Novel sulforaphane derivative

Abstract

The present invention is characterized by being a novel sulforaphane derivative comprising a complex of sulforaphane (1-isothiocyanato-4-[methylsulfinyl]butane (SFN)) and cysteine persulfide (CysSnH, n=2, 3, 4, or 5) or glutathione persulfide (GSnH or GSnG, n=2, 3, 4, or 5).

Description

Novel sulforaphane derivatives

[0001] The present invention relates to novel polysulfides, and more particularly to novel sulforaphane derivatives inherent in broccoli sprouts.

[0002] In recent years, the biological importance of polysulfides (RSnR, n>2, R=hydrogen or alkyl) has been attracting attention. For example, hydropolysulfides (RSnR) containing cysteine ​​and glutathione have been reported. ｎ H, n>2, R=alkyl) is cysteine ​​hydropersulfide (CysS ２ H) and glutathione hydropersulfide (GS ２ It has been demonstrated that endogenous cysteinyl-tRNA synthetases, such as CysH, are produced in vivo by enzymatic and / or chemical transsulfuration reactions. Recent studies have reported that recombinant cysteinyl-tRNA synthetases from Escherichia coli as well as mammals such as humans and mice can catalyze the formation of CysS2H from cysteine ​​(CysSH) used as a substrate. Furthermore, it has been revealed that these bioactive substances function as potent antioxidants and play important roles in multiple cellular processes, such as mitochondrial biogenesis and metabolic regulation. CysS ２ H and GS ２ A significant decrease in the endogenous production of H-containing polysulfides has 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 polysulfide-containing supersulfur molecules function as powerful antioxidants and as important regulators of redox signals in vivo.

[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, recent studies have shown that cysteine ​​hydroperoxide (CysS) is a cytotoxic hormone in mammals, including humans. ２H) and glutathione hydroperoxide (GS ２ It has been reported that polysulfides such as broccoli (H) are endogenously produced, and these bioactive compounds function as potent antioxidants and important regulators of redox signaling in vivo. However, few studies have focused on the endogenous polysulfide content of broccoli or the effect of germination on the polysulfide content and composition of broccoli.

[0004] "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

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

[0006] In order to achieve the above object, the present invention provides a method for the preparation of a compound having a structure comprising 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 G, n=2, 3, 4, or 5).

[0007] According to the present invention, novel polysulfides useful to humans can be provided based on research into endogenous polysulfides in broccoli.

[0008] Figure 1 shows photographs and graphs illustrating broccoli sprout germination. Figure 2 shows a graph illustrating multiple heating / cooling cycles in an acid-cycling digestion system. Figure 3 shows a graph comparing the polysulfide profiles of mature broccoli and broccoli sprouts. Figure 4 shows a graph comparing the polysulfide profiles of vegetable sprouts. Figure 5 shows a graph illustrating changes in the polysulfide profile during broccoli sprout germination and growth. Figure 6 shows a schematic diagram of targetless polysulfide omics analysis. Figure 7 shows representative results of untargeted polysulfide omics analysis of broccoli sprouts (day 2, A) and seeds (B). Figure 8 shows representative results of untargeted polysulfide omics analysis of broccoli sprouts (day 5). Figure 9 shows the results of quantitative analysis of CysSnH and GSnH. Figure 10 is a graph showing the endogenous bioavailability of oxidized glutathione polysulfides. Figure 11 is a representative MS / MS chromatogram of authentic and endogenous oxidized glutathione polysulfides. Figure 12 shows the protein expression profile (A) and polysulfidation status (B) in broccoli seeds and sprouts. Figure 13 shows the sulforaphane conjugate GS. ｎ H and CysS ｎ Figure 14 shows representative MS / MS chromatograms of authentic and endogenous sulforaphane (SFN) with glutathione or cysteine ​​hydropolysulfide. Figure 15 shows the endogenous SFN-S detected in broccoli sprouts (day 5). ｎ Cys (n=1-5) and SFN-S ｎ 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 is a graph showing the effect of SFN on the reactivity of TME-IAM toward polysulfides. Figure 18 shows the synthesis and purification of SFN-SnG and SFN-SnCys by high-resolution mass spectrometry. Figure 19 is a graph showing the radical scavenging ability of sulforaphane derivatives.

[0009] This study is based on the investigation of changes in polysulfide biosynthesis in broccoli during germination, using untargeted 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 also performed to clarify the antioxidant properties of polysulfides. The results revealed that the total polysulfide content of broccoli sprouts significantly increased during germination and growth. Cysteine ​​S-2 hydroxylase and the trisulfide cysteine ​​were the major components of organic polysulfide metabolites. Furthermore, CysS ｎ H or GS ｎ A novel sulforaphane (SFN) derivative bound to H is endogenously produced in broccoli sprouts. ｎ We found that novel SFN derivatives bound to H exhibited higher radical scavenging activity than SFN and cysteine. These results suggest that the polysulfides abundant in broccoli sprouts contribute to their health-promoting effects. This discovery provides important biological implications for the development of new pharmacological targets related to the health-promoting effects of broccoli sprouts.

[0010] High intakes 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), have been shown to reduce the risk of cardiometabolic diseases, neurological disorders, diabetes, and cancer. Cruciferous vegetables contain a variety of phytochemicals, including phenolics, carotenoids, and glucosinolates, as well as their breakdown products, such as isothiocyanates and indoles. Secondary metabolites, particularly glucosinolates, synthesized by various species of the Brassicaceae family distinguish the Brassicaceae from other plant families by imparting a distinctive flavor and providing various medicinal properties. Glucoraphanin is the major glucosinolate found in broccoli, and hydrolysis by β-thioglucosidase (myrosinase) produces the bioactive isothiocyanate sulforaphane (SFN, 1-isothiocyanato-4-[methylsulfinyl]butane). 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 diallyl trisulfide, diallyl tetrasulfide, and dimethyl trisulfide, are lipophilic polysulfides found in the essential oils of garlic (Allium sativum) and onion (A. cepa) and have anti-inflammatory, cardioprotective, and cancer-preventing properties. 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). Cruciferous vegetables, such as broccoli and white cabbage, also contain SMCSO. Although the concentration of SMCSO in cruciferous vegetables is higher than that of glucosinolates, the health-promoting effects of this compound have received little attention due to the complexity of its metabolism. Although dimethyl trisulfide is also found in mature broccoli and broccoli sprouts, no studies have focused on the content and composition of polysulfides in broccoli or the effect of germination on endogenous polysulfide production. The present inventors investigated the polysulfide content endogenous to broccoli and the effect of germination on polysulfide content and composition. First, we measured the total polysulfide content (TPsC) and total sulfur content (TSC) of broccoli sprouts and mature broccoli, and analyzed their polysulfide profiles (TPsC / TSC). Therefore, we performed a technology to comprehensively detect hydropolysulfides in broccoli sprouts, i.e., untargeted polysulfide omics analysis, using a novel alkylating agent, N-iodoacetyl-L-tyrosine methyl ester (TME-IAM). This alkylating agent can derivatize hydropolysulfides into stable adducts with minimal artificial degradation.

[0012] (Materials) CysSH (98%), cystine (95%), glutathione (GSH, 97%), glutathione disulfide (GS ２G, 95%) was purchased from Fujifilm Wako Pure Chemical Industries (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 Co., Ltd. (Tokyo, Japan). Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) and sodium disulfide (Na ２ S ２ ) were purchased from Merck (Darmstadt, Germany) and Dojindo Laboratories (Kumamoto, Japan), respectively. ３４ S-NEM-d １０ The stable isotope-labeled standard of NEM (NEM-d ５ ) and sodium sulfide. Standards of TME-IAM and its stable isotope-labeled adducts were synthesized. Stable isotope-labeled TME-[ １３ C ２ ]IAM also uses [ １３ C ２ ] was synthesized using iodoacetic acid (99% pure, Sigma-Aldrich, Burlington, MA). Broccoli seeds were purchased from Nakahara Seeds (Fukuoka, Japan). All other chemicals and reagents were purchased from general chemical suppliers and were of the highest commercially available grade.

[0013] (Vegetables) Domestically grown mature vegetables (broccoli, cabbage, garlic) and sprouts (daikon radish, red radish (R. sativus var. sativus), red cabbage (B. oleracea var. capitata f. rubra), watercress, 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] For broccoli seed germination and growth experiments, broccoli seeds (approximately 1 g) were soaked in ultrapure water for 24 hours in the dark at room temperature (24-25°C) (day 1). The seeds were then placed in a 12.5 cm diameter tray (Kitchen Farm-120, W125 x D125 x H77 mm, Yamato Plastic Co., Ltd., Nara Prefecture, Japan). The tray was placed on top of another tray containing ultrapure water and maintained at saturated humidity at room temperature (24-25°C). The water level in the bottom tray was approximately 15 cm, with only the roots of the sprouts submerged in water. Germinated sprouts were harvested every 24 hours (days 2-5), crushed in 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 broccoli sprout germination. A: Images of broccoli seeds, germinated seeds (day 1), and sprouts (days 2-5) used in the analysis. B: Length of broccoli seeds, germinated seeds (day 1), and sprouts (days 2-5). C: Fresh weight of broccoli seeds, germinated seeds (day 1), and sprouts (days 2-5). Data are shown as mean ± standard deviation (SD) (n = 20). ＊＊ p<0.01, ＊＊＊＊ *p<0.0001, compared with seeds, one-way ANOVA with Dunnett's multiple comparison test. NS, not significant. To measure the moisture content of vegetables, fresh vegetable samples (approximately 1 g) were placed in a Uniblock moisture meter (MOC63u; Shimadzu Corporation, Kyoto, Japan), and the dry weight was measured to calculate the moisture content (%).

[0015] (Quantification of Total Sulfur Content (TSC) by Acid Cycling Decomposition-Inductively Coupled Plasma Optical Emission Spectroscopy) Total sulfur content was quantified by converting all sulfur-containing molecules to sulfate ions using an acid cycling decomposition system (ECOPRE system, Agilent Technologies), and measuring the sulfate ion concentration using inductively coupled plasma optical emission spectroscopy (ICP-OES). Approximately 100 mg of each vegetable sample was mixed with 15.8 M aqueous nitric acid solution (10 mL) in an acid cycling decomposition apparatus (ACTAC), which was then combined with a concentrator (ACTAC) containing 5% (v / v) nitric acid (5 mL). The samples were then decomposed by repeated heating and cooling cycles on a graphite hot plate (AS ONE Co., Osaka, Japan), as shown in Figure 2 . After cooling to 25°C, the samples were collected and diluted to 30 mL with 1 M nitric acid solution. The samples were then further diluted with 1 M nitric acid solution to concentrations of 1 to 10 ppm. Ultrapure water containing no 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 concentrations in the vegetable samples were measured using an ICP-OES (ICPE-9000, Shimadzu Corporation, Kyoto, Japan) and quantified using a standard curve prepared from a sulfate ion standard solution (Kanto Chemical, Tokyo, Japan). The ICP-OES measurement conditions were as follows: argon gas pressure: 450 ± 10 kPa, radio frequency output: 1.2 kW, plasma gas flow rate: 14 L / min, carrier gas flow rate: 0.7 L / min, irradiation time: 30 s, and observation wavelengths: 180.731, 182.037, and 182.625 nm.

[0016] Quantification of TPsC using an alkaline / reducing sulfur removal protocol. Vegetable samples were treated with the reducing agent DTT at alkaline pH to decompose polysulfides. The freed sulfur atoms were captured by the alkylating agent 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) coupled with a stable isotope-labeled standard dilution method. Approximately 100 mg of vegetable samples were homogenized in a 20-fold volume of 100 mM N-cyclohexyl-3-aminopropanesulfonic acid-NaOH buffer solution (pH 11.0) containing 10 mM DTT and 25% (v / v) ethanol using an ULTRA-TURRAX homogenizer (T10 basic; IKA, Osaka, Japan). Thereafter, sonication was performed for 15 seconds (repeated three times at 30-second intervals), and the homogenate was incubated at 37°C for 1 hour in a heat block incubator (NICHIHIRO, Saitama, Japan).

[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 at 37°C for 1 hour in a heat block incubator. After centrifugation at 15,000 g and 4°C for 15 minutes, the resulting supernatant (10 μL) was diluted with 0.1 μM NEM- ３４ S-NEM-d １０ The resulting NEM adducts were detected by LC-ESI-MS / MS analysis using a modified version of the stable isotope-labeled standard dilution method. A buffer blank containing no vegetables was prepared, and the signal detected from the buffer blank sample was subtracted from the signal obtained from each sample.

[0018] 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 on a Mightysil-C18 column (50 mm x 2.0 mm i.d., Kanto Chemical Co.) using the Shimadzu LC-20A system and eluted with a linear gradient of methanol as the mobile phase. For the detection of NEM-S-NEM, 1% B was observed at 0-1 min, 99% B at 4-5 min, and 1% B at 5.1-8 min; for untargeted polysulfide omics analysis, 1% B was observed at 0 min, 99% B at 10-12 min, and 1% B at 13-18 min; for quantitative targeted polysulfide metabolomics and the analysis of sulforaphane derivatives and oxidized glutathione polysulfides (GS ｎ For detection of G), the adducts were detected at 1% B from 0-1 min, 99% B from 7-10 min, and 1% B from 10.1-15 min. The mobile phase contained 0.1% FA, the flow rate was 0.6 mL / min, and the temperature was 40°C. The mass spectrometer was operated in positive ion mode, with the capillary voltage and desolvation gas (nitrogen) set at 1000 V and 1000 L / h, respectively, and the temperature was 500°C. Each adduct was detected in multiple reaction monitoring mode using the parameters shown in Table 1.

[0019]

[0020] (Untargeted Omics Analysis of Polysulfides) Approximately 1 g of 2-day-old and 5-day-old broccoli seeds and sprouts was homogenized in 50 mM sodium acetate buffer (pH 6.5) using a POLYTRON homogenizer (DIAX-100; Heidolph Instruments, Schwabach, Germany) with a mixture of standard TME-IAM (lightweight, 0.8 mM), stable isotope-labeled TME-[ １３ C ２The homogenate contained 100% FA (0.2 mM weight), IAM (0.2 mM weight), 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 for 15 minutes at 4°C. The resulting supernatant was collected, diluted with 5 volumes of 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) and then with ultrapure water for 5 CVs. 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) to a sample volume of approximately 0.5-0.6 mL. A portion of the concentrate was incubated in 100 mM Tris-HCl buffer (pH 7.4) with or without 1% (v / v) 2-mercaptoethanol at 37°C for 30 minutes. After filtration through a centrifugal filter (Cosmos Spin Filter, Nacalai Tesque), the filtrate was subjected to LC-ESI-MS / MS for untargeted polysulfide omics analysis. Polysulfide candidates with an intensity at least one-tenth that of GSH were considered to have significant signals.

[0021] (Quantification of targeted polysulfide metabolomics) CysS ｎ H, G.S. ｎ H, and hydrogen polysulfides (H ２ S ｎ) (n = 1-3 for each) were quantified. Broccoli seeds, germinated seeds (day 1), and sprouts (days 2-5) were homogenized using a POLYTRON homogenizer (DIAX-100) in 20 volumes of 50 mM sodium acetate buffer (pH 6.5) containing 1 mM normal TME-IAM and 70% methanol. The homogenate was incubated at 37°C for 30 minutes and then centrifuged at 20,000 g for 15 minutes at 4°C. The resulting supernatant (100 μL) was collected, acidified with 10 μL of 10% FA, and mixed with 100 nM stable isotope-labeled TME-AM adduct standards in 0.1% FA prior to 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 to the dry weight of the sample.

[0022] (Synthesis and detection of sulforaphane derivatives) CysS ｎ H and GS ｎ To investigate the possibility of forming a conjugate between H and SFN, 1.43 mM CysSH or GSH was added to 4.29 mM Na ２ S ２ 4.29 mM Na in 85.7 mM Tris-HCl buffer (pH 7.4) containing ２ S ２ The mixture was incubated in 85.7 mM Tris-HCl buffer (pH 7.4) containing β-cysteine ​​at 37°C for 15 minutes in the dark. The resulting CysSnH and GSH were treated with 3 mM SFN at 37°C for 1 hour in the dark. The resulting mixture was diluted 20-fold with 0.1% FA to terminate the reaction, and then subjected to LC-ESI-MS / MS analysis.

[0023] To prepare the sulforaphane derivative, 1.96 mM GSH or CysSH was added to 5.88 mM Na ２ S ２The mixture was incubated in the dark at 37°C for 15 minutes in 117 mM Tris-HCl buffer (pH 7.4) containing 15 μL of 33.3 mM SFN. 15 μL of the mixture (85 μL) was added, and the mixture was incubated in the dark at 37°C for 1 hour. The reaction was stopped by diluting the mixture 10-fold with 0.1% FA, and the synthesized sulforaphane derivatives were separated by reverse-phase high-performance liquid chromatography (HPLC). HPLC was performed using a PU-2089 Plus (JASCO International Co., Tokyo, Japan) with an ultraviolet detector (UV-4075; JASCO International Co.) set at 250 nm. The reaction mixture was injected onto a C18 reversed-phase column (COSMOSIL 5C18-AR-II 10.0 mm x 150 mm; Nacalai Tesque).

[0024] A linear gradient of solvent A (water containing 0.1% FA) and solvent B (100% methanol) (gradient: 0% B in 0-4 min, 50% B in 45 min, and 100% B in 46 min) was injected at a flow rate of 3 mL / min. The eluate was concentrated under vacuum by centrifugation (175 g, 23°C). The sample was then transferred to a C18 reverse-phase column (Mightysil RP-18GP 6.0 mm x 150 mm, Kanto Chemical) using solvent A (water containing 0.1% FA) and solvent B (100% methanol) (gradient: 0% B in 0-3 min, 50% B in 15 min, 100% B in 15.1 min) at a flow rate of 3 mL / min. Fractions containing sulforaphane derivatives were concentrated under vacuum by centrifugation (175 g, 23°C). The purity of the isolated sulforaphane derivatives was evaluated by LC-ESI-MS / MS analysis, and then the concentrations of the purified sulforaphane derivatives bearing CysSnH and GSnH were determined by HPLC analysis by measuring the absorbance at 250 nm and calculating from the standard curves of the authentic CysSH conjugate of SFN (SFN-SCys) and the GSH conjugate of SFN (SFN-SG, Toronto Research Chemicals, Ontario, Canada; 95%). ｎ H and GS ｎ The concentration of the purified sulforaphane derivative bearing H was determined by HPLC analysis.

[0025] To detect the in vivo production of sulforaphane derivatives and oxidized GSH (n = 3-5), broccoli seeds and sprouts (approximately 350 mg) were homogenized using a POLYTRON homogenizer (DIAX-100) in 10 volumes 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 centrifugal filter (Cosmospin), the filtrate was subjected to LC-ESI-MS / MS to analyze sulforaphane derivatives and oxidized GSH. ｎ We detected the in vivo production of G.

[0026] (DPPH radical scavenging activity measurement) A DPPH stock solution was prepared in ethanol. 100 μM DPPH dissolved in 12 mM sodium phosphate buffer (pH 7.4) was incubated in a 96-well plate at 25°C for 20 minutes in the presence or absence of the test substance. The 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 a standard curve. The radical scavenging activity of the sample was evaluated using the inhibition rate (%). The inhibition rate was calculated using the following formula: where Ac and As represent the absorbance of the blank control (water) and the sample, respectively. The radical scavenging capacity was expressed as Trolox-equivalent antioxidant capacity and calculated using a Trolox standard curve.

[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 set at p<0.05.

[0028] Comparison of Polysulfide Profiles between Mature Broccoli and Broccoli Sprouts. Mature broccoli, broccoli sprouts, cabbage, and garlic samples were analyzed for TSC and TPsC, and their polysulfide profiles (the 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 recycling digestion system and inductively coupled plasma atomic emission spectroscopy. In (B), the total polysulfide content (TPsC) was measured using an alkaline / reductive sulfur removal protocol followed by stable isotope dilution high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS / MS). In (C), the ratio of TPsC to TSC was calculated from the values ​​shown in A and B. Data are shown as mean ± standard deviation (SD) (n = 4-6). *p<0.0001 compared with broccoli sprouts by one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. d.w. is dry weight.

[0029] As shown in Figure 3(A), there was no significant difference in the TSC of mature broccoli, cabbage, and garlic. On the other hand, the TSC of broccoli sprouts was significantly higher than that of mature broccoli; in fact, broccoli sprouts exhibited 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, green onion, water spinach, alfalfa, and okra). Quantitative analysis revealed that broccoli sprouts had the highest TPsC content 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, green onion, water spinach, alfalfa, and okra) were compared. The ratios of TPsC to TSC for the nine commercially available vegetable sprouts (white radish, red radish, red cabbage, watercress, mustard, green onion, water spinach, and alfalfa) were calculated from the values ​​shown in A and B. Data are shown as mean ± SD (n = 3-6). d. w is dry weight.

[0030] The content and composition of glucosinolates in plants can be affected by the sulfur nutritional status of the soil used for plant cultivation. Previous studies have demonstrated that supplemental sulfur fertilization of 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 careful consideration of the effects of germination and growth on the polysulfide profile of broccoli.

[0031] (Changes in Polysulfide Profiles During Broccoli Sprout Germination and Growth) In germination experiments, broccoli seeds were soaked in ultrapure water for 24 hours (Day 1) and then placed in a culture tray containing ultrapure water in the dark for 24 to 96 hours (Days 2 to 5). As shown in Figure 1, broccoli seeds germinated on Day 1, and cotyledons elongated until Day 5 in a culture time-dependent manner. Approximately 80% of the seeds consistently germinated on Day 1. The polysulfide profiles of broccoli seeds, germinated seeds, and sprouts were compared (Figure 5). Figure 5 shows the changes in polysulfide profiles during broccoli sprout germination and growth. Broccoli seeds were soaked in ultrapure water for 24 hours (Day 1). The germinated seeds were then placed in a germination tray at 24 to 25°C and cultured in the dark for an additional 96 hours (Days 2 to 5). Seeds, germinated seeds, and broccoli sprouts were quantitatively analyzed 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. Data are presented as mean ± SD (n = 4-6). ＊＊ p<0.01, ＊＊＊＊ p<0.0001 compared with seeds by one-way ANOVA with Dunnett's multiple comparison test. NS, not significant.

[0032] As shown in Figure 5A, no significant changes in TSC were observed during germination. On the other hand, when broccoli seeds were soaked in ultrapure water for 24 hours (day 1), the TPsC content in the seeds dramatically increased, and TPsC levels increased over time (Figure 5B). Calculation of the percentage of TPsC relative to TSC revealed that 15.5% of the TSC in broccoli sprouts on day 5 was TPsC (Figure 5C). Although the content and composition of glucosinolates in broccoli sprouts decrease and change compared with those in seeds, this is the first evidence that the polysulfide content in broccoli significantly increases during germination and growth. In broccoli family vegetables, volatile polysulfides such as dimethyl trisulfide and dimethyl tetrasulfide may be generated by C-S lyase using SMCSO as a substrate. In addition, cystine lyase purified from broccoli inflorescences produced CysS by β-elimination of l-cystine and SMCSO. ２ H can be produced.

[0033] (Untargeted Polysulfide Omics Analysis of Broccoli Sprouts) The present inventors have developed an untargeted polysulfide omics analysis technique that enables comprehensive detection of hydropolysulfides by LC-ESI-MS / MS analysis. The present inventors have demonstrated that chemicals containing hydroxyphenyl or hydroxyl groups can stabilize polysulfide structures. They have also successfully synthesized a novel alkylating agent, TME-IAM, that can stabilize polysulfide structures via its hydroxyphenyl group. Careful examination of the MS / MS fragmentation patterns of various TME-AM adduct standards revealed that collision-induced dissociation leads to the formation of a common fragment ion at m / z 136, which is derived from the hydroxyphenyl group of the TME-AM moiety. Based on the properties of the TME-AM adducts, we developed a nontargeted polysulfide omics analysis technique using two types of TME-IAM (normal and stable isotope-labeled) at different concentrations (normal TME-IAM to stable isotope-labeled TME-IAM ratio of 4:1) (Fig. 6A). This technique derivatizes hydropolysulfides to form stable adducts with TME-IAM, and the relative signal intensities of the hydropolysulfide adducts of normal TME-IAM (light) and stable isotope-labeled (heavy) are 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, TME-AM adducts of hydropolysulfides are susceptible to the effects of reducing agents. On the other hand, TME-AM adducts of thiols do not have a polysulfide structure and are therefore less susceptible to the effects of reducing agents. Therefore, by treating with 2-mercaptoethanol, a reducing agent, it became possible to distinguish between signals derived from hydropolysulfides and thiols. In fact, GSH and glutathione hydrotrisulfide (GS ３ Preliminary experiments using the standard product of H) confirmed that GSH and GS ３Both H and H were derivatized with two types of TME-IAM, and the relative signal intensities of each adduct were shown to be almost identical to the concentration ratio of the standard TME-IAM and the stable isotope-labeled adduct (Fig. 6B). Furthermore, the GS-adduct remained intact after treatment with 2-mercaptoethanol, whereas the GS ３ The -adducts completely disappeared after incubation with a reducing agent (Fig. 6B). These results suggest that this new technique could be a useful tool for comprehensive analysis of hydropolysulfides in biological samples.

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

[0036] Representative results of untargeted polysulfide omics analysis of broccoli seeds and sprouts (day 2 and day 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), CysS was identified as a candidate hydropolysulfide. ２ H, cysteine ​​hydrotrisulfide (CysS ３ H), cysteine ​​hydrotetrasulfide, GS ２ H, G.S. ３ Thirty-seven signals were detected, including H, glutathione hydrotetrasulfide, etc. (Figure 8). ２ H, CysS ３ H, G.S. ２ H, G.S. ３ The signal intensities of H were 55, 107, 4, and 12 times those of the corresponding thiols (i.e., CysSH and GSH), respectively. In animals (humans and mice), GSH is the predominant thiol in vivo, and GS ２ The level of H was approximately 100-fold lower than that of GSH. These results suggest that polysulfide biosynthesis may be 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 untargeted polysulfide omics analysis of broccoli sprouts (day 2, A) and seeds (B). Figure 8 shows representative results of untargeted polysulfide omics analysis of broccoli sprouts (day 5). Glutathione and cysteine ​​hydropolysulfides (GS) were analyzed. ｎ H, CysS ｎ Thirty-seven signals were detected as hydropolysulfide candidates, including hydroxylase (H; n = 2-4 for each). The gray scale of the MS / MS signals indicates the signal intensity normalized as counts per mg of dry weight (d.w.). CysSH indicates cysteine, CysS ２ H is cysteine ​​hydropersulfide, CysS ３ H is cysteine ​​hydrotrisulfide, CysS４ H is cysteine ​​hydrotetrasulfide, GS ２ H is glutathione hydropersulfide, GS ４ H is glutathione hydrotetrasulfide.

[0038] (endogenous CysS ｎ H, G.S. ｎ H, and H ２ S ｎ Quantitation of CysS ２ H and GS ２ To carefully assess the impact of H on the production of endogenous polysulfides, quantitative targeted polysulfide metabolome analysis was performed by LC-ESI-MS / MS using stable isotope dilution (SDI) (Figure 9). ｎ H, G.S. ｎ H, H ２ S ｎ (n=1-5 for each) can be specifically and quantitatively detected. Quantitative analysis revealed that in broccoli sprouts, CysSH, CysS were significantly higher than in seeds on the fifth day. ２ H, CysS ３ H, G.S. ２ H, G.S. ３ H, hydrogen sulfide (H ２ S), hydrogen disulfide (H ２ S ２ ), hydrogen trisulfide (H ２ S ３ ) was found to be significantly increased compared to that in seeds. ２ S ３ was the most abundant of the nine polysulfides and thiols analyzed, and the major organic polysulfide metabolite in broccoli sprouts was CysS. ２ H and CysS ３ H (Fig. 9, B, C, I). On the other hand, no significant change in GSH content was observed during the germination and growth of broccoli sprouts (Fig. 9, D).

[0039] GS ２ It has been demonstrated that CysS is the major polysulfide in animal tissues and cells. ２ H and CysS３ This indicates that CysS is the major organic polysulfide metabolite in broccoli sprouts. ２ H and CysS ３ H is produced by cysteinyl-tRNA synthetase using CysSH as a substrate. Furthermore, cystine lyase purified from broccoli inflorescences produces CysS by the β-elimination reaction of l-cystine and SMCSO. ２ Therefore, these enzymes are highly activated during broccoli sprout germination and growth, and CysS ２ H and CysS ３ H production can be significantly increased.

[0040] Figure 9 shows CysS ｎ H and GS ｎ The quantitative analysis results of GS in seeds, germinated seeds, and broccoli sprouts are shown below. ｎ H, CysS ｎ H, and hydrogen polysulfides (H ２ S ｎ 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, ＊＊＊＊ p<0.0001 compared to seeds, comparison by one-way ANOVA with Dunnett's multiple comparison test is shown.

[0041] GS ｎ It is well documented that G (n=3-5) is produced endogenously in a variety of organisms, including humans, mice, and yeast, but little is known about this process in plants. ｎTo detect the endogenous production of GS, broccoli seeds and sprouts were homogenized in the presence of TME-IAM. TME-IAM can convert hydropolysulfides to stable adducts without artificial decomposition through the iodoacetyl group, and stabilize the polysulfide structure by inhibiting hydrolysis through the hydroxyphenyl group, thereby minimizing the artificial formation of oxidized derivatives of polysulfides during sample preparation. As shown in Figure 10, the endogenous GS 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 results of oxidized glutathione polysulfides. Seeds, germinated seeds, and broccoli sprouts were subjected to LC-ESI-MS / MS analysis to determine the endogenous levels of oxidized glutathione polysulfides (GS). ｎ G, n=3-5) were detected. ｎ A representative MS / MS chromatogram of G is shown in Figure 7, as previously described. Data are shown as mean ± SD (n = 3). Figure 10 shows ＊ p<0.05, ＊＊＊ p<0.001, ＊＊＊＊ p<0.0001 compared to seeds, comparison by one-way ANOVA with Dunnett's multiple comparison test. GS ３ G is oxidized glutathione trisulfide, GS ４ G is oxidized glutathione tetrasulfide, GS ５ G is oxidized glutathione pentasulfide.

[0043] Endogenous and authentic GS ｎ Representative MS / MS chromatograms of GSnG are shown in Figure 11. Figure 11 shows representative MS / MS chromatograms of authentic and endogenous oxidized glutathione polysulfides. The arrows indicate the signals of each oxidized glutathione polysulfide (GSnG, n = 3-5) detected in the authentic standard (authentic) and 5-day-old broccoli sprouts (endogenous). This indicates the abundance of various GSnG in broccoli seeds and sprouts. ｎThis is the first evidence of endogenous production of G type. ３ G) functions as an excellent radical scavenger in the presence of GSH, and GS ２ Furthermore, O-silyl mercaptan-based H ２ H released from S donor ２ S ２ However, O-silyl mercaptan-based H ２ H released from S donor ２ Sodium polysulfide (NaS) can reduce the true radical DPPH more effectively than sodium polysulfide (NaS). ｎ , n=-4) exerts potent neuroprotective effects against oxidative stress-induced toxicity in midbrain dopaminergic neurons treated with 1-methyl-4-phenylpyridinium ion, cerebellar granule cells treated with methylmercury, and neuroblastoma cells treated with methylglyoxal or tert-butylhydroperoxide. Therefore, GS, which is abundant in broccoli sprouts, exerts potent neuroprotective effects against oxidative stress-induced toxicity in midbrain dopaminergic neurons treated with 1-methyl-4-phenylpyridinium ion, cerebellar granule cells treated with methylmercury, and neuroblastoma cells treated with methylglyoxal or tert-butylhydroperoxide. ｎ H, CysS ｎ H, H ２ S ｎ , G.S. ｎ G may contribute to a wide range of biological activities, including antioxidant activity.

[0044] Accumulating evidence indicates that numerous proteins are endogenously polysulfated in both prokaryotes and eukaryotes (including mammals and plants). The inventors have developed a simple, reliable, and reproducible assay method, the polyethylene glycol-linked 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 thiol and polysulfide groups of proteins can be alkylated while minimizing the artificial degradation of polysulfides. Biotin-polyethylene glycol ３６ BPM reacts with polysulfidated cysteine ​​residues containing sulfur atoms, resulting in an upward shift of the band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0045] Therefore, this assay revealed the degree of protein polysulfidation as a change in the mobility of the SDS-PAGE bands, demonstrating its suitability for examining the degree of polysulfidation of individual proteins. However, it is not suitable for comprehensive analysis of the polysulfidation state of proteins. 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), but only a faint signal was observed in broccoli sprouts (days 2-5). Meanwhile, Coomassie Brilliant Blue staining following SDS-PAGE revealed dramatic changes in protein expression patterns during germination and growth (Figure 12, B). These results suggest that polysulfide-rich proteins may be used as potential sources for protein and polysulfide biosynthesis.

[0046] Figure 12 shows the protein expression profile (A) and polysulfidation status (B) in broccoli seeds and sprouts. (A) is a representative image of the detection of polysulfidated proteins. Broccoli seeds (S), germinated seeds (day 1, D1), and sprouts (days 2 to 5, D2 to D5) were lysed in 1 mM biotin-maleimide (BM, 95% or higher, Sigma-Aldrich) or 1 mM BM containing 1 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-KOH buffer (pH 7.0), 1% (v / v) sodium dodecyl sulfate (SDS) 1% (v / v), pepstatin A (Peptide Institute, Osaka, Japan) 10 μM, leupeptin (Peptide Institute) 10 μM, ubenimex (Sigma-Aldrich, St. Louis, MO) 10 μM, and E-64 (Sigma-Aldrich) 10 μM. The protein concentration was adjusted to 3 mg / mL using an UltraTurrax homogenizer (model: T10 basic, IKA, Osaka, Japan) containing N-iodoacetyl-l-tyrosine methyl ester (TME-IAM). After centrifugation at 20,380 g for 15 min at 4°C, the resulting supernatant was incubated at 37°C for 30 min, diluted two-fold with lysis buffer containing 3 mM BM, and further incubated at 37°C for 1 h.

[0047] Proteins were denatured by boiling in 1x Laemmli sample buffer for 5 minutes and separated by SDS-PAGE on a 12% polyacrylamide gel. Biotinylated proteins were detected by Western blotting (WB) using horseradish peroxidase-conjugated streptavidin (Invitrogen, Carlsbad, CA) or total proteins were visualized by Coomassie Brilliant Blue (CBB) staining. (B) Representative images of CBB staining for protein expression profiling in broccoli seeds, germinated seeds, and sprouts. Broccoli seeds and sprouts were homogenized in an Ultra-Turks homogenizer with 20 volumes of lysis buffer. After centrifugation at 20,380 g for 30 minutes at 10°C, the protein concentration of the supernatant was measured using a biphenotypic acid kit. After boiling for 5 min in 1x Laemmli sample buffer, proteins were separated by SDS-PAGE on 12% polyacrylamide gels in the absence or presence of 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 by the hydrolysis of glucoraphanin 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. GS ２ The pKa of H is 5.45, which is 3.49 lower than the pKa of GSH. ２ H increases the availability of anions, resulting in peroxynitrite and hydrogen peroxide (H ２ O ２ ) reacts faster than GSH with electrophiles such as SFN and GS, and its reactivity increases with some electrophiles. ｎ H or CysS ｎ A reaction with H occurs, and GS of SFNｎ H or CysS ｎ H-conjugates (SFN-S ｎ G or SFN-S ｎ Cys) may be formed.

[0049] To verify this prediction, we first performed GS with SFN. ｎ H or CysS ｎ In vitro experiments were performed using H, followed by LC-ESI-MS / MS 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) were produced in vitro. Next, the endogenous production of these sulforaphane derivatives in broccoli seeds and sprouts was analyzed (Figure 13). Figure 14 shows representative MS / MS chromatograms and MS / MS spectra of authentic and endogenous sulforaphane (SFN) with glutathione or cysteine ​​hydropolysulfide (SFN-S). ｎ G and SFN-S ｎ Cys). Arrows indicate the common SFN and each 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 The signal of Cys (n=1-5, B) is shown.

[0050] FIG. 13 shows the sulforaphane complex GS ｎ H and CysS ｎ The results of the endogenous production of H are shown. Seeds, germinated seeds, and broccoli sprouts were analyzed by LC-ESI-MS / MS to detect sulforaphane (SFN, A) and GS. ｎ H conjugate (SFN-S ｎ G, B) and CysS ｎ H conjugate (SFN-S ｎRepresentative 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 presented as mean ± SD (n = 3). ＊ p<0.05, ＊＊＊ p<0.001, ＊＊＊＊ p<0.0001 compared to seeds, one-way ANOVA with Dunnett's multiple comparison test.

[0051] FIG. 15 shows endogenous SFN-S detected in broccoli sprouts (day 5). ｎ Cys (n=1-5) and SFN-S ｎ Representative MS / MS spectra of SFN-SnG (n = 1-4) and SFN-SnCys (n = 1-5) are shown, consistent with authentic standards. Figure 15 shows 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. Cleavage sites are indicated by dashed lines. LC-ESI-MS / MS analysis demonstrated the presence of SFN-SnG in broccoli sprouts. ｎ Cys (n=1-5) and SFN-S ｎ Endogenous production of G (n = 1-4) was detected, and a time-dependent increase in SFN-SnCys (n = 2-5) was observed (Figure 13, G-J). This correlated with the temporal changes in 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 ｎTo detect the in vivo production of G, broccoli seeds and sprouts were homogenized in the presence of TME-IAM, which may enhance the production of SFN-S by trapping hydropolysulfides to form stable TME-IAM adducts. ｎ Cys and SFN-S ｎ The artificial formation of G was minimized. In fact, SFN was ｎ It was confirmed that the reactivity of TME-IAM to H was not hindered (Figure 17). This indicates that the use of TME-IAM allows the detection of sulforaphane derivatives and GS. ｎ This suggests that both oxidized polysulfides such as G and hydropolysulfides can be accurately detected with minimal artificial alteration. Therefore, the SFN-S detected in broccoli seeds and sprouts ｎ Cys and SFN-S ｎ It is highly likely that G was produced endogenously. Figure 17 shows the effect of SFN on the reactivity of TME-IAM to polysulfides. ２ S ２ CysS, which was prepared by incubating at 37°C for 5 hours. ２ H and CysS ３ Cysteine ​​hydropolysulfides containing H (CysS ｎ H) was 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 minutes. The reaction mixture was then subjected to LC-ESI-MS / MS analysis to identify CysS. ｎ H (n=1-3) and H ２ S ｎ Quantitative detection of each adduct was performed (n = 1, 2). Data are shown as mean ± SD (n = 4).

[0053] SFN is known to function 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, although SFN can directly scavenge radicals, studies using the DPPH radical scavenging assay have shown that its radical scavenging activity is much weaker than that of ascorbic acid. The free radical scavenging activity of broccoli seed extracts is not correlated with the SFN content in the seeds. This indicates that SFN is not the major antioxidant component of broccoli seeds. On the other hand, SFN is more potent than glutathione (GSH) in scavenging glutathione sulfatase (GS). ２ H) is better than hydrogen peroxide (H ２ O ２ Therefore, SFN has a high scavenging ability of GSH or CysS. ｎ When bound to H, it is possible that it may exhibit a higher radical scavenging ability than SFN.

[0054] To test this hypothesis, SFN-S ｎ G and SFN-S ｎ DPPH radical scavenging assay was performed using Cys standard. Various SFN-S were obtained by chemical reaction of SFN with GSH or CysSH. ｎ G (n = 1 to 4) and SFN-S ｎ It was confirmed that SFN-S with n = 1 or 2 was produced (Fig. 14 and Fig. 15). However, sulforaphane derivatives with three or more polysulfide chains (n ​​> 3) were easily decomposed during HPLC purification. ｎ G and SFN-S ｎ As shown in Figure 18, the isolated SFN-SG and SFN-S ２ G, SFN-SCys, and SFN-S ２ Cys was detected as a protonated molecule at m / z 485.1193, 517.0914, 299.0552, and 331.0270, respectively. Product ion assignments also demonstrated the successful synthesis of these novel sulforaphane derivatives (FIG. 18).

[0055] FIG. 18 shows the SFN-S by high-resolution mass spectrometry. ｎ G and SFN-S ｎ The synthesis and purification of Cys are shown. ｎ G and SFN-S ｎ The synthesis and purification of Cys (n = 1 or 2, respectively) were performed by a liquid chromatography electrospray ionization-quadrupole-time of flight (LC-ESI-Q-TOF)-tandem mass spectrometer (G6545XT; Agilent Technologies, Tokyo, Japan) and an Agilent 1260 Infinity II Prime LC system (G7104C, G7129C, G7130A, G7117C, and G1310A, Agilent Technologies). Samples were separated on a reversed-phase Mightysil RP-18 GP column (50 mm length × 2.0 mm inner diameter, Kanto Chemical Co., Tokyo, Japan) using a mobile phase A (0.1% formic acid) and B (methanol) with a linear gradient from 1% to 99% B over 2–7 min. The flow rate was 0.6 mL / min. The ESI-Q-TOF instrument was operated in positive ion mode with an m / z scan range of 100–1000 in MS mode and 50–1000 in MS / MS mode, and a scan rate of 3 spectra / s. For collision-induced dissociation, the collision energy range was set to 10–40 V. A Q-TOF-MA was used. Vcap: 4000 V, nebulizer pressure: 55 psi, drying gas flow rate: 11 L / min, gas temperature: 350°C, fragmentor: 125 V, skimmer: 65 V, octopole RF peak: 750 V. 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 (GSｎ H or GS ｎ G, n=2, 3, 4, or 5) is bonded to a novel sulforaphane derivative. For example, it is any of the following:

[0057]

[0058] As shown in Figure 19, when a DPPH radical scavenging activity assay was performed, no significant radical scavenging activity of SFN was observed within the concentration range examined (100-1000 μM). ２ The order of radical scavenging ability was G≧GSH>SFN-SG>>SFN=disulfide of oxidized glutathione. ｎ The order of radical scavenging ability in the H bond is SFN-S ２ Cys>SFN-SCys≈CysSH>>SFN=cystine.

[0059] 19 shows the radical scavenging ability of sulforaphane derivatives. The radical scavenging ability of sulforaphane derivatives was evaluated by a 1,1-diphenyl-2-picrylhydrazyl radical scavenging assay. Data are expressed as mean ± SD (n = 3). ＊ p<0.05, ＊＊＊ p<0.001, ＊＊＊＊ p<0.0001 vs. SFN; ++p<0.01, ++++p<0.0001 vs. corresponding thiol; compared to the SFN conjugate of the corresponding hydropersulfide ＄＄＄＄ p<0.0001 compared with SFN-SCys ｑｑｑｑ p<0.0001, compared by one-way ANOVA with Tukey's multiple comparison test. GS ２ G is oxidized glutathione disulfide; TEAC, Trolox-equivalent antioxidant activity.

[0060] These results suggest that the novel sulforaphane derivative functions as a potent antioxidant. 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, indicating that the activity of SFN is not significantly altered when SFN is conjugated with GSH to form dithiocarbamic acid. Therefore, the novel conjugates of SFN with GSnH or CysSnH can effectively inhibit the detoxification of SFN and GS in vivo. ｎ H or CysS ｎ It may exert multiple biological functions derived from potential crosstalk with H.

[0061] Although various embodiments have been described above, the present invention is not limited to these. Other embodiments that are 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 G, n=2, 3, 4, or 5).

2. A complex of the sulforaphane and the cysteine ​​persulfide, having the following structural formula: The novel sulforaphane derivative according to claim 1, wherein n=2, 3, 4, or 5.

3. The complex has the following structural formula: The novel sulforaphane derivative according to claim 1, wherein n=2, 3, 4, or 5.

4. The complex is at least one of the following formulas 3, 4, and 5: The novel sulforaphane derivative according to claim 3.

5. The complex is at least one of the following formulas 6, 7, and 8: The novel sulforaphane derivative according to claim 2.

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