Quantification of fatty acids, phenolic acids, and flavonoids, antioxidant and enzyme inhibition activity of anise (Pimpinella anisum L.) seeds was the subject of this study. In fixed oil were found two saturated and four unsaturated fatty acids and linoleic acid were majors. Successive extraction of seed with 80% methanol was used to prepare four different fractions of phenolic compounds that is, free phenolics (PA-M1), esters (PA-M2), phenolic glycosides (PA-M3), and bounded phenolics (PA-M4). Six phenolic acids (5-hydroxyferulic, 3-hydroxybenzoic, ferulic, sinapinic, salicylic, and trans-cinnamic acid) were found in four different fractions. Apigenin was the only identified flavonoid. The antioxidant capacity of anise seed extracts was evaluated by four methods. Total phenolic content showed a correlation with antioxidant activity. Determination of butyrylcholinesterase inhibition was performed by modified Ellman's assay, while monitoring of hydrogen peroxide consumption was used for catalase inhibition assessment. Seed extracts showed potent inhibition of both enzymes. Novelty impact statement This study was evaluated chemical profile and biological activities of imported Pimpinella anisum L. seed, where linoleic acid was found as a major fatty acids, and ferulic acid and sinapinic acid as major phenolic compounds. Also, were demonstrated butyrylcholinesterase and catalase inhibition activities, as well as antioxidant activities. Furthermore, this is the first study describing the catalase inhibitory activity of anise seed extracts.

The choroid plexus form cerebrospinal fluid (CSF) within four ventricles the brain. The composition of CSF is very similar to the of the brain’s extracellular, and its composition depends on filtration and diffusion from the blood. Few conditions cause CSF volume and pressure to fall below normal (dehydration may be one), but many conditions can cause it to increase, sometimes under pressure. This may lead to blocked flow CSF from one or more ventricles, which causes an increase in intracranial pressure, and CSF buildup i.e. to hydrocephalus. Hydrocephalus results from one of three possible causes: over secretion of CSF, impaired absorption of CSF or obstruction of CSF pathways. The concentration of metabolites of the monoamines homovanillic acid (HVA) and 5-hydrocephalus acetic acid (5-HIAA) were determined with HPLC-ED in the CSF of 17 patients (children of 1 age) with hydrocephalus. Determined 5-HIAA concentrations are in the range of 0,99–113,89 ng/mL and HVA 0,98–165,92 ng/mL in CSF due to pressure changes occurring in hydrocephalus is increasing the turnover of serotonin and dopamine to its metabolites. The correlation no could be established between the levels of 5-HIAA and HVA and type of hydrocephalus, and the duration of disease.

Rutin is quercetin heteroside widely present in plants that exhibit many health-beneficial effects, such as strengthening the capillary walls, reducing the harmful effects of LDL cholesterol, and reducing the risk of cardiovascular diseases. Rutin was determined from three Allium species (A. cepa, A. sativum, and A. ursinum) collected in Bosnia and Herzegovina by the HPLC method associated with electrochemical detection. The analysis was performed from methanol extracts of bulb and leaf of garlic, bulb and leaf of onion, and leaves of wood garlic. Rutin was present in all of the examined samples. The highest rutin content was found in garlic leaves (0.78 ± 0.09 mg g–1), and the lowest in onion bulbs (0.04 ± 0.10 mg g–1). The contents of rutin were higher in leaf samples, suggesting that leaves of onion and garlic are recommended as a better natural source of this glycoside.

Summary Background: The production of erythrocytes is regulated by the hormone erythropoietin (EPO), which maintains the blood haemoglobin (Hb) levels constant under normal conditions. Human EPO is a glycoprotein hormone and its synthesis is controlled by the hypoxia-inducible transcription factor. The aim of this study was to establish EPO and Hb levels in patients with chronic kidney disease (CKD), as well as in control subjects, and to investigate the relationship between these parameters. Methods: This cross-sectional, observational study included 356 subjects with CKD divided into 4 subgroups according to their glomerular filtration rate (GFR). The control group consisted of 206 age and sex matched healthy subjects with GFR rate ≥90 mL/min/1.73 m 2 . EPO, Hb and serum creatinine levels were determined by using immunochemical and spectrophotometric methods. GFR was determined using the MDRD formula. Results: The CKD patients had significantly lower levels of haemoglobin (p<0.0005) and hematocrit (p<0.0005) compared to control group. Our results showed that Hb levels decreased, whereas serum creatinine increased with the increasing renal failure. The CKD patients in all four groups had significantly lower (p<0.0005) Hb levels, and significantly higher (p<0.0005) creatinine levels compared to the control group. The median EPO in group I and II were significantly higher (p=0.002; p=0.018), while median EPO in group III and IV were significantly lower (p=0.03; p=0.011) compared to the control group. Conclusions: In patients with CKD, GFR positively correlated with Hb and EPO, while the correlation between GFR and serum creatinine was negative.

The concentration of metabolites of neurohormones in cerebrospinal fluid (CSF) is an index of turnover of substances in brain. 5-Hydroxyindoleacetic acid (5-HIAA) - serotonine metabolite and homovanillic acid (HVA) - dopamine metabolite, were studied in CSF of patients with coma cerebri, hydrocephalus and tumor cerebri. CSF concentrations of 5-HIAA and HVA were determined in 140 patients of different age and sex. The samples were analyzed for 5-HIAA and HVA by high pressure liquid chromatography with electrochemical detector (HPLC-ED). Average concentration of 5-HIAA was 9.54 +/- 2.09 ng/mL, and for HVA it was 27.17 +/- 4.39 ng/mL. Average concentration of 5-HIAA in CSF of male subjects was 13.39 +/- 3.30 ng/mL, and for female subjects 4.23 +/- 1.34 ng/mL. Average concentration of HVA in CSF of male subjects was 31.52 +/- 4.86 ng/mL, and for female subjects 21.19 +/- 3.70 ng/mL (p<0.05). Results increased 5-HIAA and HVA concentration in CSF in comparison with other pathochemical conditions, such as acute lymphocyte leukemia, encephalitis, meningitis serosus, multiple sclerosis, quadriplegia and sepsis. Long-term follow-up is required to assess if they could be of prognostic significance as regards to long term attainment of brain functions in coma cerebri, hydrocephalus and tumor cerebri patients.

Ephedra is a plant with a long history of medicinal use, the main indications being asthma, rhinitis and bronchospasm. However, nowadays it is often misused/abused for weight-loss, performance enhancing and doping. The pharamcological effects are ascribed to the ephedrine alkaloids present in the plant. The aim of the study was to quantify Norephedrine (NE), Ephedrine (E), Pseudoephedrine (PE) and Methylephedrine (ME) in herb extracts of nine different Ephedra species using high performance liquid chromatography with UV detection (HPLC-UV). The alkaloids were separated on the column Symmetry C18 (5 µm, 3.9 x 150 mm). The mobile phase was delivered at the flow rate of 1 ml/min. The mobile phase was prepared with 390 ml of acetonitrile, 610 ml of pure water, 0.8 ml of o-phosphoric acid and 4.8 g of sodium lauryl sulfate. The UV detector operated at 208 nm. We found that the utilized HPLC-UV method for separation and quantification of ephedrine alkaloids was very selective, sensitive and simple. The total content of NE, E, PE and ME ranged from 0.4 mg/g dry weight (Ephedra foeminea, from Austria) to 71.9 mg/g dry weight (Ephedra major, from Morocco). None of the investigated alkaloids were detected in Ephedra altissima.

Black cumin (Nigella sativa L. [N.sativa]) seed extracts demonstrated numerous beneficial biological effects including, among others, antidiabetic, anticancer, immunomodulatory, antimicrobial, anti-inflammatory, antihypertensive, and antioxidant activity. To better understand the phytochemical composition of N. sativa seeds, methanol seed extracts were analyzed for phenolic acid and flavonoid content. Furthermore, we tested N. sativa methanol, n-hexane, and aqueous seed extracts for their inhibitory activity against butyrylcholinesterase (BChE) and catalase (CAT) as well as for antimicrobial activity against several bacterial and a yeast strains. The phenolic content of N. sativa was analyzed using ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). The inhibition of BChE was assessed by modified Ellman’s method, and the inhibition of CAT was determined by monitoring hydrogen peroxide consumption. The extracts were tested against Bacillus subtilis, Staphylococcus aureus, Salmonella enterica, and Escherichia coli using the agar diffusion method. The UHPLC-MS/MS method allowed the identification and quantification of 23 phenolic compounds within 15 minutes. The major components found in N. sativa seed extract were sinapinic acid (7.22 ± 0.73 µg/mg) as a phenolic acid and kaempferol (11.74 ± 0.92 µg/mg) as a flavonoid. All extracts showed inhibitory activity against BChE, with methanol seed extract demonstrating the highest inhibitory activity (inhibitory concentration 50% [IC50] 79.11 ± 6.06 µg/ml). The methanol seed extract also showed strong inhibitory activity against CAT with an IC50 value of 6.61 ± 0.27 µg/ml. Finally, the methanol extract exhibited considerable inhibitory activity against the tested microbial strains. Overall, this is the first study to investigate the ability of black cumin seed extracts to inhibit CAT. Our results indicate that N. sativa seed can be considered as an effective inhibitor of CAT activity.

Despite its toxicity, botulinum neurotoxin type A (BTX-A) is a valuable therapeutic agent for several motor, autonomic and pain disorders. Numerous studies have described its peripheral as well as central effects. Using reversed-phase High Performance Liquid Chromatography with Electrochemical Detection (HPLC-ED) and gradient elution, we quantified the concentrations of dopamine (DA), noradrenaline (NA), serotonin (5-HT) and their metabolites in 10 brain regions, ipsilateral and contralateral from the site of unilateral BTX-A administration (5 U/kg) into the rat whisker pad. In regions associated with nociception and pain processing we also examined possible BTX-A effects in combination with formalin-induced inflammatory orofacial pain. The dominant BTX-A effects on the monoamines and their metabolites were insignificant. The only significant increase caused by BTX-A alone was that of NA in striatum and serotonin in hypothalamus. While antinociceptive effects of BTX-A are most probably not related to central monoamine concentrations, the localized increased NA and 5-HT concentrations might play a role in reported BTX-A efficacy for the treatment of depression.

INTRODUCTION:Hypoxia is a basic stimulant in production of erythropoietin (EPO). The primary function of erythrocytes is the transport of oxygen to tissues. Erythropoietin stimulates erythropoiesis which leads to increased production of erythrocytes- their total mass. This increases the capacity of the blood to carry oxygen, reduces the hypoxic stimulus and provides a negative feedback of stopping EPO production. The aim of this study was to establish a quantitative relationship between the concentration of erythropoietin, hemoglobin and hematocrit in different values of renal insufficiency. MATERIAL AND METHODS:The survey was conducted on 562 subjects divided into two groups: with and without renal insufficiency. EPO, hemoglobin, hematocrit, serum creatinine and additional parameters iron, vitamin B12, and folic acid were determined by using immunochemical and spectrophotometric methods and glomerular filtration rate (GFR) was calculated as well. RESULTS:EPO values (median) grow to the first degree of renal insufficiency, as compared to EPO values of healthy subjects, this increase is statistically significant, p=0.002. With further deterioration of renal function the values of EPO between all pathological groups are decreasing, and this decrease is statistically significant between first and second degree of renal insufficiency (RI) p<0.001. In the group of healthy subjects EPO is correlated rho = -0.532, p <0.0005 with hematocrit. The correlations are negative and strong and can be predicted by regression line (EP0 = 41.375- Hct * .649; EPO = 61.41-Hb * 0.355). In the group of subjects with the first degree of renal insufficiency EPO is in correlation with hematocrit rho=-0.574, p<0, 0005. It is also correlated with hemoglobin rho=-0.580, p< 0.0005. The correlation is negative (EP0= 42.168- Hct * 0.678). In the group of subjects with the third degree of renal insufficiency EPO is in correlation with hemoglobin rho=0.257, p=0.028. The correlation is medium strong and positive. In the group of subjects with third and fourth degree of renal insufficiency EPO is not in correlation with hemoglobin and hematocrit p>0.05. CONCLUSION:Renal dysfunction, depending on the level of RI effects differently on the biosynthesis of EPO in a diseased kidney, and consequently it also has a different effect on biosynthesis of HB in bone marrow and its content in the blood.

Platelet hyperactivity and platelet interaction with endothelial cells contribute to the development and progression of many cardiovascular diseases such as atherosclerosis and thrombosis. The impact of platelet activity with different pharmacological agents, such as acetylsalicylic acid and coumarin derivatives, has been shown to be effective in the prevention of cardiovascular disease. Artemisia dracunculus, L. Asteraceae (Tarragon) is used for centuries in the daily diet in many Middle Eastern countries, and it is well known for its anticoagulant activity. The present study investigates the presence of coumarins in tarragon leaves and subsequently determines the extract with a major amount of coumarin derivatives. The solvents of different polarities and different pH values were used for the purpose of purifying the primary extract in order to obtain fractions with the highest coumarin content. Those extracts and fractions were investigated for their anticoagulant activity by determining prothrombin time (PT) and the international normalized ratio (INR), expressed in relation to the coagulation time of the healthy person. Purified extracts and fractions obtained from plant residue after essential oil distillation, concentrated in coumarin derivatives, showed the best anticoagulant activity, using samples of human blood. INR maximum value (2.34) and consequently the best anticoagulant activity showed the methanol extract at concentration of 5%. The INR value of normal plasma in testing this extract was 1.05.

Because of the great pharmacological potential of the pyrimidine motif, novel C-5 substituted N-3 acyclic and O-4 acyclic pyrimidine derivatives were prepared as an interesting class of compounds for biological evaluation. Introduction of the 2,3-dihydroxypropyl (DHP) and penciclovir (PCV)-like side chains to 2-methoxypyrimidin-4-one (2) afforded a mixture of N- and O-acyclic pyrimidine nucleosides in the ratio of 54: 29 (3:4) and 57:21 (5:6) with N-3 isomer being dominant. Distinction between N- and O-alkylated pyrimidine moiety was deduced from extensive experimental FT-IR, HPLC-MS and 1D (1H, 13C) and 2D (COSY, HMQC and HMBC) NMR analyses. The N-, O-regioisomers were also examined by computational method at density functional theory (DFT) RB3LYP/6-31G(d), 6-31G∗∗ and 6-31+G∗ levels. DFT global chemical reactivity descriptors (total energy, chemical hardness, electronic chemical potential and electrophilicity) were calculated for the isomers and used to predict and describe their relative stability and reactivity. The chemical reactivity indices were related to the C2N3C4 bond angle. Theoretical predictions can be used to compare chemical reactivity and stability with future biological evaluation and behaviour of these compounds.

The medicinal significance of Ephedra is based on the sympathomimetic properties of ephedrine (E) alkaloids. Pharmacological effects depend on the phytocomposition of individual Ephedra species. The aim of this study was to measure the total alkaloids content (TAC), total phenolics content (TPC), and total flavonoids content (TFC) and determine their relationship in dry herb of Ephedra major, Ephedra distachya subsp. helvetica, Ephedra monosperma, Ephedra fragilis, Ephedra foeminea, Ephedra alata, Ephedra altissima and Ephedra foliata. Nowadays, medicinal use of Ephedrae herba is limited, but the abuse of its psychostimulants is rising. In this study, TAC, TPC and TFC were determined using spectrophotometric methods. For the first time, ultra-performance liquid chromatography with ultraviolet detection (UPLC-UV) was used for separation and quantification of E-type alkaloids of various Ephedra species. The highest TPC and TFC were found in E. alata (53.3 ± 0.1 mg Gallic acid equivalents/g dry weight, 2.8 mg quercetin equivalents/g dry weight, respectively). The total content of E and pseudoephedrine determined by UPLC-UV varied between 20.8 mg/g dry weight (E. distachya subsp. helvetica) and 34.7 mg/g dry weight (E. monosperma). The variable content and ratio between secondary metabolites determined in different Ephedra species reflects their metabolic activities. Utilization of UPLC-UV unveiled that this technique is sensitive, selective, and useful for separation and quantification of different alkaloids in complex biological matrixes. The limit of detection was 5 ng. Application of UPLC-UV can be recommended in quick analyses of E-type alkaloids in forensic medicine and quality control of pharmaceutical preparations.

Low intracerebroventricular (icv) doses of streptozotocin (STZ) produce regionally specific brain neurochemical changes in rats that are similar to those found in the brain of patients with sporadic Alzheimer's disease (sAD). Since oxidative stress is thought to be one of the major pathologic processes in sAD, catalase (CAT) activity was estimated in the regional brain tissue of animals treated intracerebroventricularly with STZ and the multitarget iron chelator, antioxidant and MAO-inhibitor M30 [5-(N-methyl-N-propargylaminomethyl)-8-hydroxyquinoline]. Five-day oral pre-treatment of adult male Wistar rats with 10 mg/kg/day M30 dose was followed by a single injection of STZ (1 mg/kg, icv). CAT activity was measured colorimetrically in the hippocampus (HPC), brain stem (BS) and cerebellum (CB) of the control, STZ-, M30- and STZ + M30-treated rats, respectively, 4 weeks after the STZ treatment. STZ-treated rats demonstrated significantly lower CAT activity in all three brain regions in comparison to the controls (p < 0.05 for BS and CB, p < 0.01 for HPC). M30 pre-treatment of the control rats did not influence the CAT activity in HPC and CB, but significantly increased it in BS (p < 0.05). M30 pre-treatment of STZ-treated rats significantly increased CAT activity in the HPC in comparison to the STZ treatment alone (p < 0.05) and normalized to the control values. These findings are in line with the assumption that reactive oxygen species contribute to the pathogenesis of STZ in a rat model of sAD and indicate that multifunctional iron chelators such as M30 might also have beneficial effects in this non-transgenic sAD model.

Aims: Study investigates relation of sulfur compounds content in garlic and different type of manures that were used for rearing of this vegetable. Measured parameter was total sulfur which could be a valid parameter of content of aromatic and medicinal sulfur compounds in garlic (alliin etc.). Study Design: Quality of garlic mainly depends from content of sulfur compounds, there are articles related to influence of mineral fertilizer on the quality of garlic bulbs but effect of manures hasn’t been enough investigated. This research should give an estimation of effect of some manures on the quantity of sulfur compounds in garlic.

Five methanolic extracts obtained from different parts of birch, Betula pendula, Roth.(external and internal bark, flowers, leaves and buds), were evaluated for their antibacterial activity in this study.Triterpene compounds, betulin, betulinic acid, oleanolic acid and lupeol, were isolated from the external parts of birch bark using the method of dry column chromatography (DCC) as well as preparative thin layer chromatography (TLC).These compounds were also investigated for their antibacterial activity.Taking into account that decoction is the most commonly used pharmaceutical form of herbal drug, decoctions made from external bark, leaf, flower and bud were investigated for their antibacterial activity.Antibacterial screening, against selected Gram-positive bacteria, Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 6538P and Gram-negative bacteria, Escherichia coli ATCC 8739 and Pseudomonas aeruginosa ATCC 9027 was carried out.The methods of diffusion and dilution were used for this investigation according to European Pharmacopoea, 1996.The most prominent antibacterial activity showed oleanolic acid against bacterial species Staphylococcus aureus, expressed as minimal inhibitory concentration (MIC): 1.25% and Bacillus subtilis MIC: 0.625%.Escherichia coli showed resistance on all investigated samples.

Nitric oxide (NO), a neurotransmitter and a free radical, has been purported to be involved in numerous neurological diseases. We investigated the serum nitric oxide concentration in 30 patients with multiple sclerosis (MS), in 30 patients with epilepsy and in 30 control subjects. The aim was also to determine whether a statistically significant difference in serum NO concentrations exists between the groups of interest. The total serum nitric oxide concentration was measured using the Griess reaction after reducing nitrates to nitrites with elemental zinc. In the group multiple sclerosis, the mean NO concentrations were X ± SEM = 31.02 ± 1.79 μmol/l, in the control group X ± SEM = 25.31 ± 1.44 μmol/l and in the group epilepsy X ± SEM = 22.51 ± 1.28 μmol/l. Student's t test showed a statistically significant difference between subjects with multiple sclerosis and the control group (p = 0.013), as well as between the groups multiple sclerosis and epilepsy (p = 0.0002). This data confirms that NO may play an important role in the pathogenesis of multiple sclerosis, whereas its role in epilepsy still remains unclear.

Artemisia annua L. (Asteraceae) contains various phytochemical compounds such as monoterpenoids, sesquiterpenoids, flavonoids, coumarins, sterols etc. Scopoletin (7-hydroxy-6-methoxycoumarin) is coumarin which can be found in plants of family Asteraceae. In this study, using HPLC-ED system, quantitave analysis of scopoletin was carried out in water extracts of A. annua. The shade dried plant material (10g) was powdered and subjected to extraction with petroleum ether in Soxhlet apparatus. The defatted marc was then extracted with ethanol till complete extraction. 100mg of ethanolic extract was weighed accurately and dissolved in 10 mL of water with vigorous shaking. It was then filtered and the volume made up to 100 mL with water (stock solution). HPLC conditions were following: Mobile phase: methanol-acetonitrile-water-acetic acid (20+10+70+1); electrochemical detector with range 50 nA, potential +0.840 V, filter 0.02Hz; flow rate 1 mL/min; temperature 25°C, column: ODS hypersil. Injection volume was 20µl. Concentrations of standard were: 10ng/20µL, 20ng/20µL and 40ng/20µL. Retention time of scopoletin in standard solution and extracts of A.annua was 11.4 minutes. Content of scopoletin in extracts of two samples of A. annua were 109µg/g and 534µg/g dry plant. The result showed that very low concentration of scopoletin in plant extracts can be indentified and quantified.