Purification and characterization of polyphenol oxidase from nettle (Urtica dioica L.) and inhibitory effects of some chemicals on enzyme activity

Polyphenol oxidase (PPO) of nettle (Urtica dioica L.) was extracted and purified through (NH₄)₂SO₄ precipitation, dialysis, and CM-Sephadex ion-exchange chromatography and was used for its characterization. The PPO showed activity to catechol, 4-methylcatechol, L-3,4-dihydroxyphenylalanine (L-DOPA), L-tyrosine, p-cresol, pyrogallol, catechin and trans-cinnamic acid. For each of these eight substrates, optimum conditions such as pH and temperature were determined and L-tyrosine was found to be one of the most suitable substrates. Optimum pH and temperature were found at pH 4.5 and 30°C respectively and K_m and V_max values were 7.90?×?10^?4?M, and 11290?EU/mL for with L-tyrosine as substrate. The inhibitory effect of several inhibitors, L-cysteine chloride, sodium azide, sodium cyanide, benzoic acid, salicylic acid, L-ascorbic acid, glutathione, thiourea, sodium diethyl dithiocarbamate, β-mercaptoethanol and sodium metabisulfite were tested. The most effective was found to be sodium diethyl dithiocarbamate which acted as a competitive inhibitor with a K_i value of 1.79?×?10^?9?M. In addition one isoenzyme of PPO was detected by native polacrylamide slab gel electrophoresis.     

Assessment of Aflatoxin M1 and Heavy Metal Levels in Mothers Breast Milk in Famagusta,Cyprus

Breast milk contributes towards optimal nutrition for infants. However, studies showed that it can also contain different toxins and heavy metals, which reduce its health benefits. The aim of this study is to determine the level of contaminants such as aflatoxin M1 (AFM1), Pb, Cd, As, and Hg in breast milk samples from Famagusta, Cyprus. Correlations between moldy food consumption, smoking habits of the mothers, and contaminant levels in breast milk were also investigated. Breast milk samples from 50 lactating mothers in rural and urban areas of Famagusta District were analyzed for AFM1 by ELISA. Eighty percent of them were found to be contaminated with AFM1 with the mean measurement of 7.84?±?1.72 ng/l. Socio-demographic status, moldy food consumption habits, and smoking status do not have any effect on the AFM1 levels observed in breast milk. Heavy metal levels in breast milk were examined by inductively coupled plasma mass spectrometry, and the mean measurements were1.19?±?1.53 ppm for Pb, 0.73?±?0.58 ppm for As, 0?±?0.20 ppm for Hg, and 0.45?±?0.23 ppm for Cd. This study indicates that the levels of these contaminants in breast milk samples obtained in Famagusta District are well within the acceptable levels. However, the presence of AFM1 and heavy metals still may pose risks for infant health.     

Plant growth-promoting bacteria (PGPBs) and copper (II) oxide (CuO) nanoparticle ameliorates DNA damage and DNA Methylation in wheat (Triticum aestivum L.) exposed to NaCl stress

Journal of Plant Biochemistry and Biotechnology - Wheat is the second important cereal crop worldwide due to nutritional composition and role in meeting daily energy needs. Salinity is an abiotic...     

Analyzing the Impact of Ramalina digitellata,R. fastigiata,R. fraxinea,and R. polymorpha's Usnic Acid Concentration on Antioxidant,DNA-Protective,Antimicrobial, and Cytotoxic Properties

The present study is focused on the antimicrobial, antioxidant, cytotoxic, and DNA protective effects of methanol extract obtained from R. digitellata, R. fastigiata, R. fraxinea, and R. polymorpha species that are distributed in Turkey. The highest total phenol content was determined in R. digitellata (144.6 mgGAE/g_extract), and the highest total amount of flavonoids was found in R. fastigiata (20.40 mgGAE/g_extract). The content of usnic acid was determined by High-Performance Liquid Chromatography (HPLC) and the highest amount was found in R. digitellata. DPPH (1,1-diphenyl-2-picrylhydrazyl) and ABTS [2,2’-azinobis(3-ethylbenzathiazoline-6-sulfonic acid)] radical scavenging methods were used for antioxidant activity. R. fraxinea showed the highest DPPH⋅ and ABTS⁺⋅ scavenging activity. In addition, the DNA protective effect was investigated using pBR322 plasmid DNA, and; all studied species were found to have DNA protective effects. The antibacterial activity was investigated using the disc diffusion method, and the R. digitellata methanol extract showed the best results with a 12.35 mm zone on Proteus mirabilis. On the human lung cancer (A549) and breast cancer (MDA-MB-231) cell lines, cytotoxic activity was assessed using an MTT assay. All lichen extracts were found to have a significant cytotoxic effect on both cancer cell lines at 1000 μg/mL concentration. These results suggest that Ramalina species may be potential candidates for developing new phytopharmaceuticals and functional components.     

Screening of in Vitro Inhibition of Lactoperoxidase Enzyme by Methyl Benzoate Derivatives with Molecular Docking Studies

Lactoperoxidase enzyme (LPO) is secreted from salivary, mammary, and other mucosal glands including the bronchi, lungs, and nose, which had functions as a natural and the first line of defense towards viruses and bacteria. In this study, methyl benzoates were examined in LPO enzyme activity. Methyl benzoates are used as precursors in the synthesis of aminobenzohydrazides used as LPO inhibitors. For this purpose, LPO was purified in a single step using sepharose-4B-l-tyrosine-sulfanilamide affinity gel chromatography with a yield of 9.91 % from cow milk. Also, some inhibition parameters including the half maximal inhibitory concentration (IC₅₀) value and an inhibition constant (K_i) values of methyl benzoates were determined. These compounds inhibited LPO with K_i values ranging from 0.033±0.004 to 1540.011±460.020 μM. Compound 1 a (methyl 2-amino-3-bromobenzoate) showed the best inhibition (K_i=0.033±0.004 μM). The most potent inhibitor ( 1 a ) showed with a docking score of −3.36 kcal/mol and an MM-GBSA value of −25.05 kcal/mol, of these methyl benzoate derivatives ( 1 a – 16 a ) series are established H-bond within the binding cavity with residues Asp108 (distance of 1.79 Å), Ala114 (distance of 2.64 Å), and His351 (distance of 2.12 Å).     

Novel Quinazolinone Derivatives: Potential Synthetic Analogs for the Treatment of Glaucoma,Alzheimer's Disease and Diabetes Mellitus

Quinazolinones, which represent an important part of nitrogen-containing six-membered heterocyclic compounds, are frequently used in drug design due to their wide biological activity properties. Therefore, the novel quinazolinones were synthesized from the reaction of acylated derivatives of 4-hydroxy benzaldehyde with 3-amino-2-alkylquinazolin-4(3H)-ones with good yields (85–94 %) and their structures were characterized using Fourier-transform Infrared (FT-IR), Nuclear Magnetic Resonance (¹H-NMR, ¹³C-NMR), and High-Resolution Mass Spectroscopy (HR-MS). As the application of the synthesized compounds, their inhibition properties of the synthesized compounds on α-Glucosidase (α-Glu), Acetylcholinesterase (AChE), Butyrylcholinesterase (BChE), and Carbonic anhydrase I–II (hCA I–II) metabolic enzymes were investigated. All compounds showed inhibition at nanomolar level with the K_i values in the range of 12.73±1.26–93.42±9.44 nM for AChE, 8.48±0.92–25.84±2.59 nM for BChE, 66.17±5.16–818.06±44.41 for α-Glu, 2.56±0.26–88.23±9.72 nM for hCA I, and 1.68±0.14–85.43±7.41 nM for hCA II. Molecular docking study was performed to understand the interactions of the most potent compounds with corresponding enzymes. Also, absorption, distribution, metabolism, excretion, and toxicity (ADME/T) properties of the compounds were investigated.     

Aglycemia induces apoptosis under hypoxic conditions in A549 cells

Many of the cancer cells produce energy with accelerated glycolysis and perform lactic acid production even under normoxic conditions called the “Warburg effect”. Metabolism can directly or indirectly regulate the apoptotic mechanism so that cancer cells take advantage of reprogrammed metabolism to avoid apoptosis. The aim of this study is to examine the mechanism of apoptosis by incubating human lung carcinoma cells (A549) under different metabolic conditions in hypoxia or normoxia environments. A549 cells were incubated in the normoxic or hypoxic condition that contained 5 mM glucose (Glc 5), 25 mM glucose (Glc 25), or 10 mM galactose (OXPHOS/aglycemic), and the mechanism of apoptosis was investigated. In the hypoxia condition, the rate of early apoptosis in aglycemic OXPHOS cells was increased (15.5% ±7.1). In addition, the activity of caspase-3 (6.1% ± 0.9), caspase-9 (30.4% ± 0.9), and cytochrome c expression level increased; however, the mitochondrial membrane potential (51.9% ± 0.4) was found to be decreased. Changing the amount of oxygen in glycolytic cells had no effect on apoptosis. However, it has been determined that apoptosis is stimulated under hypoxia conditions in aglycemic cells in which galactose is used instead of glucose. Considering that the majority of cancer cells are hypoxic, these data are important in determining targets in therapeutic intervention.     

Apoptosis‐inducing effects of Morinda citrifolia L. and doxorubicin on the Ehrlich ascites tumor in Balb‐c mice

Morinda citrifolia L. (Noni) is a herbal remedy with promising anti‐cancer properties. However, its effects on various cancers are to be investigated to make a firm conclusion before implementing it into the clinical practice. Therefore, we investigated the cytotoxic potential of noni on Ehrlich ascites tumor grown in female Balb‐c mice and also combined it with a potent anti‐cancer agent, doxorubicin. One group received noni only (n = 8), another one doxorubicin (n = 8), and the other one noni + doxorubicin (n = 8) for 14 days after the inoculation of cells. The control group (n = 7) received 0.9% NaCl only. We found that short and long diameters of the tumor tissues were about 40–50% smaller, compared to those in control group. This anti‐growth effect resulted from the induction of apoptosis, which was confirmed by the positive results from the Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) analysis and the active caspase‐3 cells in tissues. Apoptosis also confirmed by caspase‐cleaved cytokeratin 18 elevation in serum of the treated groups. Further, the proliferation was decreased, which was immunohistochemically shown by the PCNA staining. We conclude that noni may be useful in the treatment of breast cancer either on its own or in combination with doxorubicin. Further studies are warranted to assess the dosage and safety of using noni fruit juice in conjuction with anti‐cancer drugs against breast cancer. Copyright © 2009 John Wiley & Sons, Ltd.     

Spectroscopic,kinetic and dosimetric features of the radical species produced after radiodegradation of solid triclosan

In the present work, spectroscopic, kinetic and dosimetric features of the radicalic intermediates produced after gamma irradiation at room temperature of solid triclosan (2,4,4-trichloro-2-hydroxydiphenyl ether; TCS) were investigated by means of electron spin resonance spectroscopy (ESR) at various temperatures. The same material was also irradiated with UV light, and an ESR spectrum very similar to that obtained for gamma-irradiated TCS, was recorded. The ESR spectrum of TCS is characterized by an unresolved doublet with resonance lines split into other doublets. An evaluation technique based on variations of four assigned peak-to-peak amplitudes and signal intensity was adopted, to monitor the evolution of the spectrum under different experimental conditions. Radicals of one type were proposed to be created upon irradiation exhibiting decays via intra-track and inter-track recombination reactions with activation energies of 43 ± 2 and 139 ± 6 kJ/mol, respectively. A radical exhibiting axial g anisotropy and interacting with two un-equivalent protons was found to describe the experimental spectrum well. The sensitivity of TCS to gamma radiation was high (G = 0.12) suggesting TCS to be a suitable dosimetric material in measuring normal and accidental radiation doses in the range of (1–25 kGy).