A method for extracting natural sulforaphane and its application in antibacterial gel

Abstract

The present invention discloses a method for extracting natural sulforaphane and its application in antibacterial gel, belonging to the technical field of plant extracts. The present invention utilizes the enzymatic hydrolysis of a homemade exogenous mustard seed enzyme solution, combined with ultrasound and microwave assistance, and then combined with ethyl acetate extraction and resin separation and purification processes, to finally extract sulforaphane from broccoli seeds. After testing, the purity of the extracted natural sulforaphane is 87.5% to 88.2%. Then, the present invention utilizes the extracted sulforaphane to prepare an antibacterial gel, which is composed of a gel, an auxiliary agent glycerol, and a solvent ultrapure water. After testing, the antibacterial gel added with the natural sulforaphane extracted by the present invention exhibits excellent antibacterial performance and pH responsiveness.

Description

Technical Field

[0001] The invention belongs to the technical field of plant extracts, and particularly relates to a method for extracting natural sulforaphane and application of the same in antibacterial gel. Background Art

[0002] Sulforaphane is a sulfur-rich dietary phytochemical found in cruciferous vegetables such as broccoli, cauliflower, kale, Brussels sprouts and cabbage. Sulforaphane has multiple activities such as antioxidant, anti-inflammatory, antibacterial and antiviral, and can promote wound healing. It also plays an important role in the prevention and treatment of diseases such as central nervous system diseases and autism spectrum disorders.

[0003] Due to the advantages of simple process, low cost, natural product, high yield, no toxic residue, green and safe, the extraction and purification technology of natural sulforaphane has become a very promising functional ingredient in functional foods, beverages, cosmetics and medicines.

[0004] Natural sulforaphane is prone to decomposition under conditions of pH>9 and high temperature. Therefore, many improvements have been made in the prior art, such as the preparation of microcapsules and composites. For example, Chinese invention patent application number 201710915662.5 discloses a method for preparing sulforaphane microcapsules. The invention uses sulforaphane as a core material and grass jelly-gelatin as a wall material to prepare microcapsules with light-proof, heat-resistant, acid-resistant, alkali-resistant and antioxidant effects, thereby improving the bioavailability and stability of sulforaphane. Another example is Chinese invention patent application number 201710440340.X, which discloses a sulforaphane-containing gastric retention composition and a preparation method thereof. The composition prepared by the invention patent can achieve the effect of sustained release of sulforaphane, reduce the decomposition of sulforaphane, and improve its bioavailability.

[0005] After searching, it is found that the existing natural sulforaphane is mainly used in oral medicines and the like, but no natural sulforaphane is used in gel dressings. Based on the excellent antioxidant, anti-inflammatory and antibacterial effects of natural sulforaphane, the present invention aims to develop its application in antibacterial gel. Summary of the invention

[0006] The purpose of the present invention is to develop the application of natural sulforaphane in antibacterial gel. At the same time, the present invention discloses a method for extracting natural sulforaphane used in the above antibacterial gel, aiming to improve the extraction purity of natural sulforaphane and develop an antibacterial gel with excellent antibacterial properties.

[0007] The purpose of the present invention can be achieved through the following technical solutions:

[0008] Application of natural sulforaphane in antibacterial gel, wherein the antibacterial gel is prepared by the following steps:

[0009] Itaconic acid, acrylamide, allyltrimethylammonium chloride, N,N-methylenebisacrylamide, ammonium persulfate, tetramethylethylenediamine and natural sulforaphane are added to ultrapure water, mixed and stirred for 10 to 20 minutes, and then glycerol is added thereto, stirred until gel-like, filled, and sterilized to obtain an antibacterial gel.

[0010] Furthermore, the antibacterial gel is composed of the following components in percentage by mass:

[0011] Itaconic acid 5.5%~6.0%;

[0012] Acrylamide 27.0%~28.5%;

[0013] Allyltrimethylammonium chloride 9.5%~11.0%;

[0014] N, N-methylenebisacrylamide 0.20%~0.35%;

[0015] Ammonium persulfate 0.30%~0.45%;

[0016] Tetramethylethylenediamine 1.5% to 2.0%;

[0017] Natural sulforaphane 4.0%-5.0%;

[0018] Glycerin 1.2%~2.0%;

[0019] The balance was ultrapure water.

[0020] Furthermore, a method for extracting natural sulforaphane comprises the following steps:

[0021] S1: Take broccoli seeds, dry them, grind them through a 40-mesh sieve, dry them and freeze them to obtain seed powder, soak the seed powder in n-hexane and heat it to 55-60° C., stir and reflux for 4-5 hours, and then heat it to 70-75° C. to separate the n-hexane, take out the solid component, and air-dry it to obtain defatted seed powder;

[0022] S2: Take defatted seed powder, add exogenous mustard seed enzyme solution, pH=7.0 potassium phosphate buffer and ascorbic acid thereto in sequence with stirring, raise the system temperature to 40-42°C, perform constant temperature enzymolysis for 20-25 minutes, and after the enzymolysis is completed, cool to room temperature to obtain an enzymolysis solution;

[0023] S3: subjecting the enzymatic hydrolysate to ultrasonic extraction for three times, each time with an interval of 5 minutes, and each ultrasonic extraction for 30 minutes. After the ultrasonic extraction is completed, an ultrasonic extract is obtained. The ultrasonic extract is subjected to microwave extraction for 5 to 7 minutes to obtain a microwave extract. The microwave extract is extracted with ethyl acetate at a volume ratio of 1:1 for three consecutive extractions. The ethyl acetate is combined and centrifuged at a centrifugal speed of 3000 to 4000 r / min for 20 to 30 minutes. The ethyl acetate layer is collected and filtered and evaporated to obtain a crude sulforaphane extract.

[0024] S4: Add the crude sulforaphane extract to acetone and stir to obtain a mixed solution. The mixed solution is filtered through a microporous filter membrane with a pore size of 0.45 μm, and the filtrate is collected. A macroporous separation resin is added thereto, and the mixture is placed in a shaking incubator at 30° C. and a rotation speed of 180 r / min for oscillation adsorption for 12 hours. After the adsorption is completed, the resin is collected by vacuum filtration, and an eluent is added to the resin, and the mixture is also placed in a shaking incubator at 30° C. and a rotation speed of 180 r / min for elution for 12 hours. After the elution is completed, the eluent is collected by vacuum filtration, and then the eluent is concentrated under reduced pressure, and then vacuum freeze-dried to constant weight to obtain purified natural sulforaphane.

[0025] Furthermore, the exogenous mustard seed enzyme solution in S2 is prepared by the following steps: adding mustard seed powder to distilled water with stirring, placing at 40-50° C., ultrasonically oscillating at a power of 60-120 W for 20-30 min, filtering to remove impurities, and obtaining the exogenous mustard seed enzyme solution.

[0026] Furthermore, the usage ratio of the distilled water to the mustard seed powder is 500 mL: 10-12 g.

[0027] Furthermore, the dosage ratio of the defatted seed powder, exogenous mustard seed enzyme solution, potassium phosphate buffer and ascorbic acid in S2 is 20g:200mL:200-300mL:16-20mg.

[0028] Furthermore, the power of the ultrasound in S3 is 60-120W; the temperature of the microwave extraction is 37-40°C, and the power of the microwave is 450-550W.

[0029] Furthermore, the usage ratio of acetone, sulforaphane crude extract, macroporous separation resin and eluent in S4 is 10-20 mL: 5 g: 1.5-2.0 g: 40-50 mL.

[0030] Furthermore, the macroporous separation resin in S4 is a XAD-2 type macroporous separation resin.

[0031] Furthermore, the eluent in S4 is a 95% by mass ethanol solution.

[0032] Beneficial effects of the present invention:

[0033] First, the present invention prepares an antibacterial gel, which is composed of a gel, an auxiliary agent glycerol and a solvent ultrapure water, wherein the present invention introduces itaconic acid and a quaternary ammonium salt allyltrimethylammonium chloride with a positive charge into acrylamide, and then forms a gel with a three-dimensional network structure at a suitable temperature through the initiation of ammonium sulfate and tetramethylethylenediamine and the cross-linking of N,N-methylenebisacrylamide. The gel prepared by the present invention is rich in carboxyl groups, amino groups and quaternary ammonium cations, and then the present invention introduces sulforaphane extracted from broccoli seeds into the above gel. Specifically, sulforaphane undergoes electrostatic adsorption with quaternary ammonium cations and the like in the gel through the isothiocyanate group with high reactivity. At the same time, some positively charged nitrogen atoms in the isothiocyanate group (the difference in electronegativity between the sulfur atom and the nitrogen atom in the isothiocyanate group causes the sulfur atom to be partially negatively charged, while the nitrogen atom is partially positively charged) can form hydrogen bonds with the abundant amino and carboxyl groups in the gel. Through the above effects, the present invention stably loads the natural sulforaphane extracted from broccoli seeds inside the gel with a three-dimensional network structure, and finally prepares the antibacterial gel, thereby realizing the application of natural sulforaphane in the antibacterial gel.

[0034] Then, after testing, the antibacterial gel prepared by the present invention has excellent antibacterial properties. Specifically, the antibacterial effect comes from the quaternary ammonium cations in the gel and the natural sulforaphane carried by it, wherein the quaternary ammonium cations mainly change the integrity and permeability of the cell membrane, causing the leakage of cell contents, thereby causing bacterial death; sulforaphane mainly inhibits bacterial nucleic acid and protein synthesis and reacts with specific enzymes inside the bacteria to inactivate the enzymes, thereby inhibiting bacterial growth and reproduction. The present invention comprehensively utilizes the synergistic effect of cationic bactericidal and natural antibacterial, and finally prepares an antibacterial gel with excellent antibacterial properties. The prepared antibacterial gel is mainly used for skin surface antibacterial.

[0035] At the same time, the antibacterial effect of the antibacterial gel prepared by the present invention is pH responsive and stable. First, the present invention introduces pH-sensitive itaconic acid into the gel. The pKa of itaconic acid is 3.5. Itaconic acid will dehydrogenate when the pH value is higher than the pKa value. +, resulting in an increase in the repulsive force between the structures of the gel, the gel will absorb water (ultrapure water) and swell, promoting the release of natural sulforaphane in the gel, and thus achieving enhanced antibacterial performance. Correspondingly, the antibacterial gel prepared by the present invention is mainly used for skin surface antibacterial, specifically including the sterilization of intact skin and the antibacterial repair of wounds. The surface of intact human skin is acidic, with a pH value of 4 to 6. When the skin is damaged, the pH value of the wound increases due to the leakage of microvessels, close to the physiological pH value (7.4) that is conducive to bacterial infection. As the pH of the wound increases, the antibacterial gel prepared by the present invention can release more natural sulforaphane, and the antibacterial performance is stronger, that is, the antibacterial effect of the antibacterial gel prepared by the present invention is pH responsive. At the same time, since sulforaphane is easily decomposed at a pH greater than 9 and at high temperatures, the present invention carries the extracted natural sulforaphane inside and on the surface of the gel that is acidic as a whole, which helps to improve the stability of natural sulforaphane, that is, the antibacterial effect of the antibacterial gel prepared by the present invention is stable in macroscopic terms, and through reasonable use, the antibacterial gel can achieve the purpose of intelligent drug release, reduce drug abuse and improve treatment effects.

[0036] Finally, the present invention provides a method for extracting the above-mentioned natural sulforaphane. The present invention utilizes the enzymatic hydrolysis of a homemade exogenous mustard seed enzyme solution, combined with ultrasound and microwave assistance, and then combined with ethyl acetate extraction and resin separation and purification technology, to finally achieve the extraction of natural sulforaphane from broccoli seeds. After testing, the purity of the extracted natural sulforaphane is 87.5% to 88.2%. DETAILED DESCRIPTION

[0037] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0038] Example 1

[0039] A method for extracting natural sulforaphane comprises the following steps:

[0040] S1 pretreatment: Take 200g of broccoli seeds (purchased from Xingning Qingfeng Yingke Seed Co., Ltd.) and dry them at 50℃ for 1h to remove excess moisture absorbed during storage and exposure to air. After drying, obtain dry seeds. After the dry seeds are cooled to room temperature, place them in a grinder and grind them through a 40-mesh sieve. Then place them at 45℃ and dry them for 5min to remove excess moisture generated by grinding. Then place them at 0℃ and store them in a frozen state to obtain seed powder. Soak the seed powder in n-hexane (AR; purchased from Sinopharm Chemical Reagent Co., Ltd.) and heat to 55℃, stir and reflux for 4h, then heat to 70℃ to separate n-hexane, take out the solid component, and continue to air-dry for 12h at room temperature to obtain defatted seed powder.

[0041] S2 exogenous mustard seed enzyme solution: mustard seeds (purchased from Xingning Qingfeng Yingke Seed Co., Ltd.) were crushed through a 60-mesh sieve to obtain mustard seed powder, 10 g of mustard seed powder was added to 500 mL of distilled water with stirring, and the mixture was placed at 40°C and ultrasonically oscillated at 60 W for 20 min, and impurities were removed by filtration to obtain exogenous mustard seed enzyme solution, which was stored at -10°C for later use;

[0042] S3 enzymolysis: 20 g of defatted seed powder was taken, 200 mL of exogenous mustard seed enzyme solution was added thereto with stirring, 200 mL of potassium phosphate buffer solution (pH=7.0; purchased from Shanghai MacLean Biochemical Technology Co., Ltd.) was added thereto with stirring, and 16 mg of ascorbic acid (97%; purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) was added thereto with stirring, the system was heated to 40°C, and enzymolysis was performed at a constant temperature for 20 min. After the enzymolysis was completed, the solution was cooled to room temperature to obtain an enzymolysis solution;

[0043] S4 extraction: The enzymatic hydrolyzate was subjected to ultrasonic extraction three times, each time with an interval of 5 minutes, each ultrasonic extraction for 30 minutes, and the ultrasonic power was 60W. After the ultrasonic extraction was completed, an ultrasonic extract was obtained, and the ultrasonic extract was subjected to microwave extraction. The microwave extraction temperature was 37°C, the microwave power was 450W, and the microwave extraction time was 5 minutes to obtain a microwave extract. The microwave extract was extracted with ethyl acetate (AR; purchased from Sinopharm Chemical Reagent Co., Ltd.) at a volume ratio of 1:1. The extraction was continued for three times, the ethyl acetate was combined, and then centrifuged at a centrifugal speed of 3000r / min for 20 minutes. The ethyl acetate layer was collected, filtered, and evaporated to obtain a crude sulforaphane extract;

[0044] S5 purification: 5 g of crude sulforaphane extract was added to 10 mL of acetone and stirred at room temperature for 15 min to obtain a mixed solution. The mixed solution was filtered through a microporous filter membrane with a pore size of 0.45 μm (φ25; purchased from Sinopharm Chemical Reagent Co., Ltd.), and the filtrate was collected. 1.5 g of macroporous separation resin (XAD-2; purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.) was added thereto, and the mixture was placed in a shaking incubator at 30°C and a rotation speed of 180 r / min for 12 h of oscillation adsorption. After the adsorption was completed, the mixture was filtered under reduced pressure. , collect the resin, and then add 40 mL of eluent to the resin, the eluent is a 95% by mass ethanol solution (CP; purchased from Sinopharm Chemical Reagent Co., Ltd.), and also place it in a shaking incubator at 30°C and a speed of 180 r / min for elution for 12 hours. After the elution is completed, vacuum filtration is performed, the eluent is collected, and then the eluent is concentrated under reduced pressure, and then placed in a vacuum freeze dryer (FD-1B-50; purchased from Jiangsu Tianling Co., Ltd.) for vacuum freeze drying to constant weight to obtain purified natural sulforaphane.

[0045] Example 2

[0046] A method for extracting natural sulforaphane comprises the following steps:

[0047] S1 pretreatment: Take 200g of broccoli seeds (purchased from Xingning Qingfeng Yingke Seed Co., Ltd.) and dry them at 80℃ for 2h to remove excess moisture absorbed during storage and exposure to air. After drying, obtain dry seeds. After the dry seeds are cooled to room temperature, place them in a grinder and grind them through a 40-mesh sieve, then place them at 50℃ and dry them for 10min to remove excess moisture generated by grinding, and then place them at -10℃ and freeze them for storage to obtain seed powder. Soak the seed powder in n-hexane (AR; purchased from Sinopharm Chemical Reagent Co., Ltd.) and heat to 60℃, stir and reflux for 5h, and then heat to 75℃ to separate n-hexane, take out the solid component, and continue to air-dry for 12h at room temperature to obtain defatted seed powder;

[0048] S2 exogenous mustard seed enzyme solution: take mustard seeds (purchased from Xingning Qingfeng Yingke Seed Co., Ltd.) and crush them through a 60-mesh sieve to obtain mustard seed powder. Stir and add 11 g of mustard seed powder to 500 mL of distilled water. Place the mixture at 50°C and ultrasonically oscillate at 100 W for 30 min. Filter to remove impurities to obtain exogenous mustard seed enzyme solution. Store the exogenous mustard seed enzyme solution at -20°C for later use.

[0049] S3 enzymolysis: 20 g of defatted seed powder was taken, 200 mL of exogenous mustard seed enzyme solution was added thereto with stirring, 280 mL of potassium phosphate buffer solution (pH=7.0; purchased from Shanghai MacLean Biochemical Technology Co., Ltd.) was added thereto with stirring, and 18 mg of ascorbic acid (97%; purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) was added thereto with stirring, the system was heated to 42°C, and enzymolysis was performed at constant temperature for 25 min. After the enzymolysis was completed, the solution was cooled to room temperature to obtain an enzymolysis solution;

[0050] S4 extraction: The enzymatic hydrolyzate was subjected to ultrasonic extraction three times, each time with an interval of 5 minutes, each ultrasonic extraction for 30 minutes, and the ultrasonic power was 100W. After the ultrasonic extraction was completed, an ultrasonic extract was obtained, and the ultrasonic extract was subjected to microwave extraction. The microwave extraction temperature was 40°C, the microwave power was 550W, and the microwave extraction time was 7 minutes to obtain a microwave extract. The microwave extract was extracted with ethyl acetate (AR; purchased from Sinopharm Chemical Reagent Co., Ltd.) at a volume ratio of 1:1. The extraction was continued for three times, the ethyl acetate was combined, and then centrifuged at a centrifugal speed of 4000r / min for 30 minutes. The ethyl acetate layer was collected, filtered, and evaporated to obtain a crude sulforaphane extract;

[0051] S5 purification: 5 g of crude sulforaphane extract was added to 15 mL of acetone and stirred at room temperature for 20 min to obtain a mixed solution. The mixed solution was filtered through a microporous filter membrane with a pore size of 0.45 μm (φ25; purchased from Sinopharm Chemical Reagent Co., Ltd.), and the filtrate was collected. 2.0 g of macroporous separation resin (XAD-2; purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.) was added thereto, and the mixture was placed in a shaking incubator at 30°C and a rotation speed of 180 r / min for 12 h of oscillation adsorption. After the adsorption was completed, the mixture was filtered under reduced pressure. , collect the resin, and then add 50 mL of eluent to the resin, the eluent is a 95% by mass ethanol solution (CP; purchased from Sinopharm Chemical Reagent Co., Ltd.), and also place it in a shaking incubator at 30°C and a rotation speed of 180 r / min for elution for 12 hours. After the elution is completed, vacuum filtration is performed, the eluent is collected, and then the eluent is concentrated under reduced pressure, and then placed in a vacuum freeze dryer (FD-1B-50; purchased from Jiangsu Tianling Co., Ltd.) for vacuum freeze drying to constant weight to obtain purified natural sulforaphane.

[0052] Example 3

[0053] A method for extracting natural sulforaphane comprises the following steps:

[0054] S1 pretreatment: Take 200g of broccoli seeds (purchased from Xingning Qingfeng Yingke Seed Co., Ltd.) and dry them at 80℃ for 2h to remove excess moisture absorbed during storage and exposure to air. After drying, obtain dry seeds. After the dry seeds are cooled to room temperature, place them in a grinder and grind them through a 40-mesh sieve, then place them at 50℃ and dry them for 10min to remove excess moisture generated by grinding, and then place them at -10℃ and freeze them for storage to obtain seed powder. Soak the seed powder in n-hexane (AR; purchased from Sinopharm Chemical Reagent Co., Ltd.) and heat to 60℃, stir and reflux for 5h, and then heat to 75℃ to separate n-hexane, take out the solid component, and continue to air-dry for 12h at room temperature to obtain defatted seed powder;

[0055] S2 exogenous mustard seed enzyme solution: mustard seeds (purchased from Xingning Qingfeng Yingke Seed Co., Ltd.) were crushed through a 60-mesh sieve to obtain mustard seed powder, 12 g of mustard seed powder was added to 500 mL of distilled water with stirring, and the mixture was placed at 50°C and ultrasonically oscillated at 120 W for 30 min, and impurities were removed by filtration to obtain exogenous mustard seed enzyme solution, which was stored at -20°C for later use;

[0056] S3 enzymolysis: 20 g of defatted seed powder was taken, 200 mL of exogenous mustard seed enzyme solution was added thereto with stirring, 300 mL of potassium phosphate buffer (pH=7.0; purchased from Shanghai MacLean Biochemical Technology Co., Ltd.) was added thereto with stirring, and 20 mg of ascorbic acid (97%; purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) was added thereto with stirring, the system was heated to 42°C, and enzymolysis was performed at constant temperature for 25 min. After the enzymolysis was completed, the solution was cooled to room temperature to obtain an enzymolysis solution;

[0057] S4 extraction: The enzymatic hydrolyzate was subjected to ultrasonic extraction three times, each time with an interval of 5 minutes, each ultrasonic extraction for 30 minutes, and the ultrasonic power was 120W. After the ultrasonic extraction was completed, an ultrasonic extract was obtained, and the ultrasonic extract was subjected to microwave extraction. The microwave extraction temperature was 40°C, the microwave power was 550W, and the microwave extraction time was 7 minutes to obtain a microwave extract. The microwave extract was extracted with ethyl acetate (AR; purchased from Sinopharm Chemical Reagent Co., Ltd.) at a volume ratio of 1:1. The extraction was continued for three times, the ethyl acetate was combined, and then centrifuged at a centrifugal speed of 4000r / min for 30 minutes. The ethyl acetate layer was collected, filtered, and evaporated to obtain a crude sulforaphane extract;

[0058] S5 purification: 5 g of crude sulforaphane extract was added to 20 mL of acetone and stirred at room temperature for 20 min to obtain a mixed solution. The mixed solution was filtered through a microporous filter membrane with a pore size of 0.45 μm (φ25; purchased from Sinopharm Chemical Reagent Co., Ltd.), and the filtrate was collected. 2.0 g of macroporous separation resin (XAD-2; purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.) was added thereto, and the mixture was placed in a shaking incubator at 30°C and a rotation speed of 180 r / min for 12 h of oscillation adsorption. After the adsorption was completed, the mixture was filtered under reduced pressure. , collect the resin, and then add 50 mL of eluent to the resin, the eluent is a 95% by mass ethanol solution (CP; purchased from Sinopharm Chemical Reagent Co., Ltd.), and also place it in a shaking incubator at 30°C and a rotation speed of 180 r / min for elution for 12 hours. After the elution is completed, vacuum filtration is performed, the eluent is collected, and then the eluent is concentrated under reduced pressure, and then placed in a vacuum freeze dryer (FD-1B-50; purchased from Jiangsu Tianling Co., Ltd.) for vacuum freeze drying to constant weight to obtain purified natural sulforaphane.

[0059] Test Example 1

[0060] The purity of the purified natural sulforaphane in Examples 1 to 3 was determined by high performance liquid chromatography. The determination process is as follows. The determination results are shown in Table 1:

[0061] (1) Chromatographic conditions: The chromatographic column is ZORBAX Eclipse XDB-C 18 (Agilent; 150 mm long × 4.6 mm inner diameter; filling particle diameter 5 μm); mobile phase is acetonitrile-water (the volume ratio of acetonitrile and water is 77:23); flow rate is 0.6 mL / min; injection volume is 10 μL; column temperature is 20°C; detection wavelength is 254 nm.

[0062] (2) Standard curve: 5 mg of sulforaphane standard (HPLC; purchased from Tianjin Jianfeng Natural Products Research and Development Co., Ltd.) was dissolved in 5 mL of methanol (AR; purchased from Sinopharm Chemical Reagent Co., Ltd.) to prepare standard solutions with final concentrations of 500, 250, 125, 62.5, 31.25, and 0 μg / mL. The concentrations were injected in order from low to high, with an injection volume of 10 μL, and each concentration was repeated 3 times. The analysis was performed under the above chromatographic conditions. With the mass concentration of sulforaphane as the ordinate Y and the peak area of ​​sulforaphane as the abscissa X, a linear regression was performed to obtain the regression equation:

[0063] Y=0.00003X-0.3102, R 2 =0.992.

[0064] (3) Based on the above chromatographic conditions and standard curve, the purity of the natural sulforaphane purified in Examples 1 to 3 was measured respectively, and recorded in the following Table 1:

[0065] Table 1

[0066] project Example 1 Example 2 Example 3 purity / % 87.5 88.2 88.0

[0067] It can be seen from Table 1 that the extraction method of the present invention can obtain high-purity sulforaphane.

[0068] Example 4

[0069] Application of natural sulforaphane in antibacterial gel:

[0070] First, the antibacterial gel is composed of the following components by mass percentage:

[0071] Itaconic acid 5.5%;

[0072] Acrylamide 27.0%;

[0073] Allyltrimethylammonium chloride 9.5%;

[0074] N, N-methylenebisacrylamide 0.20%;

[0075] Ammonium persulfate 0.30%;

[0076] Tetramethylethylenediamine 1.5%;

[0077] 4.0% natural sulforaphane prepared in Example 1;

[0078] Glycerol 1.2%;

[0079] The balance was ultrapure water.

[0080] Among them, itaconic acid has a purity of 99%; acrylamide is of reagent grade with a purity of 99%; allyltrimethylammonium chloride has a purity of 98%; N,N-methylenebisacrylamide is of reagent grade with a purity of 97%; ammonium persulfate is of reagent grade with a purity of 98.5%; tetramethylethylenediamine has a purity of 99%; glycerol is of reagent grade with a purity of 99%; the above raw materials were all purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0081] Then, the antibacterial gel was prepared by the following steps:

[0082] Itaconic acid, acrylamide, allyltrimethylammonium chloride, N,N-methylenebisacrylamide, ammonium persulfate, tetramethylethylenediamine and the natural sulforaphane prepared in Example 1 were added to ultrapure water, and the mixture was stirred for 10 minutes. Glycerol was then added thereto, and the mixture was stirred until it was in a gel state. The mixture was then filled and sterilized to obtain an antibacterial gel.

[0083] Example 5

[0084] Application of natural sulforaphane in antibacterial gel:

[0085] First, the antibacterial gel is composed of the following components by mass percentage:

[0086] Itaconic acid 5.5%;

[0087] Acrylamide 28.0%;

[0088] Allyltrimethylammonium chloride 10.0%;

[0089] N, N-methylenebisacrylamide 0.30%;

[0090] Ammonium persulfate 0.35%;

[0091] Tetramethylethylenediamine 1.8%;

[0092] 4.8% natural sulforaphane prepared in Example 2;

[0093] Glycerol 1.8%;

[0094] The balance was ultrapure water.

[0095] Among them, itaconic acid has a purity of 99%; acrylamide is of reagent grade with a purity of 99%; allyltrimethylammonium chloride has a purity of 98%; N,N-methylenebisacrylamide is of reagent grade with a purity of 97%; ammonium persulfate is of reagent grade with a purity of 98.5%; tetramethylethylenediamine has a purity of 99%; glycerol is of reagent grade with a purity of 99%; the above raw materials were all purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0096] Then, the antibacterial gel was prepared by the following steps:

[0097] Itaconic acid, acrylamide, allyltrimethylammonium chloride, N,N-methylenebisacrylamide, ammonium persulfate, tetramethylethylenediamine and the natural sulforaphane prepared in Example 2 were added to ultrapure water, and the mixture was stirred for 20 minutes. Glycerol was then added thereto, and the mixture was stirred until it was in a gel state. The mixture was then filled and sterilized to obtain an antibacterial gel.

[0098] Example 6

[0099] Application of natural sulforaphane in antibacterial gel:

[0100] First, the antibacterial gel is composed of the following components by mass percentage:

[0101] Itaconic acid 6.0%;

[0102] Acrylamide 28.5%;

[0103] Allyltrimethylammonium chloride 11.0%;

[0104] N, N-methylenebisacrylamide 0.35%;

[0105] Ammonium persulfate 0.45%;

[0106] Tetramethylethylenediamine 2.0%;

[0107] 5.0% natural sulforaphane prepared in Example 3;

[0108] Glycerol 2.0%;

[0109] The balance was ultrapure water.

[0110] Among them, itaconic acid has a purity of 99%; acrylamide is of reagent grade with a purity of 99%; allyltrimethylammonium chloride has a purity of 98%; N,N-methylenebisacrylamide is of reagent grade with a purity of 97%; ammonium persulfate is of reagent grade with a purity of 98.5%; tetramethylethylenediamine has a purity of 99%; glycerol is of reagent grade with a purity of 99%; the above raw materials were all purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0111] Then, the antibacterial gel was prepared by the following steps:

[0112] Itaconic acid, acrylamide, allyltrimethylammonium chloride, N,N-methylenebisacrylamide, ammonium persulfate, tetramethylethylenediamine and the natural sulforaphane prepared in Example 3 were added to ultrapure water, and the mixture was stirred for 20 minutes. Glycerol was then added thereto, and the mixture was stirred until it was in a gel state. The mixture was then filled and sterilized to obtain an antibacterial gel.

[0113] Comparative Example 1

[0114] Comparative Example 1 is the control group of Example 5, in which the natural sulforaphane prepared in Example 2 of the antibacterial gel component in Example 5 is replaced with ultrapure water, and the other raw materials and preparation steps remain unchanged, thereby finally obtaining the antibacterial gel.

[0115] Comparative Example 2

[0116] Comparative Example 2 is the control group of Example 5, in which the itaconic acid component of the antibacterial gel in Example 5 is replaced with ultrapure water, and the other raw materials and preparation steps remain unchanged, and the antibacterial gel is finally obtained.

[0117] Test Example 2

[0118] The antibacterial performance and pH response performance of the antibacterial gels prepared in Examples 4 to 6 and Comparative Examples 1 to 2 were tested. The performance test process is as follows, and the test results are shown in Table 2:

[0119] (1) Antibacterial performance: 0.1 g of the antibacterial gel prepared in Examples 4 to 6 and Comparative Examples 1 to 2 was mixed with 10 mL of 1×10 7The mixture of Escherichia coli liquid and Staphylococcus aureus liquid was placed in a test tube and placed on a constant temperature shaker at 37°C and 120 r / min for 4 h. This was used as the experimental group, and the liquid without antibacterial gel was used as the blank group. The liquid of the experimental group and the blank group was diluted to 1×10 5 CFU / mL, 100 μL of bacterial solution was applied to the surface of the agar plate, and placed in a shaking incubator at 37°C for 24 hours. The antibacterial rate was calculated by the plate colony counting method. The antibacterial rate calculation formula is as follows, and the calculation results are recorded in the following Table 2:

[0120] Antibacterial rate = [(Na-Nb) / Na] × 100%;

[0121] Wherein, Na is the colony count of the blank group; Nb is the colony count of the experimental group.

[0122] (2) pH response performance: The antibacterial gels prepared in Examples 4 to 6 and Comparative Examples 1 to 2 were placed in a vacuum freeze dryer (FD-1B-50; purchased from Jiangsu Tianling Co., Ltd.) and vacuum freeze-dried to constant weight to obtain a gel solid sample. 100 g of the gel solid sample was placed in 100 mL of PBS buffer solutions of pH = 5.0, pH = 7.4 and pH = 8.5, respectively, and placed on a constant temperature shaker at 37° C. for 1 h. The mixture was filtered and the filtrate was taken. The mass concentration (mg / μL) of sulforaphane in the filtrate was determined by the high performance liquid chromatography method in Test Example 1.

[0123] Table 2

[0124] project Example 4 Example 5 Example 6 Comparative Example 1 Comparative Example 2 Antibacterial rate (Escherichia coli) / % 97.7 98.5 98.2 75.4 99.0 Antibacterial rate (Staphylococcus aureus) / % 96.0 97.2 96.8 70.8 97.5 Sulforaphane mass concentration (mg / μL) / pH=5.0 0.0115 0.0045 0.0085 0 0.0375 Sulforaphane mass concentration (mg / μL) / pH=7.4 0.0235 0.0110 0.0170 0 0.0420 Sulforaphane mass concentration (mg / μL) / pH=8.5 0.0310 0.0175 0.0255 0 0.0435

[0125] It can be seen from Table 2 that by adding the natural sulforaphane extracted by the present invention, the prepared gel material has excellent antibacterial properties. At the same time, by introducing itaconic acid, the response of the antibacterial gel to pH is achieved.

[0126] It should be noted that, in this article, terms such as "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or equipment that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or equipment.

[0127] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A use of natural sulforaphane in the preparation of antibacterial gel, characterized in that: The antibacterial gel is prepared by the following steps: Itaconic acid, acrylamide, allyltrimethylammonium chloride, N,N-methylenebisacrylamide, ammonium persulfate, tetramethylethylenediamine and natural sulforaphane are added to ultrapure water, mixed and stirred for 10 to 20 minutes, and then glycerol is added thereto, stirred until gel-like, filled, and sterilized to obtain an antibacterial gel, wherein the bacteria are bacteria.

2. The use of natural sulforaphane in the preparation of antibacterial gel according to claim 1, characterized in that: The antibacterial gel is composed of the following components in percentage by weight:

3. The use of natural sulforaphane in the preparation of antibacterial gel according to claim 1, characterized in that: The natural sulforaphane is extracted by the following steps: S1: Take broccoli seeds, dry them, grind them through a 40-mesh sieve, dry them and freeze them to obtain seed powder, soak the seed powder in n-hexane and heat it to 55-60° C., stir and reflux for 4-5 hours, and then heat it to 70-75° C. to separate the n-hexane, take out the solid component, and air-dry it to obtain defatted seed powder; S2: Take defatted seed powder, add exogenous mustard seed enzyme solution, potassium phosphate buffer solution with pH=7.0 and ascorbic acid thereto in sequence with stirring, raise the system temperature to 40-42°C, perform enzymolysis at constant temperature for 20-25 minutes, and after the enzymolysis is completed, cool to room temperature to obtain an enzymolysis solution; S3: subjecting the enzymatic hydrolysate to ultrasonic extraction for three times, each time with an interval of 5 minutes, and each ultrasonic extraction for 30 minutes. After the ultrasonic extraction is completed, an ultrasonic extract is obtained. The ultrasonic extract is subjected to microwave extraction for 5 to 7 minutes to obtain a microwave extract. The microwave extract is extracted with ethyl acetate at a volume ratio of 1:1 for three consecutive extractions. The ethyl acetate is combined and centrifuged at a centrifugal speed of 3000 to 4000 r / min for 20 to 30 minutes. The ethyl acetate layer is collected and filtered and evaporated to obtain a crude sulforaphane extract. S4: Add the crude sulforaphane extract to acetone and stir to obtain a mixed solution. The mixed solution is filtered through a microporous filter membrane with a pore size of 0.45 μm, and the filtrate is collected. A macroporous separation resin is added thereto, and the mixture is placed in a shaking incubator at 30° C. and a rotation speed of 180 r / min for oscillation adsorption for 12 hours. After the adsorption is completed, the resin is collected by vacuum filtration, and an eluent is added to the resin, and the mixture is also placed in a shaking incubator at 30° C. and a rotation speed of 180 r / min for elution for 12 hours. After the elution is completed, the eluent is collected by vacuum filtration, and then the eluent is concentrated under reduced pressure, and then vacuum freeze-dried to constant weight to obtain purified natural sulforaphane.

4. The use of natural sulforaphane in the preparation of antibacterial gel according to claim 3, characterized in that: The exogenous mustard seed enzyme solution in S2 is prepared by the following steps: adding mustard seed powder to distilled water with stirring, placing at 40-50° C., ultrasonically oscillating at a power of 60-120 W for 20-30 minutes, filtering to remove impurities, and obtaining the exogenous mustard seed enzyme solution.

5. The use of natural sulforaphane in the preparation of antibacterial gel according to claim 4, characterized in that: The usage ratio of the distilled water to the mustard seed powder is 500 mL:10-12 g.

6. The use of natural sulforaphane in the preparation of antibacterial gel according to claim 3, characterized in that: The usage ratio of the defatted seed powder, exogenous mustard seed enzyme solution, potassium phosphate buffer and ascorbic acid in S2 is 20g:200mL:200-300mL:16-20mg.

7. The use of natural sulforaphane in the preparation of antibacterial gel according to claim 3, characterized in that: The power of the ultrasound in S3 is 60-120W; the temperature of the microwave extraction is 37-40°C, and the power of the microwave is 450-550W.

8. The use of natural sulforaphane in the preparation of antibacterial gel according to claim 3, characterized in that: The usage ratio of acetone, sulforaphane crude extract, macroporous separation resin and eluent in S4 is 10-20 mL: 5 g: 1.5-2.0 g: 40-50 mL.

9. The use of natural sulforaphane in preparing antibacterial gel according to claim 3, characterized in that: The macroporous separation resin in S4 is XAD-2 type macroporous separation resin.

10. The use of natural sulforaphane in the preparation of antibacterial gel according to claim 3, characterized in that: The eluent in S4 is a 95% by mass ethanol solution.

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