Method for preparing mustard alkaline double-protein composite gel

Abstract

The application discloses a preparation method of a loaded mustard alkaline double-protein composite gel. The method takes a mixed solution of gelatin and mustard alkaline as an aqueous phase, takes rapeseed oil as an oil phase, and through emulsification and cross-linking, takes soybean protein isolate as a plant cross-linking agent to be cross-linked and solidified, and then isopropanol is used to remove oil to obtain the loaded mustard alkaline double-protein composite gel. The mustard alkaline and the gelatin in the aqueous phase are made into a water-in-oil emulsion system through the emulsification and cross-linking method, and the biologically safe and non-toxic soybean protein isolate is used as the cross-linking agent to increase the viscosity of the system and stabilize the system. The animal protein and the plant protein are combined to serve as a delivery system of the mustard alkaline, and the bioavailability and the bioaccessibility of the mustard alkaline are improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of drug carriers, and relates to a preparation method of a double-protein composite gel loaded with sinapine. BACKGROUND

[0002] Sinapine has significant antioxidant, anti-radiation, anti-aging, antihypertensive, anti-inflammatory, anti-diarrhea and anti-androgen activities, but is unstable in alkaline solution, is easy to decompose into sinapinic acid and choline, is easy to hydrolyze, has poor ultraviolet stability, and is mostly decomposed during the digestive process, resulting in poor bioavailability. Therefore, targeted delivery is beneficial to expanding the application range by increasing the bioavailability and utilization.

[0003] Gelatin is an animal protein, and gelatin microspheres have the advantages of simple preparation, low price, high drug loading capacity, good dispersibility, good histocompatibility and biodegradability.

[0004] The emulsion crosslinking method is to prepare O / W, W / O, W / O / W and O / W / O type single emulsion and multiple emulsion emulsion systems according to the properties of drugs and natural or artificial high molecular materials. After forming a stable emulsion, a crosslinking agent is added by continuous stirring or other methods. The active groups (aldehyde groups) in the crosslinking agent can react with the amino groups or alcohol groups of the high molecular material to crosslink and prepare microspheres. At the same time, the microspheres are gradually solidified. After filtration, washing and drying, the final drug-loaded microspheres are obtained, and the particle size is usually in the range of 1-100 mu m. According to the different emulsification and crosslinking methods, the emulsion crosslinking method can be divided into emulsion ion crosslinking method, emulsion chemical crosslinking method and multiple emulsion method. However, the commonly used crosslinking agents such as glutaraldehyde, formaldehyde and EDA have the disadvantages of high toxicity and high price, so it is helpful to find suitable substitutes to prepare safer delivery products. In addition, the oil phase used at present is mostly a mixture of liquid paraffin and a surfactant, but some surfactants such as span 80, stearic acid, soft phospholipid and tween may cause harm to the body, which does not meet the development concept of green health. SUMMARY

[0005] In view of the problem of low bioavailability of sinapine at present, the application provides a preparation method of a double-protein composite gel loaded with sinapine, which solves the problems of instability of sinapine in alkaline environment, easy decomposition, poor ultraviolet stability and high toxicity of crosslinking agents, and improves the bioavailability of sinapine.

[0006] The technical scheme of the application is as follows:

[0007] The preparation method of the double-protein composite gel loaded with sinapine uses rapeseed oil as the oil phase, and prepares a water-in-oil emulsion system of sinapine and gelatin in the water phase by the emulsion crosslinking method. A crosslinking agent is added to increase the viscosity of the system and stabilize the system. The specific steps are as follows:

[0008] (1) Under water bath heating condition, 60% gelatin solution is mixed with 200-300 μg / mL sinapine solution uniformly at a volume ratio of 1:1 to obtain an aqueous phase;

[0009] (2) Under water bath heating condition, rapeseed oil is stirred as an oil phase;

[0010] (3) The aqueous phase is slowly added to the oil phase at a water to oil ratio of 1:3-1:4, and the mixture is stirred to be uniform and then placed in an ice water bath to obtain a water-in-oil emulsion;

[0011] (4) Soybean protein isolate is added to the water-in-oil emulsion at a mass ratio of 0.01%-0.05% of the soybean protein isolate to the volume of the water-in-oil emulsion, g:mL, and the mixture is emulsified uniformly and then placed at 4°C for cross-linking and solidification for 24 hours or more. After the cross-linking is completed, isopropanol is added for washing, centrifugation and water washing to obtain a deoiled sinapine-loaded double-protein composite gel.

[0012] Preferably, in step (1), the gelatin solution is prepared as follows: the gelatin is pre-soaked in normal temperature water for 10 minutes or more, and then dissolved in the water at 40-60°C to form a gelatin solution.

[0013] Preferably, in step (1) or (2), the water bath heating temperature is 40-60°C.

[0014] Preferably, in step (3), the stirring time is 30 minutes or more.

[0015] Preferably, in step (4), the emulsification method is magnetic stirring or vortex.

[0016] Preferably, in step (4), the specific washing method is as follows: isopropanol is added to the mixture after the cross-linking is completed for vortex washing, and then centrifuged at 8000 r / min for 5 minutes. The vortex washing and centrifugation steps are repeated, and finally the mixture is washed with water.

[0017] Compared with the prior art, the present application has the following advantages:

[0018] (1) The present application utilizes the negative charge of gelatin and the permanent positive charge of sinapine to enhance the interaction between them through electrostatic adsorption. Meanwhile, rapeseed oil is used as the oil phase, and the sinapine and gelatin in the aqueous phase are prepared into a water-in-oil emulsion system through emulsification and cross-linking. Soybean protein isolate with good biocompatibility is used as a cross-linking agent to increase the viscosity of the system and stabilize the system. The use of animal protein and plant protein composite as the delivery system of sinapine improves the bioavailability and bioaccessibility of sinapine.

[0019] (2) The prepared loaded mustard alkali double-protein composite gel has high nutritional functions such as antioxidation, anticancer, and anti-inflammation, and the functions of easy digestion and absorption, promotes further development of the mustard alkali in the field of food and medicine, and improves comprehensive utilization of soybean protein isolate. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 It is a flowchart of the preparation method of the loaded mustard alkali double-protein composite gel.

[0021] Figure 2 It is a graph of the influence of different mustard alkali concentrations on the particle size and potential of the composite gel.

[0022] Figure 3 It is a graph of the influence of different mustard alkali concentrations on the embedding rate and drug loading of the composite gel.

[0023] Figure 4 It is a graph of the influence of different water-oil ratios on the particle size and potential of the composite gel.

[0024] Figure 5 It is a graph of the influence of different water-oil ratios on the embedding rate and drug loading of the composite gel.

[0025] Figure 6 It is a graph of the influence of different protein concentrations on the particle size and potential of the composite gel.

[0026] Figure 7 It is a graph of the influence of different protein concentrations on the embedding rate and drug loading of the composite gel.

[0027] Figure 8 It is a graph of the influence of different water-oil ratios, mustard alkali concentrations, and protein concentrations on the swelling degree of the composite gel.

[0028] Figure 9 It is an optical microscope graph of the composite gel prepared in different emulsification modes.

[0029] Figure 10 It is a transmission electron microscope graph of the composite gel.

[0030] Figure 11 It is a Fourier infrared spectrum graph of uncrosslinked mustard alkali gelatin gel, soybean protein isolate, loaded mustard alkali double-protein gel, and blank double-protein composite gel.

[0031] Figure 12 It is an in-vitro digestion broken line graph of glutaraldehyde control group, soybean protein isolate experimental group, and free mustard alkali group.

[0032] Figure 13 It is an ABTS free radical scavenging capacity graph of glutaraldehyde control group and soybean protein isolate experimental group.

[0033] Figure 14The DPPH free radical scavenging capacity diagram of the glutaraldehyde control group and the soybean protein isolate experimental group. DETAILED DESCRIPTION

[0034] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions of the present application will be described clearly and completely below in combination with specific embodiments. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. All other embodiments obtained by those skilled in the art based on the embodiments in the present application without creative labor are within the protection scope of the present application.

[0035] Embodiment 1

[0036] The preparation method of the loaded mustard alkaline double-protein composite gel comprises the following steps:

[0037] (1) Gelatin is pre-soaked in normal temperature water for 10 min, and then dissolved in a 40℃ water bath to form a gelatin solution with a mass concentration of 60%, and then mixed with different concentrations of mustard alkaline solution at a volume ratio of 1:1 to prepare an aqueous phase, wherein the concentration of mustard alkaline is 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL and 500 μg / mL, respectively;

[0038] (2) Rapeseed oil is magnetically stirred in a 40℃ water bath as an oil phase;

[0039] (3) The water / oil ratio is 1:3 by volume, and the aqueous phase is slowly added to the oil phase, stirred for 30 min, and then cooled to 4℃ in an ice water bath to obtain a water-in-oil emulsion;

[0040] (4) 0.01% (g:mL, i.e. 0.01 g of soybean protein isolate is added to every 100 mL of water-in-oil emulsion) of soybean protein isolate is added to the above water-in-oil emulsion, stirred uniformly, and stored in a 4℃ refrigerator for cross-linking and solidification for 24 h, and precipitates will be precipitated;

[0041] (5) The above mixture is added with isopropyl alcohol for vortex washing, centrifuged at 8000 r / min for 5 min, washed again, centrifuged at 8000 r / min for 5 min again, and then washed with water to obtain a deoiled composite gel. The changes of particle size, zeta potential and drug loading and encapsulation efficiency of the composite gel prepared at different concentrations of mustard alkaline are detected, and the results are shown in Figure 2 and Figure 3 .

[0042] Embodiment 2

[0043] The preparation method of the loaded mustard alkaline double-protein composite gel comprises the following steps:

[0044] (1) Gelatin was pre-soaked in normal temperature water for 10 min, then heated and dissolved in 40℃ water bath to form a gelatin solution with a mass concentration of 60%, and then mixed with a mustard alkali solution with a concentration of 300 μg / mL at a volume ratio of 1:1 to prepare a water phase;

[0045] (2) Rapeseed oil was magnetically stirred under 40℃ water bath as an oil phase;

[0046] (3) The water phase was slowly added to the oil phase at different water / oil ratios, stirred for 30 min, and then cooled to 4℃ in an ice water bath to obtain a water-in-oil emulsion, wherein the water / oil ratios were 1:2, 1:3, 1:4, 1:5, and 1:6, respectively.

[0047] (4) 0.01% soybean protein isolate was added to the above water-in-oil emulsion, stirred uniformly, and stored in a 4℃ refrigerator for cross-linking and solidification for 24 h, and precipitates were separated out;

[0048] (5) The above mixture was added with isopropyl alcohol, vortex washed, centrifuged at 8000 r / min for 5 min, washed again, centrifuged at 8000 r / min for 5 min again, and then washed with water to obtain a deoiled composite gel.

[0049] The particle size, zeta potential, drug loading, and encapsulation efficiency of the composite gels prepared at different water / oil ratios were detected, and the results are shown in Figure 4 and Figure 5 .

[0050] Example 3

[0051] The preparation method of the mustard alkali-loaded double-protein composite gel comprises the following steps:

[0052] (1) Gelatin was pre-soaked in normal temperature water for 10 min, then heated and dissolved in 40℃ water bath to form a gelatin solution with a mass concentration of 60%, and then mixed with a mustard alkali solution with a concentration of 300 μg / mL at a volume ratio of 1:1 to prepare a water phase;

[0053] (2) Rapeseed oil was magnetically stirred under 40℃ water bath as an oil phase;

[0054] (3) The water phase was slowly added to the oil phase at a water / oil ratio of 1:3, stirred for 30 min, and then cooled to 4℃ in an ice water bath to obtain a water-in-oil emulsion by emulsification and cross-linking;

[0055] (4) Different amounts of soybean protein isolate were added to the above water-in-oil emulsion, stirred uniformly, and stored in a 4℃ refrigerator for cross-linking and solidification for 24 h, and precipitates were separated out;

[0056] (5) The above mixture is added with isopropyl alcohol for vortex washing, centrifuged at 8000 r / min for 5 min, washed again, centrifuged at 8000 r / min for 5 min again, and washed with water again to obtain the deoiled composite gel. The addition amount of soybean protein isolate is 0.5%, 0.1%, 0.05%, 0.01%, and 0.005%, respectively.

[0057] The changes of particle size, potential, drug loading amount, and encapsulation rate of the composite gel are detected at different concentrations of soybean protein isolate, and the results are shown in Figure 6 and Figure 7 .

[0058] Example 4

[0059] The preparation method of the double-protein composite gel loaded with sinapine includes the following steps:

[0060] (1) The gelatin is pre-soaked in normal temperature water for 10 min, and then dissolved in a water bath at 40℃ to form a gelatin solution with a mass concentration of 60%, which is then mixed with a sinapine solution with a concentration of 300 μg / mL at a volume ratio of 1:1 to prepare an aqueous phase;

[0061] (2) The rapeseed oil is magnetically stirred in a water bath at 40℃ to serve as an oil phase;

[0062] (3) The aqueous phase is slowly added to the oil phase at a water / oil ratio of 1:3, emulsified for 30 min, and cooled to 4℃ in an ice water bath to obtain a water-in-oil emulsion by emulsion crosslinking;

[0063] (4) 0.01% soybean protein isolate is added to the above water-in-oil emulsion, stirred uniformly, and stored in a 4℃ refrigerator for crosslinking and solidification for 24 h, during which precipitates are precipitated, and the emulsification methods are magnetic stirring and vortex, respectively.

[0064] (5) The above mixture is added with isopropyl alcohol for vortex washing, centrifuged at 8000 r / min for 5 min, washed again, centrifuged at 8000 r / min for 5 min again, and washed with water again to obtain the deoiled composite gel.

[0065] The changes of the morphology of the composite gel under an optical microscope are detected at different emulsification methods, and the results are shown in Figure 9 .

[0066] Test Example

[0067] The physicochemical characteristics of the double-protein composite gel loaded with sinapine prepared in Examples 1-3 are determined, and the specific method is as follows:

[0068] (1) Particle size and potential determination: The gel is diluted 50 times with water, and the particle size and potential are detected by a Mastersizer 3000 laser diffraction particle size analyzer at a data acquisition rate of 10 kHz and 25℃.

[0069] (2) Drug loading and encapsulation efficiency determination: 1 g of protein gel was ultrasonically dispersed for 30 min, centrifuged at 8000 r / min for 5 min, and 1 mL of supernatant was taken and added with 1 mL of pH = 10 buffer. The absorbance was determined at 326 nm using a UV-5000 ultraviolet-visible spectrophotometer, and the concentration was calculated according to the standard curve.

[0070] (3) Swelling degree determination: A certain amount of microspheres was dissolved in a phosphate buffer with pH 7.4. After being placed at room temperature for 24 h, the precipitate was taken out by centrifugation, and the surface moisture of the microspheres was dried and weighed. The test was repeated three times. The swelling degree of the microspheres was calculated according to the following formula, and the results are shown in Figure 8

[0071] (4) Optical microscope morphology observation: A small amount of emulsion was taken to the glass slide and covered with a cover glass. The emulsion treated by magnetic stirring and vortex was observed under 100x and 400x, respectively. The results are shown in Figure 9

[0072] (5) Transmission electron microscope morphology observation: A certain amount of hydrogel was diluted with water, dropped onto a copper mesh, and observed under an infrared lamp. The results are shown in Figure 10

[0073] (6) Fourier infrared spectrum analysis: The meson alkali double protein composite gel, blank gel, soybean protein isolate, and gel without soybean protein isolate were scanned by Fourier infrared spectrum. The infrared spectrum is shown in Figure 11

[0074] (7) In vitro digestion of gel: Gastric juice was prepared by adding 3.2 mg / mL pepsin and 2 mg / mL sodium chloride, and intestinal juice was prepared by adding 6.8 mg / mL k2HPO4, 0.8 mg / mL intestinal protease, 20 mg / mL bile salt, and 8.8 mg / mL NaCl. The temperature was set to 37°C using a water bath constant temperature oscillator. The gel was dissolved in deionized water, and the gastric juice was added for sufficient reaction. During the reaction, samples were taken every 30 minutes. After completing the gastric digestion, the intestinal juice was added, and the pH was adjusted to 7.8. Samples were taken every 30 minutes, and the absorbance was measured. Finally, the bioaccessibility, bioavailability, and gastrointestinal release were measured, and the results are shown in Figure 12

[0075] ​​​​​(8) ABTS antioxidant assay: Preparation of ABTS working solution: a mixture solution containing potassium persulfate (2.5 mmol / L) and ABTS (7 mmol / L) was prepared with pure water, and was placed in the dark at a constant temperature of 25°C for 12-16 h to obtain ABTS cation radical solution. The ABTS cation radical working solution was obtained by diluting the solution with pure water to an absorbance value of 0.7±0.005 at 734 nm. 0.1 mL of sample solution was accurately transferred, mixed with 3.9 mL of working solution, and incubated at 25°C for 6 min, and then the absorbance value A was immediately measured at 734 nm. Methanol solution was used as a blank control (A0). The ABTS radical scavenging capacity was calculated according to the following formula. The results are shown in Table 2. Figure 13

[0076] (9) DPPH antioxidant assay: 2 mL of sample solution and 2 mL of 300 μmol / L freshly prepared DPPH solution were added to test tubes, respectively, and the absorbance was measured at 517 nm after 30 min of reaction in the dark. In addition, a control group without sample and a blank group without DPPH were used. Each group of samples was measured in triplicate, and the DPPH radical scavenging capacity of the samples was calculated according to the following formula. The results are shown in Table 3. Figure 14

[0077]

[0078]

[0079]

[0080]

[0081]

[0082] As can be seen from the figure, with the increase of sinapine concentration, more sinapine is adsorbed with gelatin, thus increasing the droplet size of the emulsion, and finally increasing the size of the gel particle size Figure 2 . However, with the increase of sinapine concentration, the embedding rate decreases Figure 3 , and the embedding rate is the largest at 300 μg / mL. The reason may be that the amount of soybean protein isolate is not appropriate, the concentration of crosslinking agent is too high, and the viscosity of gelatin increases, resulting in fewer voids of the gel, and thus the embedding rate of the gel decreases. With the decrease of the water-oil ratio, the sinapine embedding rate decreases Figure 5 . The reason may be that with the increase of the volume of the oil phase, the dispersion of the emulsion becomes worse, resulting in a decrease in the encapsulation efficiency of the microspheres. The particle size of the gel is the smallest at 1:3, and then shows a growing trend Figure 4 ​​), indicating that with the increase of oil phase volume, the adhesion degree of the gel increases, resulting in the increase of particle size. And in the preparation of the gel, the more plant oil needed, the more detergent needed in washing, also increased the preparation cost. Considering comprehensively, 1:3 is the optimal value. With the decrease of protein concentration, the particle size of the microspheres first increases and then decreases( Figure 6 ), and at 0.01%, the particle size is small, which may be due to the decrease of protein concentration, the cross-linking degree between the microspheres decreases, which is more conducive to the formation of microspheres, resulting in the decrease of particle size. And at 0.05%, the drug loading is the highest( Figure 7 ), but considering comprehensively, 0.01% drug loading and embedding rate are the largest, and the embedding effect of sinapine is better.

[0083] After the gel is placed in phosphate buffer solution for 24 hours, the swelling degree is calculated. The concentration of soybean protein isolate has no significant effect on the swelling degree, but with the decrease of water-oil ratio and the increase of sinapine concentration( Figure 8 ), the swelling degree first increases and then decreases. This is because gelatin, sinapine and soybean protein isolate slowly shrink during the solidification process, expelling water in the gel. It leaves many microchannels in the gel. With the increase of sinapine concentration, the cross-linking strength between gelatin and sinapine is greater, and the formed gel is more stable. The greater the density of gelatin skeleton in the cross-linking process, the smaller the pore size of the microchannels. The more hydroxyl and amino groups in unit volume, the stronger the water absorption capacity, and the greater the swelling degree. At the same time, due to the decrease of microchannel pore size, the amount of water entering the microchannel decreases. The drug release rate slows down, and the sustained-release performance is better.

[0084] Through the morphology observation of emulsion by optical microscope( Figure 9 ), after adding crosslinking agent, comparing the two emulsification methods of magnetic stirring and vortex, it can be seen that the particle size of emulsion after magnetic stirring is more uniform. After dilution of hydrogel with water, it is observed by transmission electron microscope, and soybean protein isolate is attached to the surface of gelatin ball, and small sinapine particles are loaded on it( Figure 10 ). Secondly, by analyzing the Fourier infrared spectrum( Figure 11 ), from bottom to top, they are unadded soybean protein isolate hydrogel, soybean protein isolate, sinapine double protein composite gel, and blank double protein composite gel. Compared with d, the addition of sinapine makes the O-H bond form a wide and strong absorption peak at 3300 cm -1 , which also contains the stretching vibration absorption peak of O-H in gelatin and the stretching vibration absorption peak of free amino, that is, corresponding to the hydroxyl and amino groups in gelatin. The absorption peak at 2810-2880 cm -1 corresponds to CH2 asymmetric stretching. According to literature investigation, the interaction between soybean protein isolate and gelatin is the formation of hydrogen bond between amino and carboxyl, which improves the mechanical properties of composite protein fiber. 1630 cm -1After the absorption peak is added with the soybean protein isolate crosslinking agent, the peak area is reduced, which is probably the result of the hydroxyl group contained in the residual water in the gel, and the hydrogen bond, dipole-dipole and hydrophobic interaction between sinapine and double proteins, so that the intermolecular interaction is strengthened.

[0085] After in vitro gastrointestinal digestion for 6h Figure 12 The bioavailability of sinapine alone in vivo is low, and after 2h of gastric digestion, sinapine has been digested and decomposed into sinapinic acid; the embedded sinapine is slowly released in the intestinal juice after the completion of gastric digestion, and the release rate is about 90%; in the control group using glutaraldehyde as a crosslinking agent, sinapine can also be slowly released in the small intestine, and the release rate is about 80%, which shows that the gel prepared in the application can significantly improve the bioavailability of sinapine. Therefore, the double protein composite gel plays the role of a carrier. After in vitro digestion, sinapine is released, and through the determination of ABTS free radical scavenging capacity Figure 13 ) and DPPH free radical scavenging capacity Figure 14 ), the scavenging capacity of sinapine is stronger compared with the control group.

Claims

1. A process for the preparation of a loaded myosmine diprotein complex gel, characterized in that, The specific steps are as follows: (1) Under the condition of water bath heating, 60% gelatin solution and 200-300 μg / mL sinapine solution are mixed uniformly at a volume ratio of 1:1 to obtain an aqueous phase; (2) Under the condition of water bath heating, rapeseed oil is stirred as an oil phase; (3) The aqueous phase is slowly added to the oil phase at a water to oil ratio of 1:3-1:4, and the mixture is stirred until uniform and then placed in an ice water bath to obtain a water-in-oil emulsion; (4) Soybean protein isolate is added to the water-in-oil emulsion at a mass ratio of 0.01%-0.05% of the soybean protein isolate to the volume of the water-in-oil emulsion, g:mL, and the mixture is emulsified uniformly and then placed at 4°C for cross-linking and solidification for 24 hours or more. After cross-linking, isopropanol is added for washing, centrifugation, and water washing to obtain a deoiled sinapine-loaded double-protein composite gel.

2. The production method according to claim 1, characterized by, In step (1), the gelatin solution is prepared as follows: gelatin is pre-soaked in normal temperature water for 10 minutes or more, and then dissolved into a gelatin solution under water bath heating at 40-60°C.

3. The preparation method according to claim 1, characterized in that, In step (1) or (2), the water bath heating temperature is 40-60°C.

4. The method of claim 1, wherein, In step (3), the stirring time is 30 minutes or more.

5. The preparation method according to claim 1, characterized in that, In step (4), the emulsification method is magnetic stirring or vortexing.

6. The method of claim 1, wherein, In step (4), the specific washing method is as follows: isopropanol is added to the mixture after cross-linking, vortexed, then centrifuged at 8000 r / min for 5 minutes, the vortexing and centrifugation steps are repeated, and finally washed with water.

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