhibition, acacetin, and chrysin showed combined irreversible and reversible inhibition while chrysin dimethylether, isorhamnetin, pinocembrin, and tangeretin showed pure irreversible inhibition. These results alert on possible flavonoid⁻drug interactions on the level of CYP3A4.

The Effect of Short-Toothed and DalmatianSage Extracts on Platelet Aggregation.

Sage (Salvia sp.) is a genus of a native Mediterranean plant used for aromatization of foods. In this study, total polyphenolic profile, antioxidant properties and antiplatelet effect of short-toothed sage (Salvia brachyodon Vandas) are analyzed and compared to most commonly used Salvia species (S. officinalis, Dalmatian sage). Content of total flavonoids was 0.08-0.23% and of phenolic acids 0.47-3.04%. Antioxidant DPPH assay showed higher antioxidant capacity of S. brachyodon (29-36 mg/mL of gallic acid equivalents) than of S. officinalis. In functional test of primary haemostasis, extracts of S. brachyodon have prevented platelet aggregation in nanomolar concentration (21 nM), thus showing potential in prevention of thrombus formation as functional food or dietary supplement. Antiplatelet activity was related to antioxidant capacity (r=0.7014, p=0.0352) indicating that prevention of aggregation is not caused by an individual component, but it is rather a result of synergistic effect of polyphenols. The obtained results are preliminary and a more detailed study of the possibility of applying the investigated plant species as supplements for the prevention of the problem of cardiovascular system and platelet aggregation is needed.

Propolis ethanolic extracts reduce adenosine diphosphate induced platelet aggregation determined on whole blood.

BACKGROUND: Propolis is a well-known bee product containing more than 2000 identified compounds. It has many beneficial effects on human health that include antibacterial, antiviral, anticancer and hepatoprotective justifying its use as a dietary supplement. Platelet aggregation plays crucial role in thrombus formation that can cause stroke or heart attacks. As cardiovascular diseases, including those caused by thrombus formation, are related to 50% of deaths of Western population, the objective of this study was to determine antiaggregatory activity of propolis on platelet aggregation on the whole blood samples. METHODS: Twenty one propolis samples from Southeast Europe were characterized by spectrophotometric methods to determine content of the total flavonoids and phenolic acids. High performance liquid chromatography coupled with diode array detection was used to identify and quantify individual polyphenols. Platelet aggregation was tested by impedance aggregometry on the whole blood samples of ten healthy volunteers. RESULTS: The mean content of total polyphenols was 136.14 mg/g and ranged from 59.23 to 277.39 mg/g. Content of total flavonoids ranged between 6.83 and 55.44 mg/g with the mean value of 19.28 mg/g. Percentage of total phenolic acids was in the range 8.79 to 45.67% (mean 26.63%). Minimal antiaggregatory concentration, representing the lowest concentration of propolis extract sample that can cause statistically significant reduction of aggregation, ranged from 5 µM to 10.4 mM. Samples of propolis with lower content of luteolin and higher content of pinocembrin-7-methyleter showed better antiplatelet activity i.e. lower values of minimal antiaggregatory concentration. CONCLUSIONS: This is the first study that shows antiaggregatory potential of propolis ethanolic extracts on the whole blood samples in the low micromolar concentrations suggesting that propolis supplementation may influence platelet aggregation and consequently thrombus formation. Further in vivo studies are needed to confirm the beneficial effects in prevention of cardiovascular diseases.

Quantitative Analysis of Phenolic Acids and Antiplatelet Activity of Melissa officinalis Leaf Extracts.

The contents of total phenolic acids and rosmarinic acid were determined and the antiplatelet potential assessed of extracts of lemon balm (Melissa officinalis L., Lamiaceae) leaf samples collected during 2014 and 2015. The quantity of total phenolic acids ranged from 4.8 to 8.5%, while the rosmarinic acid content ranged from 1.78 to 4.35%. Results of statistical analysis showed that the undiluted extracts have antiplatelet activity in the ADP test (final concentration 31.25 mg/L). While there are reports on antiplatelet activity of lemon balm essential oil, to the best of our knowledge, this is the first report on antiplatelet activity of lemon balm leaf extracts.