Characterization of phytochemicals from twisted-leaf garlic (Allium obliquum L.) using liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry.

INTRODUCTION: Twisted-leaf garlic (Allium obliquum L.) is a wild Allium species, which is traditionally used as aroma plant for culinary purposes due to its unique, garlic-like flavor. It represents an interesting candidate for domestication, breeding and cultivation. OBJECTIVES: The objective of this work was to explore and comprehensively characterize polar and semi-polar phytochemicals accumulating in leaves and bulbs of A. obliquum. METHOD: Plant material obtained from a multiyear field trial was analyzed using a metabolite profiling workflow based on ultra-high performance liquid chromatography-coupled electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC/ESI-QTOFMS) and two chromatographic methods. For annotation of metabolites, tandem mass spectrometry experiments were carried out and the resulting accurate-mass collision-induced dissociation (CID) mass spectra interpreted. Onion and garlic bulb extracts were used as reference samples. RESULTS: Important metabolite classes influencing nutritional, sensory and technological properties were detected and structurally characterized including fructooligosaccharides with a degree of polymerization of 3-5, S-alk(en)ylcysteine sulfoxides and other S-substituted cysteine conjugates, flavonoids including O- and C-glycosylated flavones as well as O-glycosylated flavonols, steroidal saponins, hydroxycinnamic acid conjugates, phenylethanoids and free sphingoid bases. In addition, quantitative data for non-structural carbohydrates, S-alk(en)ylcysteine sulfoxides and flavonoids are provided. CONCLUSION: The compiled analytical data including CID mass spectra of more than 160 annotated metabolites provide for the first time a phytochemical inventory of A. obliquum and lay the foundation for its further use as aroma plant in food industry.

Exploring an Alternative Cysteine-Reactive Chemistry to Enable Proteome-Wide PPI Analysis by Cross-Linking Mass Spectrometry.

The development of MS-cleavable cross-linking mass spectrometry (XL-MS) has enabled the effective capture and identification of endogenous protein-protein interactions (PPIs) and their residue contacts at the global scale without cell engineering. So far, only lysine-reactive cross-linkers have been successfully applied for proteome-wide PPI profiling. However, lysine cross-linkers alone cannot uncover the complete PPI map in cells. Previously, we have developed a maleimide-based cysteine-reactive MS-cleavable cross-linker (bismaleimide sulfoxide (BMSO)) that is effective for mapping PPIs of protein complexes to yield interaction contacts complementary to lysine-reactive reagents. While successful, the hydrolysis and limited selectivity of maleimides at physiological pH make their applications in proteome-wide XL-MS challenging. To enable global PPI mapping, we have explored an alternative cysteine-labeling chemistry and thus designed and synthesized a sulfoxide-containing MS-cleavable haloacetamide-based cross-linker, Dibromoacetamide sulfoxide (DBrASO). Our results have demonstrated that DBrASO cross-linked peptides display the same fragmentation characteristics as other sulfoxide-containing MS-cleavable cross-linkers, permitting their unambiguous identification by MSn. In combination with a newly developed two-dimensional peptide fractionation method, we have successfully performed DBrASO-based XL-MS analysis of HEK293 cell lysates and demonstrated its capability to complement lysine-reactive reagents and expand PPI coverage at the systems-level.

The functional role of sulforaphane in intestinal inflammation: a review.

Intestinal inflammation represented by inflammatory bowel disease (IBD) has become a global epidemic disease and the number of patients with IBD continues to increase. This digestive tract disease not only affects the absorption of food components by destroying the intestinal epithelial structure, but also can induce diseases in remote organs via the gut-organ axis, seriously harming human health. Nowadays, increasing attention is being paid to the nutritional and medicinal value of food components with increasing awareness among the general public regarding health. As an important member of the isothiocyanates, sulforaphane (SFN) is abundant in cruciferous plants and is famous for its excellent anti-cancer effects. With the development of clinical research, more physiological activities of SFN, such as antidepressant, hypoglycemic and anti-inflammatory activities, have been discovered, supporting the fact that SFN and SFN-rich sources have great potential to be dietary supplements that are beneficial to health. This review summarizes the characteristics of intestinal inflammation, the anti-inflammatory mechanism of SFN and its various protective effects on intestinal inflammation, and the possible future applications of SFN for promoting intestinal health have also been discussed.

Phytochemicals in Cancer Immune Checkpoint Inhibitor Therapy.

The interaction of immune checkpoint molecules in the tumor microenvironment reduces the anti-tumor immune response by suppressing the recognition of T cells to tumor cells. Immune checkpoint inhibitor (ICI) therapy is emerging as a promising therapeutic option for cancer treatment. However, modulating the immune system with ICIs still faces obstacles with severe immunogenic side effects and a lack of response against many cancer types. Plant-derived natural compounds offer regulation on various signaling cascades and have been applied for the treatment of multiple diseases, including cancer. Accumulated evidence provides the possibility of efficacy of phytochemicals in combinational with other therapeutic agents of ICIs, effectively modulating immune checkpoint-related signaling molecules. Recently, several phytochemicals have been reported to show the modulatory effects of immune checkpoints in various cancers in in vivo or in vitro models. This review summarizes druggable immune checkpoints and their regulatory factors. In addition, phytochemicals that are capable of suppressing PD-1/PD-L1 binding, the best-studied target of ICI therapy, were comprehensively summarized and classified according to chemical structure subgroups. It may help extend further research on phytochemicals as candidates of combinational adjuvants. Future clinical trials may validate the synergetic effects of preclinically investigated phytochemicals with ICI therapy.

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.

Thermosonication for the Production of Sulforaphane Rich Broccoli Ingredients.

A large proportion of broccoli biomass is lost during primary production, distribution, processing, and consumption. This biomass is rich in polyphenols and glucosinolates and can be used for the production of bioactive rich ingredients for food and nutraceutical applications. This study evaluated thermosonication (TS) (18 kHz, 0.6 W/g, 40-60 °C, 3-7 min) for the pre-treatment of broccoli florets to enhance enzymatic conversion of glucoraphanin into the bioactive sulforaphane. TS significantly increased sulforaphane yield, despite a decrease in myrosinase activity with increasing treatment intensity. The highest sulforaphane yield of ~2.9 times that of untreated broccoli was observed for broccoli thermosonicated for 7 min at 60 °C, which was 15.8% higher than the corresponding yield for thermal processing without sonication (TP) at the same condition. This was accompanied by increase in the residual level of glucoraphanin (~1.8 and 2.3 time respectively after TP and TS at 60 °C for 7 min compared to control samples) indicating that treatment-induced release of bound glucoraphanin from the cell wall matrix and improved accessibility could be at least partially responsible for the enhanced sulforaphane yield. The result indicates the potential of TS for the conversion of broccoli biomass into high sulforaphane broccoli-based ingredients.

Cystoisospora suis merozoite development assay for screening of drug efficacy in vitro.

Cystoisospora suis is a common diarrheal pathogen of piglets and typically controlled by metaphylactic toltrazuril application. Recently, toltrazuril resistance has been reported in the field; however, both evaluation of toltrazuril efficacy against field isolates and the anticoccidial drug development for pigs is hampered by costs and labor of animal experimentation. Therefore an in vitro merozoite development assay was developed to evaluate the efficacy of compounds against C. suis in vitro. Monolayers of IPEC-1 cells were infected with sporozoites derived from oocysts of defined C. suis laboratory strains and the optimal infection dose as well as concentration, time point and duration of treatment were evaluated by quantitative real-time PCR. Cell cultures were treated with bumped kinase inhibitor (BKI) 1369 at different time points to evaluate the possibility to delineate effects on different developmental stages in vitro during invasion and early infection, and to determine different inhibitory concentrations (IC50, IC95). BKI 1369 had an IC50 of 35 nM and an IC95 of 350 nM. Dose- and duration-dependent efficacy was seen when developing stages were treated with BKI 1369 after infection (days 0-1, 2-3 and 2-5) but not when sporozoites were pre-incubated with BKI 1369 before infection. Efficacies of further BKIs were also evaluated at 200 nM. BKI 1318, 1708, 1748 and 1862 had an efficacy comparable to that of BKI 1369 (which is also effective in vivo). BKI 1862 showed a more pronounced loss of efficacy in lower concentrations than BKI 1369, signifying pharmacokinetic differences of similar compounds detectable in vitro. In addition, the effects of toltrazuril and its metabolites, toltrazuril sulfoxide and toltrazuril sulfone, on a toltrazuril sensitive and a resistant strain of C. suis were evaluated. Inhibition of merozoite growth in vitro by toltrazuril and its metabolites was dose-dependent only for toltrazuril. Clear differences were noted for the effect on a toltrazuril-sensitive vs. a resistant strain, indicating that this in vitro assay has the capacity to delineate susceptible from resistant strains in vitro. It could also be used to evaluate and compare the efficacy of novel compounds against C. suis and support the determination of the optimal time point of treatment in vivo.

Unconventional Biocatalytic Approaches to the Synthesis of Chiral Sulfoxides.

Sulfoxides are a class of organic compounds that find wide application in medicinal and organic chemistry. Several biocatalytic approaches have been developed to synthesise enantioenriched sulfoxides, mainly by exploiting oxidative enzymes. Recently, the use of reductive enzymes such as Msr and Dms has emerged as a new, alternative method to obtain enantiopure sulfoxides from racemic mixtures. In parallel, novel oxidative approaches, employing nonclassical solvents such as ionic liquids (ILs) and deep eutectic solvents (DESs), have been developed as greener and more sustainable biocatalytic synthetic pathways. This minireview aims highlights the recent advances made in the biocatalytic synthesis of enantioenriched sulfoxides by employing such unconventional approaches.

Prevailing Knowledge on the Bioavailability and Biological Activities of Sulphur Compounds from Alliums: A Potential Drug Candidate.

Allium sativum (garlic) is widely known and is consumed as a natural prophylactic worldwide. It produces more than 200 identified chemical compounds, with more than 20 different kinds of sulfide compounds. The sulfide compounds particularly are proven to contribute to its various biological roles and pharmacological properties such as antimicrobial, antithrombotic, hypoglycemic, antitumour, and hypolipidemic. Therefore, it is often referred as disease-preventive food. Sulphur-containing compounds from A. sativum are derivatives of S-alkenyl-l-cysteine sulfoxides, ajoene molecules, thiosulfinates, sulfides, and S-allylcysteine. This review presents an overview of the water-soluble and oil-soluble sulphur based phytochemical compounds present in garlic, highlighting their mechanism of action in treating various health conditions. However, its role as a therapeutic agent should be extensively studied as it depends on factors such as the effective dosage and the suitable method of preparation.

S-Alk(en)ylcysteine sulfoxides in the genus Allium: proposed biosynthesis, chemical conversion, and bioactivities.

S-Alk(en)ylcysteine sulfoxides are sulfur-containing natural products characteristic of the genus Allium. Both the flavor and medicinal properties of Allium plants are attributed to a wide variety of sulfur-containing compounds that are generated from S-alk(en)ylcysteine sulfoxides. Previous radiotracer experiments proposed that S-alk(en)ylcysteine sulfoxides are biosynthesized from glutathione. The recent identification of γ-glutamyl transpeptidases and a flavin-containing S-oxygenase involved in the biosynthesis of S-allylcysteine sulfoxide (alliin) in garlic (Allium sativum) provided insights into the reaction order of deglutamylation and S-oxygenation together with the localization of the biosynthesis, although the rest of the enzymes in the pathway still await discovery. In intact plants, S-alk(en)ylcysteine sulfoxides are stored in the cytosol of storage mesophyll cells. During tissue damage, the vacuolar enzyme alliinase contacts and hydrolyzes S-alk(en)ylcysteine sulfoxides to produce the corresponding sulfenic acids, which are further converted into various sulfur-containing bioactive compounds mainly via spontaneous reactions. The formed sulfur-containing compounds exhibit bioactivities related to pathogen defense, the prevention and alleviation of cancer and cardiovascular diseases, and neuroprotection. This review summarizes the current understanding of the occurrence, biosynthesis, and alliinase-triggered chemical conversion of S-alk(en)ylcysteine sulfoxides in Allium plants as well as the impact of S-alk(en)ylcysteine sulfoxides and their derivatives on medicinal, food, and agricultural sciences.

Cyclic sulfur-containing compounds from Allium fistulosum 'Kujou'.

Sulfur-containing compounds, allicin and ajoene, etc., were isolated from Allium species. In a recent study, some sulfur-containing cyclic compounds were isolated from A. sativum, A. cepa, and A. fistulosum. Four new compounds with multiple rings with methyl disulfide or propyl disulfide at the side chain of the 7-position, kujounins A3 (1), B1 (2), B2 (3) and B3 (4), and two new thiolane type compounds with methoxy and methyl sulfoxide moiety at the 2- and 5-positions, and allium sulfoxides A2 (5) and A3 (6), were isolated from the acetone extract of the fresh white parts of Allium fistulosum 'Kujou' with three known compounds, kujounin A1 (7) and A2 (8), and allium sulfoxide A1 (9). The chemical structures of the new compounds were elucidated on the basis of physicochemical evidence. The kujounins had a rare molecular skeleton, which was tetrahydro-2H-difuro[3,2-b:2',3'-c]furan-5(5aH)-one.

Effect of Cultivar and Cultivation Year on the Metabolite Profile of Onion Bulbs ( Allium cepa L.).

This study investigated the variation of metabolite profiles of onion bulbs ( Allium cepa L.) depending on genetic and environmental factors. Nine onion cultivars ("Corrado", "Cupido", "Forum", "Hytech", "Picador", "Redlight", "Snowpack", "Stardust", "Sturon") with different scale color and dry matter content were grown in a two-year field trial. Using a recently established metabolite profiling approach based on liquid chromatography-coupled electrospray ionization quadrupole time-of-flight mass spectrometry, 106 polar and semipolar metabolites which belong to compound classes determining nutritional, sensory, and technological quality of onion bulbs such as saccharides, flavonoids, S-substitued cysteine conjugates, amino acids, and derived γ-glutamyl peptides were relatively quantitated in parallel. Statistical analyses of the obtained data indicated that depending on the compound class genetic and environmental factors differently contributed to variation of metabolite levels. For saccharides and flavonoids the genetic factor was the major source of variation, whereas for cysteine sulfoxides, amino acids, and peptides both genetic and environmental factors had a significant impact on corresponding metabolite levels.

Flow injection spectrophotometric determination of V(V) involving on-line separation using a poly(vinylidene fluoride-co-hexafluoropropylene)-based polymer inclusion membrane.

A poly(vinylidene fluoride-co-hexafluoropropylene)-based polymer inclusion membrane (PIM) using Cyphos® IL 101 (i.e. trihexyl(tetradecyl)phosphonium chloride) as the carrier and 2-nitrophenyl octyl ether as a plasticizer in a mass ratio of 55/35/10 was employed for the on-line extractive separation of V(V) prior to its spectrophotometric determination in a flow injection analysis (FIA) system using xylenol orange as the colorimetric reagent. The selectivity of the membrane allowed the determination of V(V) in sulfate solutions in the presence of a variety of cations and anions. The interference of molybdenum(VI) was eliminated by off-line extraction using the same PIM. A univariate sequential optimization of the newly developed FIA system was conducted and under optimal conditions the system is characterized by a linear concentration range of 0.5-8.0mgL-1, detection limit of 0.08mgL-1 and sample throughput of 4h-1. The relative standard deviation at the 3mgL-1 level of V(V) was 2.9% based on 8 replicate determinations. The membrane was stable, which was reflected by the standard deviation value for determinations over three consecutive days (24 determinations of 3mgL-1 V(V)) of 3.6%. The newly developed FIA system was applied to the determination of V(V) in water and dietary supplements samples and a good agreement with inductively coupled plasma optical emission spectrometry was observed.

Changes in trans-S-1-Propenyl-l-cysteine Sulfoxide and Related Sulfur-Containing Amino Acids during Onion Storage.

trans-S-1-Propenyl-l-cysteine sulfoxide (PRENCSO) is the most abundant flavor precursor in onions. The changes in PRENCSO, its biosynthetic precursors, and cycloalliin contents in bulbs stored at 0 °C [i.e., recommended by the Food and Agriculture Organization of the United Nations (FAO)] are investigated. Ion-exchange high-performance liquid chromatography analysis revealed larger amounts of PRENCSO and cycloalliin, which were stoichiometrically balanced by the decrease in the combined amounts of the three biosynthetic precursors. The molar balance of the PRENCSO biosynthesis process during onion storage was explained well by the changes of these five compounds. High-throughput integrated liquid chromatography-tandem mass spectrometry analysis was employed to analyze multiple samples at 5 and 20 °C. The molar concentration data, converted using linear regression equations, showed a similar molar balance for the PRENCSO biosynthesis process comprising these five compounds during storage at all temperatures tested. Furthermore, the content of cycloalliin significantly increased at the expense of that of PRENCSO during storage at 20 °C.

Onionin A, a sulfur-containing compound isolated from onions, impairs tumor development and lung metastasis by inhibiting the protumoral and immunosuppressive functions of myeloid cells.

SCOPE: Recent studies have demonstrated that myeloid lineage cells, such as macrophages and myeloid suppressor cells (MDSCs), are major components exhibiting protumoral functions in the setting of tumor progression. Tumor-associated macrophages polarized to the protumoral M2 phenotype promote tumor proliferation and are considered to be a therapeutic target in patients with malignant tumors. METHODS AND RESULTS: We identified a new candidate compound, called onionin A (ONA) isolated from onions, that inhibits macrophage polarization into the M2 phenotype, as well as the immunosuppressive activity of MDSCs and tumor proliferation, by suppressing signal transducer and activator of transcription-3 (Stat3) activation. Furthermore, ONA administration was found to significantly suppress subcutaneous tumor development and lung metastasis in tumor-bearing mice. ONA administration also inhibited Stat3 activation and increased the number of infiltrating lymphocytes in tumor tissues, and an ex vivo analysis showed that the immunosuppressive effect of MDSCs in tumor-bearing mice is impaired by ONA. Moreover, ONA regulated tumor proliferation by inhibiting cell-cell interactions between macrophages and tumor cells, and ONA administration enhanced the antitumor effects of cisplatin in the tumor-bearing mice. CONCLUSIONS: These findings demonstrate that ONA may be a potential new tool for antitumor therapy and also for tumor prevention.

Production and characterization of tearless and non-pungent onion.

The onion lachrymatory factor (LF) is produced from trans-S-1-propenyl-L-cysteine sulfoxide (PRENCSO) through successive reactions catalyzed by alliinase (EC 4.4.1.4) and lachrymatory factor synthase (LFS), and is responsible for the tear inducing-property and the pungency of fresh onions. We developed tearless, non-pungent onions non-transgenically by irradiating seeds with neon-ion at 20 Gy. The bulbs obtained from the irradiated seeds and their offspring bulbs produced by selfing were screened by organoleptic assessment of tear-inducing property or HPLC analysis of LF production. After repeated screening and seed production by selfing, two tearless, non-pungent bulbs were identified in the third generation (M3) bulbs. Twenty M4 bulbs obtained from each of them showed no tear-inducing property or pungency when evaluated by 20 sensory panelists. The LF production levels in these bulbs were approximately 7.5-fold lower than those of the normal onion. The low LF production levels were due to reduction in alliinase activity, which was a result of low alliinase mRNA expression (less than 1% of that in the normal onion) and consequent low amounts of the alliinase protein. These tearless, non-pungent onions should be welcomed by all who tear while chopping onions and those who work in facilities where fresh onions are processed.

Two new bicyclic sulfoxides from Welsh onion.

Newly identified bicyclic sulfoxides, welsonins A1 (1) and A2 (2), were isolated from acetone extracts of the bulbs of the Welsh onion (Allium fistulosum). In this study, the structures of 1 and 2, which are tetrahydrothiophene-S-oxide derivatives, were characterized by spectroscopic analysis. These compounds appeared to be derived from the coupling of 1-propenyl sulfenic acid and uronic acid. Welsonin A1 (1) showed the potential to suppress tumor-cell proliferation by inhibiting the polarization of alternatively activated M2 macrophages.

DFT characterization on the mechanism of sulfoxidation with H2O2 catalyzed by tetranuclear peroxotungstates [XO4{WO(O2)2}4](n-) (X = Si(IV), P(V), S(VI), As(V), and Se(VI)).

A thorough theoretical analysis was carried out on the sulfoxidation with H2O2 catalyzed by a tetranuclear peroxotungstate [SiO4{WO(O2)2}4](4-). The active species is the [SiO4{WO(O2)2}4(H2O2)](4-) (SiW4(H2O2)) complex rather than [SiO4{WO(O2)2}4](4-) (SiW4). The catalytic cycle consists of three elementary processes: oxygen transfer, sulfoxide dissociation, and catalyst regeneration. The oxygen transfer occurs from the peroxo oxygen atom O1 of SiW4(H2O2) to the sulfur center of dimethyl sulfide with a moderate Gibbs activation energy (ΔG°(‡)) of 17.1 kcal mol(-1). By comparing potential energy surfaces and condensed Fukui functions (ƒ(+)), the electrophilicity of the outer peroxo atoms in SiW4(H2O2) determines which oxygen transfers to the dimethyl sulfide. Then, the sulfoxide dissociation proceeds with a small ΔG°(‡) value of 2.3 kcal mol(-1) by elongation of the peroxo O1-O4 distance and elimination of the product dimethylsulfoxide. Finally, the catalyst regeneration is found to occur via two successive proton transfers from H2O2 to the oxygen atoms of peroxotungstates with the ΔG°(‡) values of 15.9 and 15.3 kcal mol(-1), which has been firstly examined in the present study. All of these steps occur easily with moderate ΔG°(‡) values, but the oxygen transfer is the rate-determining step of this catalytic reaction. In addition, the catalytic activity of peroxotungstates can be effectively tuned by changing the heteroatom X of [XO4{WO(O2)2}4(H2O2)](n-) in the order: Se(VI) ≈ S(VI) > As(V) ≈ P(V) > Si(IV).

Cyclic sulfoxides-garlicnins K1, K2, and H1-extracted from Allium sativum.

Newly identified cyclic sulfoxides-garlicnins K1 (1), K2 (2), and H1 (3)-were isolated from the acetone extracts of the bulbs of garlic, Allium sativum. Garlicnin H1 (3) demonstrated potential to suppress tumor cell proliferation by regulating macrophage activation. The structures of garlicnins K1 and K2, 3,4-dimethyl-5-allyl-tetrahydrothiophen-2-one-S-oxides, and the structure of garlicnin H1, 3-carboxy-3-hydroxy-4-methyl-5-allylsulfoxide-tetrahydrothiophen-2-(ethane-1,2-diol)-S-oxide were characterized by spectroscopic analysis.

Onion: nature protection against physiological threats.

Onion (Allium cepa L.) is found in various regions of Europe, North America, Asia, and Africa. It is one of the classic examples of Allium species used not only for culinary preparations but also for medicinal purposes. Onion with a variety of purposes is often used as a raw material in many dishes and accepts almost all of the traditions and culture. Owing to its storage characteristics and durability of shipping, onions have been traded more widely than most vegetables. The pungent fractions of garlic are mostly sulfur-containing moieties while its two chemical groups have marked effect on human health. These are flavonoids and ALK (EN)-based cysteine sulfoxides (ACSOs). Compounds in onions have been reported with a range of health benefits, including anticancer properties, antiplatelet activity, antithrombotic activity, antiasthmatic activity, and antibiotic effects.