Comparative Analysis of Volatile Compounds from Four Radish Microgreen Cultivars Based on Ultrasonic Cell Disruption and HS-SPME/GC-MS.

The ultrasonic cell disruption method was used to efficiently extract isothiocyanates and other volatile compounds from radish microgreens. A total of 51 volatiles were identified and quantified by headspace solid-phase micro-extraction and gas chromatography-mass spectrometry (HS-SPME/GC-MS) in four radish microgreen cultivars, mainly including alcohols, aldehydes, isothiocyanates, sulfides, ketones, esters, terpenes, and hydrocarbons. The correlation between cultivars and volatile compounds was determined by chemometrics analysis, including principal component analysis (PCA) and hierarchical clustering heat maps. The aroma profiles were distinguished based on the odor activity value (OAV), odor contribution rate (OCR), and radar fingerprint chart (RFC) of volatile compounds. This study not only revealed the different flavor characteristics in four cultivars but also established a theoretical basis for the genetic improvement of radish microgreen flavors.

Antifungal mechanism of isothiocyanates against Cochliobolus heterostrophus.

BACKGROUND: Isothiocyanates (ITCs) generated from the 'glucosinolates-myrosinase' defense system in the Brassicaceae exhibit broad antagonistic activity to various fungal pathogens. Nevertheless, the antifungal activity of ITCs to non-adapted fungi of Brassicaceae plants were seldom determined. The inhibitory effects of ITCs on Cochliobolus heterostrophus were evaluated and the antagonistic mechanism was explored. RESULTS: The mycelium growth of C. heterostrophus was hindered significantly by allyl, 4-(methylthio)-butyl, and phenyethyl ITCs, 4MTB-ITC exhibited the highest inhibitory effect on mycelium growth with an IC50 value of 53.4 µmol L-1 . In addition, ITCs exhibited obvious inhibitory effect on conidia germination and pathogenicity of C. heterostrophus. Proteomic analysis indicated that the inhibition of C. heterostrophus by A-ITC downregulated the expression of genes related to energy metabolism, oxidoreductase activity, melanin biosynthesis, and cell wall-degrading enzymes. Furthermore, mutants ΔChtrx2 and ΔChnox1 showed increased sensitivity to ITCs, and melanin biosynthesis was inhibited significantly in C. heterostrophus in response to A-ITC. Interestingly, unlike other pathogens that infected Brassicaceae plants, the SaxA in C. heterostrophus displayed no function in ITC degradation. In addition, the ITCs also exhibited obvious inhibitory effect on mycelium growth of Setosphaeria turcica, Fusarium graminearum, and Magnaporthe oryzae. CONCLUSION: This study indicated that non-Brassicaceae-adapted pathogens are more sensitive to ITCs, and ITCs could have applications in protecting non-Brassicaceae crops in future. In addition, loss of ChNOX1 and ChTRX2 increased the sensitivity of C. heterostrophus to ITCs. Our results provided potential utilization of ITCs to control diseases caused by non-Brassicaceae pathogenic fungi. © 2022 Society of Chemical Industry.

Role of Sulfur Compounds in Vegetable and Mushroom Aroma.

At the base of the food pyramid is vegetables, which should be consumed most often of all food products, especially in raw and unprocessed form. Vegetables and mushrooms are rich sources of bioactive compounds that can fulfill various functions in plants, starting from protection against herbivores and being natural insecticides to pro-health functions in human nutrition. Many of these compounds contain sulfur in their structure. From the point of view of food producers, it is extremely important to know that some of them have flavor properties. Volatile sulfur compounds are often potent odorants, and in many vegetables, belonging mainly to Brassicaeae and Allium (Amaryllidaceae), sulfur compounds determine their specific flavor. Interestingly, some of the pathways that form volatile sulfur compounds in vegetables are also found in selected edible mushrooms. The most important odor-active organosulfur compounds can be divided into isothiocyanates, nitriles, epithionitriles, thiols, sulfides, and polysulfides, as well as others, such as sulfur containing carbonyl compounds and esters, R-L-cysteine sulfoxides, and finally heterocyclic sulfur compounds found in shiitake mushrooms or truffles. This review paper summarizes their precursors and biosynthesis, as well as their sensory properties and changes in selected technological processes.

Consequences of zinc deficiency on zinc localization, taurine transport, and zinc transporters in rat retina.

The colocalization of taurine and zinc transporters (TAUT, ZnTs) has not been explored in retina. Our objective is to evaluate the effect of the intracellular zinc chelator N,N,N,N-tetrakis-(2-pyridylmethyl) ethylenediamine (TPEN) on zinc localization and colocalization TAUT and ZnT-1 (of plasma membrane), 3 (vesicular), and 7 (vesicular and golgi apparatus) in layers of retina by immunohistochemistry. To mark zinc, it was used cell-permeable fluorescent Zinquin ethyl ester. Specific first and secondary antibodies, conjugated with rhodamine or fluorescein-isothiocyanate were used to mark TAUT and ZnTs. The fluorescence results were reported as integrated optical density (IOD). Zinc was detected in all layers of the retina. The treatment with TPEN produced changes in the distribution of zinc in layers of retina less in the outer nuclear layer compared with the control. TAUT was detected in all layers of retina and TPEN chelator produced decrease of IOD in all layers of retina except in the photoreceptor compared with the control. ZnT 1, 3, and 7 were distributed in all retina layers, with more intensity in ganglion cell layer (GCL) and in the layers where there is synaptic connection. For all transporters, the treatment with TPEN produced significant decrease of IOD in layers of retina least in the inner nuclear layer for ZnT1, in the photoreceptor for ZnT3 and in the GCL and outer plexiform layer for ZnT7. The distribution of zinc, TAUT, and ZnTs in the layers of retina is indicative of the interaction of taurine and zinc for the function of the retina and normal operation of said layers. HIGHLIGHTS: Taurine and zinc are two molecules highly concentrated in the retina and with relevant functions in this structure. Maintaining zinc homeostasis in this tissue is necessary for the normal function of the taurine system in the retina. The study of the taurine transporter and the different zinc transporters in the retina (responsible for maintaining adequate levels of taurine and zinc) is relevant and novel, since it is indicative of the interactions between both molecules in this structure.

Evidence on the Bioaccessibility of Glucosinolates and Breakdown Products of Cruciferous Sprouts by Simulated In Vitro Gastrointestinal Digestion.

Cruciferous vegetables are gaining importance as nutritious and sustainable foods, rich in phytochemical compounds such as glucosinolates (GSLs). However, the breakdown products of these sulfur-based compounds, mainly represented by isothiocyanates (ITC) and indoles, can contribute to human health. In the human digestive system, the formation of these compounds continues to varying extents in the different stages of digestion, due to the contact of GSLs with different gastric fluids and enzymes under the physicochemical conditions of the gastrointestinal tract. Therefore, the aim of the present work was to uncover the effect of gastrointestinal digestion on the release of glucosinolates and their transformation into their bioactive counterparts by applying a simulated in vitro static model on a range of brassica (red radish, red cabbage, broccoli, and mustard) sprouts. In this sense, significantly higher bioaccessibility of ITC and indoles from GSLs of red cabbage sprouts was observed in comparison with broccoli, red radish, and mustard sprouts, due to the aliphatic GSLs proportion present in the different sprouts. This indicates that the bioaccessibility of GSLs from Brasicaceae sprouts is not exclusively associated with the initial content of these compounds in the plant material (almost negligible), but also with the release of GSLs and the ongoing breakdown reactions during the gastric and intestinal phases of digestion, respectively. Additionally, aliphatic GSLs provided higher bioaccessibility of their corresponding ITC in comparison to indolic and aromatic GSLs.

Benzyl isothiocyanate-modified α-lactalbumin - Two-dimensional high-performance thin-layer chromatography for analyzing modified peptides.

In complex food matrices, non-directed reactions between food proteins and secondary plant metabolites (SPM) are conceivable. In this study, the interaction between the bioactive metabolite from garden cress (Lepidium sativum) and selected Brassicaceae - benzyl isothiocyanate (BITC) - and the dairy protein α-lactalbumin (α-LA) was investigated. It was focused on monitoring the proteolytic degradation behaviour of unmodified and BITC-modified α-LA with two-dimensional high-performance thin-layer chromatography (2D-HPTLC). The two-dimensional approach of HPTLC offers high resolution in the separation of complex peptide mixtures and might enable differentiation of protein modifications. Based on the specific peptide patterns of native and modified peptides, conclusions can be drawn about differences in protein/peptide polarity, location of a modification, and digestibility. The aim was to characterize tryptically hydrolyzed unmodified and BITC-modified peptides using the 2D method and to investigate the influence of BITC modification of α-LA on polarity and digestibility. To determine the repeatability of peptide separation by 2D-HPTLC, the unmodified and BITC-modified protein hydrolyzates were separated six times. The absolute standard deviations between the retardation factors of the individual peptide spots varied between 0.52 and 4.79 mm for the x-coordinates and between 0.41 and 6.47 mm for the y-coordinates for all three samples. Here, the mean relative standard deviations ranged from 5.80 to 10.4% for the x-coordinates and from 5.91 to 18.3% for the y-coordinates. The results of the tryptic hydrolysis indicated that, depending on the concentration of BITC used, the modification sterically hinders the cleavage sites for the enzyme, resulting in a reduced digestibility. Covalent binding of the hydrophobic BITC altered the digestibility and polarity of the protein, leading to a difference in peptide patterns between the unmodified and modified α-LA. It was concluded that the reaction was undirected, resulting in a mixture of unmodified and modified peptides, and that elongated modified peptides were formed by BITC blocking of trypsin cleavage sites.

Optimization of an Extraction Process to Obtain a Food-Grade Sulforaphane-Rich Extract from Broccoli (Brassica oleracea var. italica).

Sulforaphane (SFN) is a powerful health-promoting compound found in broccoli in the form of its inactive precursor, glucoraphanin (GFN). SFN formation occurs through the enzymatic hydrolysis of glucoraphanin by myrosinase under specific chemical conditions. Its incorporation in food formulations has been hindered by the thermal instability of SFN and low concentration in Brassicaceae. Then, extracting SFN from broccoli at a temperature below 40 °C appears as an option to recover and stabilize SFN, aiming at delivering it as a nutraceutical. We studied an eco-friendly extraction process to obtain an SFN-rich extract from broccoli. The effect of the broccoli mass/solvent ratio, ethanol concentration in the extractant solution, and extraction time on the recovery of SFN, GFN, phenolic compounds, and antioxidant activity were studied through a Box-Behnken design. The regression models explained more than 70% of the variability in the responses, adequately representing the system. The experimental factors differently affected the bioactive compound recovery and antioxidant activity of the extracts. The extraction conditions that allowed the highest recovery of bioactive compounds and antioxidant activity were identified and experimentally validated. The results may provide the basis for the design of a process to produce a sulforaphane-rich food supplement or nutraceutical by using a GRAS extractant.

Synthesis of Isothiocyanates Using DMT/NMM/TsO- as a New Desulfurization Reagent.

Thirty-three alkyl and aryl isothiocyanates, as well as isothiocyanate derivatives from esters of coded amino acids and from esters of unnatural amino acids (6-aminocaproic, 4-(aminomethyl)benzoic, and tranexamic acids), were synthesized with satisfactory or very good yields (25-97%). Synthesis was performed in a "one-pot", two-step procedure, in the presence of organic base (Et3N, DBU or NMM), and carbon disulfide via dithiocarbamates, with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium toluene-4-sulfonate (DMT/NMM/TsO-) as a desulfurization reagent. For the synthesis of aliphatic and aromatic isothiocyanates, reactions were carried out in a microwave reactor, and selected alkyl isothiocyanates were also synthesized in aqueous medium with high yields (72-96%). Isothiocyanate derivatives of L- and D-amino acid methyl esters were synthesized, under conditions without microwave radiation assistance, with low racemization (er 99 > 1), and their absolute configuration was confirmed by circular dichroism. Isothiocyanate derivatives of natural and unnatural amino acids were evaluated for antibacterial activity on E. coli and S. aureus bacterial strains, where the most active was ITC 9e.

The effect of processing and cooking on glucoraphanin and sulforaphane in brassica vegetables.

Brassica vegetables are widely consumed mostly after processing and cooking. These processing and cooking methods not only can affect the taste, texture, flavor and nutrients of these vegetables, but also influence the levels of some important bioactive compounds, such as glucosinolates (GLSs). Glucoraphanin (GLR) is the most abundant GLSs and its hydrolyzed component, sulforaphane (SLR), is the most powerful anti-cancer compound in brassica vegetables. In this review, we find out that varied treatments impact the retention of GLR and the formation of SLR differently. Be specific, 1) freezing can avoid the losses of GLR while short-time microwaving, short-time steaming and fermentation promote the biotransformation from GLR to SLR; 2) Boiling and blanching cause the largest losses of GLR and SLR, while freezing significantly protect their losses.; 3) Stir-frying varies the levels of GLR and SLR in different cooking conditions.

Rapid analysis of phenyl isothiocyanate derivatives of amino acids present in Czech meads.

Amino acids (AAs) are minor compounds occurring in meads contributing to their final organoleptic properties. Determination of AAs profile and content can help to assess the mead authenticity, adulteration and thus its quality. This work deals with the optimization of rapid analysis of 21 AAs present in mead using reversed-phase high performance liquid chromatography with spectrophotometric detection after simple derivatization procedure with phenyl isothiocyanate agent without any sample pre-treatment. Optimized derivatization and separation conditions have been successfully applied to the quantification of AAs present in five Czech meads using the multiple point standard addition method. The total amino acid content was in the range of 134-828 mg/L. The content of proline was confirmed by Harmonised spectrophotometric method. Both chromatographic and spectrophotometric methods provided overlapping results in the range of 30-266 mg/L.

Influence of Allyl Isothiocyanate on the Soil Microbial Community Structure and Composition during Pepper Cultivation.

Allyl isothiocyanate (AITC), as a fumigant, plays an important role in soil control of nematodes, soilborne pathogens, and weeds, but its effects on soil microorganisms are unclear. In this study, the effects of AITC on microbial diversity and community composition of Capsicum annuum L. soil were investigated through Illumina high-throughput sequencing. The results showed that microbial diversity and community structure were significantly influenced by AITC. AITC reduced the diversity of soil bacteria, stimulated the diversity of the soil fungal community, and significantly changed the structure of fungal community. AITC decreased the relative abundance of dominant bacteria Planctomycetes, Acinetobacter, Pseudodeganella, and RB41, but increased that of Lysobacter, Sphingomonas, Pseudomonas, Luteimonas, Pseudoxanthomonas, and Bacillus at the genera level, while for fungi, Trichoderma, Neurospora, and Lasiodiplodia decreased significantly and Aspergillus, Cladosporium, Fusarium, Penicillium, and Saccharomyces were higher than the control. The correlation analysis suggested cellulase had a significant correlation with fungal operational taxonomic units and there was a significant correlation between cellulase and fungal diversity, while catalase, cellulose, sucrase, and urease were the major contributors in the shift of the community structure. Our results will provide useful information for the use of AITC in the assessment of environmental and ecological security.

Identification of Phytochemical-Based ß-Catenin Nuclear Localization Inhibitor in NSCLC: Differential Targeting Population from Member of Isothiocyanates.

Decades of research has convinced us that phytochemical compounds contained within the plant products are the real deal, and they provide benefits such as health maintenance an d cure to illnesses. One of the deadliest noncommunicable diseases today is lung cancer, hence its disease management still deserves attention. Wnt/ß-catenin pathway activation conferring cancer stem cell (CSC) activities to non-small cell lung carcinomas (NSCLCs) may explain why the disease is still difficult to cure. In the present study, we assessed several representatives of phytochemical categories consisting of alkaloids, chalcones and isothiocyanates for their inhibitory activity to nuclear localization of ß-catenin-an important event for Wnt/ß-catenin pathway activation, in lung cancer cell lines. Real-time cell analyzer confirmed that evodiamine (EVO), chelidonine (CHE), isoliquiritigenin (ISO), licochalcone-A (LICO), benzyl isothiocyanate (BI) and phenethylisothiocyanate (PI) exhibited anti-proliferative activities and cytotoxicities to adenocarcinoma cell line SK-LU-1 and human lung CSC primary cell line (HLCSC). Immunofluorescence assay identified that CHE, ISO, LICO, BI and PI were capable of reducing the number of cells harboring ß-catenin within the nuclei of these cells. We extended the characterizations of BI and PI in Wnt-dependent squamous cell carcinoma cell line NCI-H1703 on several CSC functions and found that BI was better at inhibiting soft agar colony formation as an output of self-renewal ability, whereas PI was more effective in inhibiting the growth of multicellular tumor spheroid model mimicking micrometastases. Both however were not able to inhibit migration and invasion of NCI-H1703. In conclusion, BI could potentially be used as a safer alternative to target undifferentiated CSCs as adjuvant therapy, whereas PI could be used as chemotherapy to remove bulk tumor.

Relationship between mustard pungency and allyl-isothiocyanate content: A comparison of sensory and chemical evaluations.

The correlation of sensory and chemically evaluated pungency of mustard products was investigated via a time-intensity (TI) study and quantification of allyl isothiocyanate (AITC) contents using high-performance liquid chromatography (HPLC). Sweet, medium hot, hot, and extra hot commercial mustard products from different brands were examined. Notably, we found significant differences (p < 0.05) between the maximum perceived pungency intensity of various mustard products. The maximum perceived intensity (Imax ), the duration of the decreasing phase (DURDec ), and the area under the curve (AUC) values increased proportionally to the increase in the sample AITC content and were also higher in products classified as hot than in sweet mustards. The AITC concentration varied greatly between products from different brands and also between different sensory evaluated pungency levels. Furthermore, sensory evaluations and analytical results were correlated using regression analysis. The best correlation (correlation coefficient 0.891) was observed between the AITC concentration and AUC, when compared to that between the AITC concentration and DURDec (correlation coefficient 0.856) or the Imax value (correlation coefficient 0.803). The calculated regression model indicates that a higher AITC content induces an intensified trigeminal pungency sensation and that the sensory and chemical evaluations of mustard products were positively correlated. Therefore, by using this regression model, the sensory rating of mustard products may be predicted by chemical analysis of the AITC contents. PRACTICAL APPLICATION: This research paper provides a method to quantify the pungency inducing irritant allyl isothiocyanate in commercial mustard products and demonstrates a correlation between sensory and chemical data. Therefore, the amounts of sensory tests in product quality assurance can be reduced and replaced or at least supported by chemical quantification of pungent substances (especially AITC) in mustard products.

Science and Healthy Meals in the World: Nutritional Epigenomics and Nutrigenetics of the Mediterranean Diet.

The Mediterranean Diet (MD), UNESCO Intangible Cultural Heritage of Humanity, has become a scientific topic of high interest due to its health benefits. The aim of this review is to pick up selected studies that report nutrigenomic or nutrigenetic data and recapitulate some of the biochemical/genomic/genetic aspects involved in the positive health effects of the MD. These include (i) the antioxidative potential of its constituents with protective effects against several diseases; (ii) the epigenetic and epigenomic effects exerted by food components, such as Indacaxanthin, Sulforaphane, and 3-Hydroxytyrosol among others, and their involvement in the modulation of miRNA expression; (iii) the existence of predisposing or protective human genotypes due to allelic diversities and the impact of the MD on disease risk. A part of the review is dedicated to the nutrigenomic effects of the main cooking methods used in the MD and also to a comparative analysis of the nutrigenomic properties of the MD and other diet regimens and non-MD-related aliments. Taking all the data into account, the traditional MD emerges as a diet with a high antioxidant and nutrigenomic modulation power, which is an example of the "Environment-Livings-Environment" relationship and an excellent patchwork of interconnected biological actions working toward human health.

Intake of total cruciferous vegetable and its contents of glucosinolates and isothiocyanates, glutathione S-transferases polymorphisms and breast cancer risk: a case-control study in China.

Cruciferous vegetables contain high levels of glucosinolates (GSL) and isothiocyanates (ITC). ITC are known to induce glutathione S-transferases (GST) and thus exert their anticarcinogenic effects. This study explored the combined effects of cruciferous vegetable, GSL and ITC intake and GST polymorphisms on breast cancer risk. A total of 737 breast cancer cases and 756 controls were recruited into this case-control study. OR and 95 % CI were assessed by multivariable logistic regression. Higher cruciferous vegetable, GSL and ITC intakes were inversely associated with breast cancer risk, with adjusted OR of 0·48 (95 % CI 0·35, 0·65), 0·54 (95 % CI 0·40, 0·74) and 0·62 (95 % CI 0·45, 0·84), respectively. Compared with women carrying the GSTP1 rs1695 wild AA genotype and high cruciferous vegetable, GSL or ITC intake, carriers of the AA genotype with low cruciferous vegetable, GSL and ITC intake had greater risk of breast cancer, with adjusted OR of 1·43 (95 % CI 1·01, 1·87), 1·34 (95 % CI 1·02, 1·75) and 1·37 (95 % CI 1·05, 1·80), respectively. Persons with the GSTM1-null genotype and lower intake of cruciferous vegetables, GSL and ITC had higher risk of breast cancer than those with the GSTM1-present genotype and higher intake, with OR of 1·42 (95 % CI 1·04, 1·95), 1·43 (95 % CI 1·05, 1·96) and 1·45 (95 % CI 1·06, 1·98), respectively. Among women possessing the GSTT1-present genotype, low intake of cruciferous vegetables, GSL or ITC was associated with higher risk of breast cancer. But these interactions were non-significant. This study indicated that there were no significant interactions between cruciferous vegetable, GSL or ITC intake and GST polymorphisms on breast cancer risk.

Consumers acceptance and volatile profile of wall rocket (Diplotaxis erucoides).

Wall rocket (Diplotaxis erucoides) is a wild edible herb traditionally consumed in the Mediterranean regions with a characteristic, pungent flavour. However, little is known about its acceptance as a potential new crop. In the present study, an hedonic test with 98 volunteers was performed in order to evaluate the potential of wall rocket as a new crop. Three products were tested corresponding to microgreens, seedlings and baby-leaves. The volatile constituents were also studied due to their probable influence on acceptance, and compared to Dijon's mustard and wasabi. The degree of acceptance was mainly related to taste and pungency. Microgreens were well accepted, whereas seedlings and baby-leaves were mainly appreciated by individuals that enjoy pungent tastes. The purchase intent was also highly related to the acceptance of taste and pungency. The volatiles profile revealed that wall rocket was rich in allyl isothiocyanate, like mustard and wasabi. This compound may be greatly responsible of the relationship between the acceptance of mustard, wasabi and wall rocket. Microgreens displayed the highest levels of isothiocyanates, although the quantity of product tested by panellists did not probably allow the appreciation of such compounds. In baby-leaves, a significant decrease in isothiocyanates GC area and relative abundances was observed. These results suggest that wall rocket microgreens would be accepted by a significant proportion of the general public since pungency is lowly perceived in the product, despite its high levels of isothiocyanates. By contrast, baby-leaves may become a crop for a cohort of consumers that enjoy pungent flavours.

Soil properties, presence of microorganisms, application dose, soil moisture and temperature influence the degradation rate of Allyl isothiocyanate in soil.

Allyl isothiocyanate (AITC) is a soil fumigant derived from plants that can effectively control soil-borne diseases. Fully understanding the impact of various factors on its degradation can contribute to its effectiveness against pests and diseases. First, orthogonal design determined the extraction method of AITC in soil, that is using ethyl acetate as the extraction reagent, vortexing for 1 min as the extraction method and holding for 30 min as the method time. Then we studied the effects of soil texture and environmental factors on the rate and extent of AITC degradation in soil. The half-lives of nine origins soils varied from 12.2 to 71.8 h that were affected by the soil's electrical conductivity, available nitrogen, pH and organic matter content. Biotic degradation of AITC contributed significantly (68%-90%) of the total AITC degradation in six soil types. The degradation rate of AITC decreased as the initial dose of AITC increased. The degradation rate of AITC in Suihua soil generally increased with increasing temperature and soil moisture. The effect of temperature on AITC degradation was more pronounced when the soil was moist, which has practical implications for the control of soil pests and diseases. In agricultural soil, the soil's characteristics and environmental factors should be considered when determining the appropriate AITC dose suitable for soil borne disease while at the same time minimizing emissions and impact on the environment.

Development and validation of a HPLC-UV method for the simultaneous detection and quantification of paclitaxel and sulforaphane in lipid based self-microemulsifying formulation.

Paclitaxel (PTX) and sulforaphane (SFN) are known anticancer molecules. Their activity was found to be potentiated when tested concurrently. Only recently, however, a novel SFN enabled PTX self-microemulsifying formulation (SMEDDS) was developed for their simultaneous delivery. This necessitated the development of an analytical method for the simultaneous detection and quantitation of PTX and SFN. In this study, a simple and sensitive isocratic high performance liquid chromatography-ultraviolet (HPLC-UV) analytical method was developed and validated per International Conference on Harmonization guidelines to satisfy this objective. Its application was demonstrated when quantifying the amount of PTX and SFN released from the SMEDDS in various dissolution media. The separation of the analytes was performed with the aid of a reversed phase C18 column at ambient temperature using a 60:40 mixture of acetonitrile and KH2PO4 buffer (pH 5.0) as the mobile phase. PTX and SFN peaks were detected at 202 nm with high resolution without interference from excipients. This method showed linearity within 2.5-100 µg/mL range with r2 > 0.999. The limit of detection and lower limit of quantitation were 0.1638 and 0.4964 µg/mL for PTX and 0.4419 and 1.3389 µg/mL for SFN, respectively. A total of 98-101% of the injected samples was recovered with RSD of 0.06-0.68% indicating the suitability of the method for the simultaneous detection and quantitation of the molecules in dissolution media.

A bioinspired in vitro bioelectronic tongue with human T2R38 receptor for high-specificity detection of N-C=S-containing compounds.

Detection and identification of bitter compounds draw great attention in pharmaceutical and food industry. Several well-known agonists of specific bitter taste receptors have been found to exhibit anti-cancer effects. For example, N-C=S-containing compounds, such as allyl-isothiocyanates, have shown cancer chemo-preventive effects. It is worth noting that human T2R38 receptor is specific for compounds containing N-C=S moiety. Here, a bioinspired cell-based bioelctronic tongue (BioET) is developed for the high-specificity isothiocyanate-induced bitter detection, utilizing human Caco-2 cells as a primary sensing element and interdigitated impedance sensor as a secondary transducer. As an intestinal carcinoma cell line, Caco-2 endogenously expresses human bitter receptor T2R38, and the activation of T2R38 induces the changes of cellular morphology which can be detected by electric cell-substrate impedance sensing (ECIS). After configuration and optimization of parameters including timing of compound administration and cell density, quantitative bitter evaluation models were built for two well-known bitter compounds, phenylthiocarbamide (PTC) and propylthiouracil (PROP). The bitter specific detection of this BioET is inhibited by probenecid and U-73122, and is not elicited by other taste modalities or bitter ligands that do not activate T2R38. Moreover, by combining different computational tools, we designed a ligand-based virtual screening (LBVS) protocol to select ligands that are likely to activate T2R38 receptor. Three computationally predicted agonists of T2R38 were selected using the LBVS protocol, and the BioET presented response to the predicted agonists, validating the capability of the LBVS protocol. This study suggests this unique cell-based BioET paves a general and promising way to specifically detect N-C=S-containing compounds that can be used for pharmaceutical study and drug development.

Covalent Modification of Phosphatidylethanolamine by Benzyl Isothiocyanate and the Resultant Generation of Ethanolamine Adduct as Its Metabolite.

Benzyl isothiocyanate (BITC), a dietary isothiocyanate (ITC) derived from cruciferous vegetables, has anticancer properties. It is believed that the ITC moiety (-N═C═S) that reacts predominantly with thiol compounds plays a central role in triggering the activities resulting from these properties. Recent studies have demonstrated that ITCs also covalently modify amino moieties in a protein. In this study, we examined the chemical reaction between BITC and the aminophospholipid, phosphatidylethanolamine (PE), in the cell membrane or lipoprotein particle. To detect the BITC-modified PE, the bond between ethanolamine (EA) and phosphatidic acid in PE was cleaved using phospholipase D to form the BITC-EA adduct, which was then measured. BITC-EA was detected from the BITC-treated unilamellar liposome and low-density lipoprotein even with only a few micromoles of BITC treatment, suggesting that BITC might react with not only a thiol/amino group of a protein but also an amino moiety of an aminophospholipid. Moreover, after incorporating BITC-PE included in the liposomes into the cultured cells or after direct exposure of BITC to the cells, free BITC-EA was excreted and accumulated in the medium in a time-dependent manner. It indicates that an intracellular enzyme catalyzes the cleavage of BITC-PE to produce BITC-EA. Because the ITC-amine adduct is stable, the ITC-EA adduct could be a promising indicator of ITC exposure in vivo.