Phytochemical and Nutraceutical Screening of Ethanol and Ethyl Acetate Phases of Romanian Galium verum Herba (Rubiaceae).

Galium species are used worldwide for their antioxidant, antibacterial, antifungal, and antiparasitic properties. Although this plant has demonstrated its antitumor properties on various types of cancer, its biological activity on cutaneous melanoma has not been established so far. Therefore, the present study was designed to investigate the phytochemical profile of two extracts of G. verum L. herba (ethanolic and ethyl acetate) as well as the biological profile (antioxidant, antimicrobial, and antitumor effects) on human skin cancer. The extracts showed similar FT-IR phenolic profiles (high chlorogenic acid, isoquercitrin, quercitrin, and rutin), with high antioxidant capacity (EC50 of ethyl acetate phase (0.074 ± 0.01 mg/mL) > ethanol phase (0.136 ± 0.03 mg/mL)). Both extracts showed antimicrobial activity, especially against Gram-positive Streptococcus pyogenes and Staphylococcus aureus bacilli strains, the ethyl acetate phase being more active. Regarding the in vitro antitumor test, the results revealed a dose-dependent cytotoxic effect against A375 melanoma cell lines, more pronounced in the case of the ethyl acetate phase. In addition, the ethyl acetate phase stimulated the proliferation of human keratinocytes (HaCaT), while this effect was not evident in the case of the ethanolic phase at 24 h post-stimulation. Consequently, G. verum l. could be considered a promising phytocompound for the antitumor approach of cutaneous melanoma.

Antiangiogenic, Anti-Inflammatory and Antioxidant Properties of Bidens tripartite Herb, Galium verum Herb and Rumex hydrolapathum Root.

Plants are commonly used in folk medicine. Research indicates that the mechanisms of biological activity of plant extracts may be essential in the treatment of various diseases. In this respect, we decided to test the ethanolic extracts of Bidens tripartita herb (BTH), Galium verum herb (GVH), and Rumicis hydrolapathum root (RHR) on angiogenic, anti-inflammatory, and antioxidant properties and their total polyphenols content. In vitro studies using endothelial cells were used to see tested extracts' angiogenic/angiostatic and anti-inflammatory properties. The DPPH assay and FRAP analysis were used to detect antioxidant properties of extracts. The Folin-Ciocalteu analysis was used to determine the content of total polyphenols. The results of gas chromatography-mass spectrometry analysis was also presented. In vitro study demonstrated that BTH, GVH, and RHR ethanolic extracts significantly increased cell invasiveness, compared with the control group. Increased endothelial proangiogenic invasiveness was accompanied by reduced metalloproteinase inhibitor 1 (TIMP-1) and raised in metalloproteinase 9 (MMP-9). Only BTH and GVH significantly reduced cell proliferation, while BTH and RHR facilitated migration. Additionally, tested extracts reduced the production of proangiogenic platelet-derived growth factor (PDGF) and hepatocyte growth factor (HGF). The most potent anti-inflammatory capacity showed BTH and GVH, reducing proinflammatory interleukin 8 (CXCL8) and interleukin 6 (Il-6), compared to RHR extract that has slightly less inhibited CXCL8 production without affecting IL-6 production. Moreover, we confirmed the antioxidant properties of all examined extracts. The highest activity was characterized by RHR, which has been correlated with the high content of polyphenols. In conclusion, the modifying influence of examined extracts can be promising in disorders with pathogenesis related to angiogenesis, inflammation and free radicals formation. BTH is the best choice among the three tested extracts with its antiangiogenic and anti-inflammatory properties.

Phytochemical Screening and Antioxidant Activity of Selected Estonian Galium Species.

The aim of the present study was to examine three different Galium species from the native population of Estonia, Galium verum, Galium aparine, and Galium mollugo, to characterise their non-volatile and volatile phytochemical composition and antioxidant activity. The main groups of bioactive compounds in the plants were quantified by colorimetric tests, showing high concentrations of polyphenols (up to 27.2 ± 1.5 mg GAE/g), flavonoids (up to 7.3 ± 0.5 mg QE/g) and iridoids (up to 40.8 ± 2.9 mg AE/g). The species were compared using HPLC-DAD-MS/MS, revealing some key differences in the phytochemical makeup of the extracts. The most abundant compound in the extracts of Galium verum blossoms and herb was found to be asperuloside, in Galium aparine herb, asperulosidic acid, and in Galium mollugo herb, chlorogenic acid. Additionally, the composition of volatile compounds was analysed by SPME-GC-MS. The degree of variability between the samples was high, but three volatiles, hexanal, anethole, and ß-caryophyllene, were quantified (≥1%) in all analysed samples. The antioxidative activity of all extracts was evaluated using the ORACFL method, demonstrating that the Galium species from Estonia all exhibit strong antioxidant capacity (up to 9.3 ± 1.2 mg TE/g). Out of the extracts studied, Galium verum blossoms contained the highest amounts of bioactives and had the strongest antioxidant capacity.

GCMS characterization and biological potential of the seeds and aerial part of Galium tricorne Stokes.

Natural products have long been proven very effective against various challenging diseases including cancer and bacterial infections. Galium tricorne is one of the important source of natural products, which has not been explored till date in spite of its profound ethnomedicinal prominence. The current study has been designed to explore the biological potential of G. tricorne and to extract and isolate chemical constituents from its aerial part and seeds respectively along with identification of their chemical constituents. Phytochemical screening was performed to figure out the presence of secondary metabolite in G. tricorne. Crude Methanolic extract (Gt.Crd), which was obtained from the aerial part while the fatty acids were extracted from the seeds, which were later on analyzed by GCMS. Similarly, Well Diffusion and MTT method were used for antibacterial activity and cancer cell line assay respectively. To evaluate the cytotoxic potential, brine shrimps were used. Likewise, in Gas Chromatography-Mass Spectroscopy (GC-MS) analysis a total number of 23 compounds were identified in Gt.Crd extract out of which 7 compounds were sorted out to have some sort of toxicity profile. In the same fashion, 5 fatty acids were identified in the seeds of G. tricorne. Moreover, among the fractions, chloroform fraction (Gt.Chf) exhibited greater zone of inhibition (ZOI) 20.37 mm followed by Gt.Crd 18.40 mm against S. aureus and S. pyogenes respectively. In cytotoxicity Gt.Chf was more active followed by ethyl acetate fraction (Gt.Eta) by exhibiting 88.32±0.62% (LC50=60 µg/mL) and 73.95±2.25% (LC50=80 µg/mL) respectively at 1000 µg/mL concentration of the tested sample. Gt.Chf exhibited greater cell line inhibitory activity (IC50=61 µg/mL) against HeLa cell line. Similarly, Gt.Crd displayed IC50 values of 167.84 µg/mL and 175.46 µg/mL against HeLa and NIH/3T3 cell line respectively. Based on the literature review and screenings, it may be concluded that the aerial part and seeds of G. tricorne are the rich sources of bioactive compounds. The results of the current study also authenticate the scientific background for the ethnomedicinal uses of G. tricorne.

The effects of methanol extract of Galium verum L on cardiac redox state in hypertensive rats

Abstract The aim of this study was to assess the effects of methanol extract of G. verum on redox status of isolated heart of spontaneously hypertensive rats after ischemia. Twenty-four Wistar albino rats were divided into three groups: untreated control rats and rats that received 125 and 250 mg/kg G. verum extract for 4 weeks per os. Index of lipid peroxidation (measured as TBARS) and parameters of antioxidative defence system such as level of reduced glutathione (GSH) and activities of catalase (CAT) and superoxide dismutase (SOD) were spectrophotometrically determined in heart homogenate. The index of lipid peroxidation in heart tissue was lower in both treated groups compared to the control group. On the other hand, the activity of SOD was significantly higher after consumption of both doses, while the activity of CAT was significantly higher only after treatment with a higher dose of extract. Based on our results we might conclude that 4-week treatment with methanol extracts of G. verum has the potential to modulate myocardial redox signaling after ischemia, thus significantly alleviating cardiac oxidative stress and exerting dose-dependent antioxidant properties. Future studies are certainly necessary to fully clarify the role of this plant species in myocardial I-R injury.

Immunomodulatory Activity and Phytochemical Profile of Infusions from Cleavers Herb.

Extracts from aerial parts of G. aparine (cleavers) constitute a herbal remedy with monography in British Herbal Pharmacopeia. On the European market, there are several drugs and food supplements consisting of Galium extracts. In folk medicine, cleavers was used topically in Europe, Asia, and the Americas to treat skin diseases. In several remedies, cleavers is also listed as an immunomodulatory active herb influencing the defense response of the human body. The aim of this study was to investigate the immunostimulatory activity and antioxidant potential in vitro of a raw infusion of cleavers and bioactive fractions. The functional activity of lymphocytes in the reaction of the lymphocyte blast transformation (RLBT) method was used for immunomodulatory activity assays and direct scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH), nitric oxide (NO), and hydrogen peroxide (H2O2) was chosen for the examination of antioxidant activity. It was shown that both the raw extract and fractions show significant immunostimulatory and scavenging activities. The obtained data partially justify the traditional use of cleavers as topical remedy for skin infections and for wounds.

Phylogeny, character evolution and spatiotemporal diversification of the species-rich and world-wide distributed tribe Rubieae (Rubiaceae).

The Rubiaceae tribe Rubieae has a world-wide distribution with up to 1,000 species. These collectively exhibit an enormous ecological and morphological diversity, making Rubieae an excellent group for macro- and microevolutionary studies. Previous molecular phylogenetic analyses used only a limited sampling within the tribe or missed lineages crucial for understanding character evolution in this group. Here, we analyze sequences from two plastid spacer regions as well as morphological and biogeographic data from an extensive and evenly distributed sampling to establish a sound phylogenetic framework. This framework serves as a basis for our investigation of the evolution of important morphological characters and the biogeographic history of the Rubieae. The tribe includes three major clades, the Kelloggiinae Clade (Kelloggia), the Rubiinae Clade (Didymaea, Rubia) and the most species-rich Galiinae Clade (Asperula, Callipeltis, Crucianella, Cruciata, Galium, Mericarpaea, Phuopsis, Sherardia, Valantia). Within the Galiinae Clade, the largest genera Galium and Asperula are para- and polyphyletic, respectively. Smaller clades, however, usually correspond to currently recognized taxa (small genera or sections within genera), which may be used as starting points for a refined classification in this clade. Life-form (perennial versus annual), flower shape (long versus short corolla tube) and fruit characters (dry versus fleshy, with or without uncinate hairs) are highly homoplasious and have changed multiple times independently. Inference on the evolution of leaf whorls, a characteristic feature of the tribe, is sensitive to model choice. Multi-parted leaf whorls appear to have originated from opposite leaves with two small interpetiolar stipules that are subsequently enlarged and increased in number. Early diversification of Rubieae probably started during the Miocene in western Eurasia. Disjunctions between the Old and the New World possibly are due to connections via a North Atlantic land bridge. Diversification of the Galiineae Clade started later in the Miocene, probably in the Mediterranean, from where lineages reached, often multiple times, Africa, eastern Asia and further on the Americas and Australia.

The immunomodulatory activity of the extracts and complexes of biologically active compounds of Galium verum L. herb.

The present article discusses the results of an immunomodulatory activity of Galii veri herba, Galium verum herb (Galium verum L., Rubiaceae) fluid water extract and the effect of the polysaccharide and phenolic complexes of the letter on the immunomodulatory activity. The same extract was fractionated into a polysaccharide complex (PSC) and a polyphenolic complex (PPC). In the obtained substances, the contents of hydroxycinnamic derivates, flavonoids and polyphenols were determined spectrophotometrically; polysaccharides were quantified gravimetrically; the immunomodulatory activity of the substances was determined in the reaction of lymphocyte blast transformation. It has been established that the fluid extract of Galium verum herb contains 6.3% polysaccharides, 4.2% hydroxycinnamic derivates expressed as chlorogenic acid, 0.4% flavonoids expressed as rutin, 3.7% polyphenols expressed as gallic acid. PPC contains 4.48% hydroxycinnamic derivates expressed as chlorogenic acid, 0.43% flavonoids expressed as rutin, and 3.95% polyphenols expressed as gallic acid. The lowest immunomodulatory activity was found for PPC. A significantly higher activity was determined for PSC. The highest immunomodulatory activity was established for the fluid extract at a dilution of 1/20, its activity being by 59.4% higher compared with the lymphocyte spontaneous transformation and by 18.5% higher than that of the reference substance PHA. The obtained results enable an assumption of a synergistic effect of PPC and PSC of Galium verum herb fluid extract on the potency of its immunomodulatory activity. Key words: Galium verum L. • immunomodulatory activity • lymphocyte blast transformation • fluid water extract • polysaccharide complex • polyphenols complex.

Chemo-mapping and biochemical-modulatory and antioxidant/prooxidant effect of Galium verum extract during acute restraint and dark stress in female rats.

Galium verum is a well-known medicinal plant which is used in various pathologies. G. verum extracts are characterized here using chromatography, where among the rich pool of phenolic acids of flavonoids two known anti-stress modulators, chlorogenic acid and rutin are identified in high quantities. Additionally, the extracts are characterized using a series of in vitro assays (EPR, DPPH, TPC and TEAC). Considering the chemical findings, the potential beneficial effects of the G. verum extract are explored here in a living organism exposed to stress induced oxidative damages. Thus, the biochemical-modulatory and antioxidant roles of two doses of G. verum extract are examined in animals exposed to acute restraint and dark stress (S). The animals were divided in groups [control, S, SG1 (exposed to 25 mg G. verum extract), SG2 (50 mg extract)]. Increased levels of lipid peroxidation (TBARS from 4.43 to 8.06 nmol/mL), corticosterone from 0.43 to 1.96 µg/dL and epinephrine from 44.43 to 126.7 µg/mL, as well as decreased antioxidant enzymes activities (SOD/CAT) were observed in the S group. The G. verum extract afforded a near-normal equilibrium within the biochemical parameters of animals exposed to RS, by reducing oxidative damage (TBARS at a 3.73 nmol/mL; CS at 0.90 µg/dL; EP at 63.72 µg/mL) and by restoring the antioxidant balance.

Expression, activation and processing of a novel plant milk-clotting aspartic protease in Pichia pastoris.

Galium verum, also known as Lady's Bedstraw or Cheese Rennet, is an herbaceous perennial plant traditionally used in cheese-making. We used RACE PCR to isolate novel enzymes from Galium verum with the ability to clot milk. This approach generated two cDNA sequences (named preprogaline A and B) encoding proteins displaying the typical plant aspartic protease primary structure. Preprogaline B was expressed in the yeast Pichia pastoris, after deleting and replacing its original signal peptide with the yeast α-factor signal peptide from Saccharomyces cerevisiae. The secreted recombinant protein was obtained by growing P. pastoris in YPD medium and had the ability to clot milk. The mature form of progaline B is a heterodimeric glycosylated enzyme, with a molecular weight of approximately 48 kDa, that contains a heavy (30.7 kDa) and a light (13.5 kDa) polypeptide chains linked by disulfide bonds. Western blot analysis revealed that progaline B is activated by the acidification of the yeast culture medium and that enzymatic activation requires two steps. First the precursor protein is cleaved into two polypeptide chains by partial removal of the plant-specific insert (PSI) present in plant aspartic proteases; this is later followed by propeptide removal. By altering the pH of the P. pastoris culture medium, we were able to obtain either active or inactive forms of the enzyme. Recombinant progaline B displayed a κ-casein hydrolysis pattern analogous to those produced by the animal and microbial coagulants currently used in the dairy industry, but it exhibited a different digestion profile on α- and ß-caseins. The plant protease progaline B displays milk-clotting activities suitable for the production of novel dairy products.

Decorated Superparamagnetic Iron Oxide Nanoparticles with Monoclonal Antibody and Diethylene-Triamine-Pentaacetic Acid Labeled with Thechnetium-99m and Galium-68 for Breast Cancer Imaging.

PURPOSE: In this study we developed and tested an iron oxide nanoparticle conjugated with DTPA and Trastuzumab, which can efficiently be radiolabeled with 99m-Tc and Ga-68, generating a nanoradiopharmaceutical agent to be used for SPECT and PET imaging. METHODS: The production of iron oxide nanoparticle conjugated with DTPA and Trastuzumab was made using phosphorylethanolamine (PEA) surface modification. Both radiolabeling process was made by the direct radiolabeling of the nanoparticles. The in vivo assay was done in female Balb/c nude mice xenografted with breast cancer. Also a planar imaging using the radiolabeled nanoparticle was performed. RESULTS: No thrombus and immune response leading to unwanted interaction and incorporation of nanoparticles by endothelium and organs, except filtration by the kidneys, was observed. In fact, more than 80% of 99mTc-DTPA-TZMB@Fe3O4 nanoparticles seems to be cleared by the renal pathway but the implanted tumor whose seems to increase the expression of HER2 receptors enhancing the uptake by all other organs. CONCLUSION: However, even in this unfavorable situation the tumor bioconcentrated much larger amounts of the nano-agent than normal tissues giving clear enough contrast for breast cancer imaging for diagnostics purpose by both SPECT and PET technique. Graphical Abstract ᅟ.

The immunomodulatory activity of ethanolic extracts from Galium verum L. herb.

The present article discusses the results of the research into the phytochemical profile and immunomodulatory activity of Galii veri herba (Galium verum L., Rubiaceae) ethanolic extracts. The extracts under study were obtained from the plant material by means of maceration technique with 20% aqueous ethanol solution (fluid extract I), 60% aqueous ethanol solution (fluid extract II) and 96% ethanol (fluid extract III) on heating. In the substances obtained, the content of hydroxycinnamic derivatives, flavonoids and polyphenols were determined spectrophotometrically; polysaccharides were quantified gravimetrically; the immunomodulatory activity of the substances under study was determined in the reaction of lymphocyte blast transformation (RLBT). It was established that fluid extract I contained 2.4% polysaccharides, 3.1% hydroxycinnamic derivatives, 0.24% flavonoids, and 2.9% polyphenols; the fluid extract II contained 4.13% hydroxycinnamic derivatives, 0.16% flavonoids, and 3.84% polyphenols; fluid extract III contained 2.7% hydroxycinnamic derivatives, 0.18% flavonoids and 2.7% polyphenols. All the extracts under study possessed a marked stimulant effect on the transformation activity of the immunocompetent blood cells. The highest immunomodulatory activity was established for 96% ethanol extract: the percentage of lymphocytes proliferating in RLBT under the influence of this extract increased by 6.77- 8.04 times in comparison with their spontaneous transformation and by 1.14-1.36 times in comparison with phytohemagglutinin. The results obtained give grounds for further research in the mechanisms of the immunomodulatory activity of the extracts of G. verum herb. Key words: ethanolic extracts Galium verum L. immunomodulatory activity lymphocyte blast transformation.

Effects of mutual intercropping on the cadmium accumulation in accumulator plants Stellaria media, Malachium aquaticum, and Galium aparine.

Intercropping affects heavy-metal uptake in plants. In this study, three cadmium (Cd)-accumulator species (Stellaria media, Malachium aquaticum, and Galium aparine) were intercropped together in Cd-contaminated soil to study the effects of intercropping on co-remediation. Mutual intercropping significantly increased the biomasses of S. media, M. aquaticum, and G. aparine compared with their respective monocultures. The photosynthetic pigment contents of three species were not significantly affected by mutual intercropping. Mutual intercropping did not increase the Cd contents in roots and shoots of G. aparine, but it increased the Cd contents in roots of S. media and M. aquaticum. It also decreased the Cd contents in shoots of S. media and M. aquaticum. Only G. aparine intercropped with M. aquaticum and three-species intercropping increased Cd-accumulation levels in whole plants of each species. Only S. media intercropped with M. aquaticum and three-species intercropping increased the Cd accumulation at the whole plant level in every plant in a single pot, with S. media intercropped with M. aquaticum showing the greatest increase. Therefore, S. media intercropped with M. aquaticum and three-species intercropping may improve the efficiency of phytoremediation of Cd-contaminated soil, with S. media intercropped with M. aquaticum representing the best combination.

Investigation of the halo-artifact in 68Ga-PSMA-11-PET/MRI.

OBJECTIVES: Combined positron emission tomography (PET) and magnetic resonance imaging (MRI) targeting the prostate-specific membrane antigen (PSMA) with a 68Ga-labelled PSMA-analog (68Ga-PSMA-11) is discussed as a promising diagnostic method for patients with suspicion or history of prostate cancer. One potential drawback of this method are severe photopenic (halo-) artifacts surrounding the bladder and the kidneys in the scatter-corrected PET images, which have been reported to occur frequently in clinical practice. The goal of this work was to investigate the occurrence and impact of these artifacts and, secondly, to evaluate variants of the standard scatter correction method with regard to halo-artifact suppression. METHODS: Experiments using a dedicated pelvis phantom were conducted to investigate whether the halo-artifact is modality-, tracer-, and/or concentration-dependent. Furthermore, 31 patients with history of prostate cancer were selected from an ongoing 68Ga-PSMA-11-PET/MRI study. For each patient, PET raw data were reconstructed employing six different variants of PET scatter correction: absolute scatter scaling, relative scatter scaling, and relative scatter scaling combined with prompt gamma correction, each of which was combined with a maximum scatter fraction (MaxSF) of MaxSF = 75% or MaxSF = 40%. Evaluation of the reconstructed images with regard to halo-artifact suppression was performed both quantitatively using statistical analysis and qualitatively by two independent readers. RESULTS: The phantom experiments did not reveal any modality-dependency (PET/MRI vs. PET/CT) or tracer-dependency (68Ga vs. 18F-FDG). Patient- and phantom-based data indicated that halo-artifacts derive from high organ-to-background activity ratios (OBR) between bladder/kidneys and surrounding soft tissue, with a positive correlation between OBR and halo size. Comparing different variants of scatter correction, reducing the maximum scatter fraction from the default value MaxSF = 75% to MaxSF = 40% was found to efficiently suppress halo-artifacts in both phantom and patient data. In 1 of 31 patients, reducing the maximum scatter fraction provided new PET-based information changing the patient's diagnosis. CONCLUSION: Halo-artifacts are particularly observed for 68Ga-PSMA-11-PET/MRI due to 1) the biodistribution of the PSMA-11-tracer resulting in large OBRs for bladder and kidneys and 2) inaccurate scatter correction methods currently used in clinical routine, which tend to overestimate the scatter contribution. If not compensated for, 68Ga-PSMA-11 uptake pathologies may be masked by halo-artifacts leading to false-negative diagnoses. Reducing the maximum scatter fraction was found to efficiently suppress halo-artifacts.

Examination of secondary metabolites and antioxidant capacity of Anthyllis vulneraria, Fuchsia sp., Galium mollugo and Veronica beccabunga.

Anthyllis vulneraria L., Fuchsia sp., Galium mollugo L., and Veronica beccabunga L. were selected to analyse the phenolic content and the antioxidant activity by ferric ion reducing antioxidant power (FRAP) and trolox equivalent antioxidant capacity (TEAC) assays. The highest polyphenol, tannin, and flavonoid contents were measured in Fuchsia species (7.40 ± 0.8, 5.62 ± 0.7 and 0.72 ± 0.1 g/100 g dry weight), while the lowest values were detected in Anthyllis vulneraria (0.68 ± 0.02, 0.17 ± 0.03 and 0.45 ± 0.01 g/100 g dry weight) and Galium mollugo (1.77 ± 0.05, 0.49 ± 0.04 and 0.16 ± 0.06 g/100 g dry weight). The leaf extract of Fuchsia sp. had the highest, while the herb of A. vulneraria had the lowest antioxidant effect measured by both methods, which is probably related to total polyphenol, tannin, and flavonoid contents.

[Effects and related mechanism of flavone from Galium verum L on peroxide induced oxidative injury in human umbilical vein endothelial cells].

OBJECTIVE: To investigate the effects of flavone from Galium verum L (FGVL) on hydrogen peroxide induced oxidative injury in human umbilical vein endothelial cells (HUVEC), and explore related mechanisms. METHODS: HUVEC were divided into five groups: control group (1640 complete medium), injured group (HUVEC treated with 100 µmol/L hydrogen peroxide for 4 h), FGVL group (HUVEC treated with 12.5 mg/L FGVL (group F1), 25.0 mg/L (group F2), 50.0 mg/L (group F3) for 24 h before hydrogen peroxide). The nitric oxide content was measured by nitric acid reductase method. The 6-keto-Prostacyclin-F1α (6-keto-PGF1α), thromboxane B2 (TXB2), interleukin(IL)-6 and IL-22 were determined by ELISA.mRNA expression of phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt) and endothelial nitric oxide synthase (eNOS) was detected by RT-PCR.Protein expression of p-Akt (ser(473)) and p-eNOS (ser(1177)) was determined by Western blot.Cell apoptosis was observed with fluorescence microscope after Hoechst33258 staining. RESULTS: (1) The contents of nitric oxide were significantly lower in the injured group than in the control group ((34.11±1.78) µmol/L vs. (74.81±2.93) µmol/L, P<0.05), which was significantly increased in group F2 ((41.86±2.32) µmol/L) and group F3 ((62.79±1.16) µmol/L) compared with injured group (both P<0.05). (2)The secretion level of 6-keto-PGF1α was significantly lower in the injured group ((44.84±3.87) ng/L) than in the control group ((82.38±3.98) ng/L, P<0.05), which was significantly increased in group F1 ((52.76±1.78) ng/L), FGVL 2 group which was(56.58±1.44) ng/L and FGVL 3 group which was(67.78±2.02) ng/L than that of injured group(all P<0.05). The secretion level of TXB2 was significantly higher in the injured group((43.37±3.96) ng/L) than in the control group ((25.56±1.75) ng/L, P<0.05), which was significantly reduced group F2 group ((32.41±1.68) ng/L) and group F3 ((28.23±2.15) ng/L) than that of injured group(both P<0.05). (3) The contents of IL-6 and IL-22 were significantly higher in the injured group ((539.74±11.63) ng/L) and ((23.70±3.05) ng/L, respectively) than in the control group ((288.67±19.52) ng/L) and ((23.70±3.05) ng/L, respectively, both P<0.05). The contents of IL-6 were significantly lower in group F1, F2 and F3 compared to that of injured group(all P<0.05). The contents of IL-22 were significantly lower in group F2 and F3 than that of injured group(both P<0.05). (4)The relative levels of PI3K mRNA and eNOS mRNA in injured group (0.68±0.09 and 0.22±0.03, respectively) were significantly lower compared to control group(0.81±0.12 and 0.63±0.11, respectively, both P<0.05), PI3K mRNA in group F2 (0.76±0.03) and group F3 (PI3K mRNA 0.83±0.06) as well as eNOS mRNA in group F1 (0.37±0.08), F2 (0.53±0.04) and F3 (0.56±0.09) than those of injured group(all P<0.05). The mRNA expression of Akt was similar among groups (P>0.05). (5) The relative levels of p-Akt (ser(473)) and p-eNOS (ser(1177)) in injured group (0.48±0.05 and 0.23±0.03, respectively) were significantly lower compared to control group (0.71±0.12 and 0.66±0.05, respectively, both P<0.05), which was up-regulated in group F1, F2 and F3 groups compared to injured group(all P<0.05). (6) The cell apoptosis rate in injured groups was significantly higher compared to control group which ((63.67±11.37)% vs. (4.67±1.15)%, P<0.05) which was significantly reduced in group F1((43.33±4.16)%), F2((18.33±4.93)%) and F3((15.67±2.08)%) compared to injured group(all P<0.05). CONCLUSION: The FGVL can reduce hydrogen peroxide induced oxidative injury in HUVEC by increasing the level of nitric oxide through PI3K/Akt/eNOS pathway.

Effects of Galium aparine extract on the cell viability, cell cycle and cell death in breast cancer cell lines.

ETHNOPHARMACOLOGICAL RELEVANCE: Galium species have been traditionally used for its anti-cancer, antioxidant, anti-inflammatory, antimicrobial and cardioprotective effects in the folk medicine. Galium aparine (GA) is a typical climbing plant growing widespread in Anatolia. AIM OF THE STUDY: To investigate the potential anti-proliferative and apoptotic effect of GA methanol (MeOH) extract on MCF-7 and MDA-MB-231 human breast cancer cells and MCF-10A untransformed breast epithelial cells. MATERIALS AND METHODS: First, the extract was characterized by both liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/Q-TOF/MS) and gas chromatography-mass spectrometry (GC-MS) analyses. Then, cell viability and cell cycle distribution were investigated by XTT assay and PI staining by flow cytometry, respectively. Cell death was determined by Annexin V FITC/7-AAD staining. RESULTS: A total of 14 major phytochemicals were identified by LC/Q-TOF/MS and 34 volatile compounds were determined by GC-MS. The extract was cytotoxic in both breast cancer cell lines in a concentration and time dependent manner and showed G1 block after 72h extract treatment. However, it was not cytotoxic to MCF-10A breast epithelial cells. Flow cytometry analyses revealed that apoptosis was induced in MDA-MB-231 cells; however, necrosis was induced in MCF-7 cells. CONCLUSION: Our study suggests that GA MeOH extract may have potential anti-cancer effects against breast cancer cells without impairing normal breast epithelial cells. Ability to induction of non-apoptotic cell death besides apoptotic cell death by this complex plant-derived mixture may enable the killing of apoptosis resistant breast cancer cells but further studies should be conducted to investigate the bioavailability and metabolism of it in vivo.

The Effect of Laser Fiber Cleave Technique and Lithotripsy Time on Power Output.

PURPOSE: Various cleave techniques have recently been shown to significantly impact initial laser fiber power output during holmium laser lithotripsy. The impact of cleave technique on long-term power output has not been well characterized. The purpose of this study was to determine the effect of laser cleave technique on power output over time. MATERIALS AND METHODS: In this randomized single-blinded study, five cleave techniques were tested on two holmium laser fiber diameters (200, 365 µm) over 15 minutes of laser lithotripsy with calcium oxalate monohydrate stones. Comparisons between cleave techniques and fiber diameters were performed using independent samples Mann-Whitney U, Kruskal-Wallis, and homogeneity of variance tests with a significance of p < 0.05. RESULTS: The 365-µm fiber was more durable and less affected by burnback degradation than the 200-µm fiber (p < 0.05). While initial power output varied between cleave techniques, all significance disappeared by 3 minutes. Power output decreased rapidly by a mean of 0.62 W over 4 minutes (p < 0.05), following which there was no significant change. CONCLUSION: These findings confirm that initial laser fiber power output is significantly influenced by cleave technique, and the ceramic scissor is the optimal tool for cleaving between procedures. However, because of rapid fiber tip degradation and power loss, this study argues against routine cleaving to improve procedural efficiency in lengthy ureteroscopy cases.

The origin of unique diversity in deglaciated areas: traces of Pleistocene processes in north-European endemics from the Galium pusillum polyploid complex (Rubiaceae).

The role of glacial oscillations in shaping plant diversity has been only rarely addressed in endemics of formerly glaciated areas. The Galium pusillum group represents a rare example of an ecologically diverse and ploidy-variable species complex that exhibits substantial diversity in deglaciated northern Europe. Using AFLP and plastid and nuclear DNA sequences of 67 populations from northern, central, and western Europe with known ecological preferences, we elucidate the evolutionary history of lineages restricted to deglaciated areas and identify the eco-geographic partitioning of their genetic variation. We reveal three distinct endemic northern lineages: (i) diploids from southern Sweden + the British Isles, (ii) tetraploids from southern Scandinavia and the British Isles that show signs of ancient hybridization between the first lineage and populations from unglaciated central Europe, and (iii) tetraploids from Iceland + central Norway. Available evidence supports a stepwise differentiation of these three lineages that started at least before the last glacial maximum by processes of genome duplication, interlineage hybridization and/or allopatric evolution in distinct periglacial refugia. We reject the hypothesis of more recent postglacial speciation. Ecological characteristics of the populations under study only partly reflect genetic variation and suggest broad niches of postglacial colonizers. Despite their largely allopatric modern distributions, the north-European lineages of the G. pusillum group do not show signs of rapid postglacial divergence, in contrast to most other northern endemics. Our study suggests that plants inhabiting deglaciated areas outside the Arctic may exhibit very different evolutionary histories compared with their more thoroughly investigated high-arctic counterparts.

Galium verum aqueous extract strongly inhibits the motility of head and neck cancer cell lines and protects mucosal keratinocytes against toxic DNA damage.

Galium verum, also known as Lady's Bedstraw, is an herbaceous plant native to Europe and Asia, and has been used in traditional medicine as an anticancer medicine applied in most cases as a decoction. The influence of a Galium verum decoction on the head and neck cancer cell lines HLaC78 and FADU was analyzed and proved to be toxic in high doses on both cell lines. Cytotoxicity appeared to be influenced by expression of p-glycoprotein (MDR-1) in the carcinoma cell lines. Mucosal keratinocytes, although void of MDR-1 expression, showed only low sensitivity against high Galium concentrations. Sublethal doses of Galium extract acted as strong inhibitors of motility, as shown by a spheroid-based invasion analysis on Matrigel-coated surfaces. Inhibition of invasion was significantly more pronounced in the invasive HLaC78 cell line. mRNA expression analysis of matrix metalloproteinases MMP-2 and MMP-9 and their inhibitors TIMP-1/-2 revealed significant TIMP-1 upregulation after an 8-h Galium exposition in FADU cells. Gelatinolytic activity, however, was not influenced by Galium extract in HLaC78, in the FADU cells MMP-2/-9 activity was slightly increased after incubation with Galium extract. In primary mucosal keratinocytes, Galium decoction protected DNA against benz[a]pyrene, one of the most DNA toxic agents in cigarette smoke. In conclusion Galium extract may be useful as a preventive and/or a concomitant therapeutic approach in head and neck cancer.