Bioactive compounds from Taraxacum officinale extracts obtained by optimized ultrasound-assisted extraction

In this study, the extraction of plant species Taraxacum officinale (F. H. Wigg) was optimized providing the maximum extraction of phenolic compounds using Response Surface Methodology (RSM). The optimum extraction parameters were as follows: extraction temperature of 60°C, extraction time of 30 min and 1:32.7 g/mL solid-liquid ratio for ethanol extract, and 60°C, 30 min, 1:14.2 g/mL ratio for acetone extract. The optimized extracts exhibited different biological activities. Ethanol extract showed better DPPH radicals scavenging activity and reducing power compared to acetone extract. The extracts had low antibacterial activity. In addition, the extracts had no effect on the healthy MRC-5 cell line, while acetone extract reduced the viability of HCT-116 carcinoma cells. This paper provides a basis for further research on the optimization of dandelion extraction under different conditions and testing the bioactivity of this medicinal plant.Publishe

Comparative analysis of human DNA extraction methods and mitochondrial DNA HV1 and HV2 haplogroup determination

The high integrity of extracted DNA is necessary for the determination of human haplogroups, based on the mitochondrial DNA (mtDNA) marker sequences. The study aims were to isolate the total DNA from selected human samples and establish a protocol that gives the best yield and proper purity of DNA material for further analysis. Furthermore, human hypervariable regions - HV1 and HV2, located on mtDNA were sequenced to define human haplogroups. For extraction, samples of buccal mucosa from ten human volunteers were taken and three different protocols were used (a method with ammonium acetate, a salting-out method and GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific)). Concentration and purity of DNA were measured on BioPhotometer. Sequencing was performed by Sanger method on 3130 Genetic Analyzer (Applied Biosystems) with commercial BigDye™ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Sequences were processed using Chromas and MEGA software and compared with sequence databases with BLAST tool on NCBI. Haplogroups and mutations were determined in the MITOMAP online database. Results of extraction indicated that the Purification Kit gives the highest concentration of DNA (11.86±0.63) and low purity (1.17±0.02), while a method with ammonium acetate gave the best purity (1.67±0.17). Sequencing obtained on DNA extracted with a commercial kit, gave 5 different human haplogroups U2e, T1a, J1c, H2a, K. In conclusion, it is recommended to use the Purification Kit for obtaining high integrity DNA that can be used for effective sequencing. Determination of haplogroups and haplotypes can reveal our ancestry and have important implications in molecular systematics, phylogenetics and phylogeography.Publishe

Synthesis, characterization, DFT study, DNA/BSA-binding affinity, and cytotoxicity of some dinuclear and trinuclear gold(III) complexes

Abstract: In this study, we have synthesized a series of dinuclear and trinuclear gold(III) complexes of the general formula [Au2(N–N)Cl6] (1–3) for dinuclear and [Au3(N–N)2Cl8]+ (4–6) for trinuclear compounds, respectively, in which N–N is a bidentate ligand (1,4-diaminobutane; 1,6-diaminohexane or 1,8-diaminooctane). These complexes were characterized by elemental analysis, molar conductivity, and spectroscopic techniques (IR, UV–Vis, 1H NMR, ESI–MS). We performed DFT calculations to get insight into the geometry of the studies complexes. DNA-binding studies were performed by UV–Vis spectrophotometry and fluorescence spectroscopy. The results of competitive reactions between gold(III) complexes and ethidium bromide (EB) towards DNA have shown that selected complexes can displace EB from DNA-EB adduct. In addition, these experiments confirm that polynuclear gold(III) complexes interact with DNA covalently or via intercalation. Furthermore, high values of binding constants of gold(III) complexes towards bovine serum albumin (BSA) protein indicate good binding affinity. In addition, redox stability of complexes in the presence of DNA/BSA was confirmed by cyclic voltammetry. Results of the interactions between gold(III) complexes with DNA/BSA were discussed in reference to molecular docking data obtain by Molegro virtual docker. The cytotoxic activity of synthesized gold(III) complexes was evaluated on human breast cancer cell line (MDA-MB-231), human colorectal cancer cell line (HCT-116), and normal human lung fibroblast cell line (MRC-5). All complexes dose-dependently reduced cancer and normal cells viabilities, with significant cytotoxic effects (IC50 < 25 μM) for trinuclear gold(III) complexes (4, 5) on HCT-116 cells. Graphic abstract: [Figure not available: see fulltext.]

Matična mliječ i trans-10-hidroksi-2-decenska kiselina inhibiraju migraciju i invaziju stanica kolorektalnog karcinoma

Research background. Acquisition of migratory potential is pivotal for cancer cells, enabling invasion and metastasis of colorectal carcinoma. Royal jelly and its bioactive component trans-10-hydroxy-2-decenoic acid (10H2DA) showed remarkable antimetastatic potential, but the molecular mechanism underlying this activity is unclear. Experimental approach. Identification and quantification of 10H2DA in royal jelly originating from Serbia was done by HPLC method. Cytotoxicity of 10H2DA was measured by tetrazolium dye MTT test in concentration range 1-500 μg/mL after 24 and 72 h. Its effect on the collective and single-cell migration was measured by wound healing and transwell migration assays. Invasive potential of cancer cells was evaluated by a transwell method modified with collagen. Immunofluorescence was used for migratory and invasive protein expression, while the gene expression of these markers was evaluated by quantitative real time polymerase chain reaction (qRT-PCR). All assays were applied on human colorectal carcinoma HCT-116 and SW-480 cell lines and, except for MTT, evaluated after 24 h of treatment with two selected concentrations of royal jelly and 10H2DA. Results and conclusions. According to HPLC, the mass fraction of 10H2DA in royal jelly was 0.92% (m/m). Treatment with 10H2DA showed no cytotoxic effect; however, significant inhibitory potential of royal jelly and 10H2DA on the motility and invasiveness of colorectal cancer cells was observed. More pronounced effect was exerted by 10H2DA, which significantly suppressed collective cell migration and invasiveness of SW-480 cells, as well as single- and collective cell migration and invasive potential of HCT-116 cell line. Treatments increased epithelial markers E-cadherin and cytoplasmic β-catenin in HCT-116 cells, thus stabilizing intercellular connections. In SW-480 cells, 10H2DA increased E-cadherin on protein and gene level, and suppressed epithelial-mesenchymal transition (EMT) markers. In both cell lines, treatments induced significant suppression of promigratory/proinvasive markers: N-cadherin, vimentin and Snail on protein and gene level, which explains decreased migratory and invasive potential of HCT-116 and SW-480 cells. Novelty and scientific contribution. Our study presents new findings and elucidation of royal jelly and 10H2DA molecular mechanism that underlies their antimigratory/antiinvasive activity on colorectal cancer cells. These findings are shown for the first time indicating that these natural products are a valuable source of anticancer potential and should be reconsidered for further antitumour therapy.Pozadina istraživanja. Sposobnost migracije stanica jе ključna za invaziju i mеtastaziranje kolorektalnog karcinoma. Matična mlijеč te njezin bioaktivni sastojak trans-10-hidroksi-2-dеcеnska kisеlina (10H2DA) imaju izuzеtan antimеtastatski potеncijal, no molеkularni mеhanizam ovе aktivnosti još uvijek nije jasan. Eksperimentalni pristup. Prisutnost i količina 10H2DA u matičnoj mlijеči porijеklom iz Srbijе utvrđeni su mеtodom HPLC. Citotoksičnost 10H2DA ispitana jе nakon 24 i 72 h pomoću MTT tеsta s 1−500 μg/mL tetrazolijeve soli. Utjecaj 10H2DA na kolеktivnu migraciju i onu pojedinih stanica određen je praćenjem procesa cijeljenja rana te migracije stanica na Transwell pločama. Invazivni potеncijal stanica karcinoma ispitan jе na Transwell pločama s kolagеnom. Za određivanje еksprеsije protеina uključenih u procese migracije i invazije upotrijebljena jе imunofluorеscеntna metoda, dok jе gеnska еksprеsija tih markеra procijenjena kvantitativnom lančanom rеakcijom polimеrazе u stvarnom vrеmеnu (qRT-PCR). U svim su testovima korištene dvije stanične linije humanog kolorеktalnog karcinoma: HCT-116 i SW-480, tretirane s dvijе odabranе koncеntracijе matične mlijеči i 10H2DA, čiji je učinak mjeren 24 h nakon tretmana, osim u MTT testu. Rezultati i zaključci. Metodom HPLC utvrđeno je da matična mliječ sadržava 0,92 % (m/m) 10H2DA. Ispitivanjem utjecaja 10H2DA na stanice karcinoma utvrđeno je da kiselina nijе imala citotoksični učinak, no opažen jе znatan potеncijal matične mlijеči i 10H2DA da inhibiraju pokrеtljivost i invazivnost stanica karcinoma dеbеlog crijеva. Izražеnije je djelovanje imala 10H2DA, koja jе bitno smanjila kolеktivnu migraciju i invazivnost SW-480 stanica, kao i kolеktivnu migraciju, migraciju pojеdinih stanica i invazivni potеncijal stanične linijе HCT-116. Nakon obrade povеćala se ekspresija еpitеlnih markеra E-kadhеrina i citoplazmatskog β-katеnina u HCT-116 stanicama, što je dovelo do stabilizacije međustaničnih vеza. U stanicama SW-480 je 10H2DA povеćala ekspresiju E-kadhеrina na razini proteina i gena te inhibirala markеrе еpitеlno-mеzеnhimalnе tranzicijе (EMT). Obrada matičnom mliječi i 10H2DA je u objе stanične linijе u većoj mjeri potaknula suprеsiju promigracijskih/proinvazivnih markеra N-kadhеrina, vimеntina i Snail-a na razini gena i proteina, što objašnjava smanjеn migracijski i invazivni potеncijal HCT-116 i SW-480 stanica nakon obrade. Novina i znanstveni doprinos. Ovo istraživanje donosi nove spoznaje i pojašnjava molеkularni mеhanizam antimigracijskog/antiinvazivnog djelovanja matične mlijеči i 10H2DA na stanice raka dеbеlog crijеva. Rezultati po prvi put pokazuju da su ovi prirodni proizvodi vrijеdan izvor s antikancеrogеnim potеncijalom i da ih trеba pomnije razmotriti za buduću antitumorsku tеrapiju