Performance Liquid Chromatography-mass Spectrometry Research Articles

Screening and genetic engineering of marine-derived Aspergillus terreus for high-efficient production of lovastatin

BackgroundLovastatin has widespread applications thanks to its multiple pharmacological effects. Fermentation by filamentous fungi represents the major way of lovastatin production. However, the current lovastatin productivity by fungal fermentation is limited and needs to be improved.ResultsIn this study, the lovastatin-producing strains of Aspergillus terreus from marine environment were screened, and their lovastatin productions were further improved by genetic engineering. Five strains of A. terreus were isolated from various marine environments. Their secondary metabolites were profiled by metabolomics analysis using Ultra Performance Liquid Chromatography–Mass spectrometry (UPLC–MS) with Global Natural Products Social Molecular Networking (GNPS), revealing that the production of secondary metabolites was variable among different strains. Remarkably, the strain of A. terreus MJ106 could principally biosynthesize the target drug lovastatin, which was confirmed by High Performance Liquid Chromatography (HPLC) and gene expression analysis. By one-factor experiment, lactose was found to be the best carbon source for A. terreus MJ106 to produce lovastatin. To improve the lovastatin titer in A. terreus MJ106, genetic engineering was applied to this strain. Firstly, a series of strong promoters was identified by transcriptomic and green fluorescent protein reporter analysis. Then, three selected strong promoters were used to overexpress the transcription factor gene lovE encoding the major transactivator for lov gene cluster expression. The results revealed that compared to A. terreus MJ106, all lovE over-expression mutants exhibited significantly more production of lovastatin and higher gene expression. One of them, LovE-b19, showed the highest lovastatin productivity at a titer of 1512 mg/L, which represents the highest production level reported in A. terreus.ConclusionOur data suggested that combination of strain screen and genetic engineering represents a powerful tool for improving the productivity of fungal secondary metabolites, which could be adopted for large-scale production of lovastatin in marine-derived A. terreus.

Nutritional/Chemical Constituents and Free Radical Scavenging Potentials of the Aqueous Extract of Phoenix dactylifera Fruit

Aims: Phoenix dactylifera fruit, grown majorly in northern part of Nigeria is acclaimed to possess numerous nutritional and medicinal properties. The study was carried out to investigate the nutritional/chemical constituents and the free radical scavenging potentials of the aqueous extract of Phoenix dactylifera fruit. Methods: The aqueous extract of the fruit was subjected to standard analytical test to assay for the amino acids, phytochemicals and free radical scavenging potentials: Phytochemicals and amino acids screening was carried out via High Performance Liquid Chromatography and Gas Chromatography Mass Spectrometry analyses. The antioxidant effects of the extract on free radicals were determined on radicals 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) , 2, 2-diphenyl-1-picrylhydrazyl , Nitric oxide and Hydroxyl radical. Results: Results revealed the presence of seven secondary metabolites with a high steroid and flavonoid content and low Coumarins, Glycosides, Alkaloids, Saponin, and Terpenoid concentration. The HPLC analysis of the phytochemical showed that it contained fifteen phytocompounds, seventeen amino acids while GCMS analysis reveals eleven fatty acids. For the free radical scavenging activity, it was able to inhibit ABTS, DPPH, NO, and OH-, in a concentration dependent manner. Conclusions: The results indicate that the fruit possess good nutritional properties and abundant secondary plant metabolites constituents and this might provide evidence which corroborates with the free radical scavenging potentials of the fruit.

Decolorization and degradation of crystal violet dye by electron beam radiation: Performance, degradation pathways, and synergetic effect with peroxymonosulfate

Ionizing radiation (mainly including gamma ray and electron beam) technology provides a more efficient and ecological option for dye-containing wastewater treatment, which is supported by its successful achievements in industrial-scale applications. However, the degradation pathway of triphenylmethane dyes by radiation technology is still unclear. In this study, crystal violet (CV) was selected as representative cationic triphenylmethane dye, the decolorization and degradation performance by electron beam radiation technology was systematically evaluated. The results showed that CV can be efficiently decolorized and mineralized by radiation, and its degradation kinetics followed the first-order kinetic model. The effect of inorganic anions and chelating agents commonly existed in dye-containing wastewater on CV decolorization and total organic carbon (TOC) removal was explored. Quenching experiments, density functional theory (DFT) calculation and high performance liquid chromatography mass spectrometry (HPLC-MS) analysis were employed to reveal CV decolorization and degradation mechanism and pathway, which mainly included N-demethylation, triphenylmethane chromophore cleavage, ring-opening of aromatic products and further oxidation to carboxylic acid, and mineralization to CO2 and H2O. Additionally, electron beam radiation/PMS process was explored to decrease the absorbed dose required for decolorization and degradation, and the synergetic effect of radiation with PMS was elucidated. More importantly, the findings of this study would provide the support for treating actual dyeing wastewater by electron beam radiation technology.

Solanum nigrum Toxicity and Its Neuroprotective Effect Against Retinal Ganglion Cell Death Through Modulation of Extracellular Matrix in a Glaucoma Rat Model.

Purpose: Glaucoma is a complex degenerative optic neuropathy characterized by loss of retinal ganglion cells (RGCs) leading to irreversible vision loss and blindness. Solanum nigrum has been used for decades in traditional medicine system. However, no extensive studies were reported on its antiglaucoma properties. Therefore, this study was designed to investigate the neuroprotective effects of S. nigrum extract on RGC against glaucoma rat model. Methods: High performance liquid chromatography and liquid chromatography tandem mass spectrometry was used to analyze the phytochemical profile of aqueous extract of S. nigrum (AESN). In vitro, {3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide} (MTT) and H2DCFDA assays were used to determine cell viability and reactive oxygen species (ROS) production in Statens Seruminstitut Rabbit Cornea cells. In vivo, AESN was orally administered to carbomer-induced rats for 4 weeks. Intraocular pressure, antioxidant levels, and electrolytes were determined. Histopathological and immunohistochemical analysis was carried out to evaluate the neurodegeneration of RGC. Results: MTT assay showed AESN exhibited greater cell viability and minimal ROS production at 10 μg/mL. Slit lamp and funduscopy confirmed glaucomatous changes in carbomer-induced rats. Administration of AESN showed minimal peripheral corneal vascularization and restored histopathological alterations such as minimal loss of corneal epithelium and moderate narrowing of the iridocorneal angle. Immunohistochemistry analysis showed increased expression of positive BRN3A cells and decreased matrix metalloproteinase (MMP)-9 activation in retina and cornea, whereas western blot analysis revealed downregulation of extracellular matrix proteins (COL-1 and MMP-9) in AESN-treated rats compared with the diseased group rats. Conclusions: AESN protects RGC loss through remodeling of MMPs and, therefore, can be used for the development of novel neurotherapeutics for the treatment of glaucoma.

Insight into pyrrolizidine alkaloids degradation and the chemical structures of their degradation products using ultra high performance liquid chromatography and Q-Exactive Orbitrap mass spectrometry

Pyrrolizidine alkaloids (PAs), released into the environment by donor plants, are absorbed by crops or transported by animals, posing a global food safety concern. Photolysis is an effective way to eliminate harmful substances in the environment or food. Photolysis happens as PAs move among plants, environment and crops. In this study, we first investigated the photolysis and hydrolysis of 15 PAs and identified their degradation products via ultra-high performance liquid chromatography and Q-Exactive Orbitrap mass spectrometry. PAs were degraded under UV radiation but minimally affected by visible light from a xenon lamp, and solvent pH had little impact on their photolysis. PAs were stable in neutral and acidic solutions but degraded by 50% within 24 h in alkaline conditions. The degradation products of PAs were mainly PAs/PANOs isomers and some minor byproducts. Cytotoxicity and computational analysis revealed isomers had similar toxicity, with minor products being less toxic. This study is a precursor for revealing the potential PAs degradation dynamics in the environment and food products, providing a reference for systematic evaluations of potential health and ecological risks of their degradation products.

Аминокислотный профиль при сниженном овариальном резерве

Diminished ovarian reserve (DOR) represents a relevant issue of reproductive medicine that is often associated with infertility and reduced efficacy of IVF programs. The changes in amino acid metabolism can play a role in the DOR pathogenesis as manifestations of the folliculogenesis and oogenesis epigenetic alterations. The study was aimed to assess alterations of amino acid metabolic pathways in blood plasma and follicular fluid and estimate their clinical significance in DOR. A total of 115 infertile women aged 25–42 years were included in the study. Groups were formed based on the ovarian reserve and age. Amino acid levels in blood plasma and follicular fluid were assessed by high performance liquid chromatography–mass spectrometry (HPLC-MS); bioinformatics analysis of amino acid metabolic pathways was performed. We revealed significant changes in the phenylalanine, tyrosine and tryptophan biosynthesis (effect = 0.5; p = 0.026), alanine, aspartate and glutamate metabolism (effect = 0.114; p = 0.013), and arginine biosynthesis (effect = 0.289; p < 0.001) pathways playing a role in folliculogenesis, oogenesis, and embryogenesis. The detected differences in the amino acid levels in various body fluids made it possible to construct the logistic regression models confirming DOR with the 88% probability based on the amino acid levels in follicular fluid (sensitivity 88%, specificity 84%) and 82% probability based on plasma levels (sensitivity 65%, specificity 91%). The findings can be used for further research focused on the pathogenesis of infertility associated with DOR and for selection of the most optimal diagnostic and treatment tactics.

Liquid chromatography–mass spectrometry and xCELLigence real time cell analyzer revealed anticancer and antioxidant metabolites in Trianthema portulacastrum L. (Aizoaceae)

BackgroundThe rising incidence of cancer is a major concern globally being a leading cause of early mortality in most countries. This burden is projected to increase to 28.4 million cases worldwide by 2040 and yet, may be worsened by continuous exposure to risk factors due to a sheer shift from consumption of natural nutrients. To reverse this trend, it is imperative to source novel anticancer metabolites from plants that could minimize carcinogenic effects by suppressing the proliferation and survival of cancer cells. PurposeThis study aimed at characterizing and quantifying index anticancer and antioxidant metabolites, in the cultivars of T. portulacastrum. MethodsThe Ultra-High Performance Liquid Chromatography Mass Spectroscopy (UHP-LCMS) was used to quantify and characterize index anticancer and antioxidant metabolites in shoot, root and whole plant extracts of Trianthema portulacastrum (T01,T02,T03,T04,T05,T06). The biological activity of the tested samples was monitored on the xCELLigence Real Time Cell Analyzer to determine the cytotoxicity of the extracts as a function of cell index at a specific time post dose using the concentration range of 0.16–2.5 %. ResultsBased on the responses observed 48 h post exposure, all extracts showed high levels of cytotoxicity as determined by the xCELLigence apart from the root extracts. Rapid decreases in cell index (CI) values observed at higher extract percentages are highly indicative of cell death. These biological activities may be attributed to different anticancer metabolites such as epigallocatechin, glucaric acids, byakangelicin, xanthotoxin, apaensin, acetoxy-6-gingerol among other compounds of significant therapeutic benefits that were quantified with UHP-LCMS. ConclusionThese findings suggest that T. portulacastrum contains diverse anticancer metabolites which could be leveraged to develop plant-based anticancer drugs or novel chemicals with extended therapeutic utility.

Influence of Thermal Treatment on the Composition of Alpinia officinarum Rhizome.

Alpinia officinarum is a representative of the Zingiberaceae family, which is known for its wide use in the food and pharmaceutical industries also due to its precious pharmacological potential. The major aim of the present study was to evaluate the influence of thermal treatment on the composition of the rhizome of Alpinia officinarum and its antioxidant activity. The fresh rhizome was subjected to various thermal treatment processes-boiling, frying and microwave heating during various time intervals-and their composition and antioxidant activity were determined using chromatographic (HPLC - High Performance Liquid Chromatography and HPLC-MS - High Performance Liquid Chromatography Mass Spectrometry) and spectrophotometric (DPPH and TPC - Total Phenolic Content) methods. Pinobanksin was the main compound found in the extract of the fresh rhizome (537.79 mg/kg), followed by galangin (197.7 mg/kg) and zingerone (185.5 mg/kg). The effect of thermal treatment on the rhizome composition was varied. In general, thermal processing significantly decreased the content of active compounds in the rhizome. However, there were some exceptions-boiling for 4 min significantly increased the content of pinobanksin (1162.4 mg/kg) and galangin (280.7 mg/kg), and microwave processing for 4 min increased the content of pinocembrin (213 mg/kg). It was found that boiling and microwave treatment significantly increased the antioxidant activity of the processed rhizomes.

Direct recycle waste silicon wafer as the charge transfer bridge to assemble a Z-scheme heterojunction for enhanced photocatalytic performance

The retirement wave of photovoltaic (PV) panels is expected to result in a significant number of discarded panels in the near future. Recycling PV silicon in a rational manner will not only help reduce the environmental impact but also lead to substantial economic gains. In this study, aaste solar silicon wafers were ball-milled to obtain recycled silicon (r-Si) powder. The TiO2/r-Si carrier was prepared through a simple hydrothermal process, followed by the creation of a novel Ag3PO4/TiO2/r-Si (APO/TIO/r-Si) ternary heterojunction composite using a co-precipitation method. The photocatalytic properties of the materials doped with different concentrations of Ag3PO4 were examined using Rhodamine B (RhB) degradation rate as a performance metric. Among them, 40 %APO/TIO/r-Si heterojunction exhibits excellent photodegradation activity under visible light irradiation. The photocatalytic degradation efficiency reached 93.0 % in 30 min, with a removal rate of 82.8 % after 5 cycles. It was found that superoxide radicals and hydroxyl radicals are the main drivers of RhB photodecomposition. The decomposition pathways of RhB dye were identified using high performance liquid chromatography mass spectroscopy (HPLC-MS), revealing N-de-ethylated intermediates as the main intermediate. The photoreaction mechanism and RhB degradation pathway over APO/TIO/r-Si were elucidated, showing a direct Z-Scheme heterojunction electron transfer pathway with r-Si acting as charge transfer center. The formation of the heterojunction not only minimizes interfacial resistance effectively but also enhances disintegration and preservation of photo-carriers, leading to improved photo-redox ability and increased photoactivity. This study presents a win–win approach for recycling waste silicon wafers from retired PV panels as potential heterojunction photocatalysts for efficient photocatalytic oxidation of dye contamination.

Antioxidant, Anti-Diabetic, and Anti-Inflammation Activity of Garcinia livingstonei Aqueous Leaf Extract: A Preliminary Study.

Diabetes mellitus (DM) is the second leading cause of mortality globally. The increased concern for DM is due to the underlying complications accompanying hyperglycaemia, associated with oxidative stress and consequent inflammation. The investigation of safe and effective treatments for DM is necessary. In the present study, the cytotoxicity, phytochemical analysis, antioxidant capacity, anti-inflammatory, and antidiabetic effects in an aqueous extract of Garcinia livingstonei leaves were assessed. All tested extract concentrations showed no toxicity against C3A hepatocytes. Several phenolic compounds were identified using ultra-high performance liquid chromatography mass spectrometry (UHPLC-MS). The total polyphenol content was 100.9741 mg GAE/g, 16.7712 mg CE/g flavanols, and 2.3548 mg QE/g flavonols. The antioxidant capacity values were 253.4268 mg AAE/g, 192.232 mg TE/g, and 167.8724 mg TE/g for ferric reducing antioxidant power (FRAP), Trolox equivalent antioxidant capacity (TEAC), and 2,2-diphenyl-1-pycrylhydrazyl (DPPH), respectively. The plant extract significantly (p < 0.05) demonstrated anti-inflammatory and hypoglycaemic effects in a dose-dependent manner, with the α-glucosidase inhibition of the extract being higher (p < 0.05) than in the standard conventional drug (acarbose). The findings of this study revealed the potential of the constituents of G. livingstonei aqueous leaf extract in DM treatment. Further studies on the preparation and mechanisms of action of the plant in DM treatment are recommended.

Screening, identification, and characterization of high potential bacteria for ꞵ-cryptoxanthin production from natural sources

ꞵ-Cryptoxanthin is a carotenoid compound that presents several benefits, including provitamin A, prevention of bone loss, anti-obesity, antioxidant activities, anti-inflammatory, and anti-cancer activity. Bacteria are microorganisms of significant interest for ꞵ-cryptoxanthin production due to their short cultivation times and high yields. In this study, we aimed to screen ꞵ-cryptoxanthin producing-bacteria, which were isolated from three different sources, including surface marine water, soil, and flowers. The isolated bacterium strain from Tecoma stans (yellow elder), with a yellow pigment colony that can produce ꞵ-cryptoxanthin, zeaxanthin, and ꞵ-carotene, was identified by 16S rRNA as Pantoea anthophila FL1_IS5 strain. The carotenoids produced by this strain were identified and quantified by high performance liquid chromatography (HPLC) and high-resolution mass spectrometry (LC-HRMS/MS). Furthermore, the whole genome sequencing of P. anthophila FL1_IS5 was analyzed to confirm the carotenoids synthesis pathway and revealed the presence of the genes idi and CrtBIEXYZ. The optimized concentration of glucose and yeast extract to be added as carbon and nitrogen sources, in nutrient broth (NB) medium (10 g/L and 5 g/L, respectively), resulted in the highest cell biomass of 2.03 ± 0.08 g/L and ꞵ-cryptoxanthin production at 10.59 ± 0.14 mg/L. These results indicate high potential for ꞵ-cryptoxanthin production by the isolated bacterial strain.

Hawthorn leaf flavonoids alleviate the deterioration of atherosclerosis by inhibiting SCAP-SREBP2-LDLR pathway through sPLA2-ⅡA signaling in macrophages in mice

Ethnopharmacological relevanceHawthorn leaves are a combination of the dried leaves of the Rosaceae plants, i.e., Crataegus pinnatifida Bge. or Crataegus pinnatifida Bge. var. major N. E. Br., is primarily cultivated in East Asia, North America, and Europe. hawthorn leaf flavonoids (HLF) are the main part of extraction. The HLF have demonstrated potential in preventing hypertension, inflammation, hyperlipidemia, and atherosclerosis. However, the potential pharmacological mechanism behind its anti-atherosclerotic effect has yet to be explored. Aim of the studyThe in vivo and in vitro effects of HLF on lipid-mediated foam cell formation were investigated, with a specific focus on the levels of secreted phospholipase A2 type IIA (sPLA2-II A) in macrophage cells. Materials and methodsThe primary constituents of HLF were analyzed using ultra-high performance liquid chromatography and liquid chromatography-tandem mass spectrometry. In vivo, HLF, at concentrations of 5 mg/kg, 20 mg/kg, and 40 mg/kg, were administered to apolipoprotein E knockout mice (ApoE−/−) fed by high-fat diet (HFD) for 16 weeks. Aorta and serum samples were collected to identify lesion areas and lipids through mass spectrometry analysis to dissect the pathological process. RAW264.7 cells were incubated with oxidized low-density lipoprotein (ox-LDL) alone, or ox-LDL combined with different doses of HLF (100, 50, and 25 μg/ml), or ox-LDL plus 24-h sPLA2-IIA inhibitors, for cell biology analysis. Lipids and inflammatory cytokines were detected using biochemical analyzers and ELISA, while plaque size and collagen content of plaque were assessed by HE and the Masson staining of the aorta. The lipid deposition in macrophages was observed by Oil Red O staining. The expression of sPLA2-IIA and SCAP-SREBP2-LDLR was determined by RT-qPCR and Western blot analysis. ResultsThe chemical profile of HLF was studied using UPLC-Q-TOF-MS/MS, allowing the tentative identification of 20 compounds, comprising 1 phenolic acid, 9 flavonols and 10 flavones, including isovitexin, vitexin-4″-O-glucoside, quercetin-3-O-robibioside, rutin, vitexin-2″-O-rhamnoside, quercetin, etc. HLF decreased total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and non-high-density lipoprotein cholesterol (non-HDL-C) levels in ApoE−/− mice (P < 0.05), reduced ox-LDL uptake, inhibited level of inflammatory factors, such as IL-6, IL-8, TNF-α, and IL-1ꞵ (P < 0.001), and alleviated aortic plaques with a thicker fibrous cap. HLF effectively attenuated foam cell formation in ox-LDL-treated RAW264.7 macrophages, and reduced levels of intracellular TC, free cholesterol (FC), cholesteryl ester (CE), IL-6, TNF-α, and IL-1β (P < 0.001). In both in vivo and in vitro experiments, HLF significantly downregulated the expression of sPLA2-IIA, SCAP, SREBP2, LDLR, HMGCR, and LOX-1 (P < 0.05). Furthermore, sPLA2-IIA inhibitor effectively mitigated inflammatory release in RAW264.7 macrophages and regulated SCAP-SREBP2-LDLR signaling pathway by inhibiting sPLA2-IIA secretion (P < 0.05). ConclusionHLF exerted a protective effect against atherosclerosis through inhibiting sPLA2-IIA to diminish SCAP-SREBP2-LDLR signaling pathway, to reduce LDL uptake caused foam cell formation, and to slow down the progression of atherosclerosis in mice.

Targeted screening and quantification of flavonoids in the flavedo of navel oranges at different growth stages based on metabolomics technology

Plant-based food intake is essential for health promotion, especially in preventing chronic diseases. Flavonoids, as important bioactive substances found in citrus fruits, have a variety of health benefits, including antioxidant, anti-inflammatory, and cancer-inhibiting properties. This study used ultra-high performance liquid chromatography tandem quadrupole time of flight mass spectrometry to determine the flavonoids in the flavedo of navel oranges at different growth stages. Combined with chemometric methods to distinguish flavonoids at different growth stages, 15 differential flavonoids were deduced. In addition, the biomarker flavonoids in navel orange flavedo were quantified by high performance liquid chromatography mass spectrometry. The results showed that the contents of rutin, isosinensetin, and isorhamnetin all showed a trend of increasing and then decreasing during the growing period, while the contents of tangeretin and nobiletin would gradually decrease with the increase of fruit maturity. In addition, the analysis also revealed that the content of isorhamnetin was negatively correlated with the content of both narirutin and tangeretin, and it was hypothesized that isorhamnetin was in the same metabolic branching pathway as narirutin or tangeretin. The results of this study support the practical application of orange peel as a nutritional resource in functional foods.

Impact of lipase treatment on cuticle wax structure and anthocyanin metabolism in postharvest blueberries

AbstractIn this paper, we explored the influence of lipase on cuticle wax and anthocyanin metabolism in blueberries. The results demonstrated a progressive alteration in wax crystal structure, transitions from rod‐like to short rod‐like and flaky, and even disappearance of the cuticle wax. Gas chromatography–mass spectrometry analysis revealed a reduction in esters, aldehydes, long‐chain alkanes, and total wax content following lipase treatment of blueberry fruits. Additionally, ultra high performance liquid chromatography–mass spectrometry analysis showed significant decreases in peonidin, petunidin, cyanidin, and delphinidin, accompanied by an increase in malvidin content due to lipase treatment. Subsequent investigations uncovered that lipase treatment inhibited the activities of key anthocyanin synthetic enzymes, namely phenylalanine ammonia‐lyase (PAL), chalcone isomerase (CHI), and dihydroflavonol reductase (DFR). Moreover, lipase treatment downregulated the expression of synthesis‐related genes, including PAL, 4‐coumarate: CoA ligase, chalcone synthase (CHS), CHI, and DFR. In summary, our findings indicate that lipase disrupts the integrity of cuticle wax structure, inhibits anthocyanin synthesis, and reduces anthocyanin content. Accordingly, our studies suggest that inhibiting lipase could offer a novel approach to preserving the freshness and anthocyanin concentration of blueberries.

Effect of Mexican Propolis on Wound Healing in a Murine Model of Diabetes Mellitus.

Diabetes mellitus (DM) affects the wound healing process, resulting in impaired healing or aberrant scarring. DM increases reactive oxygen species (ROS) production, fibroblast senescence and angiogenesis abnormalities, causing exacerbated inflammation accompanied by low levels of TGF-β and an increase in Matrix metalloproteinases (MMPs). Propolis has been proposed as a healing alternative for diabetic patients because it has antimicrobial, anti-inflammatory, antioxidant and proliferative effects and important properties in the healing process. An ethanolic extract of Chihuahua propolis (ChEEP) was obtained and fractionated, and the fractions were subjected to High-Performance Liquid Chromatography with diode-array (HPLC-DAD), High-Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS) and Gas Chromatography-Mass Spectrometry (GC-MS) analyses and 46 compounds were detected. Deep wounds were made in a murine DM model induced by streptozotocin, and the speed of closure and the wound tensile strength were evaluated by the tensiometric method, which showed that ChEEP had similar activity to Recoveron, improving the speed of healing and increasing the wound tensile strength needed to open the wound again. A histological analysis of the wounds was performed using H&E staining, and when Matrix metalloproteinase 9 (MMP9) and α-actin were quantified by immunohistochemistry, ChEEP was shown to be associated with improved histological healing, as indicated by the reduced MMP9 and α-actin expression. In conclusion, topical ChEEP application enhances wound healing in diabetic mice.

MALDI-MS in first-line screening of newborns for sickle cell disease: results from a prospective study in comparison to HPLC.

Newborn screening (NBS) for sickle cell disease (SCD) requires a robust, high-throughput method to detect hemoglobin S (HbS). Screening for SCD is performed by qualitative methods, such as isoelectric focusing (IEF), and both qualitative and quantitative methods such as high performance liquid chromatography (HPLC), capillary electrophoresis (CE), and tandem mass spectrometry (MS/MS). All these methods detect HbS, as well as low-level or absent HbA, and also other variants of hemoglobin. HPLC is considered as a reference method for NBS, because of its high sensitivity and specificity in detecting HbS. NeoSickle®, a fully automated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) platform, combined with automated sample processing, a laboratory information management system and NeoSickle® software for automatic data interpretation, has increased the throughput of SCD testing. The purpose of this study was to compare the performances of NeoSickle® and HPLC. A prospective study was conducted including 9,571 samples from the NBS program to compare MALDI-MS using NeoSickle® with an HPLC method. Correlation between the two methods was studied. For the MALDI-MS method, sensitivity, specificity, NPV, and PPV were calculated. We found over 99.4 % correlation between the HPLC and MALDI-MS results. NeoSickle® showed 100 % of sensitivity and specificity in detecting SCD syndrome, leading to positive and negative predictive values of 100 %. NeoSickle® is adapted to NBS for SCD, and can be used in first-line high-throughput screening to detect HbS, and beta-thalassemia major warning. When HbS is detected, second-line use of another specific method as HPLC is necessary.

The occurrence of cytokinins and their biosynthesis pathways in epithelioma papulosum cyprini cells

Cytokinins (CKs) are small signaling molecules present in all kingdoms of life; however, their biological relevance outside of plants is poorly characterized. CK detection across kingdoms has been shown to be inconsistent and is often organism or cell line dependent. To ensure optimal detection in epithelioma papulosum cyprini (EPC), a cold-blooded vertebrate cell line, a range of biomasses were evaluated. CKs were extracted by solid-phase extraction and then detected using high-performance liquid chromatography and tandem mass spectrometry. The objectives were to establish endogenous CK profiles and characterize their biosynthesis in EPC cells. Among the 14 CKs that were scanned for, only 4 forms were detected overall within the samples analyzed, including, N6-(Δ2-isopentenyl) adenine-9-riboside (iPR), N6-(Δ2-isopentenyl) adenine-9-riboside-5′ (mono/di/tri)phosphate (iPNT), and cis-zeatin-9-riboside-5′ (mono/di/tri)phosphate ( cZNT) and 2‐methylthio‐zeatin (2MeSZ). The total CK concentrations ranged from 0.03 pmol/g to 681.025 pmol/gfwt. The results from this study provided evidence that EPC cells can produce and process CKs.

Comparative variability of nutrients, minerals, phenolics and anthocyanins with antioxidant potentials during fruit development stages in five Mulberry (Morus) cultivars

BACKGROUND: Mulberry (Morus) from the Moraceae family is distributed widely in tropical to temperate regions of the world having economic and medicinal significance with exceptional nutritional characteristics. OBJECTIVES: Assessment of variations in sugars, acidity, minerals, total phenolics, anthocyanins and antioxidant potentials of three black mulberry (Morrus serrata, M. nigra and M. macroura black) and two white mulberry cultivars (M. alba and M. macroura white) during development stages. METHODS: Total sugars and acidity were evaluated with AOAC methods and total phenolic content was estimated with folin-ciocalteau method. Antioxidant activity was evaluated with DPPH assay, minerals were quantified with atomic absorption spectroscopy and anthocyanins were assessed and identified with high performance liquid chromatography and liquid chromatography-mass spectroscopy. RESULTS: Over all, M. alba (white cultivar) displayed maximum sugar (1.35%) at fruit development stage 3 (FDS-3) following M. serrata (black cultivar) at FDS-5 (1.1%) and M. macroura white (white cultivar) at FDS-4 (0.9%). Acidity decreased in M. alba (0.8–0.09%), M. nigra (0.8–0.03%) and M. macroura white (0.6–0.04%) during development. Total phenolic content was maximum (590 GAE/100 g) in M. nigra at FDS-6 and M. serrata (450 GAE/100 g) at FDS-7. Two black cultivars (M. serrata and M. nigra) showed significant increase in total anthocyanins (14.57–100.06 and 14.13–201.87 CGE/100 g FW) from FDS-1 to FDS-7 while decrease in M. macroura black and M. macroura white (17.07–11.79 and 9.03–2.68) from FDS-1 to FDS-7. The DPPH inhibition of black mulberries was pointedly greater with significant levels of iron, copper, zinc, calcium, magnesium, sodium, phosphorus and potassium than white cultivars during maturity. HPLC and LC-MS procedures identified various anthocyanins in all cultivars including cyanidin, malvidin, pelargonidin and delphinidin. CONCLUSIONS: Mulberries with improved nutrition, acceptable range of metals, bioactive compounds and higher antioxidant potentials could be significant against various diseases like cancer, cardiovascular problems, neurodegenerative disorders, obesity, diabetes and other metabolic syndromes.

Qualitative And Quantitative Analysis of Cinnamomum Tamala Leaf Extract

Objective: The purpose of the study is to assess the properties and bioactive components of Cinnamomum tamala leaves. Methods: The leaves of Cinnamomum tamala were extracted. Different experimental methods have been used to study the phytochemicals (qualitative and quantitative). The phytochemical screening was evaluated in different extractions such as aqueous, hexane, ethanol, petroleum ether and hydroethanolic to compare the solubility of various bioactive components. Further, high performance liquid chromatography (HPLC) and gas chromatography mass spectrometry (GC-MS) were also performed to study the presence of flavonoids and secondary metabolites respectively. Results: The Qualitative analysis showed the presence of phytochemical compounds in higher concentration in hydroethanolic extract of the leaves of Cinnamomum tamala. In comparison with other extracts, hydroethanolic extract had larger yields of flavonoids (186.42 ± 13.04 mg/g QE), phenols (226.34 ± 15.84 mg/g GAE), saponins (112.10 ± 7.84 mg/g) and steroids (161.30 ± 11.29 mg/g CL). HPLC analysis revealed the presence of flavonoids in hydroethanolic extracts of the leaves of Cinnamomum tamala. GC-MS analysis proved the presence of various bioactive compounds in the hydroethanolic extract of the leaves of Cinnamomum tamala. Conclusion: The results of this study demonstrated the significance of the leaves of Cinnamomum tamala. We concluded that Cinnamomum tamala leaves have various biological activities which can treat diseases..

The identification of novel stroke-related sphingolipid biomarkers using UPLC-MS/MS

BackgroundStroke is a prominent contributor to global mortality and morbidity, thus necessitating the establishment of dependable diagnostic indicators. The objective of this study was to ascertain metabolites linked to sphingolipid metabolism and assess their viability as diagnostic markers for stroke. MethodsTwo cohorts, consisting of 56 S patients and 56 healthy volunteers, were incorporated into this investigation. Metabolite data was obtained through the utilization of Ultra Performance Liquid Chromatography and Tandem Mass Spectrometry (UPLC-MS/MS). The mass spectrometry data underwent targeted analysis and quantitative evaluation utilizing the multiple reaction monitoring mode of triple quadrupole mass spectrometry. Various data analysis techniques, including Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA), least absolute shrinkage and selection operator (LASSO) regression, Support Vector Machine (SVM), logistic regression, and Receiver Operating Characteristic (ROC) curves were employed. ResultsA comprehensive analysis detected a total of 129 metabolites related to sphingolipid metabolism, encompassing ceramides, 1-phosphoceramides, phytoceramides, glycosphingolipids, sphingomyelins, and sphingomyelins. The implementation of OPLS-DA analysis revealed significant disparities between individuals with stroke and controls, as it successfully identified 31 metabolites that exhibited significant differential expression between the two groups. Furthermore, functional enrichment analysis indicated the participation of these metabolites in diverse biological processes. Six metabolic markers, namely CerP(d18:1/20:3), CerP(d18:1/18:1), CerP(d18:1/18:0), CerP(d18:1/16:0), SM(d18:1/26:1), and Cer(d18:0/20:0), were successfully validated as potential diagnostic markers for stroke. The utilization of ROC analysis further confirmed their diagnostic potential, while a logistic regression model incorporating these markers demonstrated robust efficacy in distinguishing stroke patients from healthy controls. Conclusionthese identified metabolic markers exhibit clinical significance and hold promise as valuable tools for the diagnosis of stroke.