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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.

Cosmetic products are complex mixtures of ingredients with different physical, chemical and functional properties formulated in order to obtain the product with desired activity and quality. This paper provides a comprehensive review on the effectiveness of the environmentally friendly extraction technique based on the application of GRAS solvent CO2 and high pressures which present many advantages over the conventional processes. Supercritical CO2 (SCO2) proved to be a promising extraction solvent in this process and has the potential to replace the organic toxic solvents. Due to its unique properties, the desirable texture, absence of water or solvent traces, chemical stability and presence of bioactive agents, SCO2 extracts can be easily incorporated into the cosmetic products. Depending on the plant material, the operation parameters and the chemical profile, the obtained extracts can be used as the common cosmetic ingredients (thickeners, emollients, waxes, fragrances, and colorants). Moreover, SCO2 provides a high concentration of bioactive compounds in the final extracts which exhibit many bioactivities in the cosmetic products acting as antioxidants, anti-ageing, photo-protective agents or providing antibacterial activity and inhibition of enzymes, such as tyrosinase, lipoxygenase, collagenase and hyaluronidase. This review highlights the potential application of the SCO2 extracts in the cosmetic industry. The bioactive properties of SCO2 extracts are discussed and their capability for skin health is evaluated.

Background and objectives Constipation is one of the most common gastrointestinal conditions, particularly among older individuals. This study aimed to evaluate the efficacy and safety of selected multistrain probiotics on functional constipation and laboratory blood parameters in the elderly living in a nursing home. Subjects and methods Sixty participants (42 females and 18 males) aged 77.9 ± 8.84 years with functional constipation, who met the eligibility criteria, completed the study. In a double-blind, placebo-controlled, parallel design, each participant was randomized to receive either the selected probiotic mixture (N = 28) or placebo (N = 32) for 12 weeks as an adjunct to their usual diet and medications. The liquid probiotic formulation containing Bifidobacterium animalis subsp. lactis BLC1, Lactobacillus acidophilus LA3 and Lactobacillus casei BGP93 was tested for the first time. Results Supplementation of selected probiotics resulted in a slight but nonsignificant increase in cumulative stool frequency compared with placebo. However, after the 71st day of the treatment, the cumulative number of stools was significantly higher in the probiotic group (P Conclusions Multistrain probiotic supplementation was found to be efficacious, safe and well tolerated in the elderly with functional constipation.

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.

Devil's Claw is a traditional medicine that has been long used a wide range of health conditions, including indigestion, fever, allergic reactions, and rheumatism. The main compounds are iridoid glycosides, including harpagoside, harpagide, and procumbide. However, harpagoside is the most responsible for therapeutic activity, and its content is used as reference standard. Here, we analyzed and summarized preclinical and clinical studies focusing on therapeutic efficacy of devil's claw preparations in pathological conditions in which inflammation plays a key causative role.Devil's claw (Harpagophytum procumbens) is an herbaceous plant, which can be found in the arid steppes of South Africa, particularly in the Kalahari Desert. As traditional medicine, devil's claw has been long used in the forms of infusions, decoctions, tinctures, powders, and extracts. The main compounds of devil's claw are iridoid glycosides, such as harpagoside, harpagide, and procumbide, which are present in the plant tubers. Additionally, chemical constituents such as sugars (mainly the tetrasaccharide stachyose), triterpenoids (oleanolic and ursolic acid), phytosterols (primarily β-sitosterol), aromatic acids (caffeic, cinnamic, and chlorogenic acids), and flavonoids (luteolin and kaempferol) can be found in the plant. In order to investigate the applicative potential of devil's claw as therapeutic agent, we performed a review by focusing on its potential role in managing inflammation- and oxidative stress-related diseases, including arthritis, osteoporosis, inflammatory bowel disease, low-back pain, diabetes, and neurodegeneration.

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 study of pharmacy was introduced at the University of Zagreb (Croatia) in 1882 following the Decision of the Emperor Francis Joseph I. Since then, pharmacognosy has been an important part of the pharmacy education at the University of Zagreb. Today, pharmacognosy is taught and researched at the Department of Pharmacognosy of the Faculty of Pharmacy and Biochemistry, which was established in 1896 as the first independent institute of pharmacognosy in the world [1]. At this unique Department formerly called Institute of Pharmacognosy, its founder Professor Julije Domac and his successors preserved an exceptional collection of medicinal drugs as well as a collection of very old and rare pharmaceutical and medical books and manuscripts dating from 17th to 19th centuries. The Pharmacognostic collection was renovated in 2016 on the occasion of the 120th anniversary of the Institute. Today, the collection counts more than 1200 specimens of medicinal drugs of plant and animal origin that found their use in traditional medicine and contributed to the development of modern-day pharmaceuticals. Most of the specimens are very rare and old and come from all corners of the world, such as the aloe in monkey skin from Africa or curare from South America. Beside its scientific and educational significance for new generations of pharmacists, the Pharmacognostic collection and library have an important historical and cultural value.

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.

ObjectiveThe aim of this study was to determine the frequency and type of drug therapy problems (DTPs) in older institutionalized adults.MethodWe conducted a cross-sectional observational study from February to June 2016 at a 150-bed public nursing home in Croatia, where comprehensive medication management (CMM) services were provided. A rational decision-making process, referred to as the Pharmacotherapy Workup method, was used to classify DTPs.ResultsData were prospectively collected from 73 residents, among which 71% were age 75 years or older. The median number of prescribed medications per patient was 7 (2–16) and polypharmacy (> 4) was recorded for 54 (74.0%) patients. A total 313 DTPs were identified, with an average of 4.3 ± 2 DTPs per patient. The most frequent DTP was needing additional drug therapy (n = 118; 37.7%), followed by adverse drug reaction (n = 55; 17.6%). Lactulose (14.4%), tramadol (6.7%), and potassium (6.4%) were the medications most frequently related to DTPs.ConclusionThe high prevalence of DTPs identified among older institutionalized adults strongly suggests the need to incorporate new pharmacist-led CMM services within existing institutional care facilities, to improve the care provided to nursing home residents.

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.

We investigated the effect of natural sweetener Stevia rebaudiana and its constituent stevioside in cisplatin (CP)-induced kidney injury. Male BALB/cN mice were orally administered 10, 20, and 50 mg/kg body weight of Stevia rebaudiana ethanol extract (SE) or stevioside 50 mg/kg, 48 h after intraperitoneal administration of CP (13 mg/kg). Two days later, CP treatment resulted in histopathological changes showing kidney injury. Increased expression of 4-hydroxynonenal (4-HNE), 3-nitrotyrosine (3-NT), and heme oxygenase-1 (HO-1) in mice kidneys suggested oxidative stress. CP treatment also increased renal expression of nuclear factor-kappaB (NF-κB) p65 subunit and phosphorylated inhibitor of NF-κB (IκBα), as well as expression of pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-α). Induction of apoptosis and inhibition of the cell cycle in kidneys was evidenced by increased expression of p53, Bax, caspase-9, and p21, proteolytic cleavage of poly (ADP-ribose) polymerase (PARP), with concomitant suppression of Bcl-2 and cyclin D1 expression. The number of apoptotic cells in kidneys was also assessed. CP administration resulted in activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and signal transducer and activator of transcription 3 (STAT3). Both SE and stevioside attenuated CP nephrotoxicity by suppressing oxidative stress, inflammation, and apoptosis through mechanism involving ERK1/2, STAT3, and NF-κB suppression.

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.

Although today among oncology patients use of various preparations of complementary and alternative medicine is more and more frequent, there is unequivocal scientifi c base for their use. Among the often used preparations, especially in the treatment of cancer pain, is cannabis and its derivatives. Cannabinoids act on the endogenous cannabinoid system, with widespread receptors in the central nervous system and peripheral tissues. Although the pharmacology of the cannabinoids is still largely unknown, numerous of their eff ects were investigated. In oncology, studies have been conducted on the effect of cannabinoids on nausea and vomiting during the oncological treatment, the cancer pain and neuropathy, on appetite and weight loss, and the impact on mood, depression and anxiety. It is also observed that some of the cannabinoids have antitumor, but also protumorous activity. There have been many diff erent side eff ects of cannabinoids detected, and in a smaller percentage also the development of addiction. Best known preparations nowadays are dronabinol, nabilon and nabiximol. At the moment, the evidence lack strenght, and large randomized clinical trials are required, which would confi rm predominatly positive results of the research., Iako je danas među onkološkim bolesnicima sve učestalija uporaba različitih pripravaka komplementarne i alternativne medicine, za njihovu uporabu nema nedvojbene znanstvene potvrde. Među češće primjenjivanim pripravcima, osobito u liječenju karcinomske boli, je i kanabis i njegovi derivati. Kanabinoidi djeluju u organizmu preko endokanabinoidnog sustava, s rasprostranjenim receptorima u središnjem živčanom sustavu i perifernim tkivima. Iako je farmakologija kanabinoida još uvijek uglavnom nepoznata, do sada su istraživani njihovi brojni učinci. U onkologiji su provedena istraživanja utjecaja na mučninu i povraćanje prilikom onkološkog liječenja, na karcinomsku bol te neuropatiju, na apetit i gubitak tjelesne mase te utjecaj na raspoloženje, depresiju i tjeskobu. Također je opažen antitumorski, ali i protumorski učinak nekih kanabinoida. Zabilježeni su brojni različiti neželjeni učinci kanabinoida, a u manjem postotku i razvoj ovisnosti. Najpoznatiji pripravci danas jesu dronabinol, nabilon i nabiksimol. Sveukupno, za sada nisu osigurani dovoljno snažni i nedvojbeni dokazi i potrebne su velike randomizirane kliničke studije, koje bi potvrdile do sada opažene pozitivne rezultate istraživanja.

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.

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.

A study on the type and distribution of trichomes, morphological traits of the calyx and content of antioxidant compounds was conducted on Teucrium arduini L. populations in Croatia and in Bosnia and Herzegovina. Non-glandular trichomes, peltate trichomes and small capitate trichomes with four secretory head cells were observed on leaves, stems and calyx, whereas long capitate trichomes with a unicellular head cell were observed exclusively on the calyx. Average calyx length was 5.23–6.51 mm, calyx tube length 2.92–3.68 mm, lower lip length 1.25–2.42 mm and upper lip length 1.89–2.99 mm. The concentration of antioxidant compounds in all populations was the highest in leaves: total polyphenols (TP): 8.43–13.55%; tannins (T): 1.79–7.33%; flavonoids (TF): 0.28–0.40%; phenolic acids (TPA): 2.90–5.07%. Inflorescences generally contained the smallest amount of TP (5.89–11.42%), T (0.87–6.50%) and TPA (1.66–2.27%), while the smallest content of TF was obtained for the majority of stem samples (0.06–0.12...

Total polyphenols, tannins, β-sitosterol, and bitterness values were determined in native and cultivated populations of Teucrium L. species from Croatia (T. arduini L., T. botrys L., T. chamaedrys L., T. flavum L., T. montanum L., T. polium L., and T. scordium L. subsp. scordioides Schreb.). Teucrium species have long been present in folk medicine for diverse medicinal uses, but little is known about polyphenols, phytosterols, and bitter principles. Spectrophotometrically determined contents of total polyphenols (TP) and tannins (T) significantly varied among investigated Teucrium species and were somewhat higher in native populations. The highest TP and T contents were measured in native specimens of T. montanum (TP: 13.68%; T: 3.48%). Scanning densitometry was used for measurements of β-sitosterol levels in plant samples ranged from 0.056% (T. montanum) to 0.129% (T. botrys), and it was not significantly different between native and cultivated specimens of the same plant species. Bitterness values were similar for native and cultivated samples of the same plant species; the highest was measured for T. montanum (15659). The present study suggests that Teucrium species growing in Croatia have potential for cultivation and might be a valuable source of natural bioactive compounds.

Micromorphological investigation of the types, dimensions and distribution of characteristic trichomes in leaves and stems in Teucrium L. species (T. arduini L., T. chamaedrys L., T. flavum L., T. montanum L., T. polium L., and T. scordium L. subsp. scordioides Schreb.) distributed in Croatia was carried out as part of the taxonomical study of the genus Teucrium. Secretory types of hairs, peltate and capitate hairs were observed on the epidermis of stems and leaves of all investigated species. Non-secretory, acicular hairs were almost completely lacking on stems of T. scordium subsp. scordioides. Flagelliform hairs were not found in T. flavum and T. polium. Cladose hairs were present only in T. polium. The largest micromorphological variability was established between wild and cultivated samples of T. arduini and T. scordium subsp. scordioides, while cultivated and wild specimens of T. polium were almost identical. Differences were primarily observed in trichome dimensions and much less in micromorphological features.

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.

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.

Widely distributed in plant kingdom and abundant in our diet plant polyphenols are today among the most talked about concerning the classes of phytochemicals. There are several thousand plant-derived compounds of biogical interest that have more than one phenolic hydroxyl group attached to one or more benzene rings, thus qualifying as polyphenols. In recent years, polyphenols have gained a lot of importance because of their potential use as prophylactic and therapeutic agents in many diseases, and much work has been presented by the scientific community which focuses on their antioxidant effects. Traditionally, herbal medicines with antioxidant properties have been used for various purposes and epidemiological data also point at widespread acceptance and use of these agents. Plant polyphenols have been studied with intention to find compounds protecting against a number of diseases related to oxidative stress and free radical-induced damage, such as cardiovascular and neurodegenerative diseases, cancer, diabetes, autoimmune disorders and some inflammatory diseases. In order to evaluate the efficacy of polyphenols as antioxidants as well as to elucidate the mode of their action, researchers today are using a wide range of experimental models, from the simplest chemical antioxidant assays through the biologically more relevant cellular-based assays to the most accurate animal models, and ultimately clinical studies in humans. The latest scientific knowledge offers a more detailed understanding of the biological effects of polyphenols and their role in human health promotion and disease prevention.

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.

Owing to their richness in secondary metabolites exhibiting remarkable diversity of both chemical structures and biological activities, medicinal plants are being recognized as promising sources of lead compounds for new drugs targeting neurodegenerative diseases. Alzheimer’s disease is the most common age-related dementia with a steadily increasing prevalence, but its treatment still remains an area of significant unmet need, with therapies based largely on the acetylcholinesterase (AChE) inhibitors. Therefore, the present study aimed to evaluate selected Croatian medicinal plants belonging to the Lamiaceae family as a potential source of natural inhibitors of this target enzyme. The ethanolic extracts of 27 plant species were screened for their antiacetylcholinesterase activity by in vitro Ellman method at concentration range of 0.25-1 mg/mL. Among them, remarkable inhibitory activity above 80% inhibition rate on AChE at 1 mg/mL showed the extracts of Mentha x piperita L., M. longifolia (L.) Huds. Salvia officinalis L., Teucrium chamaedrys L., T. montanum L., T. polium L. and Thymus longicaulis C. Presl. On the other hand, seven tested plant extracts (Acinos arvensis (Lam.) Dandy, Calamintha sylvatica Bromf., Clinopodium vulgare L., Marrubium incanum Desr., Mentha pulegium L., Micromeria juliana (L.) Benth. ex Rchb. and Origanum vulgare L.) did not reach the 50% inhibition of enzyme activity. The results of this study indicated that there is a great potential of Lamiaceae species growing in Croatia to provide novel drug leads for the treatment of Alzheimer's and other neurological disorders.