Behavior, antioxidant, and metabolomics effects of Allium tuncelianum.

Allium species are consumed extensively as folkloric medicine and dietary elements, but limited studies have been conducted on them. In this study, the effects of an ethanol-water extract obtained from the underground bulb of Allium tuncelianum (Kollmann) Özhatay, B. Mathew & Siraneci (AT) on the behavioral, antioxidant, and metabolite parameters in rats were evaluated. AT was administered orally once a day at doses of 100 and 400 mg/kg to male Wistar albino rats for 10 consecutive days. The elevated plus maze, rotarod, and hotplate tests were used to examine anxiety-like behaviors, locomotor activities, and pain perception in the rats, respectively. Additionally, untargeted metabolomic analyses were performed on plasma samples and AT extracts using two orthogonal analytical platforms. The phenolic components, mainly fumaric acid, malic acid, vanillic acid, quercetin-3-arabinoside, hydrocinnamic acid, and gallocatechin, were determined in the extract. In addition, arbutin, salicylic acid, trehalose, and nicotinic acid were analyzed in the extract for the first time. The AT extract did not decrease the catalase, glutathione peroxidase, or superoxide dismutase levels; however, diazepam decreased some of those parameters significantly in the brain, liver, and kidney. Although both the AT and diazepam treatments resulted in an increase in anxiolytic-like effects compared to the control group, no significant differences were observed (p > .05). In the metabolomic analysis, significant changes were observed in the rats treated with AT and diazepam, and they caused significant changes in some metabolic pathways, including amino acid and fatty acid metabolism, compared to the control.

CB65 and novel CB65 liposomal system suppress MG63 and Saos-2 osteosarcoma cell growth in vitro.

Curable approaches for primary osteosarcoma are inadequate and urge investigation of novel therapeutic formulations. Cannabinoid ligands exert antiproliferative and apoptotic effect on osteosarcoma cells via cannabinoid 2 (CB2) or transient receptor potential vanilloid type (TRPV1) receptors. In this study, we confirmed CB2 receptor expression in MG63 and Saos-2 osteosarcoma cells by qRT-PCR and flow cytometry (FCM), then reported the reduction effect of synthetic specific CB2 receptor agonist CB65 on the proliferation of osteosarcoma cells by WST-1 (water-soluble tetrazolium-1) and RTCA (real-time impedance-based proliferation). CB65 revealed an IC50 (inhibitory concentration) for MG63 and Saos-2 cells as 1.11 × 10-11 and 4.95 × 10-11 M, respectively. The specific antiproliferative effect of CB65 on osteosarcoma cells was inhibited by CB2 antagonist AM630. CB65 induced late apoptosis of MG63 and Saos-2 cells at 24 and 48 h, respectively by FCM when applied submaximal concentration. A novel CB65 liposomal system was generated by a thin film hydration method with optimal particle size (141.7 ± 0.6 nm), polydispersity index (0.451 ± 0.026), and zeta potential (-10.9 ± 0.3 mV) values. The encapsulation efficiency (EE%) of the CB65-loaded liposomal formulation was 51.12%. The CB65 and CB65-loaded liposomal formulation releasing IC50 of CB65 reduced proliferation by RTCA and invasion by scratch assay and induced late apoptosis of MG63 and Saos-2 cells, by FCM. Our results demonstrate the CB2 receptor-mediated antiproliferative and apoptotic effect of a new liposomal CB65 delivery system on osteosarcoma cells that can be used as a targeted and intelligent tool for bone tumors to ameliorate pediatric bone cancers following in vivo validation.

GC-MS Based Metabolomics Analysis to Evaluate Short-Term Effect of Tumor Removal on Patients with Early-Stage Breast Cancer.

In this study, it was aimed to demonstrate the short-term effect of breast cancer surgery and tumor removal on the metabolomic profiles of patients with early-stage breast cancer. This cohort consisted of 18 early-stage breast carcinoma patients who had breast cancer surgery to remove tumor and surrounding tissues. The blood samples obtained preoperatively and 24 h after surgery were used in this investigation. Gas chromatography-mass spectrometry (GC-MS) based metabolomic analysis was performed to determine the metabolites. The GC-MS-based metabolomics profile enabled the identification of 162 metabolites in the plasma samples. Postoperatively, glyceric acid, phosphoric acid, O-phosphocolamine, 2-hydroxyethyliminodiacetic acid, N-acetyl-D-mannosamine, N-acetyl-5-hydroxytryptamine, methyl stearate, methyl oleate, iminodiacetic acid, glycerol 1-phosphate, ß-glycerol phosphate and aspartic acid were found to be significantly increased (P < 0.05 for all), whereas saccharic acid, leucrose, gluconic acid, citramalic acid and acetol were significantly decreased (P < 0.05 for all). Breast cancer surgery and tumor removal has an impact on the metabolomic profiles of patients with early-stage breast cancer. These findings can be used for understanding the pathogenesis of breast cancer biology and screening the success of the surgery.

Separation of the enantiomers of underivatized amino acids by using serially connected dual column high-performance liquid chromatography-tandem mass spectrometry.

In this article, a serially connected dual column liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is described for the simultaneous separation and enantioseparation of proteinogenic amino acids. For this purpose, different achiral and chiral stationary phases (CSP) and mobile phase compositions have been tested. As a result of the optimization studies, the best enatioseparation for amino acids were achieved with a combination of zwitterionic and crown ether stationary phases using a gradient of two mobile phases: A (water:TFA 99.5:0.5, % v/v) and B (acetonitrile:ethanol:TFA 85:15:0.5, % v/v/v). The developed method provided simultaneous enantioseparation of all proteinogenic amino acids under this study including isomeric and isobaric ones except for proline. The method was successfully applied to human lung adenocarcinoma cells (A549) and healthy human lung epithelial cells (BEAS-2B) cultivated with d-amino acid containing cocktails in order to evaluate d-amino acids transfer rate in normal and cancer lines. Thed/l amino acid ratios were different in cancer and normal cell lines cultivated as mentioned above for aspartic acid, cysteine, methionine, phenylalanine, and serine.

Liquid biopsy markers for early diagnosis of brain metastasis patients with breast cancer by metabolomics.

Introduction: Breast cancer is the most common cancer in women and is the second most common cause of cancer related mortality. Metabolomics, the identification of small metabolites, is a technique for determining the amount of these metabolites. Objectives: This study aimed to identify markers for the early diagnosis of brain metastasis by metabolomic methods in breast cancer patients. Methods: A total of 88 breast cancer patients with distant metastases were included in the study. The patients were divided into two groups according to their metastasis status: patients with brain metastases and distant metastases without any brain metastases. Liquid chromatography quadrupole time-of-flight mass spectrometry (LC-qTOF-MS) and gas chromatography-mass spectrometry (GC-MS) analysis methods were used for metabolomic analyses. Results: 33 of them, 88 patients had brain metastasis, and 55 patients had distant metastases without brain metastasis. A total of 72 and 35 metabolites were identified by the GC-MS and LC-qTOF-MS analysis, respectively. 47 of them were found to be significantly different in patients with brain metastasis. The pathway analysis, performed with significantly altered metabolites, showed that aminoacyl tRNA biosynthesis, valine, leucine and isoleucine biosynthesis, alanine, aspartate, and glutamate metabolism, arginine biosynthesis, glycine, serine, and threonine metabolism pathways significantly altered in patients with brain metastasis. Predictive accuracies for have identifying the brain metastasis were performed with receiver operating characteristic (ROC) analysis, and the model with fifteen metabolites has 96.9% accuracy. Conclusions: While these results should be supported by prospective studies, these data are promising for early detection of brain metastasis with markers in liquid biopsy samples.

A stability indicating RP-HPLC method for determination of the COVID-19 drug molnupiravir applied using nanoformulations in permeability studies.

Antiviral drugs have gained much more attention in recent years due to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection and many drug candidates are currently under investigation in order to end pandemic. Molnupiravir, a prodrug of the synthetic nucleoside derivative N4-hydroxycytidine, is one of the promising candidates for SARS-CoV-2 treatment. In this study, a RP-HPLC method was developed for the determination of Molnupiravir and applied for in vitro permeability studies of self-emulsifying drug delivery system (SEDDS) formulations using Caco-2 cell line. Discovery® HS C18 Column (75 ×4.6 mm, 3 µm) was used at 30 °C. Isocratic elution was performed with ACN:water (20:80 v/v) mixture. The flow rate was 0.5 mL/min and UV detection was at 240 nm. Molnupiravir eluted within 5 min. Molnupiravir was exposed to thermal, photolytic, hydrolytic, and oxidative stress conditions. Peak homogeneity data of Molnupiravir in the stressed samples peak obtained using photodiode array detector, in the stressed sample chromatograms, demonstrated the specificity of the method for their estimation in presence of degradants. The developed method was validated according to the International Council for Harmonisation (ICH) guidelines and found to be linear within the range 0.1-60.0 µg/mL. The method was simple, rapid, selective, sensitive, accurate, precise, robust and rugged. Thus, it was applied successfully for permeability quantitation of Molnupiravir in nanoformulations. The apparent permeability of Molnupiravir in SEDDS formulations, which have droplet size under 350 nm, was calculated as 3.20 ± 0.44 × 10-6 cm/s.

Metabolic Infrastructure of Pregnant Women With Trisomy 21 Fetuses; Metabolomic Analysis.

We aimed to configure impaired/altered metabolomic profiles of pregnant women carrying Down syndrome (DS) fetuses. The study involved 21 and 32 pregnant women with DS and euploid fetuses, respectively, as determined by prenatal screening and diagnosis as part of an antenatal care program. Metabolomic analyses were carried out using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-qTOF-MS) methods. A total of 95 metabolites were identified. GC-MS analysis indicated that levels of 2-hydroxybutyric acid, benzoic acid, nonanoic acid, 3-hydroxybutyric acid, and 2-ketoisocaproic acid were increased in the DS group, where beta-alanine, threonic acid, oxalic acid, alpha-tocopherol, uracil, 2-piperidone, and creatinine were decreased. However, LC-qTOF-MS analysis showed that lipid-related metabolites were decreased in women carrying DS fetuses, whereas creatine, N4-phosphoagmatine, citrate, 2,5-dioxopentanoate, 2-furoate, pyruvate, and fructose levels were increased. Pathway analysis was also performed using metabolites whose levels were significantly altered (p<0.05) between the groups, and the findings indicated that the biosynthesis pathways of aminoacyl-tRNA and "valine-leucine-isoleucine", and metabolism pathways of "glycine-serine-threonine", nitrogen, "alanine-aspartate-glutamate", propanoate, glycerophospholipid, cysteine, methionine, and phenylalanine were significantly altered. Our findings indicate a special type of metabolic status/syndrome in pregnant women with Down syndrome fetuses. It could be speculated that altered metabolic status might influence both gametogenesis and embryogenesis. Down syndrome is a complex genetic disorder that is important to detect prenatally, but may also be prevented by taking necessary precautions prior to pregnancy.

RP-HPLC method development and validation for estimation of rivaroxaban in pharmaceutical dosage forms

Rivaroxaban, an anti-clotting medication, acts at a crucial point in the blood-clotting process and stops the formation of blood clots. In this study, RP-HPLC method was developed for the determination of rivaroxaban in tablets (Xarelto® (10 mg)). Phenomenex Luna 5 µm C18 100 Å LC Column (250 x 4.6 mm) was used at 40 ºC. Isocratic elution was performed with ACN:Water (55:45 v/v) mixture. The flow rate was 1.2 mL min-1 and UV detection was at 249 nm. Internal standard (Caffeine) and rivaroxaban were eluted within 2.21 and 3.37 minutes, respectively. The developed method was validated according to the ICH guidelines and found to be linear within the range 0.005 - 40.0 µg mL-1. The method was accurate, precise, robust and rapid. Thus, it was applied successfully for the quality control assay of rivaroxaban in tablet dosage form.

Rivaroxabana, fármaco anticoagulante, atua em um ponto crucial no processo de coagulação do sangue e impede a formação de coágulos sanguíneos. Neste estudo, desenvolveu-se método de RP-HPLC para a determinação de rivaroxabana em comprimidos (Xarelto ® (10 mg)). Utilizou-se coluna LC (250 x 4,6 mm) Phenomenex Luna C18 5 mm 100 Å a 40 ºC. Realizou-se eluição isocrática com ACN: água (55:45 v/v). O fluxo foi de 1,2 mL min-1 e a detecção de UV foi a 249 nm. Padrão interno (cafeína) e rivaroxabana eluíram em 2,21 e 3,37 minutos, respectivamente. O método desenvolvido foi validado de acordo com as diretrizes do ICH e mostrou-se linear na faixa 0,005-40,0 mg mL-1. O método foi exato, preciso, robusto e rápido. Assim, foi aplicado com êxito para o ensaio de controle de qualidade da Rivaroxabana na forma de comprimidos.