CN113509450A

CN113509450A - A kind of preparation method of calcium ion-stabilized composite nanoparticles delivering quercetin

Info

Publication number: CN113509450A
Application number: CN202110779773.4A
Authority: CN
Inventors: 欧阳小琨; 张许; 章宏; 凌俊红
Original assignee: Zhejiang Ocean University ZJOU
Current assignee: Zhejiang Ocean University ZJOU
Priority date: 2021-07-09
Filing date: 2021-07-09
Publication date: 2021-10-19

Abstract

The invention provides a preparation method of calcium ion stable composite nanoparticles for delivering quercetin, and researches on Ca with different concentrations²⁺The stability of the zein/FD composite nano system loaded with quercetin is influenced, and the composite nano particles are characterized by adopting means such as SEM (scanning Electron microscope), FTIR (FTIR), Thermogravimetric (TG) analysis, X-ray diffraction spectrum (XRD), Fluorescence Spectrum (FS) and the like. The calcium ion-stabilized composite nanoparticle for delivering quercetin prepared by the invention has good pH stability and storage stability, and the ionic stability is enhanced.

Description

Preparation method of calcium ion-stabilized composite nanoparticles for delivering quercetin

Technical Field

The invention relates to a delivery nanoparticle for quercetin, in particular to a calcium ion stable composite nanoparticle for delivering quercetin and a preparation method thereof.

Background

Quercetin (Que) is a natural polyphenol compound widely existing in plants, has a certain treatment effect on diseases such as chronic bronchitis, coronary heart disease and hypertension, and is a nutritional active ingredient with high utilization value. However, the solubility and chemical stability of quercetin in water are poor, so that the application of quercetin in the fields of biology and food is limited.

In the past decades, in order to overcome the problem of poor stability, many methods have been studied, including preparation of nanoparticles using bovine serum albumin encapsulation, anti-solvent precipitation, etc., and the prior art, despite some progress, still needs to improve the solubility and chemical stability of quercetin.

Disclosure of Invention

The invention provides a preparation method of calcium ion stable composite nano particles for delivering quercetin, aiming at solving the problems in the prior art, and the preparation method comprises the following steps:

1) dissolving zein and quercetin in an ethanol water solution, magnetically stirring at 700rpm for 1 hour, and filtering with a 0.45-micrometer microporous filter membrane to remove impurities; obtaining zein and quercetin to obtain a mixed solution;

2) slowly dripping the mixed solution into the fucoidin solution by using a syringe, 6After stirring at 00rpm for 40 minutes, Ca was added dropwise to the solution²⁺Continuing stirring the solution for 20 minutes;

3) removing ethanol in the solution by using a rotary evaporator, adding a proper amount of water to ensure that the total volume of the solution is 25 times of that of the mixed solution, and then adjusting the pH value;

4) and centrifuging for 10 minutes to remove large particles to obtain composite nanoparticles.

Preferably, the weight ratio of the zein to the quercetin in the step 1) is 100: 4;

preferably, the volume concentration of the ethanol aqueous solution in the step 1) is (80% v/v), and the weight-to-volume ratio (g/ml) of the zein to the ethanol aqueous solution in the step 1) is 20: 1.

preferably, the concentration of the fucoidin solution in the step 2) is 1.67mg/ml, the pH is 4.0, and the volume ratio of the mixed solution to the fucoidin solution is 1: 24.

preferably, Ca is used in the step 2)²⁺The solution is prepared by dissolving calcium chloride powder in deionized water, and the concentration ranges from 12.5mM to 50mM respectively; the Ca²⁺The volume ratio of the solution to the mixed solution in the step 1) is 1: 1.

preferably, the pH in step 3) is adjusted to 4.0.

Preferably, the rotary evaporation conditions of the rotary evaporator in the step 3) are 40 ℃ and-0.1 MPa.

Preferably, the centrifugal speed in the step 4) is 3000 rpm.

A calcium ion stabilized composite nanoparticle for delivering quercetin, prepared by the above preparation method; the calcium ion-stabilized composite nanoparticle for delivering quercetin prepared by the invention has good pH stability and storage stability, and the ionic stability is enhanced.

Compared with the prior art, the invention has the following advantages and beneficial effects: by regulating the concentration of calcium ions, the composite nanoparticles have higher encapsulation efficiency on hydrophobic active substances, and simultaneously, the active substances have better stability and release characteristics in simulated gastrointestinal fluids.

The invention uses quercetin as a hydrophobic active substance model to further investigate Ca with different concentrations²⁺Effect on performance of active loaded zein/FD composite nanosystems. Study of Ca by various characterization means²⁺The influence on the interaction in the composite nanosystem provides more useful information for zein and fucoidan based hydrophobic active delivery systems.

Description of the drawings:

FIG. 1 shows different Ca contents²⁺The concentration of quercetin-delivering composite nanoparticles (a) PDI and average particle size, (B) zeta potential, (C) particle size distribution profile.

FIG. 2 shows different Ca²⁺EE impact profile of quercetin-delivered composite nanoparticles at concentration.

FIG. 3 shows Zein, Zein/FD and different Ca²⁺The FS map, inset is a partial magnified view of the concentration of quercetin delivering composite nanoparticles.

FIG. 4 shows Zein, fucoidan, and Ca²⁺An FTIR spectrum of quercetin-delivered composite nanoparticles at concentration.

FIG. 5 shows Zein, fucoidan, and Ca in different forms²⁺TG profile of composite nanoparticles delivering quercetin at concentration.

FIG. 6 shows Zein, fucoidan, and Ca²⁺XRD pattern of concentration of quercetin delivering composite nanoparticles.

FIG. 7 shows different Ca²⁺SEM spectra of Quercetin-delivering composite nanoparticles at concentrations (where A, B, C, D, E, F corresponds to 0mM, 0.5mM, 1mM, 1.5mM, 2mM, 2.5mM, 3mM Ca, respectively)²⁺)。

FIG. 8 shows pH values versus different Ca²⁺The concentration of the quercetin-delivering composite nanoparticle is (a) the particle diameter, (B) PDI, and (C) zeta potential diagram.

FIG. 9 shows different Ca²⁺The particle diameter (a) and the PDI (B) of the quercetin-delivering composite nanoparticle at a concentration.

FIG. 10 shows Ca at different heating temperatures²⁺(A) particle diameter, (B) PDI, (C) zeta potential, and (D) quercetin retentive of composite nanoparticles in a concentration that delivers quercetinGraph of retention.

FIG. 11 shows different Ca²⁺Photostability plot of concentration of quercetin delivered composite nanoparticles.

FIG. 12 shows long term storage (4 ℃ C., 22 days) versus different Ca²⁺The influence of (a) the particle diameter and PDI, (B) the zeta potential and the retention rate of quercetin of the quercetin-delivering composite nanoparticles at the concentration is shown.

FIG. 13 shows Ca²⁺The influence of the concentration on (a) the particle size and (B) the release characteristics of quercetin in SGIF of the quercetin-loaded composite nanosystem.

Detailed Description

The following examples are intended to further illustrate the present invention, but they are not intended to limit or restrict the scope of the invention.

Zein (zein) used in the present invention was purchased from Sigma reagent, Missouri, USA, and fucoidan was purchased from Jiejing group, Inc., Shandong.

The particle size, Polydispersity (PDI) and zeta potential of the nanoparticles are tested by a Malvern potentiostat. Before the measurement, the sample was diluted with deionized water having a pH of 4 to an appropriate concentration (a counting rate of 100-.

The method for measuring the Encapsulation Efficiency (EE) of the quercetin comprises the following steps: mixing 1mL of newly prepared composite nanoparticle dispersion with 4mL of ethanol, vortexing for 2 minutes, centrifuging at 10000g for 10 minutes, collecting supernatant, measuring absorbance of the solution at 370nm wavelength with an ultraviolet spectrophotometer, and obtaining a standard curve of quercetin (y: 0.0754512x +0.00218657, R) by referring to the same conditions²0.99987) the concentration of quercetin was calculated. All samples were tested in triplicate and the EE of quercetin was calculated using the following formula.

The present invention used a fluorescence spectrophotometer model G9800A to test the samples, and the concentration of the composite nanoparticle dispersion was diluted to about 0.25mg/mL (calculated as zein) with deionized water (pH 4.0) prior to testing. The excitation wavelength is set to be 280nm, the collection range of the emission spectrum is 290-450nm, the widths of the excitation slit and the emission slit are both 5nm, and the scanning speed is 120 nm/min.

The invention adopts a Fourier transform spectrophotometer at the wavelength of 400-4000cm^-1Resolution of 4cm^-1And (5) characterizing the sample.

Thermogravimetric (TG) analysis of the invention: the lyophilized sample (about 10mg) was taken for TG analysis with the test temperature set at 30-500 deg.C, the temperature rise rate at 10 deg.C/min and the nitrogen flow rate at 20 mL/min.

Analysis by X-ray diffraction Spectroscopy (XRD) of the invention: the lyophilized samples were tested using a wide angle XRD copper target (incident ray wavelength 0.15418 nm). The scanning range is 5-55 degrees, the accelerating voltage is 40kV, the current is 40mA, the step size is 0.02 degree, and the testing speed is 0.1 sec/step.

The SEM of the invention is characterized in that: a small amount of the freeze-dried sample was taken, fixed on a sample stand with a conductive adhesive, and subjected to vacuum gold plating, followed by observing and recording microscopic morphological characteristics of the sample at an accelerating voltage of 3.0 kv.

Study on physical and chemical stability

Stability of pH

Will contain different Ca²⁺The pH of the nanoparticle dispersion at concentrations was adjusted to 2.5, 6.0, 8.5 with 0.1M NaOH or HCl, respectively. And (3) placing the solution with the adjusted pH value at 25 ℃ for 24 hours, then testing the particle size, PDI and zeta potential of the solution, and diluting a sample to be tested by using deionized water with the same pH value before measurement.

Stability of ion

Respectively containing different Ca²⁺The stability of the nanoparticles was tested after placing the nanoparticles at 25 ℃ for 24 hours after stirring at 500rpm for 10min by adding NaCl solid to the nanoparticle dispersion at a concentration such that the solution contained 20mM, 40mM and 80mM NaCl, respectively.

Thermal stability

Respectively placing 5mL of the newly prepared composite nanoparticle dispersoid in constant-temperature water bath pots at 50 ℃, 65 ℃ and 80 ℃ for heating for 30 minutes, taking out the heated composite nanoparticle dispersoid after heating, testing the stability of the nanoparticles after cooling to 25 ℃, and testing the stability of the quercetin by the same method in the step of testing the encapsulation rate of the quercetin.

Light stability

Placing 10mL of the newly prepared composite nanoparticle dispersion in a quartz bottle, placing under a 38W ultraviolet lamp for 2 hours, sampling 1mL every 30 minutes, and testing the stability of quercetin by the method in 3.3.3.

Storage stability

After the newly prepared composite nanoparticle dispersion was stored at 4 ℃ for 22 days, its particle diameter, PDI, potential and stability of Que in the composite nanoparticles were measured.

In vitro digestion experiments

30mL of the freshly prepared dispersion was mixed with 30mL of Simulated Gastric Fluid (SGF) containing 2.0mg/mL NaCl and 3.2mg/mL pepsin, and the pH of the mixed solution was adjusted to 2.5. The mixed solution was then incubated at 37 ℃ in an incubator and after shaking at 120rpm for 60 minutes, the above solution (60mL) was mixed with 60mL of Simulated Intestinal Fluid (SIF) (containing 2.0mg/mL of pancreatin, 6.8mg/mL of K)₂HPO4, 10mg/mL porcine bile salts, and 8.8mg/mL NaCl) were mixed, the pH was adjusted to 7.4, and incubation was continued for 90 minutes. Samples were taken every 30 minutes throughout the simulated digestion experiment and were supplemented with the corresponding simulated digestion solution. The particle size of the nanoparticles was tested under different digestion conditions and the sample solution taken out was centrifuged at 10000rpm for 10 minutes and then filtered with a 0.45 μm microfiltration membrane to obtain the supernatant, followed by measurement of the quercetin release as described in the test of quercetin encapsulation efficiency.

Example 1 contains different Ca²⁺Preparation of composite nanoparticles delivering quercetin at a concentration

0.8g of zein and 32mg of quercetin were co-dissolved in 40mL of an aqueous ethanol solution (80% v/v), magnetically stirred at 700rpm for 1 hour, and then filtered through a 0.45 μm microfiltration membrane to remove impurities. Slowly adding 2mL of the above mixed solution into 48mL of fucoidan solution (containing 80mg of fucoidan and having a pH adjusted to 4.0) with a syringe, stirring at 600rpm for 40 min, and adding 2mL of Ca with different concentrations into the solution²⁺Solutions (0, 12.5mM, 25mM, 37.5m, respectively)M, 50mM, 60.5mM, 75mM) and stirring was continued for 20 minutes. The ethanol in the solution was then removed using a rotary evaporator (40 ℃, -0.1MPa) and an appropriate amount of water (pH 4.0) was added so that the total volume of the solution was 50mL, and then the pH of the solution was adjusted to 4.0. Finally, the nanoparticle dispersion was centrifuged at 3000rpm for 10 minutes to remove large particles to obtain particles containing different concentrations of Ca²⁺The dispersion of (1). And (3) carrying out vacuum freeze drying (the vacuum degree is lower than 5Pa) on the prepared part of sample liquid for subsequent characterization, and storing the rest part in a refrigerator at 4 ℃ for later use. The final solution obtained will have different Ca²⁺The composite nanoparticles delivering quercetin at concentrations (0, 0.5mM, 1.0mM, 1.5mM, 2.0mM and 3.0mM) are named as ZFQ, ZFQC0.5, ZFQC1.0, ZFQC1.5, ZFQC2.0 and ZFQC3.0, respectively, and the prepared composite nanoparticles are used for subsequent experimental studies.

Example 2 different Ca²⁺Effect of particle size, PDI and potential of Quercetin-delivering composite nanoparticles concentration

The composite nanoparticles obtained in example 1 were measured for particle size, PDI and potential, and the results are shown in FIG. 1, in which Ca was not added²⁺The particle size of the ZFQ obtained was 153.2 + -3.6 nm with Ca²⁺The concentration is gradually increased, the particle diameter of the composite nano particles is increased, and Ca is added²⁺The particle size of ZQC 3.0 prepared at a concentration of 3.0mM was 211.7 + -5.1 nm. All composite nanoparticles have very small PDI ()<0.2), and with Ca²⁺The PDI of the composite nanoparticles also gradually decreased with increasing concentration (from 0.121 to 0.068), and narrower peak widths of the particle size distribution of the formed composite nanoparticles were also observed in fig. 1C, indicating that Ca was present²⁺The addition of (2) can increase the uniformity of the composite nanoparticles. Zeta potential of the composite nanoparticles is also related to Ca²⁺The concentration showed a certain dependence from Ca²⁺The-33.8. + -. 0.6mV at concentration 0 increased to-31.5. + -. 0.8mV at 3.0 mM. Ca²⁺The addition of (a) may consume the negatively charged amino acids in the zein and the negative charges in the fucoidan, causes the total charge amount of the composite nanoparticles to be reduced, reduces the electrostatic repulsion among the particles, thereby causing the aggregation of the particles and leading the composite in the solutionThe particle size of the nanoparticles increases. At the same time, excessive Ca²⁺May also cause Ca²⁺Bridging occurs between the particles to cause an increase in particle size. Meanwhile, along with Ca²⁺The turbidity of the nanoparticle dispersion increases with increasing concentration, which is caused by the increasing particle size of the nanoparticles.

Example 3 different Ca²⁺Concentration of Quercetin EE (%) -delivering composite nanoparticles of Quercetin

EE of the nano delivery system for bioactive substances is one of important indicators for evaluating the potential for practical application thereof. FIG. 2 shows that Ca was added at different concentrations in example 1²⁺The formed composite nano particles have an encapsulation effect on quercetin. Overall, with Ca²⁺The increase of the concentration shows that the encapsulation efficiency of the compound system to quercetin is generally reduced after being increased, namely, the encapsulation efficiency is increased from 85.92 +/-0.62% of ZFQ to 90.83 +/-0.40% of ZQC 1.5 and then is reduced to 80.54 +/-0.99% of ZFQ 3.0.0. Added Ca²⁺Possibly in combination with zein and/or fucoidan, changes the structure of the composite nanoparticle. The distance between the zein and the fucoidan can be enlarged, so that the internal capacity of the composite nano particles is enlarged, and more quercetin can be loaded. When Ca is used, however²⁺When the concentration increased to 2.0mM and above, there was a significant decrease in quercetin EE, which may be an excess of Ca²⁺Adsorbed on the surface of the composite nano particle, resulting in' composite nano particle-Ca²⁺-formation of composite nanoparticles ", thereby reducing the loading space of quercetin.

Example 3 different Ca²⁺Composite nanoparticle FS analysis of concentration delivered quercetin

Interactions between proteins and other compounds may cause changes in the protein's microenvironment, which can be reflected by FS. Fig. 3 shows FS spectra of different samples (quercetin delivering composite nanoparticles prepared in example 1) at an excitation wavelength of 280 nm. For zein alone, it shows a maximum fluorescence intensity at 309nm, which is mainly derived from the fluorescence emission of tyrosine residues in zein, which is sensitive to folding and unfolding of proteins,can be used to monitor conformational changes in proteins. The addition of fucoidan resulted in a decrease in fluorescence intensity, which may be due to the interaction of positively charged amino acid residues in the zein with negatively charged fucoidan. After the quercetin is added, the fluorescence intensity is obviously reduced, which shows that the quercetin can also interact with the zein. It is noted that after addition of quercetin, a new emission peak also appeared at 298 nm. In Ca²⁺The fluorescence intensity of the composite nanoparticles increased slightly before the concentration was less than 2.0mM, indicating that Ca²⁺It is possible that electrostatic interactions cause a conformational change in the zein, exposing a more hydrophobic environment. Then at Ca²⁺At a concentration of 2.0mM, significant fluorescence quenching occurred, with Ca²⁺At concentrations of 2.0mM and 3.0mM, the particle size of the composite nanoparticles increased significantly, which may be the cause of a decrease in fluorescence intensity.

Example 4 different Ca²⁺Concentration of Quercetin-delivered composite nanoparticle FTIR analysis

FTIR can assess the interaction of the components in the composite nanoparticles. As shown in FIG. 4, different Ca prepared in example 1²⁺Quercetin-delivering composite nanoparticles at a concentration of about 3500cm^-1A relatively strong absorption peak appears on the left and right sides due to the stretching vibration of O-H. At 3000 and 2800cm^-1The absorption peak appearing in the range is attributed to the C-H stretching vibration. However, in the presence of different Ca²⁺In the concentration of the composite nanoparticles, no characteristic absorption peak of quercetin was observed, indicating that quercetin was encapsulated in the composite nanoparticles.

Compared with zein, the O-H absorption peak of ZFQ is shifted from 3439.5 to 3345.0cm^-1This indicates that hydrogen bonding and hydrophobic interactions exist in zein, fucoidan, and quercetin. The addition of calcium ions can influence hydrogen bonds or hydrophobic interaction in the composite nanoparticles, so that O-H absorption peaks in the composite nanoparticles are respectively moved to Ca²⁺3446.4cm at a concentration of 0.5-3.0mM^-1、3443.6cm^-1、3442.2cm^-1、3447.9cm^-1And 3440.7cm^-1. In addition, the absorption peak of the amide I band (C ═ O stretching vibration) was also observed from ZFQ1646.9cm^-1Respectively shifted to 1648.4cm in ZQC (0.5-3.0)^-1、1646.9cm^-1、1651.2cm^-1、1645.5cm^-1And 1646.9cm^-1(ii) a After adding Ca²⁺In the composite nanoparticle of (1), is located at 1540cm^-1No significant shift was observed in the absorption peaks of the nearby amide II band (C-N stretching vibration and N-H bending vibration), but the intensity appeared to be small, lying at 1250cm^-1The nearby absorption peak is the sulfuric acid group of fucoidan, and the intensity of the absorption peak is also changed.

Example 5 different Ca²⁺Concentration of composite nanoparticle TG analysis delivering quercetin

FIG. 5 shows different samples (different Ca prepared in example 1)²⁺Concentration of composite nanoparticles delivering quercetin). The mass loss of fucoidan at 100 ℃ (14.70%) was significantly higher than that of zein (4.87%), which is likely that fucoidan acts as a water-soluble polysaccharide and therefore contains more free water. Loss of bound water may be involved at 200 ℃ of 100-²⁺The weight loss of the post-ZFQC (0.5-3.0) was 9.66%, 17.47%, 20.01%, 24.15% and 23.12%, respectively. Ca²⁺The addition of (b) may destroy the original interaction force in the composite nanoparticle, thereby reducing the thermal stability of the composite nanoparticle.

Example 6 different Ca²⁺Concentration of Quercetin-delivered composite nanoparticle XRD analysis

FIG. 6 shows zein, fucoidan, quercetin and the composite nanoparticle prepared in example 1 (containing 0-3.0mM Ca)²⁺) XRD pattern of (a). As can be seen, zein shows characteristic absorption peaks at 9.2 ° and 21.8 ° and fucoidan shows a broad absorption peak at 22.8 °, indicating that both zein and fucoidan are present in amorphous form. The sharp absorption peaks of quercetin at 12.5 °, 13.2 °, 14.1 °, 17.2 °, 22.2 °, 26.5 ° and 27.2 ° are characteristic absorption peaks of quercetin, indicating that quercetin has a highly crystalline structure and exists in a crystalline form. However, at ZFQ, no peak was observed in the absorption of quercetin, indicating that quercetin had already been detectedThe transition to the amorphous state may be caused by intermolecular interactions of quercetin with zein and/or fucoidan during encapsulation^]. In comparison with ZFQ, Ca was added with the addition²⁺The intensities of the characteristic peaks existing in the zein and the fucoidan in the composite nano particle are gradually reduced and even disappear, which shows that the Ca²⁺The addition of (b) may cause a change in the interaction force in the composite nanoparticle. In addition, it is clearly observed that Ca is added²⁺The composite nanoparticles of (2) show some new diffraction peaks (around 27.4 degrees, 31.8 degrees and 45.5 degrees), which may be CaCl₂Caused by crystals.

Example 7 different Ca²⁺Concentration of quercetin-delivered composite nanoparticles SEM observations

By SEM, the content of different Ca was observed²⁺Concentration of composite nanoparticles delivering quercetin (prepared in example 1) microscopic morphology. All composite nanoparticles exhibited a good spherical appearance. Ca²⁺The particle diameter of the composite nano particles formed at lower concentration is less than Ca²⁺At higher concentrations, this trend is consistent with our measurement with DLS, but it is clearly observed that all samples have a smaller particle size in the SEM than that obtained with DLS, which may be the result of the difference in the measurement principles. The clumps observed in ZFQ 3.0.0 may be CaCl₂Crystals, which also indicate Ca at this time²⁺The concentration is already supersaturated.

Example 8 different Ca²⁺Physical and chemical stability of composite nanoparticles delivering quercetin in concentrations

The stability of the protein-polysaccharide system may be affected by pH. As can be seen from FIG. 8, the Ca was different²⁺The particle size of the quercetin-delivering composite nanoparticles (prepared in example 1) at a concentration slightly decreased with increasing pH, and the charge amount thereof increased, but the PDI hardly changed ((PDI))<0.20). The particle size change of the composite nanoparticles under different pH values shows that the particle size can be regulated and controlled through the pH value. The potential of the composite nanoparticles was always negative throughout the pH change, indicating that fucoidan dominates its electrical properties. p is a radical ofThe increase in the particle size of the composite nanoparticles when H is smaller can be attributed to particle aggregation caused by a decrease in electrostatic repulsion between particles in solution. While the turbidity of the solution decreases with increasing pH, which may be the result of a decrease in the particle size of the nanoparticles in the dispersion. All solutions were free of large particles and precipitation, indicating addition of Ca²⁺The latter ZFQ complex system still has good pH stability.

Example 9 different Ca²⁺Ionic stability of concentration formed composite nanoparticles

As shown in FIG. 9, different Ca²⁺The particle size of the quercetin-delivering composite nanoparticles (prepared in example 1) at concentration increased with increasing NaCl concentration, but Ca²⁺The ion stability of the composite nano particles is improved by concentration. When the NaCl concentration was 20mM, the ZFQ solution became very cloudy and precipitation occurred, which was likely to be due to the disruption of electrostatic repulsion between ZFQ nanoparticles by NaCl, resulting in instability of the solution. At this time, Ca was added although the turbidity of the solution of ZQC 0.5 was increased²⁺The ZQC nanoparticles can still keep the stability of the solution. As NaCl concentration increased, both ZQC 0.5 and ZQC 1.0 precipitated at NaCl concentrations of 40 and 80mM, and the others had Ca added²⁺The turbidity of the solution of (2) also increased, but Ca²⁺The higher concentration of the composite nanosystem of (a) shows better stability. Ca²⁺Possibly reacts with zein and fucoidin in the composite nano-particles, thereby enhancing the salt resistance of the system, namely Ca²⁺At higher concentrations, excess Ca²⁺May be attached to the surface of the composite nano particle to enhance the ionic stability.

Example 10 different Ca²⁺Thermal stability of quercetin-delivering composite nanoparticles in concentrations

As shown in FIG. 10, after heat treatment of ZQC (0-3.0mM) at 50-80 ℃, none of their particle size, PDI and zeta potential changed significantly, but the retention rate of quercetin changed. At 50 and 65 ℃ Ca²⁺The addition of (A) has little influence on the retention rate of quercetin, but at a temperature of 80 ℃, Ca²⁺The increase in concentration promotes the degradation of quercetinThe retention rate is reduced from 96.7 +/-0.86% of ZFQ to 86.47 +/-2.46% of ZFQ 3.0.0. Ca²⁺The addition of (2) may transform the structure of the composite nanoparticles, thereby reducing the thermal stability of the composite nanoparticle system at 80 ℃, resulting in a decrease in the retention rate of quercetin. This also corresponds to the result Ca obtained from the TG analysis²⁺The addition of (2) reduces the thermal stability of the composite nanoparticles.

Example 11 different Ca²⁺Photostability of quercetin-delivering composite nanoparticles in concentrations

The light stability of the quercetin is poor, and the evaluation of the stability of the quercetin of the composite system under ultraviolet light has important significance. It was found experimentally that quercetin was almost completely decomposed after uv irradiation for 60 minutes without encapsulation. In fig. 11, the encapsulated quercetin had a retention rate of 40% or more after irradiation for 120 minutes under ultraviolet light, which indicates that the encapsulated quercetin had better photostability. After 30 minutes of irradiation, there was no significant difference in the stability of quercetin in ZFQ and ZFQC (0.5-3.0). After 120 minutes of irradiation, a significant decrease in quercetin stability in ZFQC2.0 and ZFQC3.0, excess Ca, was observed²⁺May cause the structure change of the composite nano particles, weakens the binding capacity of the quercetin and the composite nano system, and reduces the protection effect of the quercetin on the quercetin.

Example 12 different Ca²⁺Storage stability of quercetin-delivering composite nanoparticles in concentrations

As shown in fig. 12, the particle size, PDI and zeta potential of the composite nanoparticle dispersion were almost unchanged after being stored at 4 ℃ for 22 days, probably because they all had a high charge amount, so that strong electrostatic repulsion between the nanoparticles in the solution was maintained, thereby ensuring the stability of the solution. After 22, the retention rate of quercetin in all the composite nanoparticles is over 90 percent, which indicates that Ca is added²⁺The compound nano system has good protection effect on the quercetin in the storage process.

Example 12 different Ca²⁺In vitro digestion study of quercetin-delivering composite nanoparticles at concentrations

As shown in fig. 13, the particle size and release characteristics of all the composite nanoparticles had similar trends in the simulated digestion experiment. In both SGF and SIF, their particle sizes are increased, and the particle size in SIF is much larger than in SGF; a large amount of quercetin was released in SGF, and then a small amount of quercetin continued to be released in SIF. This indicates that the release characteristics of quercetin in the composite nanoparticles in simulated digestion may be related to the change in particle size thereof. In SGF, pepsin may disrupt the structure of the nanoparticles causing an increase in particle size, resulting in the release of a large amount of quercetin, and a significant burst is observed at 30 minutes; subsequently in SIF, its structure is further destroyed by pancreatin, resulting in the continued release of quercetin. In addition, the different pH environments of SGF and SIF and the presence of large amounts of NaCl can also cause the particle size of the composite nanoparticles to vary. Ca is added in the whole process of simulating digestion²⁺The dispersions produced appeared to reduce quercetin release in SGF and SIF, especially in ZFQC0.5 and ZFQC1.5, indicating Ca²⁺The addition of the compound nanoparticles can change the structure of the compound nanoparticles, thereby enhancing the resistance of the compound nanoparticles to simulated gastrointestinal fluids and improving the release characteristic of quercetin.

The invention shows through the comparative study:

(1)Ca²⁺possibly in combination with zein and fucoidan by electrostatic interactions, the structure of the zein/FD based composite nanosystem is altered, thus causing changes in its properties.

(2) Adding Ca²⁺Thereafter, the composite nanosystems encapsulating quercetin still have good pH stability and storage stability, and their ionic stability is enhanced, but at Ca²⁺At higher concentrations, the photostability and thermal stability at 80 ℃ of the composite nanosystems (ZFQC2.0 and ZFQC3.0) decreased significantly, which is probably Ca²⁺Caused by excessive dosage. Ca²⁺The excess also results in a decrease in the encapsulation efficiency of quercetin.

(3) The simulated digestion experiment shows that Ca²⁺The addition of the composite nano particles can enhance the stability of the composite nano particles in simulated digestive fluidIt has good release property for active substance.

The invention researches and investigates Ca with different concentrations²⁺The stability of the zein/FD composite nano system loaded with quercetin is influenced, and the composite nano particles are characterized by adopting means such as SEM (scanning Electron microscope), FTIR (FTIR), Thermogravimetric (TG) analysis, X-ray diffraction spectrum (XRD), Fluorescence Spectrum (FS) and the like. The study showed that Ca²⁺The composite nanostructure based on zein/FD can be altered by electrostatic interaction in combination with zein and FD, thereby causing changes in its properties. Adding Ca²⁺Then, the composite nano system encapsulating quercetin shows good pH stability and storage stability, and the ionic stability is enhanced, but in Ca²⁺At higher concentrations, the photostability and thermal stability at 80 ℃ of the composite nanosystems (ZFQC2.0 and ZFQC3.0) decreased significantly, which is probably Ca²⁺Ca caused by excessive dosage²⁺The excess also results in a decrease in the encapsulation efficiency of quercetin. The simulated digestion experiment shows that Ca²⁺The addition of the compound nanoparticles can enhance the stability of the compound nanoparticles in simulated digestive juice and improve the release characteristic of quercetin in the digestive juice.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.

Claims

1. A method for preparing calcium ion-stabilized composite nanoparticles for delivery of quercetin, characterized in that the preparation method comprises the steps of:

1) dissolving zein and quercetin in an ethanol water solution, magnetically stirring at 700rpm for 1 hour, and filtering with a 0.45-micrometer microporous filter membrane to remove impurities to obtain a mixed solution of zein and quercetin;

2) slowly dropping the mixed solution into the fucoidin solution by using a syringe, stirring at 600rpm for 40 minutes, and then dropping Ca into the solution²⁺Continuing stirring the solution for 20 minutes;

2. The method of claim 1, wherein the weight ratio of zein to quercetin in step 1) is 100: 4;

3. the method of claim 1, wherein the concentration of the ethanol solution in step 1) is 80% by volume, and the weight/volume ratio g/ml of the zein to the ethanol solution in step 1) is 20: 1.

4. the method for preparing calcium ion-stabilized composite nanoparticles for delivering quercetin according to claim 1, wherein the concentration of the fucoidan solution in the step 2) is 1.67mg/ml, the pH is 4.0, and the volume ratio of the mixed solution to the fucoidan solution is 1: 24.

5. the method of claim 1, wherein the step 2) comprises Ca-ion stabilizing the composite nanoparticles with the delivery of quercetin²⁺The solution is prepared by dissolving calcium chloride powder in deionized water, and the concentration ranges from 12.5mM to 50mM respectively; the Ca²⁺The volume ratio of the solution to the mixed solution in the step 1) is 1: 1.

6. the method for preparing calcium ion-stabilized composite nanoparticles for delivering quercetin according to claim 1, characterized in that the pH in the step 3) is adjusted to 4.0.

7. The method for preparing calcium ion-stabilized composite nanoparticles for delivering quercetin according to claim 1, characterized in that the rotary evaporation condition in the step 3) is 40 ℃ and-0.1 MPa.

8. The method for preparing calcium ion-stabilized composite nanoparticles for delivering quercetin according to claim 1, characterized in that the centrifugal rotation speed in the step 4) is 3000 rpm.

9. The composite nanoparticle prepared by the method for preparing calcium ion-stabilized composite nanoparticles for delivering quercetin according to any one of claims 1-7.