Preparation method and application of apigenin-loaded cottonseed protein hydrolysate
By preparing cottonseed protein hydrolysate loaded with apigenin, the problems of low solubility and bioavailability of apigenin were solved, efficient nano-delivery and stable drug release were achieved, and it has targeted drug delivery and antioxidant activity.
Patent Information
- Application Number
- CN202511144370.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-15
- Publication Date
- 2025-10-10
AI Technical Summary
The poor solubility and low bioavailability of apigenin limit its application in food and medicine, and existing nanodelivery systems have potential toxicity risks.
The method for preparing cottonseed protein hydrolysate loaded with apigenin is adopted, and amphiphilic and phosphorylated cottonseed protein hydrolysate is prepared through the steps of adjusting pH value, dialysis and freeze drying, and apigenin is loaded to form nanoparticles with core-shell structure.
The encapsulation efficiency and drug loading of apigenin were improved, colloidal stability and antioxidant activity were ensured, the formulation had the potential for targeted drug delivery, and remained stable at gastric pH to prevent premature degradation.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of biological materials, and particularly relates to a preparation method and application of apigenin-loaded cottonseed protein hydrolysate. BACKGROUND
[0002] Apigenin (Api, 4', 5, 7-trihydroxyflavone) is a bioactive plant flavonoid belonging to the flavone subclass, and has abundant natural sources including chamomile, parsley, celery, thyme, oregano, olive, onion, kumquat, cherry, orange, broccoli, tomato, barley, carrot, and legumes. In botany, Api belongs to the genus Chrysanthemum of the family Asteraceae. Apigenin is a potential drug that can be used for the prevention and treatment of cancer. However, the poor solubility and low bioavailability of apigenin limit its application feasibility in the fields of food and medicine. Due to its low water solubility of 2.16 μg / mL at pH 7.5, the low water solubility limits its solubility and binding in the aqueous system and bioavailability, which hinders the application of apigenin to improve human health. Another reason for the low bioavailability and poor therapeutic effect of apigenin in vivo is the rapid metabolism and elimination through glucuronidation and sulfation. Therefore, it is necessary to improve the solubility and bioavailability of apigenin.
[0003] In order to solve the above problems, Api has been encapsulated into nanodelivery systems such as polymeric micelles, liposomes, and solid lipid nanoparticles. However, these materials pose potential toxicological risks to humans and the environment, and in order to achieve sustainable development and environmental protection, a non-toxic material-based delivery system and an eco-friendly encapsulation technology should be developed for Api. Proteins as natural macromolecules have attracted considerable attention in this regard, and proteins are mainly divided into two categories: animal proteins and plant proteins. Plant proteins have high biocompatibility, and are low in cost, easy to mass-produce, and renewable, especially when they are extracted from agricultural wastes and forestry residues. In contrast, the use of animal proteins is limited due to their rapid biodegradability and poor mechanical strength.
[0004] Cottonseed protein also has excellent amino acid structure, excellent foaming property, antioxidant property, water / oil retention capacity, and biocompatibility. The chemical composition and functional properties of cottonseed make it an economically attractive agricultural industrial waste, and they are high in protein content and rich in amino acids related to bioactivity, and are praised as a good source of plant proteins. In the past decade, cottonseed protein hydrolysate (CSPHs) has emerged as an innovative material for food applications.
[0005] At present, some nanomaterials pose potential toxicological risks to humans and the environment, and in order to achieve sustainable development and environmental protection, a non-toxic material-based delivery system and an eco-friendly encapsulation technology should be developed for Api. SUMMARY
[0006] An object of the present application is to solve at least the above problems and / or drawbacks, and to provide at least the advantages set out hereinafter.
[0007] To achieve these objects and other advantages of the present application, a method for preparing apigenin-loaded cottonseed protein hydrolysate is provided, characterized by comprising the following steps: Step one, dissolving cottonseed protein isolate in water, stirring overnight, adjusting pH, water bath heating, adjusting pH again and dialysis to remove by-products, freeze-drying and low-temperature storage to obtain amphiphilic cottonseed protein hydrolysate; Step two, placing the amphiphilic cottonseed protein hydrolysate in water overnight for hydration, then adding apigenin, adjusting pH in stages and stirring, finally centrifuging and freeze-drying the separated liquid to obtain apigenin-loaded cottonseed protein hydrolysate.
[0008] Preferably, the method for preparing cottonseed protein isolate in step one is as follows: adding defatted cottonseed powder to a potassium hydroxide solution, centrifuging after heating and stirring, adjusting the pH of the obtained supernatant to obtain a protein precipitate, centrifuging the protein precipitate again, washing the obtained precipitate, suspending it in water, adjusting the pH again, and freeze-drying to obtain the cottonseed protein isolate. Preferably, when preparing the cottonseed protein isolate, the ratio of the amount of defatted cottonseed powder to the amount of potassium hydroxide solution is w / v = 1:12~15, the concentration of the potassium hydroxide solution is 0.1M, the heating and stirring temperature is 50~60℃, the stirring time is 35~45min, and the centrifugation parameters are: centrifugal force 2470~2500×g, 4~5℃ centrifugation for 20~30min.
[0009] Preferably, when preparing the cottonseed protein isolate, 0.1M HCl is used to adjust the pH of the supernatant to 5.1~5.3, and 0.1M potassium hydroxide solution is used to adjust the pH of the precipitate suspension to 7.0.
[0010] Preferably, in step one, the ratio of the amount of cottonseed protein isolate to the amount of water is 10~20g:100mL, 1M sodium hydroxide solution is used to adjust the pH of the cottonseed protein isolate solution to 12.0~12.3, the water bath heating temperature is 70~90℃, the heating time is 30~60min, after cooling to room temperature, 1M HCl is used to adjust the pH to 7.0, the dialysis time is 20~30h, and the low-temperature storage temperature is -20~-30℃.
[0011] Preferably, in the step two, the use amount ratio of the amphiphilic cottonseed protein hydrolysate, apigenin, water is 10 mg:0.1-1.5 mg:1 mL, the overnight hydration temperature is 4°C, when the pH is adjusted in stages, first, 2M sodium hydroxide solution is used to adjust the pH to 13.0-13.3, after stirring for 5-10 min, 1M HCl is used to adjust the pH to 10.0-10.3, after stirring for 25-35 min, 1M HCl is used to adjust the pH to 7.0, and the centrifugal parameters are: centrifugal force 10000-11000 x g, centrifugation at 4-5°C for 10-15 min.
[0012] Preferably, before the step two, the amphiphilic cottonseed protein is subjected to phosphorylation treatment: the amphiphilic cottonseed protein hydrolysate is dissolved in water, then sodium tripolyphosphate is added, the pH of the mixed solution is adjusted, the solution is heated for reaction, cooled to room temperature, the pH is adjusted again, then dialysis and freeze-drying are performed to obtain the phosphorylated cottonseed protein hydrolysate.
[0013] Preferably, the use amount ratio of the amphiphilic cottonseed protein hydrolysate and water is 5-10 g:50 mL, the addition amount of sodium tripolyphosphate is 8-10% of the mass of the amphiphilic cottonseed protein hydrolysate, 1M sodium hydroxide solution is used to adjust the pH of the mixed solution to 9.0-9.3, the solution is heated for reaction at 40-50°C for 2-4 h, 1M HCl is used to adjust the pH of the reacted solution to 7.0, and the dialysis is performed for 45-60 h.
[0014] Preferably, in the step two, after the cottonseed protein hydrolysate is placed in water for overnight hydration, an acidic stachyose solution is added, then apigenin is added, the pH is adjusted in stages and stirred, finally, centrifugal separation is performed and the separated liquid is freeze-dried to obtain the stachyose-coated phosphorylated cottonseed protein hydrolysate loaded with apigenin, and the addition amount of the acidic stachyose solution is 5-10 wt% of the mass of water in the system.
[0015] Preferably, the preparation method of the acidic stachyose solution is as follows: stachyose is dissolved in water, then HCl is used to adjust the pH to 4.0-4.3, and the stachyose solution is stirred uniformly, and the concentration of the stachyose solution is 5-10 mg / mL.
[0016] The application further provides an application of the cottonseed protein hydrolysate loaded with apigenin, and the cottonseed protein hydrolysate loaded with apigenin is applied to the field of apigenin nano delivery.
[0017] The application has the following beneficial effects: the encapsulation efficiency of apigenin-loaded phosphorylated cottonseed protein hydrolysate (Api@PApCPH) and apigenin-loaded cottonseed protein hydrolysate (Api@ApCPH) reaches 98.84% and 98.71% respectively, and the corresponding drug loading reaches 145.46 mg / g and 145.47 mg / g; after the interaction of stachyose solution and cottonseed protein, more binding sites are provided for apigenin, and the drug loading and encapsulation efficiency are further improved, reaching 158.22 mg / g and 99.32% at the maximum; and the prepared Api@ApCPH and Api@PApCPH maintain colloidal stability under gastric juice pH, have strong particle electrostatic repulsion and high redissolution rate (>85%), indicating that the Api@ApCPH and Api@PApCPH have robust physical stability suitable for pharmaceutical processing and storage; in addition, the Api@ApCPH and Api@PApCPH have the potential for targeted drug delivery in the intestinal environment and can prevent premature gastric degradation; the Api@ApCPH and Api@PApCPH also have strong antioxidant activity, and the DPPH free radical scavenging rate of Api@PApCPH 1 is 73.99%, which is much higher than that of free apigenin (25.18%). BRIEF DESCRIPTION OF DRAWINGS
[0018] Figure 1 The projection electron microscope images of Example 1 and Example 5; Figure 2 The figure for testing and analyzing the nanoparticle size, polydispersity index and Zeta potential of Api@ApCPH and Api@PApCPH; Figure 3 The FTIR spectrum of Api@ApCPH and Api@PApCPH; Figure 4 The drug loading and encapsulation efficiency figure of Api@ApCPH and Api@PApCPH; Figure 5 The appearance figure of Api@ApCPH and Api@PApCPH nanoparticle dispersion; Figure 6 The Api retention rate of Api@ApCPH and Api@PApCPH after being redispersed in water; Figure 7 The turbidity value figure of dissociation reagent of Api@ApCPH and Api@PApCPH in different solutions; Figure 8 The antioxidant activity figure of Api@ApCPH and Api@PApCPH; Figure 9 The drug loading and encapsulation efficiency figure of the product prepared in Comparative Example 1-2; Figure 10 Figure for the separation of Api@ApCPH under 21 days storage; Figure 11 Figure for the separation of Api@PApCPH under 21 days storage; Figure 12 Api release rate of Api@ApCPH and Api@PApCPH in different buffer solutions; Figure 13 Cumulative release rate of Api of Api@ApCPH and Api@PApCPH in simulated gastric fluid and simulated intestinal fluid. DETAILED DESCRIPTION
[0019] The application will be further described in conjunction with the examples and drawings, so that those skilled in the art can implement the application according to the description and drawings.
[0020] Example 1 A preparation method of apigenin-loaded cottonseed protein hydrolysate, comprising the following steps: Step one, 10 g of defatted cottonseed powder is added into 120 mL of 0.1 M potassium hydroxide solution, heated and stirred at 55 ℃ for 40 min, then centrifuged at a centrifugal force of 2470 × g and a temperature of 4 ℃ for 20 min, the supernatant obtained by centrifugation is adjusted to a pH of 5.1 with 0.1 M HCl, and a protein precipitate is obtained, the protein precipitate is again centrifuged at a centrifugal force of 2470 × g and a temperature of 4 ℃ for 20 min, the obtained precipitate is washed and suspended in water, and then the pH is adjusted to 7 with 0.1 M potassium hydroxide solution, and then freeze-dried to obtain cottonseed protein isolate (CPI); Step two, 10 g of cottonseed protein isolate is dissolved in 100 mL of water, stirred overnight, adjusted to a pH of 12.0 with 1 M sodium hydroxide solution, heated in a water bath at 80 ℃ for 60 min, adjusted to a pH of 7.0 with 1 M HCl, and then dialyzed for 24 h to remove by-products, and then freeze-dried and stored in a refrigerator at -20 ℃ to obtain amphiphilic cottonseed protein hydrolysate (ApCPH); Step three, 10 mg of amphiphilic cottonseed protein hydrolysate is hydrated in 1 mL of water at 4 ℃ overnight, then 0.1 mg of apigenin is added, and then 2 M sodium hydroxide solution is used to adjust the pH to 13.0 and stir for 5 min, the pH is continuously adjusted to 10.0, and then the sample is continuously stirred for 30 min, and then 1 M HCl is used to adjust the pH to 7.0, the sample is centrifuged at a centrifugal force of 10000 × g and a temperature of 4 ℃ for 10 min, and finally the separated liquid is freeze-dried to obtain apigenin-loaded cottonseed protein hydrolysate, which is denoted as (Api@ApCPH 0.1).
[0021] Example 2 The difference between this embodiment and embodiment 1 is that in step three, the amount of apigenin is 0.5 mg, and other steps are consistent with embodiment 1, and the product is recorded as (Api@ApCPH 0.5).
[0022] Example 3 The difference between this embodiment and embodiment 1 is that in step three, the amount of apigenin is 1 mg, and other steps are consistent with embodiment 1, and the product is recorded as (Api@ApCPH 1).
[0023] Example 4 The difference between this embodiment and embodiment 1 is that in step three, the amount of apigenin is 1.5 mg, and other steps are consistent with embodiment 1, and the product is recorded as (Api@ApCPH 1.5).
[0024] Example 5 A method for preparing apigenin-loaded cottonseed protein hydrolysate, comprising the following steps: Step one, 10 g of defatted cottonseed powder is added to 120 mL of 0.1 M potassium hydroxide solution, heated and stirred at 55°C for 40 min, then centrifuged at 2470 x g and 4°C for 20 min, the supernatant is adjusted to pH 5.1 with 0.1 M HCl, and the protein precipitate is again centrifuged at 2470 x g and 4°C for 20 min, the precipitate is washed and suspended in water, then the pH is adjusted to 7 with 0.1 M potassium hydroxide solution, and then freeze-dried to obtain cottonseed protein isolate (CPI); Step two, 10 g of cottonseed protein isolate is dissolved in 100 mL of water, stirred overnight, the pH is adjusted to 12.0 with 1 M sodium hydroxide solution, heated in a water bath at 80°C for 60 min, then the pH is adjusted to 7.0 with 1 M HCl, and then dialyzed for 24 h to remove by-products, freeze-dried and stored in a refrigerator at -20°C to obtain amphiphilic cottonseed protein hydrolysate (ApCPH); Step three, 5 g of amphiphilic cottonseed protein hydrolysate is dissolved in 50 mL of water, then 0.4 g of sodium tripolyphosphate is added, the pH of the mixed solution is adjusted to 9.0 with 1 M sodium hydroxide solution, heated to 45°C for 3 h, then cooled to room temperature, the pH is adjusted to 7.0 with 1 M HCl, then dialyzed for 48 h to remove unreacted sodium tripolyphosphate, and finally freeze-dried to obtain phosphated cottonseed protein hydrolysate (PApCPH); Step four, 10 mg of phosphorylated cottonseed protein hydrolysate was hydrated in 1 mL of water at 4°C overnight, then 0.1 mg of apigenin was added, and then 2M sodium hydroxide solution was used to adjust the pH to 13.0 and stirred for 5 min, and then the pH was adjusted to 10.0, and then stirred for 30 min, and then 1M HCl was used to adjust the pH to 7.0, and then the sample was centrifuged at a centrifugal force of 10000 x g and a temperature of 4°C for 10 min, and finally the separated liquid was freeze-dried to obtain the apigenin-loaded phosphorylated cottonseed protein hydrolysate, denoted as (Api@PApCPH 0.1).
[0025] Example 6 The difference between this example and Example 5 is that in Step four, the amount of apigenin is 0.5 mg, and the other steps are consistent with Example 5, to obtain the apigenin-loaded phosphorylated cottonseed protein hydrolysate, denoted as (Api@PApCPH 0.5).
[0026] Example 7 The difference between this example and Example 5 is that in Step four, the amount of apigenin is 1 mg, and the other steps are consistent with Example 5, to obtain the apigenin-loaded phosphorylated cottonseed protein hydrolysate, denoted as (Api@PApCPH 1).
[0027] Example 8 The difference between this example and Example 5 is that in Step four, the amount of apigenin is 1.5 mg, and the other steps are consistent with Example 5, to obtain the apigenin-loaded phosphorylated cottonseed protein hydrolysate, denoted as (Api@PApCPH 1.5).
[0028] Example 9 A method for preparing an apigenin-loaded cottonseed protein hydrolysate, comprising the following steps: Step one, defatted cottonseed powder is added to a 0.1M potassium hydroxide solution, heated and stirred at 55°C for 40 min, then centrifuged at a centrifugal force of 2470 x g and a temperature of 4°C for 20 min, the supernatant obtained by centrifugation is adjusted to a pH of 5.1 with 0.1M HCl, a protein precipitate is obtained, the protein precipitate is again centrifuged at a centrifugal force of 2470 x g and a temperature of 4°C for 20 min, the obtained precipitate is washed, suspended in water, the pH is adjusted to 7, and then freeze-dried to obtain a cottonseed protein isolate (CPI); Step two, 10 g of cottonseed protein isolate is dissolved in 100 mL of water, stirred overnight, the pH is adjusted to 12.0 with 1M sodium hydroxide solution, then heated in a water bath at 80°C for 60 min, then the pH is adjusted to 7.0, then dialyzed for 24 h to remove by-products, and then freeze-dried and stored in a refrigerator at -20°C to obtain an amphiphilic cottonseed protein hydrolysate (ApCPH); Step three, 5 g of amphiphilic cottonseed protein hydrolysate was dissolved in 50 mL water, then 0.4 g of sodium tripolyphosphate was added, the pH of the mixed solution was adjusted to 9.0 with 1M sodium hydroxide solution, heated to 45°C for 3h, after the reaction was completed, it was cooled to room temperature, then adjusted to pH 7.0 with 1M HCl, then dialyzed for 48h to remove unreacted sodium tripolyphosphate, and finally freeze-dried to obtain phosphated cottonseed protein hydrolysate (PApCPH); Step four, first 10 mg stachyose was dissolved in 1 mL water, then the pH was adjusted to 4.0 with HCl to obtain an acidic stachyose solution, then 10 mg of phosphated cottonseed protein hydrolysate was hydrated overnight at 4°C, 0.1 mL of prepared acidic stachyose solution was added, then 1.5 mg of apigenin was added, then 2M sodium hydroxide solution was used to adjust the pH to 13.0 and stir for 5 min, then the pH was adjusted to 10.0 and continue to stir for 30 min, then the pH was adjusted to 7.0 with 1M HCl, the sample was centrifuged at 10000xg and 4°C for 10 min, and finally the separated liquid was freeze-dried to obtain apigenin-loaded phosphated cottonseed protein hydrolysate, denoted as (Stachyose-Api@PApCPH1.5).
[0029] Comparative Example 1 Apigenin / ApCPH was prepared by anti-solvent co-precipitation method (ASCP): first, amphiphilic cottonseed protein hydrolysate (ApCPH) was prepared according to the method of Example 1; then the pH of the ethanol aqueous solution (70% by volume) was adjusted to 4 with 0.1M hydrochloric acid, 1 mL of the solution was taken and 10 mg of ApCPH was added, stirred at room temperature at 600 rpm for 2h to dissolve ApCPH in the solution, then different amounts of apigenin (0.1~1.5mg) were added to the protein solution, and the stirring was continued at 600 rpm for 1h in the dark, the resulting solution was slowly injected into deionized water (using a syringe) and stirred for 30 min, then the dispersion was evaporated in a 45° rotary evaporator to remove residual ethanol, while supplementing with equal volume of deionized water, finally the dispersion was centrifuged at 2500 rpm for 20 min to remove free apigenin, and finally freeze-dried to obtain apigenin / ApCPH.
[0030] Comparative Example 2 Preparation of Apigenin / PApCPH by anti-solvent co-precipitation method (ASCP): First, phosphated cottonseed protein hydrolysate (PApCPH) was prepared according to the method of Example 1; then the pH value of the ethanol aqueous solution (70% by volume) was adjusted to 4 with 0.1M hydrochloric acid, 1 mL of the solution was taken, and 10 mg of PApCPH was added thereto, which was stirred at a speed of 600 rpm for 2 h at room temperature to dissolve PApCPH in the solution, then different amounts of apigenin (0.1-1.5 mg) were added to the protein solution, which was continuously stirred at a speed of 600 rpm for 1 h in the dark, the obtained solution was slowly injected into deionized water (using a syringe) and stirred for 30 min, then the dispersion was evaporated in a 45° rotary evaporator with a rotary evaporator to remove residual ethanol, while supplementing with an equal volume of deionized water, finally the dispersion was centrifuged at 2500 rpm for 20 min to remove free apigenin, and finally freeze-drying treatment was performed to obtain apigenin / PApCPH.
[0031] The transmission electron microscope images of Example 1 and Example 5 are shown in Figure 1 It can be seen that the obvious core-shell structure nanocomposites are successfully synthesized.
[0032] The following tests were performed on the samples prepared in the examples and comparative examples: The nanoparticle size, polydispersity index and Zeta potential of the samples were tested and analyzed, and the results are shown in Figure 2 It can be seen that the particle sizes of Api@ApCPH and Api@PApCPH are 230.8 nm and 183.4 nm, respectively, and the particle sizes of ApCPH and PApCPH are reduced from 238.6 nm and 211.2 nm to 230.8 nm and 183.4 nm, respectively, in the process of nano-encapsulation. The reduction of protein particle size after encapsulating apigenin is a highly positive result, indicating that it is a successful and efficient process, in which the flexible protein hydrolysate adapts its configuration to encapsulate the hydrophobic apigenin for the best effect, thereby producing a more compact, dense and well-structured nanocarrier. Zeta potential (ζ) is an effective indicator of the stability of nanoparticles, and the potentials of Api@ApCPH and Api@PApCPH are -27.26 and -24.44 mv, respectively. The ζ potential of the dispersion is about 20 mV at pH 7.0, and for colloidal particles, electrostatic repulsion is considered strong enough to prevent aggregation at a ζ potential of about 25 mV, which means that the prepared product can withstand aggregation for a long time and has good stability. The polydispersity indexes of Api@ApCPH and Api@PApCPH are 0.231 and 0.148, respectively, indicating that the particle size of the product prepared in the present application is uniform.
[0033] FTIR spectra of freeze-dried ApCPH, Api@ApCPH, PApCPH and Api@PApCPH powders were analyzed using KBr method with a Fourier transform infrared spectrometer: about 10 mg of sample was ground and pressed with about 100 mg of KBr into a circular pellet. The spectra of the samples were recorded in the wave number range of 4000-500 cm -1 and a resolution of 4 cm -1 . The FTIR spectra are shown in Figure 3 . It can be seen that the spectra of ApCPH, PApCPH and different concentrations of Api (from 0.1 to 1.5) complexes have a strong broad peak around 3500 cm -1 , which is usually related to O-H or N-H stretching vibration, indicating that the sample may contain hydrogen bonds or water. There is a strong absorption peak at 1700 cm -1 , which is usually related to the stretching vibration of C=O, indicating that there may be esters, carboxylic acids or other oxygen-containing functional groups. The peaks near 1400 cm -1 and 1000 cm -1 are usually related to C-H bending vibration. With the increase of the concentration of Api, some absorption peaks in the spectrum change, especially in the sample with high concentration (such as 1.5), a stronger absorption peak appears, indicating that Api has a significant effect on the spectrum. The transmittance of the sample gradually increases or decreases with the change of the concentration, indicating that Api interacts with PApCPH and APCPH, affecting the absorption intensity of the spectrum.
[0034] The encapsulation efficiency (EE) and drug loading (LC) of the sample were determined by dissolving the sample in DMSO and measuring the absorbance at 337 nm, and quantifying the unencapsulated apigenin according to the standard curve (prepared by dissolving apigenin in DMSO, the range is 0-100 μg / mL). The encapsulation efficiency (EE) and drug loading (LC) were calculated as follows: ; The results are shown in Figure 4As shown, after pH cycle treatment, for ApCPH, when the content of apigenin in the mixture increased from 0.1 mg / mL to 1.5 mg / mL, the drug loading increased from 9.45 mg / g to 145.46 mg / g, and the encapsulation efficiency increased from 94.49% to 98.71%; for PApCPH, when the content of apigenin in the mixture increased from 0.1 mg / mL to 1.5 mg / mL, the drug loading increased from 9.68 mg / g to 145.47 mg / g, and the encapsulation efficiency increased from 96.54% to 98.84%. This is because at alkaline pH, cottonseed protein is exposed to hydrophobic amino acid residues and binds to deprotonated apigenin. During the subsequent neutralization process, the denatured cottonseed protein at pH 7 is redissolved, while the apigenin becomes insoluble, attracting apigenin combined with other cottonseed proteins to form particles with apigenin as the core and cottonseed protein as the shell, thereby improving the encapsulation efficiency.
[0035] Figure 9 The drug loading and encapsulation efficiency of the product obtained by the co-solvent method for Comparative Example 1 and Comparative Example 2 can be seen from the graph that the content of apigenin mixture of the APCPH sample increased from 0.1 mg / ml to 1.5 mg / ml, the drug loading increased from 7.68 mg / g to 124.84 mg / g, and the encapsulation efficiency increased from 76.80% to 81.89%; for the PAPCPH sample, when the content of apigenin mixture increased from 0.1 mg / ml to 1.5 mg / ml, the drug loading increased from 7.81 mg / g to 123.74 mg / g, and the encapsulation efficiency increased from 78.62% to 80.49%, i.e. the product obtained by the co-solvent method has lower drug loading and encapsulation efficiency than the product obtained by the pH cycle method of the present application.
[0036] In addition, the drug loading of Stachyose-Api@PApCPH 1.5 prepared in Example 9 reached 158.22 mg / g, and the encapsulation efficiency reached 99.32%, which is due to the interaction between stachyose and cottonseed protein, which provides more binding sites for apigenin, thereby increasing the drug loading.
[0037] To test the redispersibility of the freeze-dried Api@ApCPH and Api@PApCPH nanoparticles, the method is as follows: the newly freeze-dried Api@ApCPH and Api@PApCPH will be rehydrated with ultrapure water at a concentration of 2 mg / mL for 1 h, and the appearance of the nanoparticle dispersion will be recorded; the suspension will be divided into two parts, one part will be used to determine the particle size, zeta potential and PDI; the other part will be centrifuged at 3500 rpm for 30 min, and the Api content of the supernatant will be measured to calculate the retention rate of Api. The retention rate of Api is estimated according to the following formula: ; Figure 5 The appearance of the Api@ApCPH and Api@PApCPH nanoparticle dispersions, Figure 6 The retention of the Api@ApCPH and Api@PApCPH Api, combined Figures 5-6 It can be seen that the freeze-dried powder is completely redispersible in aqueous solution; the redispersion rate of all samples is very high; this indicates that the Api@ApCPH and Api@PApCPH have excellent freeze-drying and redispersion stability; the excellent freeze-drying and redispersion stability of the Api@ApCPH and Api@PApCPH nanoparticles will be conducive to their storage and application in the food and pharmaceutical industries.
[0038] The freeze-dried sample powders of Api@PApCPH 1 and Api@ApCPH 1 were respectively prepared into 0.1, 0.25, 0.5, 1.0 mg / mL reconstituted samples with 1% sodium dodecyl sulfate (SDS), 1 mol / L NaCL solution, and into 8.0, 4.0, 2.0, 1.0 mg / mL reconstituted samples with 8 mol / l urea (Urea) solution; after centrifugation, the sample absorbance was read at 337 nm using a UV-visible spectrometer, and the amount of apigenin retained in the sample was calculated using the pre-developed calibration curve; Figure 7 The turbidity value graph of the sample in different solutions shows that with the increase of the dissociation reagent concentration, the turbidity values of NaCl and SDS show an increasing trend, while the turbidity value of urea shows a decreasing trend; under high concentration of urea, the turbidity value of urea corresponding to Api@ApCPH 1 is significantly reduced, while the turbidity value of urea corresponding to Api@PApCPH 1 is close to constant; the interaction between nanoparticles is mainly hydrophobic interaction and hydrogen bond; in the presence of NaCl, the NaCl turbidity corresponding to Api@PApCPH 1 increases slightly, indicating that there is a weak electrostatic interaction between Api and PApCPH; this weak interaction can be attributed to the negatively charged phosphate group and the negatively charged protein at pH above the isoelectric point; during the synthesis process of nano-encapsulation, the change of pH value corresponds to the decrease of Api solubility, and can induce the formation of nanoparticles through hydrophobic interaction; indicating that Api@PApCPH has good pressure resistance during storage and combination with other systems.
[0039] Free apigenin with concentration of 1 mg / ml, ApCPH with concentration of 1 mg / ml, PApCPH with concentration of 1 mg / ml, Api@ApCPH 1, Api@PApCPH 1 were diluted to different concentrations (0.5, 0.25, 0.1 mg / ml) respectively; the absorbance of reaction mixture was measured at 517 nm using UV-visible spectrophotometer (0.5 mL of sample and 0.75 mL of DPPH solution with concentration of 40 μg / mL). Before measurement, the reaction mixture was kept constant stirring in a constant temperature bath, the temperature was 25±0.1℃, the time was 40 min, and it was carried out in the dark. The maximum absorption control was considered to be 40 μg / mL of DPPH solution added to 0.75 mL of ethanol / water; Figure 8 For the antioxidant activity graph of nanoparticles at different concentrations, it can be seen that when the concentration of apigenin increases from 0.1 mg / mL to 1.0 mg / mL, the clearance rate of DPPH free radicals increases from 18.84% to 25.18%, compared with Api@ApCPH 1, Api@PApCPH 1 shows better antioxidant improvement effect, and the antioxidant capacity of Api@ApCPH 1 and Api@PApCPH 1 is higher than that of free Api.
[0040] The Api@ApCPH and Api@PApCPH samples were stored for 21 days, and the separation of Api in the samples was observed to characterize the physical stability of the samples, and the results are shown in Figures 10-11 As can be seen, under the condition of lower Api concentration, the Api@ApCPH colloidal nanocomposite has almost no separation during the 21-day storage period, showing good stability; with the increase of Api concentration, the stability of the Api@ApCPH sample decreases, among them, Api@ApCPH 1 and Api@ApCPH 1.5 separate after 21 days of storage; while the Api@PApCPH colloidal nanocomposite only has Api@PApCPH 1.5 separate after 21 days of storage, the low concentration of Api@PApCPH does not separate, showing good stability, which shows that the phosphorylation treatment of cottonseed protein hydrolysate helps to improve the stability of the product.
[0041] Api@ApCPH and Api@PApCPH were evaluated for apigenin release over 180 hours in sodium acetate buffer (pH 4.5) and phosphate (pH 7.4), respectively: 4 ml of release medium was sampled and introduced into a dialysis bag and placed in a 50 ml centrifuge tube containing the same buffer as the dialysis bag. These tubes were gently shaken at a constant temperature (37°C, 20 rpm) for approximately 7 days. At specific time points (0, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, and 168 hours), 2 ml of sample was removed from the release medium and replaced with fresh medium (2 ml). The absorbance of the samples was read at 337 nm using a UV-visible spectrometer, and the amount of apigenin released in the samples was calculated using a pre-developed calibration curve. The results are as follows Figure 12 As shown in the figure (a: sodium phosphate, b: sodium acetate), apigenin release from the Api@PApCPH nanocomposite is faster than from the Api@ApCPH nanoparticles. This is because the acidic environment of pH 4.5, close to the isoelectric point of cottonseed protein, leads to a densified particle structure and reduced apigenin solubility, ultimately resulting in a reduced release amount. Therefore, under these conditions, the release rate of the encapsulated apigenin is slow. However, in a phosphate buffer solution at pH 7.4, the pH 7.4 buffer significantly enhances drug release by altering the charge state of cottonseed protein (moving it away from the isoelectric point), inducing nanoparticle swelling, increasing apigenin solubility, and leveraging buffer ion effects. Under normal physiological conditions (pH 7.4), Api release is even more rapid, reaching 100% release from Api@PApCPH within 120 hours, but not within 168 hours. These results indicate that the release profile of Api is sensitive to pH, especially Api@PApCPH.
[0042] Api release rate test of Api@ApCPH and Api@PApCPH in simulated gastric fluid and simulated intestinal fluid, respectively: Equal volume of ethanol and simulated gastric fluid (SGF: 0.13 mg / mL pepsin and 2 mg / mL sodium chloride) or simulated intestinal fluid (SIF: 12.0 mg / mL fatty salt, 0.8 mg / mL trypsin, 6.8 mg / mL KH2PO4 and 8.8 mg / mL sodium chloride) were mixed as release medium. 4 mL dispersion was placed in dialysis bag (molecular cut-off = 8-14 kDa), immersed in a test tube containing 40 mL release medium, and incubated at 37°C with shaking at 150 rpm for 2 h. Then, the sample dialysis bag was transferred to 40 mL SIF and continued to incubate with shaking at 150 rpm for 2 h. Every 30 min, 2 mL sample was taken from the release medium and replaced with fresh medium (2 mL) to maintain a constant volume. The absorbance of the sample was read at 337 nm using a UV-visible spectrometer, and the amount of released apigenin in the sample was calculated using a pre-developed calibration curve; The results are shown in Figure 13 As can be seen, about 79% of Api was rapidly released into SGF in the first 2 h, and the remaining Api continued to be released into SIF in the next 2 h. When Api was coated in ApCPH, aggregation occurred in SGF, and the aggregates did not disintegrate after being transferred to SIF, which severely hindered the release of bioactive substances and the absorption of APCPHs in the gastrointestinal tract. Consistent with the results in the figure, the cumulative release only reached 45.92% through the gastrointestinal tract, indicating that irreversible aggregation of ApCPH can reduce the absorption of Api to some extent. For PApCPH, slow release was observed compared to free Api, indicating that PApCPH can control the release of Api, and the release rate of Api reached 62.66%, which is due to the strong hydrogen bonding and hydrophobic interaction between PApCPH and Api, indicating that PApCPH has the potential for targeted drug delivery and prevention of premature gastric degradation in the intestinal environment. At the same time, the release rate of Api was observed to be faster in SGF than in SIF, which may be due to the attack of the protein shell by pepsin in SGF, leading to the gradual disintegration of the nanoparticles. However, even after further degradation in SIF, the release was still incomplete, indicating that the shell still exists, which hinders the complete release of bioactive substances and the absorption of nanoparticles. The above results show that PApCPH not only can significantly inhibit the burst release of free Api, but also can further enhance the absorption of bioactive substances delivered by PApCPH during simulated gastrointestinal digestion.
[0043] While embodiments of the application have been disclosed in connection with the above specification and drawings this description is not intended to limit the scope of the application and many modifications, enhancements, alternatives, and variations will become apparent to those skilled in the art from this disclosure. Accordingly, it is intended that the application not be limited to the described embodiments, but that it include all variations falling within the scope of the claims, and their equivalents.
Claims
1. A method for preparing a cottonseed protein hydrolysate loaded with apigenin, characterized in that: The following steps are involved: Step 1: dissolving cottonseed protein isolate in water, stirring overnight, adjusting the pH and then heating in a water bath, adjusting the pH again and dialyzing to remove by-products, freeze-drying and storing at low temperature to obtain an amphiphilic cottonseed protein hydrolyzate; Step 2: placing the amphiphilic cottonseed protein hydrolysate in water for overnight hydration, then adding apigenin, adjusting the pH in stages and stirring, and finally centrifuging and freeze-drying the separated liquid to obtain the apigenin-loaded cottonseed protein hydrolysate.
2. The method for preparing the apigenin-loaded cottonseed protein hydrolysate according to claim 1, wherein In the step 1, the ratio of cottonseed protein isolate to water is 10-20 g:100 mL, the pH of the cottonseed protein isolate solution is adjusted to 12.0-12.3 with 1M sodium hydroxide solution, the water bath temperature is 70-90° C., the heating time is 30-60 min, and after cooling to room temperature, the pH is adjusted to 7.0 with 1M HCl, the solution is dialyzed for 20-30 h, and the low-temperature storage temperature is -20--30° C.
3. The method for preparing the apigenin-loaded cottonseed protein hydrolysate according to claim 1, wherein In the step 2, the dosage ratio of the amphiphilic cottonseed protein hydrolysate, apigenin, and water is 10 mg:0.1-1.5 mg:1 mL, the overnight hydration temperature is 4° C., and when adjusting the pH in stages, first adjust the pH to 13.0-13.3 with a 2M sodium hydroxide solution, stir for 5-10 minutes, then adjust the pH to 10.0-10.3 with 1M HCl, stir for 25-35 minutes, and then adjust the pH to 7.0 with 1M HCl. The centrifugation parameters are: centrifugal force 10,000-11,000×g, centrifugation at 4-5° C. for 10-15 minutes.
4. The method for preparing the apigenin-loaded cottonseed protein hydrolysate according to claim 1, wherein Before step 2, the amphiphilic cottonseed protein is phosphorylated: the amphiphilic cottonseed protein hydrolysate is dissolved in water, and then sodium tripolyphosphate is added, the pH of the mixed solution is adjusted, the mixture is heated for reaction, cooled to room temperature, the pH is adjusted again, and then dialyzed and freeze-dried to obtain a phosphorylated cottonseed protein hydrolysate.
5. The method for preparing the apigenin-loaded cottonseed protein hydrolysate according to claim 4, wherein: The ratio of the amphiphilic cottonseed protein hydrolysate to water is 5-10 g:50 mL, the amount of sodium tripolyphosphate added is 8-10% of the mass of the amphiphilic cottonseed protein hydrolysate, the pH of the mixed solution is adjusted to 9.0-9.3 with 1 M sodium hydroxide solution, the mixture is heated to react at 40-50° C. for 2-4 hours, the pH of the solution after the reaction is adjusted to 7.0 with 1 M HCl, and the solution is dialyzed for 45-60 hours.
6. The method for preparing the apigenin-loaded cottonseed protein hydrolysate according to claim 1, wherein In the step 2, after the cottonseed protein hydrolysate is placed in water for overnight hydration, an acidic stachyose solution is added thereto, and then apigenin is added, the pH is adjusted in stages and stirred, and finally centrifuged and the separated liquid is freeze-dried to obtain a stachyose-coated apigenin-loaded phosphorylated cottonseed protein hydrolysate, wherein the amount of the acidic stachyose solution added is 5-10 wt% of the mass of water in the system.
7. The method for preparing the apigenin-loaded cottonseed protein hydrolysate according to claim 6, wherein: The acidic stachyose solution is prepared by dissolving stachyose in water, adjusting the pH to 4.0-4.3 with HCl, and stirring evenly. The concentration of the stachyose solution is 5-10 mg / mL.
8. An application of a phosphorylated cottonseed protein hydrolysate loaded with apigenin prepared by the preparation method according to any one of claims 1 to 7, characterized in that: The phosphorylated cottonseed protein hydrolysate loaded with apigenin is applied to the field of apigenin nano-delivery.