Novel sulforaphane (1-isothiocyanato-4-[methylsulfinyl]butane:SFN) derivatives

Abstract

Based on research into endogenous polysulfides in broccoli, we provide novel polysulfides useful for animals. [Solution] Sulforaphane (1-isothiocyanato-4-[methylsulfinyl]butane (SFN)) and cysteine ​​persulfide (CysS n H, n=2,3,4, or 5), or glutathione persulfide (GS n H, or GS n This is a novel SFN derivative consisting of a complex with G, n=2,3,4, or 5). JPEG2026048471000019.jpg64170

Description

[Technical Field]

[0001] This invention relates to novel polysulfides, and more specifically to novel SFN derivatives inherent in broccoli sprouts. [Background technology]

[0002] In recent years, the biological importance of polysulfides (RSnR, n>2, R=hydrogen or alkyl) has attracted attention. For example, hydropolysulfides (RSnR) containing cysteine ​​and glutathione. n Polysulfides (H, n>2, R=alkyl), such as cysteine ​​hydropersulfide (CysS2H) and glutathione hydropersulfide (GS2H), have been shown to be endogenously produced in vivo through enzymatic and / or chemical sulfur transfer reactions. Recent studies have reported that recombinant cysteinyl-tRNA synthetases in mammals, including humans and mice, as well as Escherichia coli, can catalyze the formation of CysS2H from cysteine ​​(CysSH) used as a substrate. Furthermore, these bioactive substances have been shown to function as potent antioxidants and play important roles in multiple cellular processes, including mitochondrial biosynthesis and metabolic regulation. Significant reductions in the endogenous production of polysulfides, including CysS2H and GS2H, have been reported in patients with cardiovascular disease and chronic obstructive pulmonary disease, suggesting that these substances may play an important role in human health and disease prevention. Non-patent document 1 discloses that supersulfur molecules, including polysulfides, function as potent antioxidants and as important regulators of redox signaling in living organisms.

[0003] Increased intake of broccoli (Brassica oleracea var. italica) has been associated with a reduced risk of cardiometabolic diseases, neurological disorders, diabetes, and cancer. Broccoli is rich in various phytochemicals, including glucosinolates and isothiocyanates. Furthermore, it has recently been reported that polysulfides such as cysteine ​​hydroperoxide (CysS2H) and glutathione hydroperoxide (GS2H) are endogenously produced in mammals, including humans, and these bioactive substances have been shown to function as potent antioxidants and important regulators of redox signaling in the body. However, there has been little research focusing on the endogenous polysulfide content of broccoli or the effects of germination on the polysulfide content and composition of broccoli. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] “Cysteinyl-tRNA synthetase governs cysteine ​​polysulfidation and mitochondrial bioenergetics,” Nature Communications 8, October 27, 2017, 1177, https: / / doi.org / 10.1038 / s41467-017-01311-y [Overview of the project] [Problems that the invention aims to solve]

[0005] The present invention aims to provide novel polysulfides useful for animals, based on research into endogenous polysulfides in broccoli. [Means for solving the problem]

[0006] To achieve the above objective, the present invention provides sulforaphane ((1-isothiocyanato-4-[methylsulfinyl]butane (SFN))) and cysteine ​​persulfide (CysS n H, n=2,3,4, or 5), or glutathione persulfide (GS n H, or GS n It is characterized by being a novel SFN derivative consisting of a complex with G, n=2,3,4, or 5). [Effects of the Invention]

[0007] According to the present invention, based on research into endogenous polysulfides in broccoli, it is possible to provide novel polysulfides useful to humans. [Brief explanation of the drawing]

[0008] [Figure 1] Figure 1 shows a photograph and graph illustrating the germination of broccoli sprouts. [Figure 2] Figure 2 is a graph showing multiple heating / cooling cycles in an acid cyclic decomposition system. [Figure 3] Figure 3 is a graph showing a comparison of the polysulfide profiles of mature broccoli and broccoli sprouts. [Figure 4] Figure 4 is a graph showing a comparison of polysulfide profiles in vegetable sprouts. [Figure 5] Figure 5 is a graph showing the changes in the polysulfide profile during the germination and growth stages of broccoli sprouts. [Figure 6] Figure 6 is a schematic diagram of targetless polysulfide omics analysis. [Figure 7] Figure 7 is a graph showing representative results of non-targeted polysulfide omics analysis of broccoli sprouts (day 2, A) and seeds (B). [Figure 8] Figure 8 is a graph showing representative results of non-targeted polysulfide-omics analysis of broccoli sprouts (day 5). [Figure 9] Figure 9 is a graph showing the quantitative analysis results for CysSnH and GSnH. [Figure 10] Figure 10 is a graph showing the endogenous bioeffects of oxidized glutathione polysulfide. [Figure 11] Figure 11 shows representative MS / MS chromatograms of true and endogenous oxidized glutathione polysulfide. [Figure 12] Figure 12 shows the protein expression profiles (A) and polysulfide status (B) in broccoli seeds and sprouts. [Figure 13] Figure 13 is a graph showing the endogenous production results of sulforaphane complexes GSnH and CysSnH. [Figure 14] Figure 14 shows representative MS / MS chromatograms of true and endogenous sulforaphane (SFN) with glutathione or cysteine ​​hydropolysulfide. [Figure 15] Figure 15 shows representative MS / MS spectra of endogenous SFN-SnCys (n=1-5) and SFN-SnG (n=1-4) detected in broccoli sprouts (day 5). [Figure 16] Figure 16 is a graph showing the correlation analysis between SFN-SnCys levels and SFN levels (A-E) or CysSH content (F-J). [Figure 17] Figure 17 is a graph showing the effect of SFN on the reactivity of TME-IAM with polysulfides. [Figure 18] Figure 18 shows the synthesis and purification of SFN-SnG and SFN-SnCys by high-resolution mass spectrometry. [Figure 19] Figure 19 is a graph showing the radical scavenging ability of SFN derivatives. [Modes for carrying out the invention]

[0009] This invention is based on the investigation of changes in polysulfide biosynthesis in broccoli during germination, using non-targeted polysulfide omics analysis and quantitative targeted polysulfide metabolomics by liquid chromatography-electrospray ionization-tandem mass spectrometry. Furthermore, a 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay was performed to clarify the antioxidant properties of polysulfides. As a result, it was revealed that the total polysulfide content of broccoli sprouts increased significantly during germination and growth. Cysteine ​​S-2 hydroxylase and trisulfide cysteine ​​were the main components of organic polysulfide metabolites. n H, or GS n A novel sulforaphane (SFN) derivative bound to H is endogenously produced in broccoli sprouts, and CysS n We discovered that a novel SFN derivative bound to H exhibits higher radical scavenging activity than SFN and cysteine. These results suggest that polysulfides, which are abundant in broccoli sprouts, contribute to their health-promoting effects. This finding provides important biological implications for the development of new pharmacological targets related to the health-promoting effects of broccoli sprouts.

[0010] Studies have shown that consuming large amounts of cruciferous vegetables such as broccoli (Brassica oleracea var. italica), cabbage (B. oleracea var. capitata), cauliflower (B. oleracea var. botrytis), white radish (Raphanus sativus var. longipinnatus), kale (B. oleracea var. acephala), and watercress (Nasturtium officinale) reduces the risk of cardiometabolic diseases, neurological disorders, diabetes, and cancer. Cruciferous vegetables contain a variety of phytochemicals, including phenols, carotenoids, and glucosinolates, as well as their breakdown products such as isothiocyanates and indoles. The secondary metabolites synthesized by various species of cruciferous vegetables, particularly glucosinolates, contribute to their unique and distinctive flavor and provide various medicinal components, distinguishing them from other plant families. Glucoraphanin is the main glucosinolate found in broccoli, and hydrolysis by β-thioglucosidase (myrosinase) produces sulforaphane (SFN, 1-isothiocyanato-4-[methylsulfinyl]butane), a bioactive isothiocyanate. The bioactive components of broccoli sprouts differ from those of mature broccoli, making them more effective than mature broccoli in preventing diseases associated with oxidative stress.

[0011] Volatile polysulfides such as diaryllyl trisulfide, diaryl tetrasulfide, and dimethyl trisulfide are a type of lipophilic polysulfide found in the essential oils of garlic (Allium sativum) and onion (A. cepa), and possess anti-inflammatory, cardioprotective, and cancer-preventive effects. These volatile polysulfides are derived from (+)-S-alkyl(ene)-l-cysteine ​​sulfoxides such as (+)-S-methyl-l-cysteine ​​sulfoxide (SMCSO, methine) and S-allyl-l-cysteine ​​sulfoxide (alliin). SMCSO is also found in cruciferous vegetables such as broccoli and white cabbage. Despite the higher concentration of SMCSO in cruciferous vegetables compared to glucosinolates, the complex metabolism of SMCSO has led to little attention being paid to the health-promoting effects of this compound. Dimethyl trisulfide is present in mature broccoli and broccoli sprouts, but there has been no research focusing on the polysulfide content and composition in broccoli, or the effect of germination on the production of endogenous polysulfide. The inventors of this application investigated the polysulfide content inherent in broccoli and the effect of germination on the polysulfide content and composition. First, the total polysulfide content (TPsC) and total sulfur content (TSC) of broccoli sprouts and mature broccoli were measured, and their polysulfide profiles (TPsC / TSC) were analyzed. Then, a technique for comprehensively detecting hydropolysulfide in broccoli sprouts, i.e., non-targeted polysulfide omics analysis, was performed using a novel alkylating agent, N-iodoacetyl-L-tyrosine methyl ester (TME-IAM). This alkylating agent can derivatize hydropolysulfide into stable adducts with minimal artificial degradation.

[0012] (material) CysSH (98%), cystine (95%), glutathione (GSH, 97%), and glutathione disulfide (GS2G, 95%) were purchased from FUJIFILM Wako Pure Chemical (Osaka, Japan). N-ethylmaleimide (NEM, >99%) and dithiothreitol (DTT, >99%) were purchased from Nacalai Tesque (Kyoto, Japan). dl-SFN (>95%) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) were obtained from Tokyo Chemical Industry (Tokyo, Japan). Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) and sodium disulfide (Na2S2) were purchased from Merck (Darmstadt, Germany) and Dojindo Laboratories (Kumamoto, Japan), respectively. The stable isotope-labeled standard of bis-S-NEM adduct (NEM- 34 S-NEM-d 10 ) was prepared using stable isotope-labeled NEM (NEM-d5) and sodium sulfide. TME-IAM and its stable isotope-labeled adduct standard were synthesized. The stable isotope-labeled TME- 13 C2]IAM was also synthesized using 13 C2]iodoacetic acid (purity 99%, Sigma-Aldrich, Burlington, MA) instead of normal iodoacetic acid. Broccoli seeds were purchased from Nakahara Seedlings (Fukuoka Prefecture). All other chemicals and reagents were purchased from general chemical suppliers and the highest grade commercially available was used.

[0013] (Vegetables) Domestically cultivated mature vegetables (broccoli, cabbage, garlic) and sprouts (daikon radish, red radish (R. sativus var. sativus), red cabbage (B. oleracea var. capitata f. rubra), cress, mustard greens (B. juncea), green onion (Allium fistulosum), water spinach (Ipomoea aquatica), alfalfa (Medicago sativa), okra (Abelmoschus esculentus), broccoli) were purchased, powdered in liquid nitrogen, and stored at -80 °C until required for analysis.

[0014] In the experiment on broccoli seed germination and growth, broccoli seeds (approximately 1g) were soaked in ultrapure water at room temperature (24-25°C) in the dark for 24 hours (Day 1). The seeds were then placed in a 12.5cm diameter tray (Kitchen Farm-120, W125×D125×H77mm, Yamato Plastic Co., Ltd., Nara Prefecture). The tray was placed on top of another tray containing ultrapure water, maintaining saturated humidity at room temperature (24-25°C). The water level in the lower tray was approximately 15cm, at which point only the roots of the sprouts were submerged. Germinated sprouts were harvested every 24 hours (Days 2-5), crushed with liquid nitrogen, and stored at -80°C until analysis was required. Representative images of broccoli seeds and germinated sprouts are shown in Figure 1. Figure 1 shows the germination of broccoli sprouts. Image A shows the broccoli seeds, germinated seeds (day 1), and sprouts (days 2-5) used in the analysis. Image B shows the lengths of the broccoli seeds, germinated seeds (day 1), and sprouts (days 2-5). Image C shows the fresh weights of the broccoli seeds, germinated seeds (day 1), and sprouts (days 2-5). The data are shown as mean ± standard deviation (SD) (n=20). **p <0.01, **** p<0.0001, comparison with seeds, one-way ANOVA with Dunnett's multiple comparison test. NS, no significant difference. To measure the moisture content of vegetables, fresh vegetable samples (approx. 1g) were placed in a Uniblock moisture meter (MOC63u; Shimadzu Corporation, Kyoto, Japan), and the dry weight was measured to calculate the moisture content (%).

[0015] (Determination of total sulfur content (TSC) by acid cyclic decomposition-inductively coupled plasma emission spectroscopy) The total sulfur content was quantified by converting all sulfur-containing molecules to sulfate ions using an acid cyclic decomposition system (ECOPRE system, Agilent Technologies), and then measuring the sulfate ion concentration by inductively coupled plasma atomic emission spectroscopy (ICP-OES). Approximately 100 mg of each vegetable sample was mixed with 10 mL of 15.8 M nitric acid aqueous solution in an acid cyclic decomposition apparatus (ACTAC), and then this apparatus was combined with a concentrate (ACTAC) containing 5 mL of 5% (v / v) nitric acid. Subsequently, decomposition was carried out by repeating the heating / cooling cycle multiple times on a graphite hot plate (AS ONE Co., Osaka, Japan), as shown in Figure 2 of the multiple heating / cooling cycles in the acid cyclic decomposition system. After cooling the samples to 25°C, they were collected, diluted to 30 mL with 1 M nitric acid solution, and then further diluted to a concentration of 1-10 ppm with 1 M nitric acid solution. Ultrapure water without vegetables was used as a blank sample, and the signal detected from the blank sample was subtracted from the signal obtained from each sample. The sulfate ion concentration in vegetable samples was measured using ICP-OES (ICPE-9000, Shimadzu Corporation, Kyoto, Japan) and quantified using a standard curve obtained from sulfate ion standard solution (Kanto Chemical, Tokyo, Japan). The measurement conditions for ICP-OES were as follows: Argon gas pressure: 450±10kPa, High-frequency output: 1.2kW, Plasma gas flow rate: 14L / min, Carrier gas flow rate: 0.7L / min, Irradiation time: 30 seconds, Observation wavelengths: 180.731, 182.037, 182.625nm.

[0016] (Quantitative determination of TPsC using an alkaline / reducing sulfur removal protocol) Vegetable samples were treated at an alkaline pH with the reducing agent DTT to decompose polysulfides, and the freed sulfur atoms were captured by the alkylating reagent NEM to form a stable bis-S-NEM adduct (NEM-S-NEM). This adduct was then quantitatively detected using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS / MS) combined with stable isotope-labeled standard dilution. Approximately 100 mg of vegetable samples were homogenized using an ULTRA-TURRAX homogenizer (T10 basic; IKA, Osaka, Japan) with 20-fold addition of 100 mM N-cyclohexyl-3-aminopropanesulfonic acid-NaOH buffer (pH 11.0) containing 10 mM DTT and 25% (v / v) ethanol. Subsequently, sonication was performed for 15 seconds (repeated three times at 30-second intervals), and the homogenate was incubated in a heat block incubator (NICHIHIRO, Saitama, Japan) at 37°C for 1 hour.

[0017] The reaction mixture (10 μL) was added to 200 mM Tris(hydroxymethyl)aminomethane (Tris)-HCl buffer (pH 7.4) (90 μL) containing 20 mM NEM and 80% (v / v) methanol, and the resulting mixture was incubated in a heat block incubator at 37°C for 1 hour. After centrifugation at 15,000 g and 4°C for 15 minutes, the resulting supernatant (10 μL) was treated with 0.1 μM NEM- 34 S-NEM-d 10 The sample was diluted 10-fold with a 0.1% (v / v) formic acid (FA) solution containing the substance. The resulting NEM adduct was detected by LC-ESI-MS / MS analysis using a modified version of the dilution method for stable isotope-labeled standards. A buffer blank without vegetables was prepared, and the signal detected from the buffer blank sample was subtracted from the signals obtained from individual samples.

[0018] (LC-ESI-MS / MS) LC-ESI-MS / MS analysis was performed using a Xevo TQD triple quadrupole mass spectrometer (Waters Co., Milford, MA) coupled to a Shimadzu LC-20A system (Shimadzu Corporation) equipped with an autosampler (SIL-20A), a communication bus module (CBM-20A), an online degasser (DGU-20A5), two liquid chromatographs (LC-20AD), and a column oven (CTO-20A). Samples were evaluated using a Mightysil-C18 column (50 mm × 2.0 mm inner diameter, Kanto Chemical Co., Ltd.) with a Shimadzu LC-20A system, and eluted using a linear gradient with methanol as the mobile phase. In NEM-S-NEM detection, 1%B was detected at 0-1 min, 99%B at 4-5 min, and 1%B at 5.1-8 min; in non-targeted polysulfide omics analysis, 1%B was detected at 0 min, 99%B at 10-12 min, and 1%B at 13-18 min; quantitative targeted polysulfide metabolomics and SFN derivatives and oxidized glutathione polysulfide (GS) n In detection of G), 1% B was detected at 0-1 min, 99% B at 7-10 min, and 1% B at 10.1-15 min. The mobile phase contained 0.1% FA, with a flow rate of 0.6 mL / min and a temperature of 40°C. The mass spectrometer was operated in positive ion mode, with the capillary voltage and desolvation gas (nitrogen) set to 1000 V and 1000 L / h, respectively, and the temperature at 500°C. Each adduct was detected in multiple reaction monitoring mode using the parameters shown in Table 1.

[0019] [Table 1]

[0020] (Omics analysis of polysulfides without targeting specific molecules) Using a POLYTRON homogenizer (DIAX-100; Heidolph Instruments, Schwabach, Germany), approximately 1 g of broccoli seeds and sprouts from day 2 and day 5 were mixed in 50 mM sodium acetate buffer (pH 6.5) with standard TME-IAM (lightweight, 0.8 mM) and stable isotope-labeled TME-[ 13The mixture contained [C2]IAM (weight, 0.2 mM) and 70% methanol. The homogenate was incubated at 37°C for 30 minutes, and the reaction was stopped by adding 0.5 mL of 10% FA. The mixture was then centrifuged at 20,000 g at 4°C for 15 minutes. The supernatant was collected, diluted five-fold with 0.1% FA, and subjected to reversed-phase solid-phase extraction using a Wakogel 100C18 (Fujifilm Wako Pure Chemical Industries) open column equilibrated with 0.1% FA. The column was washed with 0.1% FA for 5 column volumes (CVs), followed by 5 CVs of ultrapure water. The TME-AM adduct was eluted with 3 CVs of methanol containing 0.01% FA. The eluate was concentrated under vacuum by centrifugation (175 g, 23°C, approximately 2.5 hours) until the sample volume was approximately 0.5-0.6 mL. A portion of the concentrate was incubated in 100 mM Tris-HCl buffer (pH 7.4) at 37°C for 30 minutes in the presence or absence of 1% (v / v) 2-mercaptoethanol. After filtration using a centrifugal filter (Cosmospin filter, Nacalai Tesque), the filtrate was subjected to LC-ESI-MS / MS for non-targeted polysulfide omics analysis. Polysulfide candidates with an intensity of 1 / 10 or greater than that of GSH were considered significant signals.

[0021] (Quantitative analysis of targeted polysulfide metabolomics) CysS n H, GS n H and hydrogen polysulfide (H2S nThe following were quantified (n=1-3 for each): Broccoli seeds, germinated seeds (day 1), and sprouts (days 2-5) were homogenized using a POLYTRON homogenizer (DIAX-100) with 20 times the volume of 50 mM sodium acetate buffer (pH 6.5) containing 1 mM standard TME-AM and 70% methanol. The homogenates were incubated at 37°C for 30 minutes, then centrifuged at 20,000 g at 4°C for 15 minutes. The resulting supernatant (100 μL) was collected, acidified with 10 μL of 10% FA, and mixed with a 100 nM stable isotope-labeled TME-AM adduct standard in 0.1% FA before LC-ESI-MS / MS analysis. The resulting TME-AM adducts were quantified by LC-ESI-MS / MS analysis using the stable isotope-labeled standard dilution method. Polysulfide concentrations were normalized by the dry weight of the sample.

[0022] (Synthesis and detection of SFN derivatives) CysS n H and GS n To investigate the possibility of H-SFN conjugate formation, 1.43 mM CysSH or GSH was incubated in 85.7 mM Tris-HCl buffer (pH 7.4) containing 4.29 mM Na2S2 in the dark at 37°C for 15 minutes. The resulting CysSnH and GSH were treated with 3 mM SFN at 37°C for 1 hour in the dark. The resulting mixtures were diluted 20-fold with 0.1% FA to stop the reaction, and then subjected to LC-ESI-MS / MS analysis.

[0023] For the preparation of SFN derivatives, 1.96 mM GSH or CysSH was incubated in 117 mM Tris-HCl buffer (pH 7.4) containing 5.88 mM Na2S2 at 37°C for 15 minutes in the dark. 15 μL of 33.3 mM SFN was added to the mixture (85 μL) and incubated at 37°C for 1 hour in the dark. The reaction was stopped by diluting the mixture 10-fold with 0.1% FA, and the synthesized SFN derivatives were separated by reversed-phase high-performance liquid chromatography (HPLC). HPLC was performed using a PU-2089 Plus (JASCO International Co., Tokyo, Japan) with a UV detector (UV-4075; JASCO International Co.) set to 250 nm. The reaction mixture was injected into a C18 reversed-phase column (COSMOSIL 5C18-AR-II 10.0 mm × 150 mm; Nacalai Tesque).

[0024] The samples were injected at a flow rate of 3 mL / min using a linear gradient of solvent A (water containing 0.1% FA) and solvent B (100% methanol) (gradient = 0% B / 0-4 min, 50% B / 45 min, and 100% B / 46 min). After concentrating the eluate under vacuum by centrifugation (175 g, 23°C), the samples were eluted using a C18 reversed-phase column (Mightysil RP-18GP 6.0 mm × 150 mm, Kanto Chemical) with solvent A (water containing 0.1% FA) and solvent B (100% methanol) (gradient = B=0% at 0-3 min, B=50% at 15 min, and B=100% at 15.1 min) at a flow rate of 3 mL / min. The fraction containing the SFN derivative was concentrated under vacuum by centrifugation (175 g at 23°C). After evaluating the purity of isolated SFN derivatives by LC-ESI-MS / MS analysis, the concentrations of purified SFN derivatives containing CysSnH and GSnH were determined by HPLC analysis, measuring the absorbance at 250 nm. The concentrations were then calculated from the standard curves for the true CysSH conjugate of SFN (SFN-SCys) and the GSH conjugate of SFN (SFN-SG, Toronto Research Chemicals, Ontario, Canada; 95%), respectively. n H and GS n The concentration of the purified SFN derivative containing H was determined by HPLC analysis.

[0025] To detect the in vivo production of SFN derivatives and oxidized GSH (n=3-5), broccoli seeds and sprouts (approximately 350 mg) were homogenized using a POLYTRON homogenizer (DIAX-100) with 10 times the volume of 50 mM sodium acetate buffer (pH 6.5) containing 0.1 mM normal TME-IAM and 70% methanol. After centrifugation at 20,000 g for 15 minutes at 4°C, the resulting supernatant (700 μL) was concentrated by centrifugation under vacuum (175 g at 23°C) until the sample volume was approximately 100 μL. After filtration through a centrifuge filter (Cosmospin), the filtrate was subjected to LC-ESI-MS / MS to detect SFN derivatives and GS. n We detected the in vivo production of G.

[0026] (Measurement of DPPH radical scavenging activity) DPPH stock solution was prepared in ethanol. In a 96-well plate, 100 μM DPPH dissolved in 12 mM sodium phosphate buffer (pH 7.4) was incubated at 25°C for 20 minutes, either in the presence or absence of the test substance. Absorbance at 517 nm was measured using an Infinite200PRO microplate reader (Tecan Trading, Mannedorf, Switzerland). Trolox solution (final concentration: 5–40 μM) was used to determine the standard curve. The radical scavenging ability of the samples was evaluated using the inhibition rate (%). The inhibition rate was calculated using the following formula.

number

[0027] (statistical analysis) Data are presented as the mean ± standard deviation (SD) of at least three independent experiments. All analyses were performed using GraphPad Prism software (GraphPad Software, La Jolla, CA). Statistical significance was defined as p<0.05.

[0028] (Comparison of polysulfide profiles of mature broccoli and broccoli sprouts) Total sulfur content (TSC) and total polysulfide content (TPsC) were measured for samples of mature broccoli, broccoli sprouts, cabbage, and garlic, and their polysulfide profiles (ratio of TPsC to TSC) were analyzed (Figure 3). Figure 3 shows a comparison of the polysulfide profiles of mature broccoli and broccoli sprouts. In (A), the total sulfur content (TSC) of commercially available vegetables (garlic, cabbage, broccoli, and broccoli sprouts) was quantified using an acid cyclic decomposition system and inductively coupled plasma atomic emission spectrometry. In (B), the total polysulfide content (TPsC) was measured using an alkaline / reducing sulfur removal protocol preceding high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS / MS) with stable isotope dilution. In (C), the ratio of TPsC to TSC was calculated from the values ​​shown in A and B. Data are presented as mean ± standard deviation (SD) (n=4-6). p<0.0001. Comparison with broccoli sprouts was performed using one-way ANOVA and Dunnett's multiple comparison test. dw is dry weight.

[0029] As shown in Figure 3(A), there was no significant difference in TSC between mature broccoli, cabbage, and garlic. On the other hand, the TSC of broccoli sprouts was significantly higher than that of mature broccoli, and in fact, broccoli sprouts showed the highest TPsC (Figure 3(B)). The TPsC of these vegetables accounted for 14.6%, 4.2%, 3.9%, and 2.4% of the TSC of the broccoli sprout, mature broccoli, garlic, and cabbage samples, respectively (Figure 3(C)). This is the first evidence that broccoli sprouts are richer in polysulfides than mature broccoli. Furthermore, the polysulfide profile of broccoli sprouts was compared with that of nine other vegetable sprouts (white radish, red cabbage, red radish, watercress, mustard greens, leeks, water spinach, alfalfa, and okra). Quantitative analysis revealed that broccoli sprouts had the highest TPsC value among vegetable sprouts, followed by red cabbage (8.2%) and mustard (4.9%) sprouts (Figure 4). Figure 4 shows a comparison of polysulfide profiles in vegetable sprouts. The polysulfide profiles of nine commercially available vegetable sprouts (white radish, red radish, red cabbage, watercress, mustard, leek, water spinach, alfalfa, and okra) were compared. The ratio of TPsC to TSC for the nine commercially available vegetable sprouts (white radish, red radish, red cabbage, watercress, mustard, leek, water spinach, and alfalfa) was calculated from the values ​​shown in A and B. Data are shown as mean ± SD (n=3-6). dw is dry weight.

[0030] The glucosinolate content and composition in plants can be influenced by the sulfur nutrient status in the soil used for cultivation. Previous studies have demonstrated that sulfur supplementation in cruciferous vegetables in greenhouse and field experiments can affect glucosinolate content by -25% to over +1200%. Therefore, changes in the polysulfide profile of broccoli during germination and growth are important for carefully considering the effects of germination and growth on the polysulfide profile of broccoli.

[0031] (Changes in polysulfides during broccoli sprout germination and growth) In the germination experiment, broccoli seeds were immersed in ultrapure water for 24 hours (Day 1), and then placed in culture trays containing ultrapure water in the dark for 24–96 hours (Days 2–5). As shown in Figure 1, the broccoli seeds germinated on Day 1, and the cotyledons elongated in a culture time-dependent manner up to Day 5. On Day 1, approximately 80% of the seeds consistently germinated. The polysulfide profiles of broccoli seeds, germinated seeds, and sprouts were compared (Figure 5). Figure 5 shows the changes in the polysulfide profile during the germination and growth process of broccoli sprouts. Broccoli seeds were immersed in ultrapure water for 24 hours (Day 1), and then the germinated seeds were placed in germination trays at a temperature of 24–25°C and cultured in the dark for a further 96 hours (Days 2–5). Seeds, germinated seeds, and broccoli sprouts were subjected to quantitative analysis for TSC(A) and TPsC(B), respectively. In (C), the ratio of TPsC to TSC was calculated from the values ​​shown in A and B. The data are shown as mean ± SD (n=4-6). ** p<0.01, **** p<0.0001, compared to seeds using one-way ANOVA with Dunnett's multiple comparison test. NS, not statistically significant.

[0032] As shown in Figure 5A, no significant changes were observed in TSC during germination. On the other hand, when broccoli seeds were immersed in ultrapure water for 24 hours (day 1), the TPsC of the seeds increased dramatically, and the TPsC continued to increase over time (Figure 5B). When the percentage of TPsC relative to TSC was calculated, it was found that TPsC accounted for 15.5% of the TSC of broccoli sprouts on day 5 (Figure 5(C)). While the glucosinolate content and composition in broccoli sprouts decrease and change compared to those in seeds, this invention provides the first evidence that the polysulfide content of broccoli increases significantly during germination and growth. In broccoliaceae vegetables, volatile polysulfides such as dimethyl trisulfide and dimethyl tetrasulfide may be produced by CS lyase using SMCSO as a substrate. Furthermore, cystine lyase purified from broccoli inflorescences can produce CysS2H through β-elimination of l-cystine and SMCSO.

[0033] (Non-targeted polysulfide omics analysis of broccoli sprouts) The inventors of this application have developed a non-targeted polysulfide omics analysis technique that can comprehensively detect hydropolysulfides by LC-ESI-MS / MS analysis. The inventors have demonstrated that chemical substances containing hydroxyphenyl or hydroxyl groups can stabilize polysulfide structures. They have also succeeded in synthesizing a novel alkylating agent, TME-IAM, that can stabilize polysulfide structures via hydroxyphenyl groups. Careful examination of the MS / MS fragmentation patterns of various TME-AM adduct standards revealed that collision-induced dissociation forms a common fragment ion with m / z 136 derived from the hydroxyphenyl group of the TME-AM moiety. Based on this characteristic of TME-AM adducts, a non-targeted polysulfide omics analysis technique was developed using two types of TME-IAM (normal and stable isotope-labeled) at different concentrations (the ratio of normal TME-IAM to stable isotope-labeled TME-IAM is 4:1) (Figure 6, A). This technology allows hydropolysulfides to be derivatized by TME-IAM to form stable adducts, and the relative signal intensity of the hydropolysulfide adduct with normal TME-IAM (light) and the hydropolysulfide adduct with stable isotope-labeled (heavy) is in the range of 2-4.

[0034] However, since TME-IAM reacts not only with hydropolysulfides but also with thiols, it is necessary to distinguish between signals derived from hydropolysulfides and signals derived from thiols. Because it contains a polysulfide structure, the TME-AM adduct of hydropolysulfide is susceptible to the effects of reducing agents. On the other hand, the TME-AM adduct of thiols does not have a polysulfide structure and is therefore less susceptible to the effects of reducing agents. For this reason, treatment with 2-mercaptoethanol, a reducing agent, made it possible to distinguish between the signals of hydropolysulfides and thiols. In fact, preliminary experiments using standards of GSH and glutathione hydrotrisulfide (GS3H) showed that both GSH and GS3H were derivatized with two types of TME-IAM, and the relative signal intensities of each adduct were in close agreement with the concentration ratio of normal TME-IAM and stable isotope-labeled adducts (Figure 6, B). Furthermore, the GS-adduct remained after treatment with 2-mercaptoethanol, but the GS3-adduct completely disappeared after incubation with a reducing agent (Figure 6, B). These results suggest that this new technique could be a useful tool for the comprehensive analysis of hydropolysulfides in biological samples.

[0035] Figure 6 shows a schematic diagram of the targetless polysulfide omics analysis. In (A), for non-targeted polysulfide omics, broccoli sprouts were homogenized in sodium acetate buffer containing 70% methanol with normal (pale, 0.8 mM) and stable isotope-labeled (heavy, 0.2 mM) N-iodoacetyl-L-tyrosine methyl ester (TME-IAM) (Step 1). After solid-phase extraction by reverse-phase column, the eluate was concentrated by centrifugation under vacuum (Step 2). The concentrate was incubated in the absence or presence of 2-mercaptoethanol (Step 3) and analyzed in MRM (multiple reaction monitoring) mode by LC-ESI-MS / MS to detect the common m / z 136 fragment ion from the TME-AM adduct (Step 4). In (B), representative MS / MS chromatograms of glutathione (GSH) and glutathione trisulfide (GS3H) are shown. The left image shows the GSH and GS3H adducts of normal TME-IAM, the center image shows the GSH and GS3H adducts of stable isotope-labeled TME-IAM, and the right image shows the GSH and GS3H adducts of normal TME-IAM after 2-mercaptoethanol treatment. An asterisk (*) indicates 13 This indicates a carbon-labeled atom.

[0036] Representative results from non-targeted polysulfide omics analysis of broccoli seeds and sprouts (days 2 and 5) are shown in Figures 7 and 8. The number of signals identified as polysulfide candidates increased in the order of seeds < day 2 sprouts < day 5 sprouts. In broccoli sprouts (day 5), 37 different signals were detected as candidate hydropolysulfides, including CysS2H, cysteine ​​hydrotrisulfide (CysS3H), cysteine ​​hydrotetrasulfide, GS2H, GS3H, and glutathione hydrotetrasulfide (Figure 8). Interestingly, the signal intensities of CysS2H, CysS3H, GS2H, and GS3H were 55, 107, 4, and 12 times, respectively, compared to their corresponding thiols (i.e., CysSH and GSH). In animals (humans and mice), GSH is the dominant thiol in vivo, and the level of GS2H is about 1 / 100th that of GSH. These results suggest that polysulfide biosynthesis is highly activated during the germination and growth of broccoli sprouts, and that polysulfides play an important physiological role in seed germination and growth.

[0037] Figure 7 shows representative results of non-targeted polysulfide-omics analysis of broccoli sprouts (day 2, A) and seeds (B). Figure 8 shows representative results of non-targeted polysulfide-omics analysis of broccoli sprouts (day 5). Glutathione and cysteine ​​hydropolysulfide (GS) n H, CysS n Thirty-seven signals, including H (n=2-4 for each), were detected as candidate hydropolysulfides. The grayscale of the MS / MS signals shows the signal intensity normalized as counts per 1 mg of dry weight (dw). CysSH is cysteine, CysS2H is cysteine ​​hydropersulfide, CysS3H is cysteine ​​hydrotrisulfide, CysS4H is cysteine ​​hydrotetrasulfide, GS2H is glutathione hydropersulfide, and GS4H is glutathione hydrotetrasulfide.

[0038] (endogenous CysS nH, GS n H, and H2S n (Quantitative determination) To carefully evaluate the effect of germination on the production of endogenous polysulfides such as CysS2H and GS2H, quantitative targeted polysulfide metabolome analysis was performed using LC-ESI-MS / MS with stable isotope dilution (Figure 9). This method allowed us to evaluate CysS n H, GS n H, H2S n (Each n=1-5) can be detected specifically and quantitatively. Quantitative analysis revealed that in broccoli sprouts, CysSH, CysS2H, CysS3H, GS2H, GS3H, hydrogen sulfide (H2S), hydrogen disulfide (H2S2), and hydrogen trisulfide (H2S3) were significantly increased on day 5 compared to seeds. Compared to those in seeds, H2S3 was the most abundant of the nine polysulfides and thiols analyzed, and the major organic polysulfide metabolites in broccoli sprouts were CysS2H and CysS3H (Figure 9, B, C, I). On the other hand, no significant changes in GSH content were observed during the germination and growth process of broccoli sprouts (Figure 9, D).

[0039] GS2H has been demonstrated to be the major polysulfide in animal tissues and cells. The results indicate that CysS2H and CysS3H are the major organic polysulfide metabolites in broccoli sprouts. CysS2H and CysS3H are produced by cysteinyl-tRNA synthetase using CysSH as a substrate. Furthermore, cystine lyase purified from broccoli inflorescences can produce CysS2H via the β-elimination reaction of l-cystine and SMCSO. Therefore, these enzymes are highly activated during broccoli sprout germination and growth, potentially leading to a significant increase in CysS2H and CysS3H production.

[0040] Figure 9 shows CysS n H and GS n The quantitative analysis results for H are shown. GS in seeds, germinated seeds, and broccoli sprouts. nH, CysS n H and hydrogen polysulfide (H2S n The endogenous levels of ) were measured by quantitative polysulfide metabolomics using LC-ESI-MS / MS. Data are shown as mean ± SD (n=3). *** p<0.001, **** The results show a comparison with seeds, with p<0.0001, and a one-way ANOVA with Dunnett's multiple comparison test.

[0041] GS n G(n=3-5) is well-established to be endogenously produced in various organisms, including humans, mice, and yeast, but little is known about this process in plants. n To detect the endogenous production of GS, broccoli seeds and sprouts were homogenized in the presence of TME-IAM. TME-IAM can convert hydropolysulfide to a stable adduct without artificial degradation via its iodoacetyl group, and stabilize the polysulfide structure by inhibiting hydrolysis via its hydroxyphenyl group, thus minimizing the artificial formation of polysulfide oxidative derivatives during sample preparation. Endogenous GS is shown in Figure 10. n G(n=3-5) was detected in broccoli seeds and sprouts, and GS n G levels were significantly higher in broccoli sprouts than in seeds.

[0042] Figure 10 shows the endogenous bioeffects of oxidized glutathione polysulfide. Seeds, germinated seeds, and broccoli sprouts were subjected to LC-ESI-MS / MS analysis to determine the levels of oxidized glutathione polysulfide (GS). n G (n=3-5) was detected. n A typical MS / MS chromatogram of G is shown in Figure 7, as previously described. The data is shown as mean ± SD (n=3). Figure 10 shows * p<0.05, *** p<0.001, ****The following comparisons are shown using one-way ANOVA with p<0.0001, comparing with seeds, and using Dunnett's multiple comparison test. GS3G is oxidized glutathione trisulfide, GS4G is oxidized glutathione tetrasulfide, and GS5G is oxidized glutathione pentasulfide.

[0043] Endogenous and genuine GS n Representative MS / MS chromatograms of G are shown in Figure 11. Figure 11 shows representative MS / MS chromatograms of true and endogenous oxidized glutathione polysulfide. Arrows indicate the signals of each oxidized glutathione polysulfide (GSnG, n=3-5) detected in true standards (true) and 5-day-old broccoli sprouts (endogenous). This shows various GS in broccoli seeds and sprouts. n This is the first evidence regarding the endogenous production of the G type. Oxidized glutathione trisulfide (GS3G) acts as an excellent radical scavenger in the presence of GSH, forming GS2H. Furthermore, H2S2 released from O-silyl mercaptan-based H2S donors can reduce the true radical DPPH more effectively than H2S released from O-silyl mercaptan-based H2S donors. Sodium polysulfide (NaS n (n=-4) exhibits potent neuroprotective effects against oxidative stress-induced toxicity in midbrain dopaminergic neurons administered with 1-methyl-4-phenylpyridinium ions, cerebellar granule cells administered with methylmercury, and neuroblastoma cells administered with methylglyoxal or tert-butyl hydroperoxide. Therefore, GS, which is abundant in broccoli sprouts, is effective. n H, CysS n H, H2S n GS n G may contribute to a wide range of biological activities, including antioxidant effects.

[0044] Accumulated evidence shows that numerous proteins exhibit endogenous polysulfidation in both prokaryotes and eukaryotes (including mammals and plants). The inventors developed a simple, reliable, and reproducible assay—a polyethylene glycol-conjugated maleimide-labeled gel shift assay—to detect protein polysulfides. By using a weak electrophile, such as 8-nitroguanosine 3′,5′-cyclic monophosphate, as the first step, both the thiol and polysulfide groups of proteins can be alkylated while minimizing artificial degradation of the polysulfide. Biotin-polyethylene glycol 36 -Maleimide (BPM) reacts with polysulfide-containing cysteine ​​residues, resulting in an upward shift of the band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0045] Therefore, this assay can reveal the degree of protein polysulfidation as a change in the mobility of SDS-PAGE bands, demonstrating its suitability for investigating the degree of polysulfidation of individual proteins. However, it is not suitable for comprehensively analyzing the state of protein polysulfidation. Therefore, to detect variations in polysulfidated proteins in broccoli seeds and sprouts, biotin-maleimide was used instead of BPM to minimize changes in SDS-PAGE band mobility (Figure 12). As shown in Figure 12, A, a strong signal of approximately 50 kDa was detected in seeds and germinated seeds (day 1), while only a faint signal was observed in broccoli sprouts (days 2-5). On the other hand, coma brilliant blue staining following SDS-PAGE revealed a dramatic change in protein expression patterns during germination and growth (Figure 12, B). These results suggest that polysulfide-rich proteins may be a potential source for the biosynthesis of proteins and polysulfides.

[0046] Figure 12 shows the protein expression profiles (A) and polysulfide status (B) in broccoli seeds and sprouts. (A) is a representative image for the detection of polysulfide proteins. Broccoli seeds (S), germinated seeds (Day 1, D1), and sprouts (Days 2-5, D2-5) are homogenized in an UltraTurrax homogenizer (Model: T10) containing either 1 mM biotin-maleimide (BM, ≥95%, Sigma-Aldrich) or 1 mM N-iodoacetyl-l-tyrosine methyl ester (TME-IAM), along with 1 mM biotin-maleimide (BM, ≥95%, Sigma-Aldrich) containing 1 mM biotin-maleimide (BM, ≥95%, Sigma-Aldrich) or 1 mM N-iodoacetyl-l-tyrosine methyl ester (TME-IAM). The protein concentration was adjusted to 3 mg / mL using basic (IKA, Osaka, Japan). After centrifugation at 20,380 g at 4°C for 15 minutes, the supernatant was incubated at 37°C for 30 minutes, diluted 2-fold with lysis buffer containing 3 mM BM, and incubated further at 37°C for 1 hour.

[0047] Proteins were denatured by boiling in 1x Laemmli sample buffer for 5 minutes, separated by SDS-PAGE using a 12% polyacrylamide gel, and biotinylated proteins were detected by Western blotting (WB) with horseradish peroxidase-conjugated streptavidin (Invitrogen, Carlsbad, CA), or all proteins were visualized by coma brilliant blue (CBB) staining. (B) is a representative image of CBB staining of protein expression profiling in broccoli seeds, germinated seeds, and sprouts. Broccoli seeds and sprouts were homogenized with 20 times the volume of lysis buffer in an Ultra Turks homogenizer. After centrifugation at 20,380 g for 30 minutes at 10°C, the protein concentration of the supernatant was measured using a bicinhontinic acid kit. After boiling in 1x Laemmli sample buffer for 5 minutes, proteins were separated by SDS-PAGE using a 12% polyacrylamide gel in or without 5% (v / v) 2-mercaptoethanol (2-ME), followed by CBB staining.

[0048] (CysS n H and Gs n Identification of the SFN complex of H and its potent radical scavenging ability. Mature broccoli and broccoli sprouts are known to be rich in SFN, which is produced when glucoraphanin is hydrolyzed by myrosinase. The central carbon atom of the isothiocyanate group of SFN is highly electrophilic and readily reacts with GSH and CysSH to form SFN-SG and SFN-SCys complexes, respectively. The pKa of GS2H is 5.45, which is 3.49 lower than the pKa of GSH. Furthermore, because GS2H has increased anion availability, it reacts faster than GSH with electrophiles such as peroxynitrite and hydrogen peroxide (H2O2), and its reactivity is increased depending on the electrophile. Therefore, in broccoli sprouts, SFN and GS n H or CysS n A reaction occurs with H, and SFN's GS n H or CysS nH-joint (SFN-S each) n G or SFN-S n Cys) may form.

[0049] To verify this prediction, we first need to compare GS data with SFN. n H or CysS n In vitro experiments were performed using H, and then LC-ESI-MS / MS was performed to analyze the reaction products of the mixture. As shown in Figure 14, SFN-S n Cys(n=1-5) and SFN-S n G(n=1-4) was generated in vitro. Next, the endogenous generation of these SFN derivatives in broccoli seeds and sprouts was analyzed (Figure 13). Figure 14 shows representative MS / MS chromatograms and MS / MS spectra (SFN-S) of true and endogenous sulforaphane (SFN) with glutathione or cysteine ​​hydropolysulfide. n G and SFN-S n The arrows indicate the common SFN and the respective SFN-S detected in the standard (Authentic) and 5-day-old broccoli sprouts (Endogenous), respectively. n G(n=1-4, A) and SFN-S n This shows the signal for Cys(n=1-5, B).

[0050] Figure 13 shows the sulforaphane complex GS. n H and CysS n The endogenous production results of H are shown. Seeds, germinated seeds, and broccoli sprouts were subjected to LC-ESI-MS / MS analysis to obtain sulforaphane (SFN, A) and GS. n H conjugate (SFN-S n G, B) and CysS n H conjugate (SFN-S n Cys (C) was detected. Representative MS / MS chromatograms and MS / MS spectra of SFN and its conjugates detected in broccoli sprouts (day 5) are shown in Figures 14 and 15. Data are shown as mean ± SD (n=3). *p < 0.05, *** p < 0.001, **** p < 0.0001, and the comparison was made by one-way ANOVA using Dunnett's multiple comparison test for comparison with the seeds.

[0051] Figure 15 shows the endogenous SFN-S detected in broccoli sprouts (day 5) n Cys (n = 1 - 5) and SFN-S n representative MS / MS spectra of G (n = 1 - 4), which are consistent with the standards. Figure 15 shows the representative MS / MS spectra and chemical structures assigned to endogenous SFN-SnG and SFN-SnCys. Representative MS / MS spectra and chemical structures of SFN (A), SFN-SnG (n = 1 - 4, B - E), and SFN-SnCys (n = 1 - 5, F - J) detected in broccoli sprouts on day 5 are shown on the left and right, respectively. The cleavage sites are indicated by dashed lines. By LC-ESI-MS / MS analysis, endogenous generation of SFN-S n Cys (n = 1 - 5) and SFN-S n G was detected, and a time-dependent increase in SFN-SnCys (n = 2 - 5) was observed (Figure 13, G - J). This correlates with the temporal changes in the SFN signal and CysSH content (Figure 16). Figure 16 shows the correlation analysis between SFN-SnCys levels and SFN levels (A - E) or CysSH content (F - J). The linearity (R2) was 0.5716 (A), 0.9202 (B), 0.9377 (C), 0.8470 (D), 0.8939 (E), 0.6809 (F), 0.6073 (G), 0.6332 (H), 0.4993 (I), and 0.5570 (J), respectively.

[0052] SFN-SnCys and SFN-S n To detect the in vivo production of G, broccoli seeds and sprouts were homogenized in the presence of TME-IAM. This captures hydroropolysulfides to form stable TME-IAM adducts, thus SFN-S n Cys and SFN-S nArtificial formation of G was minimized. In fact, it was confirmed that SFN does not interfere with the reactivity of TME-IAM with CysS n H (Figure 17). This suggests that both oxidized polysulfides such as SFN derivatives and GS n G and hydropolysulfides can be accurately detected with minimal artificial changes by using TME-IAM. Therefore, SFN-S n Cys and SFN-S n G detected in broccoli seeds and sprouts are likely to be endogenously produced. Figure 17 shows the effect of SFN on the reactivity of TME-IAM with polysulfides. Cysteine hydropolysulfides (CysS n H) containing CysS2H and CysS3H, prepared by incubating 10 μM cysteine and 10 μM Na2S2 at 37 °C for 5 h, were reacted with 1 mM TME-IAM. The reaction was carried out in 50 mM sodium acetate buffer (pH 6.5) in the presence (black) or absence (white) of 10 μM SFN at 37 °C for 30 min. Then, the reaction mixture was subjected to LC-ESI-MS / MS analysis to quantitatively detect each adduct of CysS n H (n = 1 - 3) and H2S n (n = 1, 2). Data are shown as mean ± SD (n = 4).

[0053] SFN is known to act as a potent inducer of phase II detoxification enzymes such as NAD(P)H:quinone oxidoreductase 1 and glutathione S-transferase by activating the Keap1 / Nrf2 pathway. Furthermore, while SFN can directly scavenge radicals, studies using DPPH radical scavenging assays have shown that SFN's radical scavenging ability is far weaker than that of ascorbic acid. The free radical scavenging activity of broccoli seed extract does not correlate with the SFN content in the seeds. This indicates that SFN is not the major antioxidant component of broccoli seeds. On the other hand, glutathione sulfatase (GS2H) has a higher hydrogen peroxide (H2O2) scavenging ability than glutathione (GSH). Therefore, SFN is more effective than GSH or CysS n When combined with H, it is possible that it will exhibit higher radical scavenging ability than SFN.

[0054] To test this hypothesis, SFN-S n G and SFN-S n A DPPH radical scavenging assay was performed using Cys standards. Various SFN-S reactions occurred due to the chemical reaction between SFN and GSH or CysSH. n G(n=1~4) and SFN-S n We confirmed that Cys(n=1~5) is generated (Figures 14 and 15). However, SFN derivatives with three or more polysulfide chains (n>3) are easily degraded during HPLC purification, so SFN-S with n=1 or 2 is used. n G and SFN-S n We successfully isolated only Cys. As shown in Figure 18, the isolated SFN-SG, SFN-S2G, SFN-SCys, and SFN-S2Cys were detected as protonated molecules at m / z 485.1193, 517.0914, 299.0552, and 331.0270, respectively. The assignment of product ions also revealed that these novel SFN derivatives were successfully synthesized (Figure 18).

[0055] Figure 18 shows SFN-S obtained by high-resolution mass spectrometry. nG and SFN-S n This shows the synthesis and purification of Cys. SFN-S n G and SFN-S n Cys (n=1 or 2 each) were synthesized and purified using liquid chromatography-electrospray ionization-quadrupole-time-of-flight (LC-ESI-Q-TOF)-tandem mass spectrometry (G6545XT; Agilent Technologies, Tokyo, Japan) and Agilent 1260 Infinity II Prime LC systems (G7104C, G7129C, G7130A, G7117C, and G1310A, Agilent Technologies). Samples were separated using a reversed-phase Mightysil RP-18 GP column (50 mm length × 2.0 mm inner diameter, Kanto Chemical Co., Ltd., Tokyo, Japan) with mobile phases A (0.1% formic acid) and B (methanol), applying a linear gradient from 1% to 99% B over 2–7 minutes. The flow rate was 0.6 mL / min. The ESI-Q-TOF instrument was operated in positive ion mode with a scan speed of 3 spectra / second, using a m / z scan range of 100-1000 in MS mode and 50-1000 in MS / MS mode. For collision-induced dissociation, the collision energy was set in the range of 10-40V. A Q-TOF-MA was used. Vcap: 4000V, nebulizer pressure: 55psi, dry gas flow rate: 11L / min, gas temperature: 350℃, fragmenter: 125V, skimmer: 65V, octopole RF peak: 750V. Representative MS chromatograms, MS / MS spectra, and chemical structures of SFN-SCys, SFN-S2Cys, SFN-SG, and SFN-S2G are shown on the left, center, and right, respectively. Cleavage sites are indicated by dashed lines.

[0056] The endogenous polysulfides in broccoli sprouts are sulforaphane (1-isothiocyanato-4-[methylsulfinyl]butane (SFN)) and cysteine ​​persulfide (CysS n H, n=2,3,4, or 5), or glutathione persulfide (GS n H, or GS nG, n=2,3,4, or 5) is bonded to a novel SFN derivative. For example, it is one of the following:

[0057] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0058] As shown in Figure 19, when a DPPH radical scavenging activity assay was performed, no significant radical scavenging ability of SFN was observed in the concentration range examined (100-1000 μM). On the other hand, the SFN-GSH conjugates showed radical scavenging ability in the order of SFN-S2G ≥ GSH > SFN-SG >> SFN = oxidized glutathione disulfide. Similarly, SFN and CysS n The order of radical scavenging ability in the H conjugates was SFN-S2Cys > SFN-SCys ≈ CysSH >> SFN = Cystine.

[0059] Figure 19 shows the radical scavenging ability of SFN derivatives. The radical scavenging ability of SFN derivatives was evaluated by the 1,1-diphenyl-2-picrylhydrazyl radical scavenging assay. The data are expressed as mean ± SD (n=3). * p<0.05, *** p<0.001, ****p<0.0001 SFN pair; ++p<0.01, ++++p<0.0001 corresponding thiol pair; compared with the corresponding hydropersulfide SFN conjugate $$$$ p<0.0001, compared with SFN-SCys qqqq Comparison was performed using one-way ANOVA with Tukey's multiple comparison test, with p<0.0001. GS2G has antioxidant capacity equivalent to oxidized glutathione disulfide; TEAC, or Trolox.

[0060] These results suggest that the novel SFN derivatives function as potent antioxidants. Furthermore, SFN treatment strongly induces the expression of UDP-glucuronosyltransferase 1A1 and glutathione S-transferase A1 in HePG2 cells. Similarly, SFN-SG can also induce phase II detoxification enzymes, and it has been shown that the activity of SFN does not change significantly even when SFN is conjugated with GSH to form dithiocarbamate. Therefore, the novel conjugates of SFN with GSnH or CysSnH suggest that SFN and GSnH can interact in vivo. n H or CysS n This may exhibit multiple biological functions stemming from potential crosstalk with H.

[0061] Although various embodiments have been described above, the present invention is not limited to these. Other embodiments conceivable within the scope of the technical idea of ​​the present invention are also included within the scope of the present invention.

Claims

1. Sulforaphane (1-isothiocyanato-4-[methylsulfinyl]butane (SFN)) and cysteine ​​persulfide (CysS ｎ H, n=2,3,4, or 5), or glutathione persulfide (GS ｎ H, or GS ｎ A novel SFN derivative consisting of a complex with G, n = 2, 3, 4, or 5.

2. It consists of a composite of the aforementioned SFN and the aforementioned cysteine ​​persulfide, Having the structural formula shown in Chemical Formula 1 below, 【Chemistry 1】 n = 2, 3, 4, or 5 A novel SFN derivative according to claim 1.

3. The aforementioned complex has the structural formula shown in Chemical Formula 2 below. 【Chemistry 2】 n = 2, 3, 4, or 5 A novel SFN derivative according to claim 1.

4. The composite is at least one of the following chemical formulas 3, 4, and 5. 【Transformation 3】 【Chemistry 4】 【Transformation 5】 A novel SFN derivative according to claim 3.

5. The composite is at least one of the following chemical formulas 6, 7, and 8. 【Transformation 6】 【Transformation 7】 【Transformation 8】 A novel SFN derivative according to claim 2.

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