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Recent research on the extraction of valuable natural compounds, especially plants antioxidants, was focused on the use of green solvents, including deep eutectic solvents (DES). In this paper, for the first time, selected DES solvents were applied for the extraction of phenolic acids and flavonoids from elderberry (Sambucus nigra L.) and rosemary (Rosmarinus officinalis L.). The presence of those polyphenolic compounds in elderberry flowers and rosemary leaves was detected by thin-layer chromatography, and their contents were determined spectrophotometrically. Extraction efficiencies of water, 70% ethanol and DESs based on choline chloride (ChCl) and ethylene glycol (EG) in a molar ratio of 1:3 and choline chloride and glycerol (Gly) in the same molar ratio were compared. DESs were applied in pure form and with the addition of 30% and 50% of water (V/V). Obtained results showed that 50% ChCl:Gly and 50% ChCl:EG have similar efficiency for phenolic acid extraction as commonly used 70% ethanol. The EG-based DES showed slightly higher performance for the extraction of rosemary phenolic acids, while the one based on Gly showed better results for elderberry. With respect to flavonoid extraction from elderberry flowers, 70% ChCl:EG was comparable to 70% ethanol. Our study demonstrated that tested novel and environmentally friendly DESs could replace ethanol for the extraction of plant polyphenols.

Phlomis fruticosa L. and P. herba-venti are species belonging to the Lamiaceae family, which have been traditionally used to prepare tonic and digestive drinks. Multiple studies also demonstrated the inhibitory effects of P. fruticosa extracts and essential oil against oxidative/proinflammatory pathways and bacterial strains deeply involved in ulcerative colitis. Considering these findings, the present study evaluated the effects of alcoholic P. fruticosa and P. herba-venti leaf extracts in isolated rat colon challenged with Escherichia coli lipopolysaccharide (LPS), an ex vivo experimental paradigm of ulcerative colitis. In this context, we assayed colon levels of pro-oxidant and proinflammatory biomarkers, including nitrites, malondialdehyde (MDA), lactate dehydrogenase (LDH), and serotonin (5-HT). Additionally, the extracts have been tested in order to evaluate possible inhibitory effects on specific bacterial and fungal strains involved in ulcerative colitis. Alcoholic P. fruticosa and P. herba-venti extracts were able to blunt LPS-induced nitrite, MDA, 5-HT, and LDH levels in isolated rat colon. The same extracts also inhibited the growth of Pseudomonas aeruginosa, E. coli, Staphylococcus aureus, Candida albicans and Candida tropicalis. In conclusion, our findings show a potential role exerted by alcoholic P. fruticosa and P. herba-venti in managing the clinical symptoms related to ulcerative colitis.

The polyphenolic composition and anti-inflammatory activity of selected Thymus species (T. longicaulis C. Presl, T. praecox Opiz subsp. polytrichus (A. Kerner Ex Borbas) Jalas, T. pulegioides L., T. serpyllum L. subsp. serpyllum and T. striatus Vahl) was studied in comparison with T. vulgaris, as an officinal medicinal plant, to evaluate the biomedical potential of these Croatian wild-growing species. This paper provides a first insight into the polyphenolic profiles of T. praecox subsp. polytrichus and T. striatus. Also, some flavone and hydroxycinnamic derivatives were at first identified in the other studied species by LC-DAD-ESI–MS/MS analysis. Rosmarinic acid and glycosides of scutellarein and luteolin were found to be the most abundant polyphenolic constituents. The Thymus extracts inhibited Src tyrosine kinase activity and they reduced the production of proinflammatory cytokine interleukin-6 in Balb/C mice splenocytes. The obtained results highlighted T. longicaulis as a potential source of herbal drugs with anti-inflammatory properties and/or novel functional food rich in flavonoids and phenolic acids.

In the search for new natural resources with promising biological activity, chemical composition as well as antibacterial, antifungal and antioxidant activities of the essential oil from Croatian indigenous cultivar of lavandin (Lavandula × intermedia‘Budrovka’) were studied for the first time and compared with those of lavender (L. angustifolia) cultivated under the same pedo-climatic conditions of central Croatia. Forty compounds were identified in the studied Lavandula essential oils by GC–MS analysis. Linalool (57.1%), linalyl acetate (9.8%) and 1,8-cineole (8.4%) were the most abundant compounds of lavandin ‘Budrovka’ oil while lavender oil contained mainly linalool (54.0%), linalyl acetate (11.6%) and lavandulol (6.6%). Both essential oils were characterized by a very low amount of camphor (≤0.3%). The antimicrobial activities of two Lavandula essential oils and their dominant constituents were evaluated against 30 medically important pathogens and microbial contaminants of food, cosmetic and pharmaceutical products using agar well diffusion and broth dilution methods. L. × intermedia ‘Budrovka’ essential oil showed a broad-spectrum of antibacterial (MICs 0.25–2.5 mg/mL) and antifungal (MICs 0.1–2 mg/mL) activities, being generally more than or at least as effective as those from L. angustifolia. Among the two dominant compounds which were also tested comparatively and found to exhibit antimicrobial effect, linalool has been pointed out as much more potent agent then linalyl acetate. Antioxidant studies based on DPPH assay indicated that the lavandin ‘Budrovka’ essential oil has significantly stronger antioxidant properties than lavender essential oil (IC50 21.6 mg/mL and 27.7 mg/mL, respectively). This study highlighted Croatian indigenous cultivar lavandin ‘Budrovka’ as a rich source of essential oil with pronounced antimicrobial properties due to abundance of linalool and indicated its potential use as biopreservative in food, pharmaceutical and cosmetic products or as natural remedy with promising health benefit.

The present study aimed to evaluate antioxidant and acetylcholinesterase (AChE) inhibitory activities of the ethanolic extracts of six selectedThymusspecies growing in Croatia (T. longicaulis,T. praecoxsubsp.polytrichus,T. pulegioides,T. serpyllumsubsp.serpyllum,T. striatus, andT. vulgaris). Antioxidant effectiveness was assessed using six different assays, in comparison with rosmarinic acid, luteolin, and reference antioxidants. All testedThymusextracts possessed DPPH (IC50= 3–6 μg/mL) and nitric oxide (IC50= 70–177 μg/mL) free radical scavenging activities, strong reducing properties (IC50= 11–15 μg/mL), ferrous ion chelating activity (IC50= 126–389 μg/mL), ability to inhibit lipid peroxidation (IC50= 34–80 μg/mL), and high total antioxidant capacities (238–294 mg AAE/g). AChE inhibitory activity was examined using Ellman's colorimetric method and all tested extracts showed anti-AChE activity in a dose dependent manner. The values of 10–28%, 23–39%, and 64–86% were obtained for tested concentrations of 0.25, 0.5, and 1 mg/mL, respectively. Additionally, the contents of total hydroxycinnamic derivatives, flavonoids, and tannins in dried plant samples were determined spectrophotometrically. Our results highlightedThymusspecies as a rich source of natural antioxidants and AChE inhibitors that could be useful in preventing and treating Alzheimer’s disease and other neurodegenerative disorders.

Background Combining conventional drugs and traditional medicine may represent a useful approach to combating antibiotic resistance, which has become a serious threat to global public health. This study aimed to evaluate the potential synergistic interactions between Tanreqing (TRQ) injection, a commercial traditional Chinese medicine formula used for the treatment of upper respiratory tract infection, and selected antibiotics used against methicillin-resistant Staphylococcus aureus (MRSA). Methods The minimum inhibitory concentrations (MICs) of TRQ, vancomycin and linezolid against planktonic MRSA strain were determined by the broth microdilution method. The combined effects of TRQ and antibiotics were studied by the checkerboard method and the time-kill curve assay. The 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assay was employed to determine the inhibitory effect of the test compounds alone and in combination against MRSA embedded in biofilms. Results MRSA strain was found to be susceptible to TRQ formula with MIC value 4125 μg/ml, while the MIC values for antibiotics, vancomycin and linezolid, were 2.5 μg/ml. The checkerboard analysis revealed that TRQ markedly enhanced activities of the tested antibiotics by reducing their MICs. In the time-kill analysis, TRQ at 1/2 × MIC in combination with vancomycin at 1/2 × MIC, as well as TRQ at 1/8 × MIC in combination with linezolid at 1/2 × MIC decreased the viable colonies by ≥2log10 CFU/ml, resulting in a potent synergistic effect against planktonic MRSA. In contrast to the tested antibiotics, which did not affect mature MRSA biofilms at subinhibitory concentrations, TRQ alone showed strong ability to disrupt preformed biofilms and induce biofilm cell death. The combination of TRQ with vancomycin or linezolid at sub-MIC concentrations resulted in a synergistic antibiofilm effect significantly higher than for each single agent. Conclusions This study provides the first in vitro evidence on the synergistic effects of TRQ and vancomycin or linezolid against planktonic and biofilm MRSA, and revealed their optimal combination doses, thereby providing a rational basis for the combination therapies against MRSA.

Diosmetin (DIOS) is a flavone aglycone commonly occurring in citrus species and olive leaves, in addition it is one of the active ingredients of some medications. Based on both in vitro and in vivo studies several beneficial effects are attributed to DIOS but the biochemical background of its action seems to be complex and it has not been completely explored yet. Previous investigations suggest that most of the flavonoid aglycones have negative effect on ATP synthesis in a dose dependent manner. In our study 17 flavonoids were tested and interestingly DIOS caused a significant elevation of intracellular ATP levels after 6- and 12-h incubation in MDCK kidney cells. In order to understand the mechanism of action, intracellular ATP and protein levels, ATP/ADP ratio, cell viability and ROS levels were determined after DIOS treatment. In addition, impacts of different enzyme inhibitors and effect of DIOS on isolated rat liver mitochondria were also tested. Finally, the influence of DIOS on the ATP depleting effect of the mycotoxin, ochratoxin A was also investigated. Our major conclusions are the followings: DIOS increases intracellular ATP levels both in kidney and in liver cells. Inhibition of glycolysis or citric acid cycle does not decrease the observed effect. DIOS-induced elevation of ATP levels is completely abolished by the inhibition of ATP synthase. DIOS is able to completely reverse the ATP-depleting effect of the mycotoxin, ochratoxin A. Most probably the DIOS- induced impact on ATP system does not originate from the antioxidant property of DIOS. Based on our findings DIOS may be promising agent to positively influence ATP depletion caused by some metabolic poisons.

Migraine is one of the most common neurological disorder, which has long been related to brain serotonin (5-HT) depletion and neuro-inflammation. Despite many treatment options are available, the frequent occurrence of unacceptable adverse effects further supports the research toward nutraceuticals and herbal preparations, among which Tanacetum parthenium and Salix alba showed promising anti-inflammatory and neuro-modulatory activities. The impact of extract treatment on astrocyte viability, spontaneous migration and apoptosis was evaluated. Anti-inflammatory/anti-oxidant effects were investigated on isolated rat cortexes exposed to a neurotoxic stimulus. The lactate dehydrogenase (LDH) release, nitrite levels and 5-HT turnover were evaluated, as well. A proteomic analysis was focused on specific neuronal proteins and a fingerprint analysis was carried out on selected phenolic compounds. Both extracts appeared able to exert in vitro anti-oxidant and anti-apoptotic effects. S. alba and T. parthenium extracts reduced LDH release, nitrite levels and 5-HT turnover induced by neurotoxic stimulus. The downregulation of selected proteins suggest a neurotoxicity, which could be ascribed to an elevated content of gallic acid in both S. alba and T. parthenium extracts. Concluding, both extracts exert neuroprotective effects, although the downregulation of key proteins involved in neuron physiology suggest caution in their use as food supplements.

The well-known 4-hydroxycoumarin derivative warfarin is a widespread anticoagulant drug. Besides its strong albumin binding property warfarin has a narrow therapeutic window. Therefore, a few percent of displacement from albumin can result in serious biological consequences. The flavonoid molecular group also shows very strong plasma albumin binding characteristics occupying the same binding site. It is plausible to hypothesize that flavonoid aglycones may be able to displace warfarin from human serum albumin (HSA). In our study the competing activities of different flavone (acacetin, apigenin, chrysin, luteolin), flavonol (galangin, quercetin) and flavanone (hesperetin, naringenin) aglycones were investigated using fluorescence spectroscopy. Our results represent that flavonoids are able to displace warfarin from the surface of HSA. On the other hand, when comparing flavone or flavonol groups to flavanones the latter group seems to be much weaker competitor. These observations were also supported by calculation of stability constants. Our investigations strongly suggest that we should reckon with the described molecular displacement. However, further in vivo studies are needed to support the findings of our model system.

Thymus longicaulis C. Presl is a small aromatic perennial herb used as a traditional remedy for cold, flu and cough. Composition of the essential oil of T. longicaulis from Croatia and its in vitro antimicrobial activity against the most common respiratory pathogens were evaluated. The yield of essential oil obtained by hydrodistillation from aerial plant parts was 1.2%. According to the GC-MS analysis, a total of forty one compounds (99%) were identified. Thymol (46.3%), γ-terpinene (16.2%), thymyl methyl ether (11.4%), and p-cymene (9.4%) were the main components. Antimicrobial activity of the essential oil against six clinically isolated bacterial and yeast strains was determined using standard disc agar diffusion method and microdilution broth assay. The essential oil exhibited antimicrobial activity towards all tested respiratory pathogens. The most sensitive strains were Haemophilus influenzae and Streptococcus pneumoniae(MIC=0.78 mg/mL), while Staphylococcus aureus was the most resistant (MIC>25.00 mg/mL). Our results indicate that T. longicaulis essential oil could be effective against clinically relevant respiratory pathogens which have the ability to develop resistance to antimicrobial drugs.

Ochratoxin A (OTA) is a highly toxic mycotoxin found worldwide in cereals, foods, animal feeds and different drinks. Based on previous studies, OTA is one of the major causes of the chronic tubulointerstitial nephropathy known as Balkan endemic nephropathy (BEN) and exerts several other adverse effects shown by cell and/or animal models. It is a well-known fact that OTA binds to various albumins with very high affinity. Recently, a few studies suggested that reducing the bound fraction of OTA might reduce its toxicity. Hypothetically, certain drugs can be effective competitors displacing OTA from its albumin complex. Therefore, we examined 13 different drug molecules to determine their competing abilities to displace OTA from human serum albumin (HSA). Competitors and ineffective chemicals were identified with a steady-state fluorescence polarization-based method. After characterization the competitive abilities of individual drugs, drug pairs were formed and their displacing activity were tested in OTA-HSA system. Indometacin, phenylbutazone, warfarin and furosemide showed the highest competing capacity but ibuprofen, glipizide and simvastatin represented detectable interaction too. Investigations of drug pairs raised the possibility of the presence of diverse binding sites of competing drugs. Apart from the chemical information obtained in our model, this explorative research might initiate future designs for epidemiologic studies to gain further in vivo evidence of long-term (potentially protective) effects of competing drugs administered to human patients. Copyright © 2012 John Wiley & Sons, Ltd.

To investigate the mechanisms underlying the protective effects of quercetin-rutinoside (rutin) and its aglycone quercetin against CCl(4)-induced liver damage in mice.BALB/cN mice were intraperitoneally administered rutin (10, 50, and 150 mg/kg) or quercetin (50 mg/kg) once daily for 5 consecutive days, followed by the intraperitoneal injection of CCl(4) in olive oil (2 mL/kg, 10% v/v). The animals were sacrificed 24 h later. Blood was collected for measuring the activities of ALT and AST, and the liver was excised for assessing Cu/Zn superoxide dismutase (SOD) activity, GSH and protein concentrations and also for immunoblotting. Portions of the livers were used for histology and immunohistochemistry.Pretreatment with rutin and, to a lesser extent, with quercetin significantly reduced the activity of plasma transaminases and improved the histological signs of acute liver damage in CCl(4)-intoxicated mice. Quercetin prevented the decrease in Cu/Zn SOD activity in CCl(4)-intoxicated mice more potently than rutin. However, it was less effective in the suppression of nitrotyrosine formation. Quercetin and, to a lesser extent, rutin attenuated the inflammation in the liver by down-regulating the CCl(4)-induced activation of nuclear factor-kappa B (NF-κB), tumor necrosis factor-α (TNF-α) and cyclooxygenase (COX-2). The expression of inducible nitric oxide synthase (iNOS) was more potently suppressed by rutin than by quercetin. Treatment with both flavonoids significantly increased NF-E2-related factor 2 (Nrf2) and heme oxygenase (HO-1) expression in injured livers, although quercetin was less effective than rutin at an equivalent dose. Quercetin more potently suppressed the expression of transforming growth factor-β1 (TGF-β1) than rutin.Rutin exerts stronger protection against nitrosative stress and hepatocellular damage but has weaker antioxidant and anti-inflammatory activities and antifibrotic potential than quercetin, which may be attributed to the presence of a rutinoside moiety in position 3 of the C ring.

Antioxidant activities of three selected Micromeria species growing in Croatia (M. croatica, M. juliana and M. thymifolia) were evaluated using five different antioxidant assays, in comparison with plant polyphenolic constituents and reference antioxidants. All studied ethanolic extracts exhibited considerable activity to scavenge DPPH and hydroxyl free radicals, reducing power, iron chelating ability and total antioxidant capacity in the order: M. croatica > M. juliana > M. thymifolia. Total polyphenol (9.69–13.66%), phenolic acid (5.26–6.84%), flavonoid (0.01–0.09%) and tannin (3.07–6.48%) contents in dried plant samples were determined spectrophotometrically. A strong positive correlation between antioxidant activities and contents of phenolic acids and tannins was found, indicating their responsibility for effectiveness of tested plants. Our findings established Micromeria species as a rich source of antioxidant polyphenols, especially the endemic M. croatica.

Flavonoids play an important role in the treatment of various diseases, as they are able to inhibit reactive oxygen species, which cause damage to cells and tissues which may lead to increased risk of inflammatory diseases. Baicalin and baicalein, two flavonoids found in the roots ofScutellaria baicalensis, in the leaves ofThymus vulgarisandOroxylum indicum, were tested for their anti-inflammatory activity as well as for their cytotoxicity. Thereby the two compounds were investigated on Src tyrosine kinase inhibition and inhibition of production of interleukin (IL-6) in lipopolysaccharide- (LPS-) stimulated THP-1 cells. Additionally, the THP-1 cell line was used for the determination of the cytotoxicity. Both baicalin and baicalein showed some anti-inflammatory properties, while baicalein turned out to be the more active compound with higher inhibitory activities on both Src tyrosine kinase and production of cytokine IL-6. Baicalin and baicalein showed no signs of cytotoxicity in the MTS cytotoxicity assay in THP-1 cells.

BACKGROUND: Micromeria croatica (Pers.) Schott is an aromatic plant from Lamiaceae family previously found to possess potent in vitro antioxidant activity which is mainly attributed to the high level of polyphenolic substances. The aim of this study was to investigate the hepatoprotective activity and possible underlying mechanisms of Micromeria croatica ethanolic extract (MC) using a model of carbon tetrachloride (CCl4)-induced liver injury in mice. METHODS: Male BALB/cN mice were randomly divided into seven groups: control group received saline, MC group received ethanolic extract of M. croatica in 5% DMSO (100 mg/kg b.w., p.o.), and CCl4 group was administered CCl4 dissolved in corn oil (2 mL/kg, 10% v/v, ip). MC50, MC200 and MC400 groups were treated with MC orally at doses of 50, 200 and 400 mg/kg once daily for 2 consecutive days, respectively, 6 h after CCl4 intoxication. The reference group received silymarin at dose of 400 mg/kg. At the end of experiment, blood and liver samples were collected for biochemical, histopathological, immunohistochemical and Western blot analyses. In addition, major phenolic compounds in MC were quantified by HPLC-DAD. RESULTS: CCl4 intoxication resulted in liver cells damage and oxidative stress and triggered inflammatory response in mice livers. MC treatment decreased ALT activity and prevented liver necrosis. Improved hepatic antioxidant status was evident by increased Cu/Zn SOD activity and decreased 4-hydroxynonenal (4-HNE) formation in the livers. Concomitantly, nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) were overexpressed. The hepatoprotective activity of MC was accompanied by the increase in nuclear factor-kappaB (NF-κB) activation and tumor necrosis factor-alpha (TNF-α) expression, indicating amelioration of hepatic inflammation. Additionally, MC prevented tumor growth factor-β1 (TGF-β1) and α-smooth muscle actin (α-SMA) expression, suggesting the potential for suppression of hepatic fibrogenesis. CONCLUSION: These results of the present study demonstrated that MC possesses in vivo antioxidant and anti-inflammatory activity and exhibits antifibrotic potential, which are comparable to those of standard hepatoprotective compound silymarin.

Micellar electrokinetic chromatography (MEKC) was used for the separation and determination of two iridoid glycosides, aucubin and catalpol, in several Plantago species growing in Croatia: P. altissima L., P. argentea Chaix, P. coronopus L., P. holosteum Scop. (subsp. depauperata, subsp. holosteum and subsp. scopulorum), P. lagopus L., P. lanceolata L., and P. maritima L. Hot water extraction (HWE) was applied for the isolation of iridoid substances. Significant differences appeared between the iridoid contents in the examined species. The yield of aucubin and catalpol was up to 0.27% and 1.81% of the dry mass of the leaves, respectively. Besides aucubin and catalpol, two related compounds were determined in the plant samples.

Myricetin-3-O-α-rhamnoside (myricitrin) is a naturally occurring phenolic compound which possesses antioxidant and anti-inflammatory activity. The aim of this study was to determine the hepatoprotective effects of myricitrin. Myricitrin at doses of 10, 30 and 100 mg/kg and silymarin at dose of 100 mg/kg were administered to BALB/cN mice by oral gavage, once daily for two consecutive days following carbon tetrachloride (CCl4)-intoxication. Myricitrin significantly ameliorated CCl4-induced increase in serum aspartate transaminase (AST) and alanine transaminase (ALT) levels and histopathological changes in the liver. Hepatic oxidative stress was reduced by myricitrin, as evidenced by the decrease in lipid peroxidation, with concomitant increase in glutathione (GSH) level and cytochrome P450 2E1 (CYP2E1) expression. In addition, cyclooxygenase-2 (COX-2) and tumor necrosis factor-alpha (TNF-α) overexpression in the liver was reduced, suggesting the suppression of inflammation. The expression of transforming growth factor-beta1 (TGF-β1) and alpha-smooth muscle actin (α- SMA) was markedly ameliorated, indicating the inhibition of profibrotic response. Myricitrin also improved the regeneration of hepatic tissue after CCl4- intoxication, as evidenced by increased proliferating cell nuclear antigen (PCNA) expression. The results of the current study suggest that myricitrin exhibits a significant hepatoprotective activity. Myricitrin provided better hepatoprotection when compared to silymarin, which is consistent with its higher in vitro antioxidant potential.

The present study aimed to evaluate acetylcholinesterase (AChE) inhibitory and antioxidant activities of Lamiaceae medicinal plants growing wild in Croatia. Using Ellman’s colorimetric assay all tested ethanolic extracts and their hydroxycinnamic acid constituents demonstrated in vitro AChE inhibitory properties in a dose dependent manner. The extracts of Mentha x piperita, M. longifolia, Salvia officinalis, Satureja montana, Teucrium arduini, T. chamaedrys, T. montanum, T. polium and Thymus vulgaris at 1 mg/mL showed strong inhibitory activity against AChE. The antioxidant potential of the investigated Lamiaceae species was assessed by DPPH• scavenging activity and total antioxidant capacity assays, in comparison with hydroxycinnamic acids and trolox. The extracts differed greatly in their total hydroxycinnamic derivatives content, determined spectrophotometrically. Rosmarinic acid was found to be the predominant constituent in most of the investigated medicinal plants (by RP-HPLC) and had a substantial influence on their AChE inhibitory and antioxidant properties, with the exception of Teucrium species. These findings indicate that Lamiaceae species are a rich source of various natural AChE inhibitors and antioxidants that could be useful in the prevention and treatment of Alzheimer’s and other related diseases.

In this work, qualitative and quantitative analyses of total hydroxycinnamic acid (THA) derivatives in leaves, flowers and aerial parts of 32 Lamiaceae species were carried out. HPTLC analysis of plant material indicated rosmarinic acid as the main phenolic acid in 22 Lamiaceae plant extracts, while chlorogenic acid predominated in other 10 investigated species. Determination of THA in different medicinal plants was performed by colorimetric methods using chromogenic system of HCl-NaNO2–Na2MoO4–NaOH and two standards: rosmarinic acid (R-THA procedure) and chlorogenic acid (C-THA procedure). Performance characteristics of THA assay were established, and the quality of the analytical procedures was evaluated using valuable prevalidation strategy. Good quality of the measurements at the lower analyte level, excellent resolution of blank and analyte signals, homogenous data material, ideal linear calibration and analytical evaluation functions, very low limit of detection [L D(R-THA) = 0.53 μg; L D(C-THD) = 1.75 μg] and limit of quantitation [L Q(R-THD) = 1.76 μg; L Q(C-THD) = 4.21 μg], and high precision and accuracy confirmed the high quality of investigated procedures as valuable tools in THA analysis. Application of these prevalidated, simple and rapid analytical methods revealed that the studied Lamiaceae plants differ greatly in their THA contents, ranging from 1.95 % (Acinos arvensis) to 11.03 % (Satureja subspicata). The results obtained from qualitative and quantitative analysis provided new information regarding phytochemical characterization of Lamiaceae species originating from Croatia and highlighted members of the genus Micromeria, Origanum, Melissa and Satureja as rich sources of hydroxycinnamic acids.

A rapid reversed-phase high performance liquid chromatographic method was developed and applied for simultaneous separation and determination of flavonoids and phenolic acids in eight Plantago L. taxa (P. altissima L., P. argentea Chaix, P. coronopus L., P. holosteum subsp. depauperata Pilger, P. holosteum Scop. subsp. holosteum, P. holosteum subsp. scopulorum (Degen) Horvatic, P. lagopus L., and P. maritima L.) growing in Croatia. Chromatographic separation was carried out on Zorbax Eclipse XDB-C18 using gradient elution with a water (pH=2.5, adjusted with trifluoroacetic acid) and acetonitrile mixture at 30˚C. The contents of analyzed phenolic compounds (% of the dry weight of the leaves, dw) varied among examined species: rutin (max. 0.024%, P. argentea), hyperoside (max. 0.020%, P. lagopus), quercitrin (max. 0.013%, P. holosteum subsp. holosteum), quercetin (max. 0.028%, P. holosteum subsp. scopulorum), chlorogenic acid (max. 0.115%, P. lagopus), and caffeic acid (max. 0.046%, P. coronopus). Isoquercitrin was determined only in P. argentea (0.020%), while isochlorogenic acid content was below limit of quantification in all investigated species. Multivariate analysis (UPGMA and PCA) showed significant differences in contents of investigated polyphenolic compounds between different Plantago taxa. Accordingly, investigated substances might be employed as good chemotaxonomic markers in the study of complex genus Plantago.

The mycotoxin Ochratoxin A (OTA) appears worldwide in cereals, plant products, different foods and drinks. Ochratoxin A binds to plasma albumin with a very high affinity. However, it is well known that natural flavonoids can also bind to human serum albumin (HSA) at the same binding site as OTA does (site I, subdomain IIA). A few experimental literature data suggest that reducing the bound fraction of OTA speeds up its elimination rate with a potential decrease in its toxicity. In our experimental model competitive binding properties of flavonoid aglycones were examined with a fluorescence polarization based approach. Our data show that some of the flavonoids are able to remove the toxin from HSA. We conclude that among the 13 studied flavonoid aglycones galangin and quercetin were the most effective competitors for OTA.

A Croatian indigenous cultivar of lavandin, Lavandula x intermedia 'Budrovka' (Lamiaceae) was studied for the phenolic acids, flavonoids, anthocyanins, procyanidins and total tannins, as well as total polyphenols content, in the flower, inflorescence stalk and leaf ethanolic extracts. Antioxidant potentials on these plant part extracts were assessed by the DPPH free radical scavenging activity, iron chelating activity, reducing power, lipid peroxidation inhibition properties and total antioxidant capacity assays. All results were compared with those of Lavandula angustifolia, the only member of the Lavandula genus officially used in modern phytotherapy. Based on the results of our parallel study, we may suggest that Lavandula x intermedia 'Budrovka' is as potent an antioxidant as Lavandula angustifolia and the antioxidant activity of the Lavandula extracts is mainly due to the presence of rosmarinic acid. A good correlation was found between the polyphenol contents and antioxidant activities of the extracts.

Allergic diseases are considered to be multifactorial inflammatory diseases that present a serious health problem worldwide while their prevalence have continuously increased during the past few decades. They are results of type I hypersensitivity mediated through the effects of antigen-specific IgE which binds to mast cells and, on subsequent cross-linking by allergen reexposure, causes cell degranulation with the release of preformed mediators in the early-phase as well as the production of cytokines in the late-phase. The aim of our study was to investigate the antiallergic activities of cultivated Lavandula hybrida Rev. and L. angustifolia P. Mill. using the rat basophilic leukaemia cell line (RBL-2H3), a well-characterised in vitro model of mast cells. The ethanolic extracts of flowers of L. hybrida (LHE) and L. angustifolia (LAE) were found to significantly inhibit release of β -hexosaminidase, the marker of antigen-IgE-mediated degranulation in RBL-2H3 cells, showing similar activities (IC50=37 ug/mL and IC50=42 ug/mL, respectively). Furthermore, using the enzyme-linked immunosorbent assay (ELISA), we established that LHE and LAE inhibit antigen-induced production of anti-inflammatory cytokine IL-4 (IC50=60 ug/mL and IC50=45 ug/mL, respectively). Among ten investigated principal bioactive compounds, luteolin, apigenin and ursolic acid were found to be the most potent inhibitors of degranulation as well as cytokine production. In conclusion, the tested extracts showed antiallergic effects acting on the both phases of type I reaction. Therefore, our results suggest a possible therapeutic application of Lavandula species in allergy and allergy-related diseases.

Propolis, the resinous product collected by honey bees from various plant sources, has been found to have numerous biological activities which are mainly attributed to the presence of polyphenolic compounds. Our study aimed to evaluate the polyphenolic composition and in vitro modulation effect of propolis on cytokines production. Four samples of raw propolis originated from the continental region of Croatia were analysed by reversed-phase HPLC-DAD method. Contents of flavonoids (chrysin, galangin, apigenin, kaempferol, luteolin and naringenin), phenolic acids (caffeic, ferulic and coumaric acids) and caffeic acid phenyl ester (CAPE) significantly varied among examined samples and were in a wide concentration range between 0.13 mg/g and 66.15 mg/g. Modulation of cytokine production in concavalin A-stimulated splenocytes from Balb/C mice by propolis ethanolic extracts (0.3-90 μ g/mL), chrysin (0.1-30 μ g/mL) and CAPE (0.01-3 μ g/mL) was evaluated using the enzyme-linked immunosorbent assay (ELISA). Treatment of cells with the propolis extracts (≥ 10 μ g/mL) that contained high quantities of chrysin, galangin and CAPE significantly (85-100 %) suppressed IL-4, lL-5, IL-10 and IFN-γ expression. Our results demonstrated that propolis affects the key messengers of immune response which play a very important role in the pathogenesis of immune system-associated diseases.