A water extract of broccoli seeds for improving bioavailability of sulforaphane and a preparation process thereof

Abstract

The application discloses a water extract of broccoli seeds for improving bioavailability of sulforaphane and a preparation process thereof. 2+ The application introduces phytic acid and zinc gluconate to construct a synergistic regulation system, uses the directional chelation of phytic acid to effectively deprive the essential cofactor of the ring sulfur designated protein, fundamentally blocks the generation path of the inactive nitrile, and uses the allosteric activation effect of zinc ions and the pH stability of gluconate to significantly enhance the catalytic efficiency of myrosinase.

Description

Technical Field

[0001] This invention relates to the field of plant extraction technology, and in particular to a water extract of broccoli seeds that improves the bioavailability of sulforaphane and its preparation process. Background Technology

[0002] Sulforaphane is one of the most potent plant-based active ingredients known to induce phase II detoxification enzyme activity in humans, exhibiting remarkable physiological functions in antioxidation, anti-inflammation, inhibition of tumor cell proliferation, and immune regulation. In nature, sulforaphane does not exist in a free state but is stored in large quantities in the cell vacuoles of cruciferous plants as its stable precursor, sulforaphane glycosides, with broccoli seeds being the richest source. When plant tissues are broken or mechanically damaged, sulforaphane glycosides in the vacuoles come into contact with endogenous myrosinase, undergoing hydrolysis in an aqueous environment, initially generating an unstable thiohydroxyoxime-O-sulfate intermediate. Under normal physiological pH conditions, this intermediate spontaneously transforms into highly bioactive sulforaphane through the Lossen rearrangement mechanism. However, broccoli seeds also contain another key enzyme, epithiospecifier protein (ESP), which releases ferrous ions (Fe2+) endogenously from the seeds. 2+ Under the condition that ) exists, ESP can competitively guide the above hydrolysis intermediate to another side reaction pathway, causing the sulfur atom to rearrange intramolecularly to the end of the side chain, generating sulforaphane nitrile, which has almost no anticancer activity. This endogenous competitive mechanism seriously reduces the proportion and purity of sulforaphane in the final product.

[0003] To address the aforementioned issues, existing technologies typically employ heat treatment to inactivate the enzyme and suppress ESP interference. For example, short-term heat treatment of broccoli seed powder at high temperatures preferentially inactivates ESP while retaining some black myrosinase activity. However, the thermal stability difference between ESP and black myrosinase is limited, making precise temperature gradient control difficult in practical processes. Insufficient heating results in high residual levels of nitrile byproducts, while excessive heating leads to simultaneous inactivation of black myrosinase, resulting in a significant decrease in overall hydrolysis conversion efficiency. Furthermore, some literature reports adjusting the catalytic activity of black myrosinase by adding exogenous metal ions; however, such single methods can only improve the forward reaction rate to a certain extent and cannot fundamentally block the ESP-mediated nitrile formation side reaction pathway, making it difficult to substantially improve product selectivity. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the purpose of this invention is to provide a water extract of broccoli seeds with improved bioavailability of sulforaphane and its preparation process, utilizing phytic acid to enhance the bioavailability of Fe. 2+The targeted chelation effectively deprived the essential cofactor of the cyclic sulfur-specific protein, blocking the formation pathway of inactive nitrile; the catalytic efficiency of myrosinase was enhanced by utilizing the allosteric activation effect of zinc ions and the pH stabilizing effect of gluconate.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A water extract of broccoli seeds with improved bioavailability of sulforaphane is obtained by enzymatic hydrolysis of broccoli seeds in an aqueous medium by introducing a multidentate chelating agent and a metal ion allosteric activator.

[0007] Preferably, the aforementioned multidentate chelating agent is phytic acid or phytate.

[0008] Preferably, the aforementioned metal ion allosteric activator is zinc gluconate.

[0009] The extraction process of broccoli seed water extract includes the following steps:

[0010] S1. Degreasing: Crush and defatt the broccoli seeds to obtain defatted seed powder;

[0011] S2. Preparation of conditioning solution: Add multidentate chelating agent and metal ion allosteric activator to deionized water to obtain multi-ion conditioning extract;

[0012] S3, Synergistic Enzymatic Hydrolysis: Add defatted seed powder to the controlled extract and stir for enzymatic hydrolysis;

[0013] S4. Separation and drying: After the reaction is completed, heat inactivation is performed and the supernatant is collected by centrifugation and dried to obtain broccoli seed water extract.

[0014] Preferably, in step S1, the broccoli seeds are pulverized to a mesh size of 60-80, the degreasing solvent is petroleum ether, and the degreasing is performed 1-3 times.

[0015] Preferably, in step S2, the mass percentage concentration of the multidentate chelating agent in the controlled extract is 0.1%-0.5%, the mass percentage concentration of the metal ion allosteric activator in the extract is 0.05%-0.2%, and the pH value of the controlled extract is adjusted to 4.0-6.5.

[0016] Preferably, in step S3, the enzymatic hydrolysis temperature is 25-45℃, the enzymatic hydrolysis time is 60-180 min, and the mass-volume ratio of defatted seed powder to controlled extract is 1:10-1:25.

[0017] Preferably, in step S4, the centrifugation speed is 8000-10000 rpm; the drying is vacuum freeze drying or low-temperature spray drying, and an excipient is added before drying, wherein the excipient is one or more of maltodextrin, dextrin or trehalose.

[0018] Application of broccoli seed water extract in the preparation of products with antioxidant, anti-inflammatory, tumor cell proliferation inhibition or immune function regulation properties.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] (1) This invention introduces phytic acid and zinc gluconate to construct a synergistic regulatory system, utilizing phytic acid to regulate Fe 2+ The targeted chelation effectively deprives the essential cofactor of the cyclic sulfur-specific protein, fundamentally blocking the formation pathway of inactive nitrile; at the same time, by utilizing the allosteric activation effect of zinc ions and the pH stabilizing effect of gluconate, the catalytic efficiency of myrosinase is significantly enhanced.

[0021] (2) The extract of the present invention shows a significant synergistic effect in inducing phase II detoxification enzymes and transmembrane absorption in the intestine, and effectively eliminates the pungent odor caused by nitrile impurities. The product has excellent sensory quality. The whole process conditions are mild and safe. Large-scale production can be achieved using conventional extraction equipment. It has extremely high economic benefits and broad prospects for functional food development. Detailed Implementation

[0022] To clearly illustrate the technical features of this solution, the following detailed implementation method will be used to describe the solution.

[0023] A water extract of broccoli seeds with improved bioavailability of sulforaphane is obtained by enzymatic hydrolysis of broccoli seeds in an aqueous medium by introducing a multidentate chelating agent and a metal ion allosteric activator.

[0024] The mass ratio of sulforaphane to sulforaphane nitrile in the water extract is greater than 20:1. The multidentate chelating agent is one or more of phytic acid, phytate, citric acid, tartaric acid, EDTA, and / or phytate. The metal ion allosteric activator is zinc gluconate.

[0025] The extraction process of broccoli seed water extract includes the following steps:

[0026] S1. Degreasing: Crush and defatt the broccoli seeds to obtain defatted seed powder. The crushing mesh of the broccoli seeds is 60-80 mesh. The defatting solvent is petroleum ether. The defatting is performed 1-3 times.

[0027] S2. Preparation of conditioning solution: Add a multidentate chelating agent and a metal ion allosteric activator to deionized water. The mass percentage concentration of the multidentate chelating agent in the conditioning extract is 0.1%-0.5%, and the mass percentage concentration of the metal ion allosteric activator in the extract is 0.05%-0.2%. Adjust the pH value to 4.0-6.5 to obtain the multi-ion conditioning extract.

[0028] S3. Synergistic enzymatic hydrolysis: Add defatted seed powder to the regulating extract, with a mass-to-volume ratio of defatted seed powder to regulating extract of 1:10-1:25. Stir and hydrolyze, with an enzymatic hydrolysis temperature of 25-45℃ and a hydrolysis time of 60-180 min.

[0029] S4. Separation and drying: After the reaction is completed, heat inactivation is performed and the supernatant is collected by centrifugation at a speed of 8000-10000 rpm; excipients are added, which are one or more of maltodextrin, dextrin or trehalose; vacuum freeze-drying or low-temperature spray drying is used to obtain broccoli seed water extract.

[0030] Example 1

[0031] The extraction process of broccoli seed water extract includes the following steps:

[0032] S1. Degreasing: The broccoli seeds are crushed and degreased to obtain defatted seed powder. The crushing mesh of the broccoli seeds is 60 mesh, the degreasing solvent is petroleum ether, and the degreasing is performed twice.

[0033] S2. Preparation of conditioning solution: Add 0.1% phytic acid and 0.05% zinc gluconate to deionized water to adjust the pH to 6.5 to obtain a multi-ion regulated extract.

[0034] S3, Synergistic Enzymatic Hydrolysis: Add defatted seed powder to the regulating extract, with a mass-to-volume ratio of defatted seed powder to regulating extract of 1:15. Stir and hydrolyze, with a hydrolysis temperature of 38℃ and a hydrolysis time of 90 min.

[0035] S4. Separation and drying: After the reaction is completed, heat inactivation is performed and the supernatant is collected by centrifugation at a speed of 8000 rpm; vacuum freeze drying or low-temperature spray drying is used to obtain broccoli seed water extract.

[0036] Example 2

[0037] The difference between this embodiment and Example 1 is that the amount of phytic acid added to the extract is 0.2%, the amount of zinc gluconate added is 0.10%, the initial pH value is adjusted to 6.0, the isothermal enzymatic hydrolysis temperature is 38℃, and the enzymatic hydrolysis time is 120 min.

[0038] Example 3

[0039] The difference between this embodiment and Example 1 is that the amount of phytic acid added to the extract is 0.3%, the amount of zinc gluconate added is 0.12%, the initial pH value is set to 6.0, the isothermal enzymatic hydrolysis temperature is 38℃, and the enzymatic hydrolysis time is 120 min.

[0040] Example 4

[0041] The difference between this embodiment and Example 1 is that the amount of phytic acid added to the extract is 0.4%, the amount of zinc gluconate added is 0.15%, the initial pH value is 5.5, the enzymatic hydrolysis temperature is 35℃, and the enzymatic hydrolysis time is 150 min.

[0042] Example 5

[0043] The difference between this embodiment and Example 1 is that the amount of phytic acid added to the extract is 0.5%, the amount of zinc gluconate added is 0.2%, the initial pH value is 5.5, the enzymatic hydrolysis temperature is 35℃, and the enzymatic hydrolysis time is 180 min.

[0044] Comparative Example 1

[0045] This comparative example serves as a blank control. Natural hydrolysis conditions were used, and no regulatory substances were added during the extraction process. Only pure deionized water was used as the extraction medium, and the enzyme was hydrolyzed at a constant temperature of 38°C for 120 min with stirring.

[0046] Comparative Example 2

[0047] The difference between this comparative example and Example 3 is that zinc gluconate is not added.

[0048] Comparative Example 3

[0049] The difference between this comparative example and Example 3 is that phytic acid is not added.

[0050] Comparative Example 4

[0051] This comparative example uses a physical heat treatment scheme to inactivate the specified cyclic sulfur protein. First, defatted seed powder is pretreated in a 70°C hot air environment for 30 minutes, and then enzymatically hydrolyzed with pure water at 38°C for 120 minutes.

[0052] Comparative Example 5

[0053] The difference between this comparative example and Example 3 is that another common chemical chelating agent is used instead of phytic acid, and 0.3% (w / w) of disodium ethylenediaminetetraacetate (EDTA-2Na) is added to the conditioning solution.

[0054] Performance testing

[0055] (1) Sulforaphane yield and conversion selectivity

[0056] The guiding effect of different regulatory systems was evaluated by determining the absolute content and ratio of sulforaphane (SFN) and sulforaphane nitrile (SFN-NIT) in the extract. Quantitative analysis was performed using high-performance liquid chromatography (HPLC). The sample pretreatment process was as follows: 100 mg of dried powder from Examples 1-5 and Comparative Examples 1-4 was accurately weighed, and 5 mL of ethyl acetate was added for ultrasonic extraction for 20 minutes. The organic layer was then separated by centrifugation, dehydrated with anhydrous sodium sulfate, and rotary evaporated to dryness. Finally, the residue was reconstituted with 1 mL of methanol, filtered through a 0.22 μm organic filter membrane, and injected for analysis.

[0057] Table 1. Results of determination of sulforaphane and nitrile impurities in extracts of each group

[0058]

[0059] As shown in Table 1, the sulforaphane content in Examples 1-5 was significantly higher than that in Comparative Examples 1-4. In Comparative Example 1, under natural hydrolysis conditions, the ratio of sulforaphane to sulforaphane nitrile was close to 1:1, with a nitrile content of 1.92 mg / g, fully demonstrating the strong nitrification-promoting effect of endogenous cyclic sulfur-specific proteins on hydrolysis intermediates in the presence of free iron ions. In Comparative Example 2, with the addition of only phytic acid, the selectivity increased to 87.4%, and the nitrile content decreased to 0.45 mg / g. This confirms that phytic acid, through its six negatively charged phosphate groups, efficiently chelates ferrous ions in the system, inhibiting the activity of the cyclic sulfur-specific protein due to the loss of essential cofactors, forcing the intermediate to generate sulforaphane. In Comparative Example 3, with the addition of only zinc gluconate, the selectivity was only 59.0%, and the residual nitrile content was still as high as 1.78 mg / g, indicating that a single zinc salt cannot effectively block the nitrification side reaction. Comparative Example 4 was preheated at 70℃. Although the nitrile content decreased, the sulforaphane yield was only 2.12 mg / g, and a burnt and bitter taste was produced. This indicates that although heat inactivation can partially inhibit cyclic sulfur-specific proteins, it also severely damages the catalytic activity of myrosinase.

[0060] Comparative Example 5, which used EDTA-2Na instead of phytic acid, was slightly inferior to Example 3, suggesting that phytic acid may have better complexation selectivity and compatibility with the enzyme microenvironment within a specific pH window than synthetic chelating agents. In summary, the high conversion selectivity demonstrated in Example 3 is the result of the synergistic effect of phytic acid chelating cyclic sulfur-specific proteins and zinc gluconate allosterically activating myrosinase. The former interrupts the nitrile formation pathway at the molecular level, while the latter ensures the full conversion of glucosinolates by maintaining high myrosinase activity; both are indispensable.

[0061] (2) Kinetic stability of endogenous black mustard enzyme

[0062] Enzyme activity was assessed using absorbance monitoring, based on the principle that the absorbance of the reaction system at 227 nm decreases linearly when myrosinase hydrolyzes the substrate myrosinase. The experiment was conducted in a 300 μL reaction system containing phosphate buffer (pH 6.0) and a final concentration of 0.5 mM myrosinase substrate. Intermediate enzyme solutions obtained during the extraction process of Examples 3, 1, 3, and 4 were added as enzyme sources. Specific enzyme activity (U / mg protein) was calculated by measuring the initial reaction rate. Residual activity was measured again after 120 minutes of enzymatic hydrolysis to calculate the percentage of remaining enzyme activity. Specific results are shown in Table 2.

[0063] Table 2. Specific activity and stability data of black mustard enzymes

[0064]

[0065] As shown in Table 2, the synergistic regulatory system reveals a protective effect on the activity and stability of myrosinase from an enzyme kinetic perspective. The initial specific activity of Example 3 reached 4.85 U / mg, significantly higher than 1.52 U / mg in Comparative Example 1 and 0.85 U / mg in Comparative Example 4. This indicates that the presence of zinc ions, by binding to the allosteric regulatory site of myrosinase, induces a conformational shift of the enzyme molecule to an open state with high catalytic efficiency, thereby increasing its affinity for and conversion rate of the substrate glucosinolates. More importantly, at the end of the 120-minute enzymatic hydrolysis reaction, the enzyme activity retention rate of Example 3 was still as high as 92.4%, while that of Comparative Example 1 was only 65.8%, and the initial activity of Comparative Example 4 had been significantly reduced due to preheating damage, with a retention rate of only 42.1%. This difference is closely related to the evolution of the system pH. The final pH value of Example 3 was 5.85, which is exactly within the optimal activity window of myrosinase; while the pH of Comparative Example 1 had dropped to 4.92, and the accumulation of acidic byproducts led to irreversible denaturation and loss of activity of the enzyme protein. In Example 3, the gluconate ion acted as a buffering agent for the weak acid anion, neutralizing the protons released during hydrolysis and stabilizing the microenvironment pH within a suitable range, thus delaying the acid inactivation process of the enzyme. Although Comparative Example 3 showed a higher initial specific activity under zinc ion activation, it lacked the phytic acid's balancing effect on metal ions, ultimately resulting in a pH drop to 5.24 and an enzyme activity retention rate of only 85.2%, further demonstrating the necessity of the synergistic effect of zinc activation and phytic acid pH buffering.

[0066] (3) Determination of the fold induction of phase II enzyme (NQO1) based on the HepG2 cell model

[0067] This experiment employed the Prochaska microplate bioassay, using the human hepatocellular carcinoma cell line HepG2 as the cell model. The experimental procedure was as follows: HepG2 cells were seeded at a density of 15,000 cells per well in 96-well cell culture plates and cultured for 24 hours to allow them to adhere and enter the logarithmic growth phase. Subsequently, different concentration gradients of the extract from Example 3, the extract from Comparative Example 1, and pure sulforaphane standard solution were added to each well. All concentrations were normalized to the molar concentration of sulforaphane. Cells were treated for another 48 hours. After treatment, the culture medium was removed, and the cells were lysed to release the intracellular enzyme solution. A reaction substrate mixture containing thiazolyl blue, oxidized coenzyme I, and menadione was added to the lysate. The absorbance was measured at 610 nm using a microplate reader; this absorbance was directly proportional to the NQO1 enzyme activity. Simultaneously, the total protein content in each well was determined using the diquinoline formate method to normalize the enzyme activity data. The final calculated index is the NQO1 induction fold, which is the ratio of the specific activity of the drug-treated group to that of the solvent control group. The concentration required to double the activity of NQO1, i.e., the CD value, is further calculated. The specific results are shown in Table 3.

[0068] Table 3. NQO1 induction activity data in HepG2 cells

[0069]

[0070] As shown in Table 3, in the NQO1 induction assay of HepG2 cells, the extract of Example 3 induced a 2.15-fold increase in enzyme activity at a sulforaphane concentration of 0.25 μM, with a CD value of only 0.22 μM, which was superior to the pure SFN standard. This is because, although phytic acid itself does not directly induce NQO1, as an antioxidant, it can reduce basal oxidative stress in cells, thereby making cells more sensitive to the pro-oxidative signals of sulforaphane. The effects of Comparative Examples 1 and 4 were extremely poor, not only because of the low absolute content of SFN, but also because they contained a high proportion of sulforaphane nitrile. Although nitrile substances can enter cells, their affinity for Keap1 is extremely weak, and they competitively occupy transport sites, playing an antagonistic role and thus reducing the overall induction efficacy.

[0071] (4) Permeability and absorption of Caco-2 cell monolayer membrane

[0072] This experiment used a Caco-2 cell monolayer model to simulate the absorption barrier function of the human intestinal epithelium. Caco-2 cells were seeded on Transwell porous membranes and cultured continuously for 21 days until a dense, polar, and intact cell monolayer was formed. Barrier integrity was confirmed by measuring transmembrane resistance, which was greater than 800 Ω·cm. 2The permeability experiment was designed to be a unidirectional transport from the apical side to the basal side of the cell monolayer. At the start of the experiment, each group of test samples was added to the apical side chamber at a final sulforaphane concentration of 10 μM. Samples were taken from the basal side chamber at 30, 60, 90, and 120 minutes after administration. The concentration of sulforaphane permeating the cell monolayer was quantitatively determined using liquid chromatography-tandem mass spectrometry, and the apparent permeability coefficient was calculated. The specific results are shown in Table 4.

[0073] Table 4 Comparison of Caco-2 cell transport experimental data

[0074]

[0075] As shown in Table 4, the extract of Example 3 exhibits extremely high permeability, falling into the category of highly absorbable compounds. This is because, in the system containing phytic acid and zinc gluconate, sulforaphane molecules may form quasi-molecular clusters with gluconate ions through hydrogen bonds or weak electrostatic interactions, increasing their monomolecular dispersibility in the aqueous phase and thus amplifying the concentration gradient across the membrane. In Comparative Example 1, the high content of sulforaphane nitrile not only makes it difficult to efficiently cross the membrane itself but may also interfere with non-specific transport channels or tight junction structures on the cell membrane, further reducing overall transport efficiency. In contrast, Example 3, due to its single and pure composition, allows sulforaphane molecules to smoothly cross the cell monolayer via passive diffusion, thus exhibiting excellent absorption performance. This result elucidates the important material basis for the improved bioavailability of the extract of this invention from the perspective of intestinal absorption.

[0076] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A water extract of broccoli seeds with improved bioavailability of sulforaphane, characterized in that, The aqueous extract was obtained by enzymatic hydrolysis of broccoli seeds in an aqueous medium by introducing a multidentate chelating agent and a metal ion allosteric activator.

2. The broccoli seed water extract for improving the bioavailability of sulforaphane according to claim 1, characterized in that, The multidentate chelating agent is phytic acid or phytate.

3. The broccoli seed water extract for improving the bioavailability of sulforaphane according to claim 1, characterized in that, The metal ion allosteric activator is zinc gluconate.

4. The preparation process of the broccoli seed water extract according to any one of claims 1-3, characterized in that, Includes the following steps: S1. Degreasing: Crush and defatt the broccoli seeds to obtain defatted seed powder; S2. Preparation of conditioning solution: Add multidentate chelating agent and metal ion allosteric activator to deionized water to obtain multi-ion conditioning extract; S3, Synergistic Enzymatic Hydrolysis: Add defatted seed powder to the controlled extract and stir for enzymatic hydrolysis; S4. Separation and drying: After the reaction is completed, heat inactivation is performed and the supernatant is collected by centrifugation and dried to obtain broccoli seed water extract.

5. The preparation process according to claim 4, characterized in that, In step S1, the broccoli seeds are pulverized to a mesh size of 60-80, the degreasing solvent is petroleum ether, and the degreasing is performed 1-3 times.

6. The preparation process according to claim 4, characterized in that, In step S2, the mass percentage concentration of the multidentate chelating agent in the extract is 0.1%-0.5%, and the mass percentage concentration of the metal ion allosteric activator in the extract is 0.05%-0.2%; the pH of the extract is adjusted to 4.0-6.

5.

7. The preparation process according to claim 4, characterized in that, In step S3, the enzymatic hydrolysis temperature is 25-45℃, the enzymatic hydrolysis time is 60-180 min, and the mass-volume ratio of defatted seed powder to controlled extract is 1:10-1:

25.

8. The preparation process according to claim 4, characterized in that, In step S4, the centrifugation speed is 8000-10000 rpm; the drying is vacuum freeze drying or low temperature spray drying, and an excipient is added before drying, wherein the excipient is one or more of maltodextrin, dextrin or trehalose.

9. The use of the broccoli seed water extract according to any one of claims 1-3 in the preparation of products with antioxidant, anti-inflammatory, tumor cell proliferation inhibition or immune function regulation properties.

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