Preparation of Chinese yam protein as hydrophobic drug carrier and application of Chinese yam protein in improvement of renal injury

Abstract

The invention relates to preparation of Chinese yam protein as a hydrophobic drug carrier and application of the Chinese yam protein to improvement of renal injury. The Chinese yam protein is dispersed in deionized water, hydration is performed overnight, glutamine transaminase and galactose are added, enzyme deactivation is performed after water bath, the mixture is cooled to the room temperature, centrifugation is performed, supernate is taken and dialyzed at the temperature of 4 DEG C, and freeze-drying is performed to obtain GL-YP powder; dissolving the GL-YP powder, hydrating overnight, and regulating the pH value to 11.0 by using 1M NaOH; dissolving curcumin in absolute ethyl alcohol, dropwise adding a curcumin stock solution into the GL-YP solution, and stirring; and adjusting the pH value to 7.0 by using 1M HCL, centrifuging the prepared suspension, taking the supernatant, and freeze-drying to obtain the GLYP-Cur-NPs nanoparticles. The GL-YP can be used as an effective curcumin encapsulation carrier and has good encapsulation effect and drug loading capacity, and the problem of water solubility of curcumin as a hydrophobic drug is solved; the GLYP-Cur-NPs has a protective effect in a cis-platinum induced HEK293 cell oxidative damage model, can reduce the inflammation level, has treatment potential for renal injury, and is used for preparing the medicine for treating renal injury.

Description

Technical Field

[0001] The present invention relates to an application of yam protein, and in particular to a preparation method using yam protein as a hydrophobic drug carrier and an application of the method for improving kidney damage. Background Art

[0002] The carriers for delivering functional factors are mainly prepared from food-grade natural macromolecules, such as proteins, starches, and soluble polysaccharides. According to the type of carrier, the delivery system can be divided into particulate carriers, emulsion carriers, and lipid carriers. Granular delivery carriers are mainly composed of natural biopolymers, and can be further divided into micron-scale and nano-scale delivery systems according to the particle size. Functional factors encapsulated in nano-scale particle carriers can be directly absorbed by intestinal epithelial cells in an encapsulated form through transcellular and extracellular transport, thereby improving bioavailability. However, in some cases, although high-intensity encapsulation can effectively slow down the release of these functional factors, it will also increase the difficulty of release, thereby reducing bioaccessibility and bioavailability. Therefore, appropriate carrier design and construction should be carried out according to actual needs, which is crucial for the efficient utilization of the physiological activity of functional factors.

[0003] Chinese yam (DioscoreaeRhizoma) is the underground tuber of Dioscorea opposita Thunb., a plant of the Dioscoreaceae family. It is rich in polysaccharides, sterols, saponins, flavonoids and other active compounds. A large amount of waste liquid is generated during the production and processing of yam products, and the waste liquid is rich in protein. This protein can be used as an important source of bioactive compounds. Yam protein (YP) has antioxidant, anti-mutagenic, anti-aging and immunomodulatory effects, which have been widely studied and verified. In addition to its functional properties, yam protein also has good physical and chemical properties and can be used as a raw material for food and pharmaceutical preparations. Natural yam protein has the advantage of low economic cost and can be used in drug delivery systems to effectively encapsulate hydrophobic drugs, thereby improving stability and bioavailability. However, protein-coated biocarriers are easily affected by environmental stress, and reports on the use of yam protein as a hydrophobic drug carrier to prepare nanoparticles are also rare.

[0004] Curcumin (Cur) is a polyphenol compound extracted from the traditional Chinese medicine turmeric (Curcuma longa L.), which has strong biological activities such as antioxidant, anti-inflammatory, treatment of kidney damage and anti-tumor. Although Cur has a high safety range and has multiple functional properties, its solubility in aqueous solution is extremely low, and it is easily degraded by factors such as light and temperature in vitro. Therefore, most of Cur is excreted in the body in its original form through feces, resulting in a low content in the blood and cannot be effectively utilized. Studies have shown that active protein delivery systems, such as nanoparticles, emulsions, liposomes and gels, can be used to improve the bioavailability of Cur. The development of new protein products to prepare nanoparticles loaded with Cur is expected to improve the physical and chemical properties of Cur while improving its bioaccessibility, enhance its water solubility, and provide a new theoretical basis for the utilization and development of new protein resources.

[0005] Due to their excellent properties (including biodegradability, modifiability and diverse amino acid sequences), natural macromolecular proteins are widely developed for biopharmaceutical delivery systems for transporting drugs and nutrients. Studies have shown that Cur can be combined with casein and egg white protein to form nanoparticles. However, most proteins are very unstable under gastrointestinal conditions and are easily affected by environmental stresses such as high temperature, leading to protein hydrolysis and denaturation or unfolding of protein structure, which makes the encapsulation factor lose protection and can cause premature leakage of bioactive compounds under certain conditions. Whether using yam protein as a carrier for loading Cur to prepare nanoparticles can improve the loading rate of yam protein and withstand gastrointestinal fluid digestion requires further study. Summary of the invention

[0006] In order to solve the above technical problems, the present invention modified yam protein with galactose for the first time, prepared and characterized Cur nanoparticles based on yam protein-galactose conjugate, and further explored the therapeutic effect of the nanoparticles on the cisplatin-induced HEK293 cell oxidative damage model.

[0007] The present invention provides a method for preparing a hydrophobic drug carrier using yam protein, comprising the following steps:

[0008] The yam protein was dispersed in deionized water to a protein concentration of 0.1% to 0.5%. After sufficient stirring, the mixture was hydrated at 4°C overnight and centrifuged. 5U / g glutamine transaminase and a certain mass ratio of galactose were added to the supernatant. The mixture was incubated in a 40°C water bath for a certain period of time, the enzyme was inactivated, the mixture was cooled to room temperature, and the mixture was centrifuged. The supernatant was dialyzed at 4°C for more than 48 hours and freeze-dried to obtain GL-YP powder, i.e., a hydrophobic drug carrier.

[0009] Preferably, the preparation method of the yam protein is as follows:

[0010] Fresh yam was homogenized with distilled water at a mass ratio of 1:4, and the pH was adjusted to 9.0 with 1M NaOH; after stirring for 2 hours, the mixture was centrifuged at 8000rpm at 4°C; the supernatant was taken, the pH was adjusted to 4.0 with 1M HCl, and the solution was stirred for 30 minutes and centrifuged at 10000rpm at 4°C; the precipitate was then redissolved in water, the pH was adjusted to 7.0 with 1M NaOH, and the mixture was centrifuged at 8000rpm at 4°C. The supernatant was dialyzed at 4°C for more than 48 hours and then freeze-dried to obtain yam protein.

[0011] Furthermore, the mass ratio of yam protein to galactose is 1:1-5.

[0012] The GL-YP powder prepared by the method of the present invention is tested for its effect on HEK-293 cell viability by MTT method, and the GL-YP group has no significant effect on cell viability. Therefore, the GL-YP prepared by the present invention can be used as a biological non-toxic carrier for drug delivery and can be used as an encapsulation carrier for curcumin.

[0013] The present invention also provides a method for preparing glycosylated yam protein-curcumin nanoparticles, comprising the following steps:

[0014] The GL-YP powder prepared above was dissolved in deionized water to a protein concentration of 0.1%. After sufficient stirring, it was hydrated at 4°C overnight, centrifuged at 4°C, and the pH of the supernatant was adjusted to 11.0 with 1M NaOH. At the same time, a certain amount of curcumin was dissolved in anhydrous ethanol. Then, the curcumin stock solution was slowly added dropwise to the GL-YP solution at a volume ratio of 1:20, and stirred at 400rpm in the dark for 30 minutes. Then, the pH was adjusted to 7.0 with 1M HCL, and stirred at 400rpm in the dark for 30 minutes. The prepared nanoparticle suspension was centrifuged at 4°C, the supernatant was taken, and lyophilized to obtain GLYP-Cur-NPs nanoparticles.

[0015] Furthermore, the mass of curcumin in 1 mL of anhydrous ethanol is 0.1 to 2.0.

[0016] The GLYP-Cur-NPs nanoparticles prepared by the method of the present invention are used to detect the effect of the GLYP-Cur-NPs on the viability of HEK-293 cells by MTT method. GLYP-Cur-Nps have a significant improvement effect on cell viability (P<0.05). Treatment with GLYP-Cur-Nps at a concentration of 75-200 μg / mL reverses the decrease in HEK-293 cell viability induced by cisplatin (CP) in a concentration-dependent manner. GLYP-Cur-NPs show good repair ability for the CP-induced damage model, indicating that GLYP-Cur-NPs have a protective effect in the cisplatin-induced HEK293 cell oxidative damage model and can reduce the level of inflammation. Therefore, the GLYP-Cur-NPs nanoparticles prepared by the present invention can be used to prepare drugs for treating or preventing renal injury.

[0017] Beneficial effects of the present invention:

[0018] The present invention obtains yam protein with a purity of more than 80% from fresh yam and glycosylates it, and mixes it with Cur to obtain glycosylated yam protein-curcumin nanoparticles (GLYP-Cur-NPs). This method provides a new research direction for the application of yam active ingredients, thereby contributing to the development of natural protein-based carrier nanoparticles. The present invention analyzes the grafting degree and brownness experiments. When the mass ratio of yam protein to galactose reaches 1:3, the grafting degree is the highest; the glycosylation product (GL-YP) after cross-linking of yam protein and galactose enhances the intermolecular hydrogen bonds and electrostatic interactions, thereby achieving a more stable state and good bioaccessibility, and has good compatibility with Cur. The encapsulation rate and drug loading experiments show that GL-YP can be used as an effective encapsulation carrier for curcumin and has good encapsulation effect and drug loading, which solves the water solubility problem of curcumin as a hydrophobic drug and is beneficial to improving its bioavailability; GLYP-Cur-NPs have good thermal stability; GL-YP can effectively enhance Curcumin is absorbed and released in the intestine, and GLYP-Cur-Nps3 shows good curcumin release ability in artificial intestinal fluid. GLYP-Cur-NPs is a loading and drug delivery system with good ability, which can specifically achieve sustained-controlled release and effective bioavailability in the gastrointestinal tract. In the present invention, GLYP-Cur-NPs have a protective effect and can reduce the level of inflammation in the cisplatin-induced HEK293 cell oxidative damage model, effectively improve the cellular oxidative stress level and the release of kidney injury markers and inflammatory factors, and have therapeutic potential for HEK-293 cell damage, making up for the application of yam protein and its drug carrier in preventing kidney injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a schematic diagram of determining protein molecular weight using the SDS-PAGE method in the comparative experiment of the present invention;

[0020] Figure 2 Scanning electron microscope images of the comparative experiment of the present invention; (A) Cur; (B) YP; (C) GL-YP; (DH) GLYP-Cur-NPs 1-5 ;

[0021] Figure 3 Schematic diagram of structural characterization of GLYP-Cur-NP in the comparative experiment of the present invention; (A) XRD, (B) FT-IR, (C) dTG, (D) infrared peak fitting;

[0022] Figure 4 Schematic diagram of TG structural characterization of GLYP-Cur-NP in the comparative experiment of the present invention;

[0023] Figure 5 Schematic diagram of the stability characterization of GLYP-Cur-NP in the comparative experiment of the present invention; (A) 4°C storage stability, (B) 25°C storage stability, (C) the effect of salt ions on GLYP-Cur-NP, (D) the effect of pH on GLYP-Cur-NP, (E)-(I) thermal treatment stability (20°C, 30°C, 40°C, 50°C, 60°C), (J) GLYP-Cur-NPs in vitro simulated gastrointestinal fluid results;

[0024] Figure 6 Schematic diagram of the in vitro antioxidant activity characterization of YP, GL-YP and GLYP-Cur-NP in the comparative experiment of the present invention; (A) DPPH free radical scavenging activity, (B) ABTS free radical scavenging activity;

[0025] Figure 7 Schematic diagram of molecular dynamics simulation characterization in the comparative experiment of the present invention; (A) molecular docking, (B) root mean square deviation (RMSD) of GLYP-Cur-NPs system, (C) protein-ligand contact, (D) protein-ligand contact (0-100ns), (E) ligand properties, (F) ligand torsion curve;

[0026] Figure 8 Schematic diagram of the characterization of cytotoxicity and cellular uptake in the comparative experiment of the present invention; (A) cytotoxicity of YP, (B) cytotoxicity of GL-YP, (C) cytotoxicity of Cur, (D) cytotoxicity of GLYP-Cur-NPs3, (E)-(F) uptake of GLYP-Cur-NPs3 and average fluorescence of cells at different times; *P<0.05, **P<0.01, ****P<0.0001, indicating significant differences among the groups;

[0027] Fig. 9Schematic diagram of the therapeutic effect of different concentrations of GLYP-Cur-NPs on cisplatin-induced HEK293 cell damage in the comparative experiment of the present invention; (A) Recovery of HEK293 cells damaged by cisplatin by different concentrations of YP, (B) Recovery of HEK293 cells damaged by cisplatin by different concentrations of GL-YP, (C) Recovery of HEK293 cells damaged by cisplatin by different concentrations of Cur, (D) Recovery of HEK293 cells damaged by cisplatin by different concentrations of GLYP-Cur-NPs3, (E)-(G) Effects of different concentrations of GLYP-Cur-NPs on the content of antioxidant enzymes (GSH, CAT, SOD in HEK293 cells damaged by cisplatin), (H) (I)-(L) Effects of different concentrations of GLYP-Cur-NPs on the MDA content of cisplatin-damaged HEK293 cells, (M)-(N) Effects of different concentrations of GLYP-Cur-NPs on inflammatory factors (TNF-α and IL-1β) in cisplatin-induced HEK293 cell injury; ####P<0.0001 indicates a significant difference between the model group and the control group; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 indicate a significant difference between the GLYP-Cur-NPs3 administration group and the model group. DETAILED DESCRIPTION

[0028] Embodiment 1,

[0029] This embodiment provides a preparation method using yam protein as a hydrophobic drug carrier, comprising the following steps:

[0030] (1) Preparation of yam protein

[0031] Fresh yam was homogenized with distilled water at a ratio of 1:4 (w / w), and the pH was adjusted to 9.0 with 1M NaOH; after stirring for 2 hours, the mixture was centrifuged at 8000rpm for 10 minutes at 4°C; the supernatant was taken, the pH was adjusted to 4.0 with 1M HCl, and the solution was stirred for 30 minutes, and centrifuged at 10000rpm for 15 minutes at 4°C; the precipitate was then redissolved in water, the pH was adjusted to 7.0 with 1M NaOH, and centrifuged at 8000rpm for 10 minutes at 4°C, the supernatant was dialyzed at 4°C for 48 hours, and then freeze-dried to obtain yam protein; the purity of the yam protein exceeded 80%.

[0032] (2) Preparation of glycosylated yam protein

[0033] The yam protein was dispersed in deionized water to a protein concentration of 0.1% (w / v). After stirring for 4 hours, it was hydrated overnight at 4°C and centrifuged at 8000 rpm for 10 minutes at 4°C. 5 U / g glutamine transaminase and galactose in different mass ratios (1:1, 1:2, 1:3, 1:4 and 1:5) were added to the supernatant. After incubation in a 40°C water bath for 2 hours, the enzyme was inactivated in a 90°C water bath for 10 minutes. After cooling to room temperature, the supernatant was centrifuged at 8000 rpm for 10 minutes at 4°C. The supernatant was dialyzed at 4°C for 48 hours and freeze-dried to obtain GL-YP powder, i.e., a hydrophobic drug carrier.

[0034] The hydrophobic drug carriers were labeled in order according to the added mass of galactose as GL-YP1 to GL-YP5.

[0035] Embodiment 2,

[0036] This embodiment provides a method for preparing glycosylated yam protein-curcumin nanoparticles, comprising the following steps:

[0037] The GL-YP3 powder prepared in Example 1 was dissolved in deionized water to a protein concentration of 0.1% (w / v), stirred for 4 h, hydrated overnight at 4°C, centrifuged at 8000 rpm for 10 min at 4°C, and the pH of the supernatant was adjusted to 11.0 with 1M NaOH; at the same time, a certain mass (0.1, 0.5, 1.0, 1.5 and 2.0 g) of curcumin was dissolved in 1 mL of anhydrous ethanol; then the curcumin stock solution was slowly added dropwise to the GL-YP solution at a volume ratio of 1:20, and stirred at 400 rpm in the dark for 30 min; then the pH was adjusted to 7.0 with 1M HCL, and stirred at 400 rpm in the dark for 30 min; the prepared nanoparticle suspension was centrifuged at 8000 rpm for 10 min at 4°C, the supernatant was taken, and lyophilized to obtain GLYP-Cur-NPs nanoparticles.

[0038] The nanoparticles were labeled in order according to the added mass of curcumin as GLYP-Cur-NPs1 to GLYP-Cur-NPs5.

[0039] Comparative experiment:

[0040] Cur, YP, GL-YP and GLYP-Cur-NPs were compared and analyzed. All experiments were repeated three times, and the data were expressed as the mean ± standard deviation of three independent experiments. Statistical methods used analysis of variance (ANOVA) using version SPSS21 (SPSS Inc. Chicago, IL). P < 0.05 was considered statistically significant.

[0041] 1. Determination of grafting degree and browning index:

[0042] Take 200 μL (2 mg / mL) sample solution, add 4 mL OPA solution and mix well, place in a 35°C water bath for 2 min, measure the absorbance at 340 nm, and use deionized water as a blank control. The brownness is measured by UV spectrophotometer, take 2 mL of sample and measure the absorbance at 420 nm. The calculation formula of grafting degree DG is:

[0043]

[0044] A0 is the absorbance value of the solution before reaction; A1 is the absorbance value of the sample.

[0045] The present invention analyzes the grafting degree and brownness experiment, and finds that when the mass ratio of yam protein to galactose is 1:3, the grafting degree is the highest, and when the mass ratio of yam protein to galactose is 1:2, the brownness is the lowest.

[0046] 2. Determination of encapsulation efficiency (EE) and drug loading (LA):

[0047] The encapsulation efficiency (EE) and drug loading (LA) were determined according to the methods in previous studies. The sample solution was centrifuged at 8000 rpm and 4 °C for 10 min, and the supernatant was dispersed with an equal amount of anhydrous ethanol and stirred for 10 min to ensure that Cur was completely dissolved. 2 =0.9989) standard curve, and the Cur content in the supernatant was determined by UV-visible spectrophotometer at 426nm. The calculation formula of EE and LA is as follows:

[0048]

[0049] m c is the mass of Cur in the supernatant (mg), m t is the total mass of curcumin (mg).

[0050]

[0051] m c is the mass of Cur in the supernatant (mg), m w is the total mass of the sample (mg).

[0052] Under the same experimental conditions, with the addition of curcumin, GLYP-Cur-NPs 1-3The encapsulation efficiency of GLYP-Cur-NPs1 was 87%±2%, and the encapsulation efficiency of GLYP-Cur-NPs1 was the highest, reaching 90.17%. With the continued addition of curcumin, the encapsulation efficiency of GLYP-Cur-NPs4 began to decrease significantly (P<0.05). The excessive addition of Cur changed the hydrophobicity and steric hindrance of GL-YP, thus affecting the encapsulation efficiency. The drug loading increased from 1.68% to 23.09% and reached the maximum. The encapsulation efficiency and drug loading experiments showed that GL-YP can be used as an effective encapsulation carrier of curcumin and has good encapsulation efficiency and drug loading.

[0053] 3. Particle size and zeta potential determination:

[0054] Different samples were dispersed in deionized water and placed in a Malvern Zetasizer Nano ZS90 nanoparticle size analyzer. Dynamic light scattering (DLS) was used to measure the PDI, average particle size and zeta potential distribution of the samples.

[0055] The average particle size of yam protein is 198.93nm; after enzymatic grafting of YP, some amino acid groups are detached, combined with the carbonyl group in galactose and self-aggregated, and the average particle size increases to 237.91nm; with the addition of Cur, the particle size of GLYP-Cur-NPs also gradually increases, and the average particle size ranges from 198.18 to 334.15nm. PDI gradually increases, and the ζ-potential decreases from -5.44 to -25mV; with the addition of Cur, the ζ-potential gradually decreases.

[0056] 4. Protein molecular weight determination:

[0057] like Figure 1 As shown, the protein molecular weight of YP, GL-YP, and GLYP-Cur-NPs was determined by SDS-PAGE. The band distribution of YP showed that the molecular weight was mainly concentrated at 31 kDa, and the purity was over 80%. The molecular weight of YP, GL-YP, and GLYP-Cur-NPs3 remained relatively stable.

[0058] 5. Scanning Electron Microscope (SEM):

[0059] The freeze-dried powder was placed on a conductive plate using a scanning electron microscope (Hitachi S4800), and then gold was sprayed on the surface of the sample. The microscopic morphology of Cur, YP, GL-YP and GLYP-Cur-NPs was observed under an accelerating voltage of 10 kV and a magnification of 10000×.

[0060] like Figure 2As shown in the figure, the surface structure of the nanoparticles is significantly different from that of yam protein and glycosylated yam protein. When the nanoparticles are formed, the proteins gradually disintegrate and aggregate, and re-form large aggregates. Under alkaline conditions, the solubility of the proteins changes, and electrostatic interactions drive them to undergo bridging-repolymerization. Curcumin recrystallizes and is encapsulated by the proteins and is also adsorbed by the proteins on the surface, forming a relatively compact complex. SEM results show that glycosylated YP successfully encapsulates the drug. Van der Waals forces and hydrophobic interactions play an important role in the pH self-assembly of composite nanoparticles.

[0061] 6.X-ray diffraction (XRD):

[0062] X-ray powder diffraction curves were analyzed by X-ray diffractometer (Bruker D2 PHASER) to evaluate the crystal changes of Cur and GL-YP during the assembly process. The XRD patterns of powdered GL-YP, Cur and GLYP-Cur-NPs were determined by setting the 2θ angle in the range of 5-45° and the scanning rate at 10° / min.

[0063] like Figure 3 As shown in Figure A, through X-ray diffraction analysis of the sample, Cur has many sharp diffraction peaks at diffraction angles of 17.2° and 10-30°, and the diffraction angle of Cur indicates that it is in a highly crystalline state. GL-YP has a wide diffraction peak at 20.78°, which is in an amorphous state. GLYP-Cur-NPs also has a wide diffraction peak near 20°, and does not show the characteristic peak of Cur, indicating that the nanoparticles have changed from a crystalline state to an amorphous state, which shows that glycosylated yam protein can load curcumin well and change its crystallinity, thereby improving solubility and bioavailability.

[0064] 7. FT-IR spectroscopy analysis:

[0065] The sample powder was mixed with pure potassium bromide powder at a ratio of 1:200 (w / w) and pressed into a tray to form a transparent sheet. -1 The Fourier transform infrared (FTIR) spectra of the samples were recorded under 400 nm. The FT-IR spectra of the samples were observed using a FT-IR spectrometer (Bruker alpha II) to study the molecular interactions between the samples.

[0066] like Figure 3As shown in B, based on the infrared spectroscopy (FT-IR) test results before and after the formation of the hydrophobic drug carrier, it is speculated that the glycosylation reaction is related to electrostatic interaction by analyzing the changes in the characteristic spectral peaks, and that the reaction involves changes in hydrogen bonds with the addition of sugar. The absorption peak of the amide band of curcumin shifts before and after the formation of nanoparticles, which is speculated to be related to electrostatic interaction, hydrophobic interaction and hydrogen bonding. And after the formation of nanoparticles, the curcumin 1000-2000cm -1 1000cm -1 The following infrared characteristic absorption peaks have disappeared, because Cur binds to GL-YP and restricts the molecular vibration of Cur, and this binding also changes the secondary structure of the protein. The infrared spectra of GL-YP and GLYP-Cur-NPs are roughly the same, which also confirms that Cur is successfully bound to GL-YP.

[0067] 8. Thermogravimetric analysis (TGA):

[0068] The thermal behavior of the different samples was evaluated using a Mettler (TGA / DSC 3+). The samples were sealed in an aluminum pan with a lid and heated to 800 °C at 10 °C / min using nitrogen as carrier gas with a flow rate of 20 mL / min.

[0069] like Figure 4 As shown in the figure, GL-YP has a maximum degradation rate around 601℃, which is better than YP in thermal stability and has a similar degradation pattern to nanoparticles. The maximum degradation rate of curcumin occurs at 398℃. Figure 3 As shown in Figure C, GLYP-Cur-NPs4 has the strongest resistance to thermal decomposition, while GLYP-Cur-NPs1 has the weakest. Adding too little or too much Cur is not conducive to improving the thermal stability of nanoparticles. In addition, the peak value of DTG of GLYP-Cur-NPs is smaller than that of YP and GL-YP, indicating that the thermal stability is significantly improved after combination. In addition, the endothermic peak of curcumin in the nanoparticles disappears, and is replaced by a smoother curve, which shows that GL-YP and Cur have good compatibility, and Cur is successfully encapsulated in GL-YP.

[0070] 9. Nanoparticle Stability:

[0071] 9.1 Temperature stability:

[0072] The samples were uniformly dispersed in deionized water and placed in a water bath heated to 20, 30, 40, 50, and 60° C. The EE of the nanoparticles was measured every 20 min.

[0073] 9.2 pH stability:

[0074] After the sample was evenly dispersed in deionized water, the pH of the sample was adjusted to 4.0, 5.0, 6.0, 7.0, 8.0, and the sample EE was measured after stirring at 400 rpm for 20 min.

[0075] 9.3 Storage stability:

[0076] The samples were dispersed evenly in deionized water and stored at 4°C and 25°C for 12 days. The EE of the samples was measured every 2 days. 9.4 Salt Ion Stability:

[0077] The sample was evenly dispersed in deionized water, 0, 200, 400, 600, 800, and 1000 mM NaCl solutions were added to the sample solution and stirred at 400 rpm for 20 min, and the sample EE was measured.

[0078] like Figure 5 As shown in Figure C, as the salt ion concentration gradually increases, the charge carried by the sodium ions changes the electrostatic interaction in the solution system, causing aggregation and sedimentation to easily occur in the system, destroying the spatial structure of the nanoparticles, the encapsulation rate of the drug becomes lower and lower, and the precipitate gradually precipitates. Figure 5 D shows that when the salt ion concentration is 1 mol / mL, the system is almost completely destroyed. In addition, with the change of pH, the change of electrostatic force between nanoparticles and the change of spatial structure will reduce the stability of the system. Under acidic conditions, the solution stability is better. This is because under acidic conditions, proteins will self-aggregate and wrap drugs. On the other hand, due to the presence of a large number of hydrophilic hydroxyl groups, a relatively strong spatial steric hindrance effect is formed between molecules. Under alkaline conditions, the spatial structure of proteins will unfold, and the protonation ability of carboxyl groups will increase. At this time, the transfer of charge in the solution will make the hydrophobic effect stronger. Curcumin also undergoes a conjugation effect under alkaline conditions and dissolves in the system, causing intermolecular interactions and destroying the stability of the system. With the increase of the amount of curcumin added, this intermolecular interaction becomes more obvious, and the drug retention rate under alkaline conditions becomes lower and lower. And with the increase of temperature, the stability of nanoparticles gradually decreases, and obvious precipitation occurs. This is because too high a temperature will destroy the intermolecular hydrogen bond interaction, and at the same time the hydrophobic effect becomes stronger, and the spatial structure of proteins changes irreversibly, inducing molecules to aggregate and precipitate. In addition, due to the increase in temperature, the Brownian motion of molecules becomes more intense, which is not conducive to the stability of the system. The encapsulation rate of the nanoparticle solution decreased by about 30% within 2 days at 4°C ( Figure 5 A), and dropped to about 10% after 8 days. At 25°C, the encapsulation rate dropped by about 60% in 2 days ( Figure 5B), and dropped to about 10% after 4 days. As the storage time increases and the storage temperature rises, the intermolecular thermal motion and protein structure changes will be intensified, making the hydrophobic groups more exposed and curcumin released freely. In general, the temperature range of 20-30℃ is more conducive to maintaining the structural integrity of the nanoparticles and keeping the solution stable.

[0079] 10. Simulated gastrointestinal fluid digestion:

[0080] The 1 mg / mL nanoparticle solution was dissolved in simulated gastric fluid at a ratio of 1:3 (v / v), stirred at 37°C, 300 rpm for 2 h, the digested sample solution was adjusted to pH 6.8, and the above solution was added to simulated intestinal fluid at a ratio of 1:1 (v / v), and continued to stir at 37°C, 300 rpm for 28 h. The digested solution was collected at time intervals (0.3 h, 0.6 h, 1 h, 1.3 h, 1.6 h, 2 h, 4 h, 6 h, 8 h, 18 h, 30 h) for further analysis, and the simulated gastric fluid (intestinal fluid) was supplemented to the original volume. In short, the samples with different digestion times were centrifuged at 8000 rpm for 10 min, and the supernatant was added with an equal volume of anhydrous ethanol to determine the release rate of Cur. The release rate calculation formula is as follows:

[0081]

[0082] In the formula, m r and m t represent the amount of Cur released after digestion and the initial mass of Cur, respectively.

[0083] like Figure 5 As shown in Figure J, by analyzing the release curve of Cur in the digestive juice at different digestion stages and digestion times, it can be seen that Cur is almost not released in artificial gastric juice (SGF), and the protein and the drug cannot be separated. This requires a change in pH to destroy the protein network through electrostatic interaction and hydrophobic interaction. Only a very small part of Cur will be released under the action of pepsin. In the artificial intestinal fluid (SIF) digestion stage, a better digestion release pattern is shown. Because under near-neutral conditions, proteins will gradually open from the aggregated folding state under acidic conditions, and hydrophobic effects will cause Cur to be gradually released from the embedded state. Under the action of trypsin, the protein will gradually be hydrolyzed into small molecules, and the release rate of Cur will be greatly improved. The glycosylation product (GL-YP) after cross-linking of yam protein and galactose can effectively enhance the gastrointestinal absorption and release of curcumin, and GLYP-Cur-Nps3 shows good curcumin release ability in SIF. Therefore, it can be inferred that GLYP-Cur-NPs is a drug delivery system with good capabilities, which can achieve sustained and controlled release and effective bioavailability specifically in the gastrointestinal tract.

[0084] 11. Evaluation of antioxidant activity:

[0085] Determination of DPPH and ABTS free radical scavenging activity. Sample solutions of different concentrations were incubated with 0.1 mM DPPH alcohol solution at a ratio of 1:1 (v / v) at room temperature in the dark for 30 min. black ) and deionized water instead of sample (A control ) instead of the absorbance of the alcohol solution of DPPH measured at 517 nm.

[0086] A mixed solution of 7mM ABTS and potassium persulfate (KPS) was mixed at a ratio of 1:1 (v / v) and protected from light for 12-18 hours. The solution was then diluted with anhydrous ethanol to obtain an absorbance of 0.7±0.02 at 734nm. Subsequently, 0.2mL of the sample was added to 2.0mL of the diluted ABTS solution, mixed, and incubated at room temperature in the dark for 5min, with deionized water as a blank control. The free radical scavenging rate of DPPH and ABTS was calculated as follows:

[0087]

[0088] Among them, A sample , A black , A control are the absorbance of the sample group, blank group and control group respectively; A1 and A0 are the absorbance of the sample group and blank group respectively.

[0089] like Figure 6 As shown in the data, the DPPH and ABTS free radical scavenging ability of GL-YP was slightly improved compared with YP, and the antioxidant capacity of the nanoparticles was significantly enhanced compared with GL-YP; the half-abts free radical scavenging concentrations of GLYP-Cur-Nps2 to GLYP-Cur-Nps5 were 330, 201, 131, and 127 μg / mL, respectively, and the half-dpph free radical scavenging concentrations of GLYP-Cur-Nps3 to GLYP-Cur-Nps5 were 658, 505, and 429 μg / mL, respectively. With the addition of Cur and the increase in the concentration of nanoparticles, the free radical scavenging ability gradually increased.

[0090] 12.Molecular docking and dynamics simulation:

[0091] Schrödinger molecular docking software was used to determine the ligand-protein interaction. The ligands were prepared as follows. First, the two-dimensional structure files of the main active compounds were obtained from the PubChem (https: / / pubchem.ncbi.nlm.nih.gov / ) database and then imported into The optimized ligand was obtained using the LigPrep module and the default configuration of the OPLS4 force field in the Schrödinger software, constant potential ionization at physiological pH, and optical isomers of the ligand. The receptor was prepared as follows. The amino acid sequence was converted to a PDB file using Swissmodel (PDB ID: 4TWM) and then imported into Maestro software was used, and the protein preparation wizard with default settings was used to optimize the receptor. Finally, molecular docking studies were performed using the Glide module in Schrodinger software.

[0092] The docking binding energy of Gal and YP is -6.021 Kcal / mol, and the binding energy of the complex formed after docking is -3.768 Kcal / mol. We observed that Gal and YP have a strong binding ability. Figure 6 A shows an intuitive image of the optimal docking of the receptor and the ligand. Gal is deeply embedded in YP and can form hydrogen bonds with YP's THR-206 residue and ASP-92 residue; in addition, the interaction between proteins was observed after docking of the complex formed by Gal and Cur, and Cur can form hydrogen bonds with ASN-178 and IL3-172 in its complex;

[0093] The mean square deviation (RMSD) of molecular dynamics (MD) simulation results indicates the conformational and interaction stability of the ligand and the complex. In the simulation, the average displacement change of selected atoms of the protein and ligand in a specific frame relative to the reference frame is measured by RMSD. For YP( Figure 6 A), RMSD fluctuations are considered to be completely acceptable, and GL-YP has large RMSD fluctuations ( Figure 6 B) The RMSD value of the GLYP-Cur-NPs system also showed obvious fluctuations at 40 ns.

[0094] Protein-ligand contacts are monitored during protein-ligand interactions, as well as the total number of specific protein-ligand contacts during the entire motion trajectory. These interactions are divided into four types: hydrogen bonds (green), hydrophobic bonds (light pink), ionic bonds (red), and water bridges (blue). The simulation results show that more hydrophobic interactions occur during glycosylation. In the GLYP-Cur-NPs system ( Figure 7 C), multiple amino acid sites such as GLN-235 and GLU-24 mainly interact with Cur through water bridges. With the increase of changes in amino acid side chains, amino acid residues of different subtypes also form multiple contacts with the ligand. Figure 7 D shows the interaction of the residues with the ligand in each trajectory frame, with the ligand indicated in dark orange in the scale on the right side of the figure. Figure 6D) The interaction time in the selected trajectory (0.00 to 100.00 ns) is significantly shorter than that of GLYP-Cur-NPs. Cur interacts with GL-YP with a greater number of amino acid contacts compared to Gal and YP. The reaction is also more vigorous, indicating that GL-YP is more stable than the GLYP-Cur-NPs system.

[0095] Figure 7 F shows the ligand torsion curve, summarizing the conformational evolution of each rotatable bond (RB) in the ligand throughout the simulation trajectory (0.00 to 100.00 ns). The top panel shows a 2D schematic of the ligand, the dial describes the torsion conformation throughout the simulation, and the bar graph summarizes the data on the dial by showing the probability density of the torsion. By observing the relationship between the histogram and the torsion potential, we observe that the change in RB is caused by the combination of Gal and YP. Except for the light green bonds in the figure, the rest of the RB can rotate between -180° and 180°, and all rotatable bonds change their torsion potential. It is worth noting that when the RB of Cur undergoes conformational changes, the rotation of the bond position is symmetrical, only the oxygen atom directly connected to the benzene ring undergoes a small rotation, and the rest show different torsion potentials.

[0096] The time evolution analysis of the radius of gyration (rGyr) showed that the rGyr value of the GLYP-Cur-NPs system ( Figure 7E) decreases first and then increases during the simulation, indicating the dynamic behavior of Cur in unfolding-folding during binding to GL-YP, and higher flexibility after contact with Cur during the reorganization process. The rGyr value of the GLYP-Cur-NPs system is slightly higher than that of the GL-YP system, indicating that the GLYP-Cur-NPs system is relatively disordered, similar to the results of protein secondary structure. The change in the solvent accessible surface area (SASA) of the protein when the two systems are combined is the main parameter for evaluating the surface area of ​​the biomolecule accessible to the solvent. Compared with the GLYP-Cur-NPs system, the SASA area value of the GL-YP system increases slightly, indicating that Cur increases the exposed surface area of ​​GL-YP, resulting in a larger interaction area. Intramolecular hydrogen bonding (intraHB) is used as an indicator to evaluate the interaction with other molecules. In the glycosylation process of YP, intramolecular hydrogen bonding plays an important role in the glycosylation reaction, while in GLYP, hydrogen bonding with Cur is almost not involved in the reaction, indicating that the GLYP-Cur-NPs system is mainly bound by other forces. The molecular surface area (MolSA) value is equivalent to the van der Waals surface area, which only works on the molecular surface, that is, the part of the molecule that is in close contact. The molecular surface area calculation results show that Cur and GL-YP undergo dipole-dipole interactions, resulting in changes in the polarity of the protein surface. The combination of the two and the van der Waals force play an important role. The polar surface area (PSA) is an important parameter for predicting drug transport properties, indicating that the solvent accessible surface area of ​​the molecule is only contributed by oxygen and nitrogen atoms. The PSA fluctuation of the GL-YP system is greater than that of the GLYP-Cur-NPs system, indicating that GL-YP has a greater polarity than GLYP-Cur-NPs, and there are more polar functional groups distributed in the molecule, making it more hydrophilic.

[0097] 13. Experiment on the treatment of cisplatin-induced HEK293 cell damage by GLYP-Cur-NPs: 13.1 Cell culture:

[0098] HEK-293 cells were purchased from the Chinese Academy of Sciences (Shanghai, China) and cultured in Gibco's DMEM medium supplemented with 20% fetal bovine serum and 1% penicillin / streptomycin at 37°C and 5% carbon dioxide (CO2), with subculture every 2-3 days. 13.2 Cell viability assay:

[0099] All experiments were performed when cells were grown in DMEM medium and reached 80% density. 3Cells were seeded into 96-well plates. After treating the cells with different concentrations of YP, GL-YP, Cur, and GLYP-Cur-NPs3 for 24 hours, the culture medium was discarded after 24 hours, 20 μL MTT solution (5 mg / mL) was added, and incubated at 37°C for 4 hours. Then 150 μL DMSO was added, shaken on the plate for 10 minutes, and the absorbance value was measured at 490 nm to determine the cell viability. After stimulating the cells with cisplatin (15 mg / mL) for 24 hours, the cells were treated with different concentrations of YP, GL-YP, Cur, and GLYP-Cur-NPs3 for 24 hours, and the cell viability was measured using the MTT method.

[0100] 13.3 Determination of oxidative stress indicators:

[0101] HEK293 cells were plated at 1.5 × 10 per well. 5 The cells were inoculated in 6-well plates and cultured for 24 h. After cisplatin treatment for 24 h, different doses of GLYP-Cur-Nps3 were added and cultured for another 24 h. The culture medium was discarded and the HEK 293 cells in different groups were collected in 1 mL of phosphate-buffered saline (PBS) buffer (pH 7.4) and sonicated on ice (300 W, 3 s, 10 s interval) for subsequent biochemical assays.

[0102] 13.4 Determination of cytokines in HEK-293 cells:

[0103] After the experimental treatment, the cells were sonicated on ice (300 W, 3 s, 10 s interval), and the cell lysate was centrifuged at 4°C, 3000 rpm for 10 min. The supernatant was taken and the contents of TNF-α, IL-1β, KIM-1, NGAL, ADPN, and NAG in the cell supernatant were determined.

[0104] 13.5 Cellular uptake experiment:

[0105] FI-TC staining was used to label the drug and observe the drug absorption by HEK-293 cells. 5×10 HEK293 cells were placed in each well. 5 The cells were inoculated in a 24-well plate containing cell slides and cultured for 24 hours. Then, the cells were co-incubated with FI-TC-labeled GLYP-Cur-NPs3 for different periods of time, washed 5 times with PBS (0.01M, pH 7.4), and the cell slides were fixed in 4% pre-cooled paraformaldehyde for 30 minutes and stained with DAPI containing anti-fluorescence quencher. After washing 5 times with PBS (0.01M, pH 7.4), the cell slides were taken out and photographed under a fluorescence microscope.

[0106] Statistical analysis:

[0107] The MTT method was used to detect the effects of YP, GL-YP, Cur, and GLYP-Cur-Nps3 on the viability of HEK-293 cells. Figure 8 As shown in AD, compared with the normal group, the YP and GL-YP groups had no significant effect on cell viability, making them suitable as biological non-toxic carriers for drug delivery. Cur had a dose-dependent toxicity compared with the normal group, while GLYP-Cur-Nps3 significantly improved cell viability (P<0.05).

[0108] GLYP-Cur-NPs3 were labeled with fluorescein isothiocyanate (FITC), and the uptake of nanoparticles by HEK-293 cells at different times was observed under a fluorescence microscope. Figure 8 E), when FITC-labeled nanoparticles were used, the fluorescence intensity gradually increased with the increase of treatment time from 0 to 6 h, and the average fluorescence intensity was 54.40% at 6 h ( Figure 8 F). After 8 hours, the fluorescence intensity weakened and the cell absorption metabolism reached a dynamic equilibrium.

[0109] Compared with the normal group ( Fig. 9 AD), 24 hours after CP induction, YP and GL-YP had no significant effect on cell viability, while the Cur group caused a gradual decrease in HEK-293 cell viability. However, treatment with 75–200 μg / mL concentration of GLYP-Cur-Nps3 reversed the CP-induced decrease in cell viability in a concentration-dependent manner. This indicates that GLYP-Cur-NPs exhibit good repair ability for the CP-induced damage model.

[0110] Fig. 9 EH, When HEK-293 cells were exposed to cisplatin, four levels of oxidative stress were disturbed compared to the control group. After treatment with GLYP-Cur-Nps3, MDA levels decreased and SOD levels increased. This was also evidenced by the increase in CAT activity levels, and GSH also increased significantly (P<0.01). These results suggest that the nanoparticles reduce oxidative stress-induced cell damage by increasing GSH, CAT, and SOD enzyme activities while downregulating MDA levels. These results emphasize the antioxidant role of GLYP-Cur-Nps in alleviating oxidative stress.

[0111] To investigate the protective effect of GLYP-Cur-Nps3 against renal cell injury, the levels of KIM-1, NAG, ADPN, and NGAL, which serve as early biomarkers of CP-induced injury ( Figure 8 IL). As well as the levels of inflammatory factors such as TNF-α and IL-1β ( Figure 8M, N). After CP induction, the levels of intracellular renal injury markers and inflammatory factors increased significantly. After GLYP-Cur-NPs3 treatment, the levels of KIM-1, NAG, NGAL, TNF-α, and IL-1β decreased, while ADPN increased compared with the CP group. These data indicate that GLYP-Cur-NPs have a protective effect and can reduce the level of inflammation in the CP-induced injury model.

Claims

1. A method for preparing a hydrophobic drug carrier using yam protein, characterized in that: The following steps are involved: The yam protein was dispersed in deionized water to a protein concentration of 0.1% to 0.5%. After sufficient stirring, the mixture was hydrated at 4°C overnight and centrifuged. 5U / g glutamine transaminase and a certain mass ratio of galactose were added to the supernatant. The mixture was incubated in a 40°C water bath for a certain period of time, the enzyme was inactivated, the mixture was cooled to room temperature, and the mixture was centrifuged. The supernatant was dialyzed at 4°C for more than 48 hours and freeze-dried to obtain GL-YP powder, i.e., a hydrophobic drug carrier.

2. The method for preparing a hydrophobic drug carrier using yam protein according to claim 1, characterized in that: The preparation method of the yam protein is as follows: Fresh yam was homogenized with distilled water at a mass ratio of 1:4, and the pH was adjusted to 9.0 with 1M NaOH; after stirring for 2 hours, the mixture was centrifuged at 8000rpm at 4°C; the supernatant was taken, the pH was adjusted to 4.0 with 1M HCl, and the solution was stirred for 30 minutes and centrifuged at 10000rpm at 4°C; the precipitate was then redissolved in water, the pH was adjusted to 7.0 with 1M NaOH, and the mixture was centrifuged at 8000rpm at 4°C. The supernatant was dialyzed at 4°C for more than 48 hours and then freeze-dried to obtain yam protein.

3. The method for preparing a hydrophobic drug carrier using yam protein according to claim 1, characterized in that: The mass ratio of yam protein to galactose is 1:1-5.

4. A method using yam protein as a hydrophobic drug carrier, characterized in that: Prepared according to any one of claims 1 to 3.

5. The use of yam protein as a hydrophobic drug carrier according to claim 4, characterized in that: GL-YP is used as a bio-nontoxic carrier for drug delivery.

6. A method for preparing glycosylated yam protein-curcumin nanoparticles, characterized in that: The following steps are involved: The GL-YP powder prepared by any one of the preparation methods described in claims 1 to 3 is dissolved in deionized water to make the mass concentration of protein 0.1%, after sufficient stirring, hydrated at 4°C overnight, centrifuged at 4°C, and the pH of the supernatant is adjusted to 11.0 with 1M NaOH; at the same time, a certain mass of curcumin is dissolved in anhydrous ethanol; then the curcumin stock solution is slowly added dropwise to the GL-YP solution at a volume ratio of 1:20, and stirred at 400rpm in the dark for 30 minutes; then the pH is adjusted to 7.0 with 1M HCL, and stirred at 400rpm in the dark for 30 minutes; the prepared nanoparticle suspension is centrifuged at 4°C, the supernatant is taken, and lyophilized to obtain GLYP-Cur-NPs nanoparticles.

7. The method for preparing glycosylated yam protein-curcumin nanoparticles according to claim 6, characterized in that: The mass of curcumin in 1 mL of anhydrous ethanol is 0.1-2.

0.

8. A glycosylated yam protein-curcumin nanoparticle, characterized in that: Prepared according to the preparation method according to any one of claims 6 or 7.

9. The use of a glycosylated yam protein-curcumin nanoparticle according to claim 8, characterized in that: GLYP-Cur-NPs nanoparticles are used to prepare drugs for treating or preventing renal injury.

10. The use according to claim 9, characterized in that: GLYP-Cur-NPs nanoparticles are used to prepare drugs for treating or preventing cisplatin-induced renal injury.

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