Chromatography-electrospray Ionization-tandem Mass Research Articles

Comparative Analysis of Amino Acid Profiles in Patients with Glioblastoma and Meningioma Using Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry (LC-ESI-MS/MS)

Brain tumors account for 1% of all cancers diagnosed de novo. Due to the specificity of the anatomical area in which they grow, they can cause significant neurological disorders and lead to poor functional status and disability. Regardless of the results of biochemical markers of intracranial neoplasms, they are currently of no diagnostic significance. The aim of the study was to use LC-ESI-MS/MS in conjunction with multivariate statistical analyses to examine changes in amino acid metabolic profiles between patients with glioblastoma, meningioma, and a group of patients treated for osteoarthritis of the spine as a control group. Comparative analysis of amino acids between patients with glioblastoma, meningioma, and the control group allowed for the identification of statistically significant differences in the amino acid profile, including both exogenous and endogenous amino acids. The amino acids that showed statistically significant differences (lysine, histidine, α-aminoadipic acid, phenylalanine) were evaluated for diagnostic usefulness based on the ROC curve. The best results were obtained for phenylalanine. Classification trees were used to build a model allowing for the correct classification of patients into the study group (patients with glioblastoma multiforme) and the control group, in which cysteine turned out to be the most important amino acid in the decision-making algorithm. Our results indicate amino acids that may prove valuable, used alone or in combination, toward improving the diagnosis of patients with glioma and meningioma. To better assess the potential utility of these markers, their performance requires further validation in a larger cohort of samples.

A liquid chromatography electrospray ionization tandem mass spectrometry method for quantification of up to eighteen cannabinoids in hemp-derived products

A LC-ESI/MS/MS method was developed for quantification of up to eighteen cannabinoids, the maximum number published so far. A thorough study of published LC-ESI/MS/MS methods using triple quadrupole mass spectrometers revealed a possible misconception that multiple reaction monitoring (MRM) was able to definitively differentiate structural isomers of cannabinoids, especially Δ8-/Δ9-tetrahydrocannabinol (THC), which explained why many of those methods were developed for a limited number of cannabinoids, as small as two, and did not include Δ8-THC. In this study, the use of a quadrupole time-of-flight (QTOF) mass spectrometer for targeted analysis indicated that MRM could not definitively distinguish structural isomers of Δ9-THC, with a possible exception of cannabicyclol (CBL) for less accurate quantification, so their baseline separation was essential for their accurate quantification. After the developed method was successfully validated according to the ISO 17025 guidelines, it was further applied for the analysis of eighteen hemp-derived products, including drinks, water-soluble oils, topical serum, body lotion, face cream, lip balm, gummies, hard candy, coffee, snacks, and pet treats. The LOQ was 0.00008% (w/w) for drinks with the analysis of 12.5 mg/mL extracts, while the LOQ was 0.008% (w/w) for other samples because 125 μg/mL extracts were analyzed due to higher content of cannabinoids in non-drink samples. For the first-time, extraction recovery and matrix effect were tracked in real-time for each sample being analyzed, obtaining 92.9–106.3% and 91.3–120.2% in triplicate measurements, respectively, by spiking abnormal cannabidiol (ACBD), a cannabinoid not naturally present in hemp, into each sample before extraction and ACBD-d3 into each sample after extraction.

Nyctanthes arbor-tristis bioactive extract ameliorates LPS-induced inflammation through the inhibition of NF-κB signalling pathway

Ethnopharmacological relevanceNyctanthes arbor-tristis L. is a mythical plant used in traditional Indian medicinal systems for the treatment of inflammation, rheumatoid arthritis, and pain-related responses. However, its bioactive compounds and underlying mechanism of action have not been fully elucidated. Aim of the studyThis investigation aimed to study the anti-inflammatory and anti-nociceptive effects of the bioactive extract of N. arbor-tristis (NATE), both in vitro and in vivo, elucidate the possible mechanism of action, and determine its chemicals. Materials and methodsWe studied the anti-inflammatory and anti-nociceptive activities of NATE on lipopolysaccharide-stimulated RAW264.7 macrophages, paw-ear edema, and acetic acid-induced pain in rats and analysed its chemical components using Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometric (LC-ESI-MS). ResultsNATE efficiently reduced the production of various inflammatory mediators and factors, such as free radicals, lipid peroxidation, nitrous oxide (NO), reactive oxygen species (ROS), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-alpha (TNFα), interleukin-6 (IL-6), interleukin-1beta (IL-1β), and IL-10, as well as their corresponding mRNA expression in LPS-induced RAW264.7 cells (p < 0.001). Furthermore, NATE inhibited the activation of a key signaling pathway, nuclear factor-kappa B (NF-kB), as it caused a decrease in the degradation of inhibitor of kB alpha (IkBa). Administration of NATE significantly inhibited carrageenan-induced paw edema (p < 0.001), TPA-induced ear edema, and the production of inflammatory factors (p < 0.01). NATE revealed anti-nociceptive impacts in acetic acid-induced writhing and tail immersion experiments (p < 0.001) as well as no toxicity signs. A total of six compounds, namely iridoid glycoside (6,7-di-O-benzonylnyctanthoside), arborsides A, arborsides C, betulinic acid, kaempferol 3-O-glucoside, and kaempferol 3-O-rutinoside, were characterized through the examination of their mass spectra in correlation with those documented in a database of mass spectra. ConclusionsThe present study furnishes scientific corroboration of the inhibitory potency of N. arbor-tristis as a promising herbal treatment for inflammation and pain responses without toxicity, offering a scientific basis for future drug development strategies aimed at ameliorating inflammatory diseases.

Untargeted polysulfide omics analysis of alternations in polysulfide production during the germination of broccoli sprouts

Higher consumption of broccoli (Brassica oleracea var. italica) is associated with a reduced risk of cardiometabolic diseases, neurological disorders, diabetes, and cancer. Broccoli is rich in various phytochemicals, including glucosinolates, and isothiocyanates. Moreover, it has recently reported the endogenous production of polysulfides, such as cysteine hydropersulfide (CysS2H) and glutathione hydropersulfide (GS2H), in mammals including humans, and that these bioactive substances function as potent antioxidants and important regulators of redox signaling in vivo. However, few studies have focused on the endogenous polysulfide content of broccoli and the impact of germination on the polysulfide content and composition in broccoli. In this study, we investigated the alternations in polysulfide biosynthesis in broccoli during germination by performing untargeted polysulfide omics analysis and quantitative targeted polysulfide metabolomics through liquid chromatography–electrospray ionization–tandem mass spectrometry. We also performed 2,2-diphenyl-1-picrylhydrazyl radical–scavenging assay to determine the antioxidant properties of the polysulfides. The results revealed that the total polysulfide content of broccoli sprouts significantly increased during germination and growth; CysS2H and cysteine hydrotrisulfide were the predominant organic polysulfide metabolites. Furthermore, we determined that novel sulforaphane (SFN) derivatives conjugated with CysS2H and GS2H were endogenously produced in the broccoli sprouts, and the novel SFN conjugated with CysS2H exhibited a greater radical scavenging capacity than SFN and cysteine. These results suggest that the abundance of polysulfides in broccoli sprouts contribute to their health-promoting properties. Our findings have important biological implications for the development of novel pharmacological targets for the health-promoting effects of broccoli sprouts in humans.

Lycium RIN negatively modulate the biosynthesis of kukoamine A in hairy roots through decreasing thermospermine synthase expression

Root bark (Lycii cortex) of Lycium contains high contents of characteristic bioactive compounds, including kukoamine A (KuA) and kukoamine B (KuB). RIPENING INHIBITOR (RIN) is well known as a master regulator of Solanaceaous fruit ripening. However, the role of RIN in the biosynthetic pathway of KuA in Lycium remains unclear. In this study, integrated transcriptomic, metabolomic analyses and hairy root system are used to characterize the role of RIN in KuA biosynthesis in Lycium. The ultra performance liquid chromatography electrospray ionization tandem mass spectrometry analysis revealed that KuA was significantly induced in LrRIN1 RNAi lines and not detected in overexpression lines. A total of 20,913 differentially expressed genes (DEGs) and 60 differentially accumulated metabolites (DAMs) were detected in LrRIN1 transgenic hairy roots, which were used for weighted gene co-expression network analysis. Our result reveals a high association between KuA and structural genes in the phenolamide pathway, which shows a negative correlation with LrRIN1. In addition, overexpression of the polyamine pathway gene thermospermine synthase LcTSPMS, a potential target gene of Lycium RIN, increased the contents of both KuA and KuB in L. chinense hairy root, indicating that TSPMS is responsible for KuA biosynthesis and is also the common upstream biosynthetic gene for both KuA and KuB. Our results lay a solid foundation for uncovering the biosynthetic pathway of KuA, which will facilitate the molecular breeding and genetic improvement of Lycium species.

Catalytic behavior of nitrous acid for acetaminophen transformation during the freezing process

This study demonstrates the transformation of acetaminophen by reactive nitrous acid in a frozen solution and its abnormal stoichiometry. The chemical reaction between acetaminophen and nitrous acid (AAP/NO2− system) was negligible in the aqueous solution; however, the reaction rapidly progressed if the solution started to freeze. The ultrahigh performance liquid chromatography–electrospray ionization tandem mass spectrometry measurements showed that polymerized acetaminophen and nitrated acetaminophen were formed in the proceeding reaction. Electron paramagnetic resonance spectroscopy measurements showed that nitrous acid oxidized acetaminophen via a one-electron transfer reaction producing acetaminophen-derived radical species, which is the cause of acetaminophen polymerization. We demonstrated that a relatively smaller dose of nitrite than acetaminophen caused significant acetaminophen degradation in the frozen AAP/NO2− system and revealed that the dissolved oxygen content notably affected acetaminophen degradation. We showed that the reaction occurs in a natural Arctic lake matrix (nitrite and acetaminophen spiked). Considering that the freezing phenomenon is common in the natural environment, our research provides a possible scenario for the freezing chemistry of nitrite and pharmaceuticals in environmental chemistry.

Natural Occurrence of Alternaria Toxins in Citrus-Based Products Collected from China in 2021.

A total of 181 citrus-based products, including dried fruits, canned fruits, and fruit juices, collected from China and from abroad in 2021 were analyzed for the four Alternaria toxins (ALTs): alternariol (AOH), alternariol monomethyl ether (AME), tentoxin (TEN), and tenuazonic acid (TeA) via ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS). Although the concentrations of the four ALTs varied by product and geographically, TeA was the predominant toxin followed by AOH, AME, and TEN. Products made in China showed higher levels of ALTs than those made abroad. Maximum levels of TeA, AOH, and AME in analyzed domestic samples were 4.9-fold, 1.3-fold, and 1.2-fold, respectively, higher than those in imported products. Furthermore, 83.4% (151/181) of the analyzed citrus-based products were contaminated with at least two or more ALTs. There were significant positive correlations between AOH and AME, AME and TeA, and TeA and TEN in all analyzed samples. More importantly, the solid and the condensed liquid products had higher concentrations of ALTs than the semi-solid product samples, as well as tangerines, pummelos, and grapefruits compared to the other kinds of citrus-based products. In conclusion, co-contamination with ALTs in commercially available Chinese citrus-based products was universal. Extensive and systematic surveillance of ALTs in citrus-based products, both domestic and imported, is required to obtain more scientific data for the determination of the maximum allowable concentrations of ALTs in China.

Proteomic Analysis in Valvular Cardiomyopathy: Aortic Regurgitation vs. Aortic Stenosis.

Left ventricular (LV) reverse remodeling after aortic valve (AV) surgery is less predictable in chronic aortic regurgitation (AR) than in aortic stenosis (AS). We aimed to disclose specific LV myocardial protein signatures possibly contributing to differential disease progression. Global protein profiling of LV myocardial samples excised from the subaortic interventricular septum in patients with isolated AR or AS undergoing AV surgery was performed using liquid chromatography-electrospray ionization-tandem mass spectrometry. Based on label-free quantitation protein intensities, a logistic regression model was calculated and adjusted for age, sex and protein concentration. Web-based functional enrichment analyses of phenotype-associated proteins were performed utilizing g:Profiler and STRING. Data are available via ProteomeXchange with identifier PXD039662. Lysates from 38 patients, including 25 AR and 13 AS samples, were analyzed. AR patients presented with significantly larger LV diameters and volumes (end-diastolic diameter: 61 (12) vs. 48 (13) mm, p < 0.001; end-diastolic volume: 180.0 (74.6) vs. 92.3 (78.4), p = 0.001). A total of 171 proteins were associated with patient phenotype: 117 were positively associated with AR and the enrichment of intracellular compartment proteins (i.e., assigned to carbohydrate and nucleotide metabolism, protein biosynthesis and the proteasome) was detected. Additionally, 54 were positively associated with AS and the enrichment of extracellular compartment proteins (i.e., assigned to the immune and hematopoietic system) was observed. In summary, functional enrichment analysis revealed specific AR- and AS-associated signatures of LV myocardial proteins.

Heroin-Related Fatalities in Jeddah, Saudi Arabia, between 2008 and 2018

To date, epidemiological studies have not evaluated heroin-related deaths in the Middle East and North African regions, especially Saudi Arabia. All heroin-related postmortem cases reported at the Jeddah Poison Control Center (JPCC) over a 10-year period (21 January 2008 to 31 July 2018) were reviewed. In addition, liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) was utilized to determine the 6-monoacetylmorphine (6-MAM), 6-acetylcodeine (6-AC), morphine (MOR), and codeine contents in unhydrolyzed postmortem specimens. Ninety-seven heroin-related deaths were assessed in this study, and they represented 2% of the total postmortem cases at the JPCC (median age, 38; 98% male). In the blood, urine, vitreous humor, and bile samples, the median morphine concentrations were 280 ng/mL, 1400 ng/mL, 90 ng/mL, and 2200 ng/mL, respectively; 6-MAM was detected in 60%, 100%, 99%, and 59% of the samples, respectively; and 6-AC was detected in 24%, 68%, 50%, and 30% of the samples, respectively. The highest number of deaths (33% of total cases) was observed in the 21-30 age group. In addition, 61% of cases were classified as "rapid deaths," while 24% were classified as "delayed deaths." The majority (76%) of deaths were accidental; 7% were from suicide; 5% were from homicide; and 11% were undetermined. This is the first epidemiological study to investigate heroin-related fatalities in Saudi Arabia and the Middle East and North African region. The rate of heroin-related deaths in Jeddah remained stable but increased slightly at the end of the study period. Most patients were heroin-dependent abusers and from the middle-aged group. The availability of urine, vitreous humor, and bile specimens provided valuable information regarding the opioids that were administered and the survival time following heroin injection.

Antifungal activity and application of Bacillus tequilensis A13 in biocontrol of Rehmannia glutinosa root-rot disease

BackgroundThe bacterial genus Bacillus, an important group of bacteria which can suppress phytopathogens, has been widely used in agriculture. However, different species of Bacillus often displayed significant differences in probiotic efficiency and mechanism, suggesting that it is very necessary to investigate the biocontrol potential of new Bacillus isolates, especially from under-evaluated Bacillus spp. Rehmannia glutinosa (R. glutinosa), an important traditional Chinese medicinal herb, is affected by a serious root-rot disease caused by the fungus Fusarium solani (F. solani). Biocontrol agents against this root-rot disease in R. glutinosa have yet to be developed. This study explored, for the first time, the activity and biocontrol mechanism of a new Bacillus isolate, Bacillus tequilensis A13, in antagonizing F. solani.ResultsB. tequilensis A13 displayed a strong inhibitory activity (73.49% ± 1.33%) against F. solani growth in vitro and was able to survive and multiply in the sterilized soil. The results from liquid chromatography electrospray ionization tandem mass spectrometry (LC–MS/MS) and Gene Ontology (GO) analyses indicated that B. tequilensis A13 cell-free supernatant contained six antifungal compounds, eight antifungal compound synthases, and several functional proteins involved in the processes of plant stress resistance, etc. Finally, the joint application of complex fertilizer together with B. tequilensis A13 significantly reduced the incidence of R. glutinosa root rot in the field.ConclusionB. tequilensis A13 strongly inhibited F. solani growth by producing antifungal compounds and proteins associated with plant stress resistance/tolerance, and proved to be a promising candidate biocontrol agent against R. glutinosa root-rot disease.Graphical

In-Depth Analysis of the N-Glycome of Colorectal Cancer Cell Lines.

Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the second leading cause of cancer deaths worldwide. A well-known hallmark of cancer is altered glycosylation. Analyzing the N-glycosylation of CRC cell lines may provide potential therapeutic or diagnostic targets. In this study, an in-depth N-glycomic analysis of 25 CRC cell lines was conducted using porous graphitized carbon nano-liquid chromatography coupled to electrospray ionization mass spectrometry. This method allows for the separation of isomers and performs structural characterization, revealing profound N-glycomic diversity among the studied CRC cell lines with the elucidation of a number of 139 N-glycans. A high degree of similarity between the two N-glycan datasets measured on the two different platforms (porous graphitized carbon nano-liquid chromatography electrospray ionization tandem mass spectrometry (PGC-nano-LC-ESI-MS) and matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS)) was discovered. Furthermore, we studied the associations between glycosylation features, glycosyltransferases (GTs), and transcription factors (TFs). While no significant correlations between the glycosylation features and GTs were found, the association between TF CDX1 and (s)Le antigen expression and relevant GTs FUT3/6 suggests that CDX1 contributes to the expression of the (s)Le antigen through the regulation of FUT3/6. Our study provides a comprehensive characterization of the N-glycome of CRC cell lines, which may contribute to the future discovery of novel glyco-biomarkers of CRC.

Assessing the Effectiveness of Chemical Marker Extraction from Amazonian Plant Cupuassu (Theobroma grandiflorum) by PSI-HRMS/MS and LC-HRMS/MS.

Employing a combination of liquid chromatography electrospray ionization and paper spray ionization high-resolution tandem mass spectrometry, extracts from cupuassu (Theobroma grandiflorum) pulp prepared with either water, methanol, acetonitrile or combinations thereof were subjected to metabolite fingerprinting. Among the tested extractors, 100% methanol extracted preferentially phenols and cinnamic acids derivatives, whereas acetonitrile and acetonitrile/methanol were more effective in extracting terpenoids and flavonoids, respectively. And while liquid chromatography- mass spectrometry detected twice as many metabolites as paper spray ionization tandem mass spectrometry, the latter proved its potential as a screening technique. Comprehensive structural annotation showed a high production of terpenes, mainly oleanane triterpene derivatives. of the mass spectra Further, five major metabolites with known antioxidant activity, namely catechin, citric acid, epigallocatechin-3'-glucuronide, 5,7,8-trihydroxyflavanone, and asiatic acid, were subjected to molecular docking analysis using the antioxidative enzyme peroxiredoxin 5 (PRDX5) as a model receptor. Based on its excellent docking score, a pharmacophore model of 5,7,8-trihydroxyflavanone was generated, which may help the design of new antioxidants.

Stratum corneum lipid and cytokine biomarkers at age 2 months predict the future onset of atopic dermatitis

Atopic dermatitis (AD) commonly occurs in children and can progress into severe phenotypes or atopic march, causing significant impairment in quality of life. It is important to find early biomarkers of future onset of AD before any clinical manifestations. We sought to find early predictors of future onset of AD in skin stratum corneum (SC). Skin tape strips were collected from the forearm of newborns (n= 111) with and without family history of atopic diseases at the age of 2 months before any signs of clinical AD. Children were clinically monitored until they reached age 2 years to ensure the presence or absence of AD. Skin tape strips were subjected to lipidomic analyses by the liquid chromatography electrospray ionization tandem mass spectrometry and cytokine determination by Meso Scale Discovery U-Plex assay. Overall, 22 of 74 (29.7%) and 5 of 37 (13.5%) infants developed AD in the risk group and the control group, respectively. In the SC of future AD children, protein-bound ceramides were decreased (P< .001), whereas unsaturated sphingomyelin species (P< .0001) and "short-chain" nonhydroxy fatty acid sphingosine and alpha-hydroxy fatty acid sphingosine ceramides were elevated (P< .01 and .05, respectively) as compared with healthy children. Thymic stromal lymphopoietin and IL-13 levels were increased in the SC of future AD subjects (by 74.5% and 78.3%, P= .0022 and P< .0001, respectively). Multivariable logistic regression analysis revealed strong AD predicting power of the combination of family history, type 2 cytokines, and dysregulated lipids, with an odds ratio reaching 54.0 (95% CI, 9.2-317.5). Noninvasive skin tape strip analysis at age 2 months can identify asymptomatic children at risk of future AD development with a high probability.

Comparative analysis of the metabolites in Pinellia ternata from two producing regions using ultra-high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry

Abstract The rhizomes of Pinellia ternata have a long history of being used as a traditional Chinese herb. To determine the chemical constituents of Pinelliae rhizome, Xi banxia and Jing banxia were collected from two different producing regions and subjected to analysis using ultra-high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry. A total of 573 metabolites were identified in 12 different categories. Next, cluster analysis and principal component analysis were performed, which revealed a distinct separation between the two species. The analysis of the differential metabolites revealed that among the 155 metabolites in Xi banxia, 68 metabolites were upregulated, and 87 metabolites were downregulated. The relative concentration of flavonoids in Xi banxia was higher than that in Jing banxia, while the relative concentrations of alkaloids, phenolic acids, and terpenoids were higher in Jing banxia. These results would provide a theoretical basis for the pharmacological activity analysis and functional study of P. ternata.

Aqueous Extracts of Four Medicinal Plants and Their Allelopathic Effects on Germination and Seedlings: Their Morphometric Characteristics of Three Horticultural Plant Species

Allelopathy is a biological phenomenon of synthesizing and excreting compounds that can affect the growth of various organisms, i.e., plant species. The aim of this work was to investigate the possible allelopathic influence of aqueous extracts, obtained from plant species chia (Salvia hispanica L.), black cumin (Nigella sativa L.), wormwood (Artemisia absinthium L.), and nettle (Urtica dioica L.), on the seed germination growth and morphometric characteristics of pepper (Capsicum annuum L.), spinach (Spinacia oleracea L.) and lettuce (Lactuca sativa L.) seedlings in laboratory conditions. Pepper, spinach, and lettuce seedlings were treated with aqueous extracts of chia, nettle, wormwood, and black cumin of different concentrations (2.5, 5 and 10%, respectively). The aqueous extracts were prepared according to the method developed by Norsworthy and the qualitative and quantitative analysis of phenolic compounds in aqueous extracts were performed using ultra-performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS). Phytochemical composition of chia aqueous extracts showed the highest content (above the 1 mg L−1) of epicatechin, quinic acid, caffeic acid, esculetin and cinnamic acid in the comparison with others from the total of 19 detected chemical compounds. In aqueous extracts of black cumin, there were six compounds detected in content above 1 mg L−1, i.e., epicatechin, quinic acid, caffeic acid, esculetin, cinnamic acid, and kaempferol. The same compounds were the most abundant in wormwood extracts, along with gallic acid. Epicatechin and esculetin were only two compounds detected in aqueous extract of nettle in concentration higher than 1 mg L−1. According to the results of this study, only the treatment of spinach seeds with chia aqueous extract in concentration of 2.5% before germination stimulates the germination of spinach seeds, and wormwood herbs and chia extracts in concentrations of 2.5 and 5% stimulate the germination of lettuce seeds and the growth of hypocotyl and radicle length of developed seedlings. The treatment of pepper and lettuce seeds with aqueous extract of nettle in concentration of 10% completely inhibited seed germination.

Metabolites Profiling and In Vitro Biological Characterization of Different Fractions of Cliona sp. Marine Sponge from the Red Sea Egypt.

Red Sea marine sponges are an important source of biologically active natural products. Therefore, the present study aimed to investigate, for the first time, the components of n-hexane, dichloromethane, and ethyl acetate fractions of Cliona sp. marine sponge collected from the Red Sea, Egypt using UPLC-ESI-MS/MS (Ultra-performance liquid chromatography electrospray ionization tandem mass spectrometry) analysis. The analysis revealed the tentative identification of 23, 16, and 24 compounds from the n-hexane, dichloromethane, and ethyl acetate fractions of Cliona sp., respectively. In addition, the examination of these fractions resulted in the isolation and identification of three sterols and one amino acid. The identification of the isolated compounds was confirmed by 1D and 2D NMR (Nuclear Magnetic Resonance), and MS (Mass spectrometry), and IR (Infrared) spectroscopy. The in vitro cytotoxic, antioxidant, and antimicrobial activities of the total ethanolic extract and its sub-fractions were also evaluated. Interestingly, the ethyl acetate fraction showed potent cytotoxic activity against colon (HCT-116) and human larynx carcinoma (HEP-2) cell lines with IC50 (Half-maximal Inhibitory Concentration) 6.11 ± 0.2 and 12.6 ± 0.9 µg/mL, respectively. However, the dichloromethane fraction showed strong antioxidant activity, with IC50 75.53 ± 3.41 µg/mL. Notably, the total ethanolic extract showed the strongest antibacterial activity against Staphylococcus aureus and Escherichia coli, with MIC (Minimum Inhibitory Concentration) 62.5 ± 0.82 and 125 ± 0.62 µg/mL, respectively, compared to other fractions. In conclusion, this is the first report on the secondary metabolites content and biological activities of Cliona sp. from the Red Sea, Egypt. It also highlights the need for further research on the most active fractions against various cancer cell lines and resistant bacterial and fungal strains. Cliona sp. extract and its fractions could be a potential source of novel and safe natural drugs with a wide range of medicinal and pharmaceutical applications.

Metabolomics analysis reveals the accumulation patterns of flavonoids and phenolic acids in quinoa (Chenopodium quinoa Willd.) grains of different colors

Quinoa grains are gaining increasing popularity owing to their high nutritional merits. However, only limited information is available on the metabolic profiles of quinoa grains. In this study, we determined the metabolic profiles of black, red, and white quinoa grains via an ultraperformance liquid chromatography–electrospray ionization–tandem mass spectrometry (UPLC–ESI–MS/MS)-based metabolomics. A total of 689 metabolites were identified, among which 251, 182, and 317 metabolites displayed different accumulation patterns in the three comparison groups (Black vs Red, Black vs White, and Red vs White), respectively. In particular, flavonoid and phenolic acid contents displayed considerable differences, with 22 flavonoids, 5 phenolic acids, and 1 betacyanin being differentially accumulated among the three quinoa cultivars. Additionally, correlation analysis showed that flavonoids and phenolic acids could act as betanin co-pigments in quinoa grains. In conclusion, this study provides comprehensive insights into the adequate utilization and development of novel quinoa-based functional foods.

Development of methods for quantitative determination of the total and reactive polysulfides: Reactive polysulfide profiling in vegetables

Alliaceous and cruciferous vegetables are rich in bioactive organosulfur compounds, including polysulfides, which exhibit a broad spectrum of potential health benefits. Here, we developed novel, accurate, and reproducible methods to quantify the total polysulfide content (TPsC) and the reactive polysulfide content (RPsC) using liquid chromatography–electrospray ionization–tandem mass spectrometry, and analyzed the reactive polysulfide profiles of 22 types of fresh vegetables, including onions, garlic, and broccoli. Quantitative analyses revealed that onions contained the largest amounts of polysulfides, followed by broccoli, Chinese chive, and garlic. A strong positive correlation was observed between the TPsC and RPsC, whereas only a moderate positive correlation was found between the total sulfur content and TPsC. These results suggest that reactive polysulfide profiling can be a novel criterion for evaluating the beneficial functions of vegetables and their derivatives, which may lead to an understanding of the detailed mechanisms underlying their bioactivities.

LC–MS/MS Coupled with Chemometric Analysis as an Approach for the Differentiation of Fritillariae cirrhosae Bulbus and Fritillariae pallidiflorae Bulbus

Fritillariae cirrhosae bulbus (FCB) is one of the most important traditional Chinese medicines (TCM) for the treatment of cough and phlegm. Due to increasing demand and the complexity of FCB’s botanical origin, various substitutes have appeared in the market, resulting in a major challenge to distinguish FCB and its substitutes (F. pallidiflorae bulbus, FPB). Therefore, discriminating FCB from FPB has becoming an urgent necessity. In this study, an ultra-high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (UPLC–ESI–MS/MS) method was developed for the simultaneous quantification of nine steroidal alkaloids (imperialine-3-β-D-glucoside, imperialine, verticine, verticinone, peimisine, yibeinoside A, delavine, delavinone, ebeidinone) within 8 min. According to the composition and content of the above nine compounds, multivariate chemometric analyses were applied for the classification of FCB and FPB. The quantitative results showed that there were both similarities and differences in the content of nine steroidal alkaloids between FCB and FPB, and it was difficult to directly distinguish these two species. Fortunately, with the aid of chemometric analyses, FCB and FPB were successfully differentiated by partial least squares discrimination analysis (PLS-DA) and orthogonal partial least squares discrimination analysis (OPLS-DA) models based on the nine alkaloids’ content. Moreover, four compounds (yibeinoside A, ebeiedinone, delavinone and imperialine) were discovered as potential markers for the identification and differentiation of FCB and FPB. Additionally, compared to other studies, this work collected a large number of samples (49 batches of FCB and 17 batches of FPB) to ensure the reliability of the results. In conclusion, this work established a new approach for the authentication of FCB based on its active components, which provides a good reference for the quality control of FCB and will help us to understand the chemical composition differences between FCB and its adulterants further.

Combined widely targeted metabolomics and transcriptomics analysis reveals differentially accumulated metabolites and the underlying molecular bases in fleshy taproots of distinct radish genotypes

Radish is an important taproot crop with medicinal and edible uses that is cultivated worldwide. However, the differences in metabolites and the underlying molecular bases among different radish types remain largely unknown. In the present study, a combined analysis of liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS) and RNA-Seq data was conducted to uncover important differentially accumulated metabolites (DAMs) among radish accessions with green, white and red taproot flesh colours. A total of 657 metabolites were identified and 138 DAMs were commonly present in red vs. green and red vs. white accessions. Red accessions were rich in phenolic compounds, while green and white accessions had more amino acids. Additionally, 41 metabolites and 98 genes encoding 37 enzymes were enriched in the shikimate and phenolic biosynthesis pathways. CHS is the rate-limiting enzyme determining flavonoid differences among accessions. A total of 119 candidate genes might contribute to red accession-specific accumulated metabolites. Specifically, one gene cluster consisting of 16 genes, including one RsMYB1, RsMYBL2, RsTT8, RsDFR, RsANS, Rs4CL3, RsSCPL10, Rs3AT1 and RsSAP2 gene, two RsTT19 and RsWRKY44 genes and three RsUGT genes, might be involved in anthocyanin accumulation in red radish fleshy taproots. More importantly, an InDel marker was developed based on an RsMYB1 promoter mutation, and the accuracy reached 95.9% when it was used to select red-fleshed radishes. This study provides comprehensive insights into the metabolite differences and underlying molecular mechanisms in fleshy taproots among different radish genotypes and will be beneficial for the genetic improvement of radish nutritional quality.