CN118045062A - Protein-polysaccharide ternary composite nanoparticle loaded with quercetin, and preparation method and application thereof - Google Patents
Protein-polysaccharide ternary composite nanoparticle loaded with quercetin, and preparation method and application thereof Download PDFInfo
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- CN118045062A CN118045062A CN202410147263.9A CN202410147263A CN118045062A CN 118045062 A CN118045062 A CN 118045062A CN 202410147263 A CN202410147263 A CN 202410147263A CN 118045062 A CN118045062 A CN 118045062A
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- quercetin
- zein
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- gelatin
- carboxymethyl starch
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Abstract
The invention discloses a quercetin-loaded protein-polysaccharide ternary composite nanoparticle, and a preparation method and application thereof. The quercetin nano-particles of the invention remarkably improve the thermal stability, pH stability, photostability and storage stability of the drug delivery system; the ternary composite nanoparticle (zein-gelatin-carboxymethyl starch) for delivering quercetin has higher pH stability, can be very stable at pH2-8, and further has stronger antioxidant activity and higher in-vitro bioavailability. The invention provides a new practical technology for delivering natural bioactive ingredients.
Description
Technical Field
The invention relates to the technical field of quercetin nano drug delivery systems, in particular to a protein-polysaccharide ternary composite nanoparticle of quercetin loaded by zein double modified by carboxymethyl starch and gelatin, and a preparation method and application thereof.
Background
Quercetin is a natural polyhydroxyflavonoid that is found in a variety of vegetables and fruits. Quercetin has been proved to have pharmacological activities such as antioxidation, anti-inflammation, anticancer, antiallergic, blood pressure reduction and the like, and has important application value in the fields of biological medicines, functional foods and the like. However, quercetin has very low solubility in water, is unstable in light and high temperature environments, and has very low bioavailability for direct oral administration, thus limiting the application of quercetin in practical products. Designing and developing an appropriate delivery system is an effective strategy to increase its solubility, stability, bioavailability, and bioefficacy.
Nanoparticles are a common class of nanoscale delivery systems, typically using high molecular polymers as wall materials, that can effectively encapsulate and carry natural active ingredients. The protein and the polysaccharide are used as two main biopolymers, have the advantages of wide sources, safety, no toxicity, good biocompatibility, easy preparation into nano materials and the like, and are widely applied to construction of nano-scale carriers for protection and delivery of bioactive substances.
Zein (zein) -based nano-delivery systems of bioactive components are a recent research focus. Zein is the main storage protein of corn, contains more than 50% of nonpolar amino acids (leucine, alanine, and proline), is soluble in high concentration ethanol aqueous solution, but insoluble in water, and this property makes zein suitable for embedding hydrophobic active ingredients. The quercetin and zein are co-dissolved in high concentration ethanol, and the quercetin is embedded into the hydrophobic core of the zein nano-particles by an anti-solvent method. However, zein nanoparticles are easy to aggregate due to strong hydrophobicity, and particularly have poor stability near isoelectric points, and have low embedding rate for drugs. The prior researches generally use biopolymer coating to solve the problems, and currently single polysaccharide or protein is generally used as a stabilizer of zein nano-particles, wherein the polysaccharide is commonly used as pectin, sodium alginate, hyaluronic acid, chitosan and the like; proteins for zein nanoparticle surface modification, mainly sodium caseinate, are also reported in literature as soy protein isolates and lactoferrin. The surface modification of the polysaccharide or the protein improves the embedding rate and the stability of the zein to active ingredients to a certain extent, but is easy to aggregate under the condition of lower pH, such as pH 2-3, and has the problems of low bioavailability, large particle size and the like.
Besides the binary system, there are few reports of ternary systems, mainly adopting a layer-by-layer self-assembly method, such as a protein/polysaccharide and polysaccharide/polysaccharide composite system to carry out surface modification on zein nano-particles. The protein/polysaccharide composite system is mainly sodium caseinate, and the polysaccharide can be pectin, sodium alginate, chitosan and the like. The protein/polysaccharide complex system shows good synergistic effect. In the polysaccharide/polysaccharide composite system, the surface charge of zein nano particles is positive charge, so that the first layer of modification uses anionic polysaccharide to be combined under the electrostatic action, such as sodium alginate and pectin, the second layer of modification uses cationic polysaccharide, usually chitosan, and the ternary composite nano particles with positive charges are constructed.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a quercetin-loaded protein-polysaccharide ternary composite nanoparticle, and a preparation method and application thereof.
The invention is realized by the following technical scheme:
A protein-polysaccharide ternary composite nanoparticle loaded with quercetin takes zein to wrap the quercetin as a core, gelatin and carboxymethyl starch are assembled into a shell, and the composite nanoparticle with a core-shell structure for delivering the quercetin is constructed.
A preparation method of a quercetin-loaded protein-polysaccharide ternary composite nanoparticle specifically comprises the following steps:
(1) Preparation of ethanol solution of quercetin-zein
0.6G zein was dissolved in 30mL 80% aqueous ethanol, then treated as quercetin: adding quercetin into zein the mass ratio of 1:5-1:30, and stirring thoroughly to dissolve to obtain quercetin-zein ethanol solution;
(2) Preparation of gelatin solution
Dissolving a certain amount of gelatin in 20mL of deionized water, stirring at 40 ℃ for 0.5h, and then stirring at room temperature for 0.5h to prepare gelatin solution with a certain concentration, wherein the mass of gelatin is 12.5mg-100mg;
(3) Preparation of quercetin-loaded gelatin-modified zein nanoparticle dispersion liquid
Taking 5mL of the ethanol solution of quercetin-zein obtained in the step (1), slowly dripping the solution into 20mL of the gelatin solution obtained in the step (2), and stirring at a speed of 400-1000 rpm for 10-60min;
(4) Preparation of carboxymethyl starch solution
Dissolving a certain amount of carboxymethyl starch in 25mL of deionized water, and stirring overnight to prepare carboxymethyl starch solution with a certain concentration, wherein the mass of carboxymethyl starch is 25mg-200mg;
(5) Preparation of quercetin-loaded gelatin-carboxymethyl starch double-modified zein nanoparticle dispersion liquid
Slowly dripping the quercetin-loaded gelatin-modified zein nanoparticle dispersion liquid obtained in the step (3) into the carboxymethyl starch solution obtained in the step (4), stirring at a speed of 400-1000 rpm for 30-120min, then removing ethanol under reduced pressure on a rotary evaporator, and supplementing the volume with deionized water to 50mL to obtain the quercetin-loaded gelatin-carboxymethyl starch dual-modified zein nanoparticle dispersion liquid;
and (3) carrying out freeze drying on the dispersion liquid of the quercetin-loaded gelatin-carboxymethyl starch double-modified zein nano particles obtained in the step (5) to obtain the quercetin-loaded protein-polysaccharide ternary composite nano particles.
The mass ratio of the quercetin to the zein in the step (1) is 1:5-1:20; the mass of the gelatin in the step (2) is 20mg-50mg; the mass of the carboxymethyl starch in the step (4) is 50mg-150mg.
The stirring speed in the step (3) is 500-600 rpm, and the stirring time is 10-30min; the stirring speed in the step (5) is 500-800 rpm, and the stirring time is 30-60min.
A quercetin-loaded protein-polysaccharide ternary composite nanoparticle is used for preparing antioxidant drugs and functional foods.
A preparation method of a medicine-carrying composite nanoparticle modified by calcium ions comprises the steps of stirring a quercetin-loaded gelatin-carboxymethyl starch double-modified zein nanoparticle dispersion liquid, adding 0.1-1 mL of calcium chloride solution while stirring, enabling the final calcium ion concentration of a mixed system to be 0.15-1.5 mM, and continuously stirring for 0.5-2 h; centrifuging at 3000rpm for 10 min to remove large particles, and obtaining the calcium ion modified drug-loaded composite nano particles.
The application of the calcium ion modified drug-loaded composite nano-particles in preparing antioxidant drugs and functional foods.
The technical principle of the invention is as follows:
Zein has unique self-assembly characteristics due to the large number of hydrophobic amino acids and the distinct partitioning of the hydrophilic and hydrophobic portions of the structure, and can be used to encapsulate hydrophobic materials by preparing nanoparticles via an antisolvent process. The gelatin has strong hydrophilicity and shows the characteristic of protein with weak negative electricity in deionized water, and the pH value of the aqueous solution is basically between 4.6 and 5.3 and is lower than the isoelectric point of zein, so that the pH value does not need to be regulated, and the preparation process is simpler. The zein self-assembled particles are positively charged, and gelatin is adsorbed on the surfaces of the zein particles through electrostatic interaction. Carboxymethyl starch is an anionic polysaccharide with good water solubility, and can be adsorbed on the surface of zein/gelatin nano-particles through electrostatic interaction to obtain the quercetin-loaded zein/gelatin/carboxymethyl starch nano-particles.
The invention determines double modification zein nano particles of a gelatin and carboxymethyl starch composite system through repeated screening, and the result shows that the medicament encapsulation efficiency is obviously improved, the stability under different pH environments and the stability against heat, ultraviolet rays and long-term storage are improved, and the biological accessibility is obviously improved through in vitro simulation gastrointestinal digestion experiments. The two biopolymers are not used in similar nano systems, and the combined use of the two biopolymers is not reported.
Gelatin is a linear polymer formed by crosslinking amino acid and peptides obtained by hydrolyzing and refining animal collagen. As a hydrolysate of natural protein, the gelatin has excellent biocompatibility and biodegradability, is low in price and easy to obtain, and is widely used in the fields of photosensitive materials, medical foods, daily chemical industry and the like. Gelatin is a protein macromolecule composed of 18 amino acids, wherein the molecule contains both anionic functional groups and cationic functional groups, the rest part is hydrophobic functional groups, and the ratio of three groups is about 1:1:1, the unique structure brings about various excellent properties of gelatin, such as colloid protection, film forming property, surface activity, reversible transformation of gel and sol state, etc., so that the gelatin can be widely applied in the fields of food, pharmaceutical auxiliary materials, etc.
The gelatin nanoparticles can be used as drugs and gene delivery vehicles for sustained and targeted release, such as delivery of various hydrophilic or hydrophobic anticancer drugs, such as methotrexate, cytarabine, camptothecine, curcumin, cycloheximide, doxorubicin and the like, and can enhance the cellular uptake of the substances, thereby improving the therapeutic effect of the drugs. However, the gelatin nanoparticles prepared without the crosslinking treatment are easily aggregated, and thus crosslinking is generally required to obtain nanoparticles with high stability, and the commonly used crosslinking agents mainly include aldehydes, genipin, carbodiimide, microbial transglutaminase, and the like.
The invention uses the natural hydrophilic polymer of gelatin as the first coating of zein nano particles, increases the hydrophilicity and reduces the hydrophobic aggregation of zein. However, instability at pH conditions around the isoelectric point of the protein still exists. For this purpose, a second coating is applied on the basis of which the anionic polysaccharide carboxymethyl starch is used.
Carboxymethyl starch, also called sodium carboxymethyl starch (CMS), is a water-soluble anionic polymer compound which is low in cost, good in biocompatibility, nontoxic, tasteless, and stable to light and heat, and is easily dissolved in water to form transparent liquid. Food-grade carboxymethyl starch is widely applied to products such as milk, beverage, frozen food, fast food, cake, syrup and the like. The carboxymethyl starch is used as a food additive, so that the taste of food is more viscous, the edible taste is improved, the stability of the food is also improved, and the shelf life of the food can be prolonged in the food processing process. In addition, carboxymethyl starch is physiologically inert and has no caloric value, and thus can be used to make low caloric value foods. The carboxymethyl starch is also quite widely applied in medicine, and can be used as pharmaceutical auxiliary materials, disintegrating agents, excipients and the like for preparing capsules, tablets, granules, sustained and controlled release preparations and the like in the aspect of pharmaceutical preparations.
Because the carboxymethyl starch has such excellent properties, the carboxymethyl starch is adopted as a second layer of shell material, and the carboxymethyl starch interacts with gelatin and zein under the actions of static electricity, hydrogen bonds and the like to form a ternary delivery system, so that the stability and water solubility of the quercetin are obviously improved, and in addition, the research shows that the antioxidant activity and bioavailability of the quercetin in an aqueous solution can be also obviously improved. Less carboxymethyl starch is used in nano drug-carrying systems of natural active ingredients, du Jing is a carboxymethyl starch and hydroxypropyl trimethyl ammonium chloride chitosan nano complex for oral delivery of insulin; li and the like are used for preparing carboxymethyl starch and chitosan hydrochloride composite nano gel which is used for delivering curcumin and shows strong pH sensitivity; leonida et al, polyelectrolyte complexes formed with carboxymethyl starch and chitosan, for colonic administration of acetaminophen. According to our searches, no stabilizer for zein nanoparticles using gelatin or carboxymethyl starch has been found, and the combination of the two has not been reported yet.
Calcium ions (Ca 2+) have a variety of biological properties that can be used to modulate the properties of the colloidal complex. Calcium ions are most commonly used in sodium alginate systems to enhance rheological properties or to adjust texture to produce gels. The invention further adds calcium ions with a certain concentration to electrostatically react with anionic polysaccharide on the surfaces of the nano particles to form a nano gel-like structure, thereby improving the stability of the nano particles, particularly the stability of salt concentration and having good slow release effect.
The invention has the advantages that: aiming at the problems of unstable zein nano-particles, low burying rate of the quercetin Pi Subao, low bioavailability of the quercetin and the like, the invention provides a method for efficiently preparing the three-element composite nano-particles of the zein/gelatin/carboxymethyl starch loaded with the quercetin by combining an antisolvent method with a layer-by-layer electrostatic accumulation method, and the three-element composite nano-particles have outstanding pH stability, good thermal stability, good light stability and good storage stability after analysis; in addition, the preparation also has good antioxidant activity and bioavailability; the technical scheme provided by the invention has the advantages of simple method, safe operation, green solvent and easy popularization.
Drawings
FIG. 1 is a transmission electron microscope image of nanoparticles prepared according to an embodiment of the present invention;
FIG. 2 is a photograph of nanoparticle dispersions prepared in examples 1-3 of the present invention;
FIG. 3 is a graph showing the release profile of quercetin in simulated gastrointestinal fluids for different prescriptions;
FIG. 4 shows the release profile of quercetin from example 1 (Zein/Gel/CMS-Que, drug loaded composite nanoparticle) and example 11 (Zein/Gel/CMS-Que-Ca 2+, calcium ion modified drug loaded composite nanoparticle).
Detailed Description
The following examples illustrate the process of the present invention, but are not limited thereto. Other suitable modifications and variations to these conditions and parameters, which are commonly encountered and will be apparent to those skilled in the art, are within the spirit and scope of the invention.
Example 1
1. Experimental part
(1) Preparation of zein and Quercetin ethanol solution
0.6G zein was dissolved in 30mL 80% aqueous ethanol, then treated as quercetin: zein mass ratio 1:10 adding quercetin, and stirring thoroughly to dissolve.
(2) Preparation of gelatin solution
20Mg of gelatin was dissolved in 20mL of deionized water, stirred at 40℃for 0.5h, and then stirred at room temperature for 0.5h to prepare a gelatin solution of 1 mg/mL.
(3) Preparation of quercetin-loaded gelatin-modified zein nanoparticle dispersion liquid
Taking 5mL of the ethanol solution of quercetin-zein obtained in the step (1), slowly dropwise adding the gelatin solution (volume of 20 mL) obtained in the step (2), and rotating at 500 rpm for 20min.
(4) Preparation of carboxymethyl starch solution
50Mg of carboxymethyl starch was dissolved in 25mL of deionized water and stirred overnight to prepare a 2mg/mL carboxymethyl starch solution.
(5) Preparation of quercetin-loaded gelatin-carboxymethyl starch double-modified zein nanoparticle dispersion liquid
Slowly dripping the quercetin-loaded gelatin-modified zein nano particles obtained in the step (3) into the carboxymethyl starch solution obtained in the step (4), wherein the rotating speed is 700 revolutions per minute, and the time is 30 minutes. The ethanol was then removed under reduced pressure on a rotary evaporator and the volume was made up to 50mL with deionized water. Obtaining the quercetin-loaded ternary composite nanoparticle dispersion liquid.
2. Characterization:
(1) The average particle size was 172nm and PDI was 0.18 as measured using a British Markov dynamic light scattering instrument.
(2) The quercetin encapsulation rate adopts the following method:
Freshly prepared nanoparticle dispersion was centrifuged at 12000rpm for 30min, and the supernatant was diluted with absolute ethanol to the appropriate concentration. The absorbance of the diluted solution was then measured at 373nm using an ultraviolet-visible spectrophotometer, and the concentration of quercetin was calculated as standard curve y=0.1077x+0.0056 (R 2 =0.9998), and the quercetin encapsulation rate was calculated as follows:
encapsulation Efficiency (EE) = (total quercetin-free quercetin Pi Suliang)/total quercetin x 100%
The detection shows that the encapsulation rate of quercetin is 91.6%.
And observing the morphology of the nano particles by using a transmission electron microscope. The diluted nanoparticle dispersion was dropped onto a coated copper mesh, and after several minutes, excess liquid was sucked out from the side with filter paper and dried naturally. The morphology of the samples was observed and photographed using a transmission electron microscope (JEM-1400 flash, japan) at an accelerating voltage of 120kV, as shown in FIG. 1.
3. As a comparison test, we also used an antisolvent method to prepare a zein nanoparticle dispersion loaded with quercetin, comprising the following steps:
Preparation of a quercetin-loaded zein nanoparticle dispersion: taking 5mL of the quercetin-zein ethanol solution obtained in the experimental part step (1), slowly dropwise adding the quercetin-zein ethanol solution into 20mL of deionized water (the pH is adjusted to 4.0-5.0), and stirring at a rotation speed of 500 rpm for 20min. The ethanol was then removed under reduced pressure on a rotary evaporator and the volume was made up to 25mL with deionized water. The average particle diameter was 89nm, PDI was 0.19, and quercetin encapsulation efficiency was 59.4% as measured using a British Markov dynamic light scattering instrument.
Quercetin-loaded gelatin-modified zein nanoparticles from experimental part step (3): the average particle diameter 145nm, PDI of 0.22 and quercetin encapsulation efficiency of 72.2% are detected by analysis.
From the above, it is clear that the particle size is increased and the encapsulation efficiency of quercetin is greatly improved, as compared with the binary composite nanoparticle and zein nanoparticle.
Example 2
(1) Preparation of zein and Quercetin ethanol solution
0.6G zein was dissolved in 30mL 80% aqueous ethanol, then treated as quercetin: zein mass ratio 1:15 adding quercetin, and stirring thoroughly to dissolve.
(2) Preparation of gelatin solution
30Mg of gelatin was dissolved in 20mL of deionized water, stirred at 40℃for 0.5h, and then stirred at room temperature for 0.5h to prepare a gelatin solution of 1.5 mg/mL.
(3) Preparation of quercetin-loaded gelatin-modified zein nanoparticle dispersion liquid
Taking 5mL of the ethanol solution of quercetin-zein obtained in the step (1), slowly dropwise adding the gelatin solution (volume of 20 mL) obtained in the step (2), and rotating at 500 rpm for 20min.
(4) Preparation of carboxymethyl starch solution
80Mg of carboxymethyl starch was dissolved in 25mL of deionized water and stirred overnight to prepare a carboxymethyl starch solution of 3.2 mg/mL.
(5) Preparation of quercetin-loaded gelatin-carboxymethyl starch double-modified zein nanoparticle dispersion liquid
Slowly dripping the quercetin-loaded gelatin-modified zein nano particles obtained in the step (3) into the carboxymethyl starch solution obtained in the step (4), wherein the rotating speed is 700 revolutions per minute, and the time is 45 minutes. And then removing ethanol under reduced pressure on a rotary evaporator, and complementing the volume with deionized water to 50mL to obtain the quercetin-loaded ternary composite nanoparticle dispersion liquid. Through analysis and detection, the average particle diameter is 181nm, the PDI is 0.20, and the quercetin encapsulation efficiency is 94.2%.
Example 3
(1) Preparation of zein and Quercetin ethanol solution
0.6G zein was dissolved in 30mL 80% aqueous ethanol, then treated as quercetin: zein mass ratio 1:15 adding quercetin, and stirring thoroughly to dissolve.
(2) Preparation of gelatin solution
25Mg of gelatin was dissolved in 20mL of deionized water, stirred at 40℃for 0.5h, and then stirred at room temperature for 0.5h to prepare a gelatin solution of 1.25 mg/mL.
(3) Preparation of quercetin-loaded gelatin-modified zein nanoparticle dispersion liquid
Taking 5mL of the ethanol solution of quercetin-zein obtained in the step (1), slowly dropwise adding the gelatin solution (volume of 20 mL) obtained in the step (2), and rotating at 500 rpm for 20min.
(4) Preparation of carboxymethyl starch solution
120Mg of carboxymethyl starch was dissolved in 25mL of deionized water and stirred overnight to prepare a carboxymethyl starch solution of 4.8 mg/mL.
(5) Preparation of quercetin-loaded gelatin-carboxymethyl starch double-modified zein nanoparticle dispersion liquid
Slowly dripping the quercetin-loaded gelatin-modified zein nano particles obtained in the step (3) into the carboxymethyl starch solution obtained in the step (4), wherein the rotating speed is 700 revolutions per minute, and the time is 60 minutes. The ethanol was then removed under reduced pressure on a rotary evaporator and the volume was made up to 50mL with deionized water. Obtaining the quercetin-loaded ternary composite nanoparticle dispersion liquid. As shown in FIG. 2, the average particle diameter 186nm, PDI, quercetin encapsulation efficiency was 96.1% as measured by analysis.
Example 4
Thermal stability test
Nanoparticle dispersions prepared in examples 1 to 3 were incubated in four water baths at different temperatures (40 ℃, 60 ℃, 80 ℃ and 100 ℃) for 0.5 hours and then cooled to room temperature. The particle size of the sample and the retention of quercetin were measured. A Zein-Que NPs loaded with quercetin was used as a control.
Retention of quercetin = content of quercetin in nanoparticles after treatment/content of quercetin in nanoparticles before treatment
TABLE 1 thermal stability test results
As can be seen from table 1, the ternary composite nanoparticle dispersions prepared in example 1, example 2 and example 3 show good stability at different temperatures, both in nanoparticle size and drug encapsulation efficiency. The treatment temperature was increased and the stability was only slightly lowered.
And Zein-Que NPs loaded with quercetin, the particle size obviously increases with the increase of temperature, and the amount of the loaded medicine also obviously decreases.
Example 5
Experiment of pH stability
Nanoparticle dispersions prepared in examples 1-3 were adjusted to different pH values with 1MHCl or 1 MNaOH. Then, the particle size change was detected by a dynamic light scattering instrument. A Zein-Que NPs loaded with quercetin was used as a control.
TABLE 2pH stability test results
As can be seen from table 2, the ternary composite nanoparticle dispersions prepared in example 1, example 2 and example 3 have good stability throughout the entire pH range from 2 to 8. Only at pH2 and 3 there was a small increase in particle size, but no aggregation and precipitation was observed.
The Zein-Que NPs loaded with quercetin have large particle size, large aggregation and poor stability at pH of 5-6. This pH is exactly the isoelectric point of the protein, indicating poor stability of zein nanoparticles at pH around the isoelectric point.
Example 6
Light stability test
Nanoparticle dispersions prepared in examples 1 to 3 were taken and placed under an ultraviolet lamp (363 nm, 30W) and irradiated for 2 hours. At different time points, sampling and detecting the content of quercetin in the sample at 0.5h, 1h, 1.5h and 2h respectively, and calculating the quercetin retention rate. The Zein nanoparticle dispersion (Zein-Que NPs) loaded with quercetin and the unencapsulated quercetin ethanol solution were used as controls.
Retention of quercetin = quercetin content in nanoparticles treated for a specific time/quercetin content in nanoparticles before treatment
TABLE 3 light stability test results
As can be seen from table 3 above, the ternary composite nanoparticle dispersions prepared in example 1, example 2 and example 3 were excellent in photostability and did not significantly decrease in drug encapsulation efficiency.
And Zein-Que NPs loaded with quercetin, the medicine encapsulation efficiency is reduced to a certain extent along with the extension of illumination time, and the retention rate of the medicine is 82% after illumination for 2 hours. The unencapsulated quercetin ethanol solution has the worst light stability, the medicine encapsulation efficiency is obviously reduced, and the medicine retention rate is only 61% after illumination for 2 hours.
Example 7
Storage stability test
Taking the quercetin-loaded ternary nanoparticle dispersion liquid prepared in the example 1, storing for one month at 4 ℃, sampling and detecting the particle size and the content of quercetin in the dispersion liquid, and calculating the quercetin retention rate. The particle size is increased from 172nm to 185nm, and the quercetin retention rate is 91%.
Taking the quercetin-loaded ternary nanoparticle dispersion liquid prepared in example 2, storing for one month at 4 ℃, sampling and detecting the particle size and the content of quercetin in the dispersion liquid, and calculating the quercetin retention rate. The particle size is increased from 181nm to 188nm, and the quercetin retention rate is 93%.
Taking the quercetin-loaded ternary nanoparticle dispersion liquid prepared in example 3, storing for one month at 4 ℃, sampling and detecting the particle size and the content of quercetin in the dispersion liquid, and calculating the quercetin retention rate. The particle size is increased from 186nm to 201nm, and the quercetin retention rate is 94%.
Taking Zein-Que NPs loaded with quercetin as a control, the particle size is increased from 90nm to 140nm, and the quercetin retention rate is 72%.
Example 8
DPPH free radical scavenging Activity experiment
4ML of the sample was mixed well with 4mL of DPPH solution (0.1 mM,95% ethanol), and the mixture was incubated at room temperature for 30 minutes in the absence of light. The absorbance of the mixture at 517nm was measured using an ultraviolet-visible spectrophotometer and the absorbance value was designated as a s. The absorbance of a mixture of 4mL H 2 O and 4mL DPPH was designated A c, and the absorbance of a mixture of 4mL sample and 4mL 95% ethanol was designated A 0. The DPPH radical scavenging activity was calculated as follows:
By analysis, the three-way quercetin-loaded nanoparticles prepared in example 1, example 2 and example 3 had DPPH radical scavenging rates of 91%, 93% and 92%, respectively, whereas the Zein-Que NPs-loaded Zein nanoparticle dispersion (Zein-Que NPs) had DPPH radical scavenging rates of 85% and the quercetin ethanol solution at the same concentration had DPPH radical scavenging rates of 83%.
Example 9
ABTS free radical scavenging Activity assay
ABTS aqueous solution (7.4 mM) and potassium persulfate solution (2.6 mM) were mixed in equal volumes, and the mixture was left to stand at room temperature in the dark for 14 hours. Then PBS (10 mM, pH 7.4) is used for dilution, so that the absorbance at 734nm is 0.70+/-0.02, and the ABTS working solution is obtained. 40. Mu.L of the sample was added to 4mL of freshly prepared ABTS working solution and the mixed solution was reacted for 5 minutes in the dark. The absorbance of the solution was measured at 734nm using an ultraviolet-visible spectrophotometer and the absorbance value was recorded as A s.Ac as a mixture of 40. Mu.L PBS (10 mM, pH 7.4) and 4mL ABTS working fluid at 734 nm. ABTS radical scavenging activity was calculated as follows:
By analysis, the ABTS free radical clearance of the quercetin-loaded ternary nanoparticles prepared in example 1, example 2 and example 3 was 80%, 88% and 82%, while the ABTS free radical clearance of the quercetin-loaded Zein nanoparticle dispersion (Zein-Que NPs) was 72% and the DPPH free radical clearance of the quercetin ethanol solution at the same concentration was 67%.
It can be seen from examples 8 and 9 that the antioxidant activity of the three-way zein/gelatin/carboxymethyl starch nanoparticle loaded with quercetin was higher than that of the zein nanoparticle loaded with quercetin, and also higher than that of the quercetin ethanol solution. Quercetin is a lipophilic natural active ingredient, has higher antioxidant activity, but has low free radical scavenging activity of a water system due to low solubility in water, and is reported in literature to be lower than 30％(Li H,et al.Fabrication of stable zein nanoparticles coated with soluble soybean polysaccharide for encapsulation of quercetin.Food Hydrocolloids,2019,87,342-351)., so that the antioxidant performance of the fat-soluble active ingredient in an aqueous solution can be remarkably improved through a nanoparticle drug-carrying system.
Example 10
In vitro simulated gastrointestinal digestion experiment
An in vitro Simulated Gastric Fluid (SGF) and an in vitro Simulated Intestinal Fluid (SIF) were formulated separately. The specific formula is as follows: in vitro simulated gastric fluid: 2g/L sodium chloride, 3.2g/L pepsin and hydrochloric acid to adjust the pH to 1.2. In vitro simulated intestinal juice: 1.2mM calcium chloride, 15mM sodium chloride, 4.76g/L pancreatin, 5.16g/L bile salt, sodium hydroxide (0.1M) to adjust the pH to 7.0.
25ML of a quercetin-loaded nanoparticle dispersion (example 1, example 2) was mixed well with the same volume of simulated gastric fluid, the pH was adjusted to 2.0, and then incubated for 2h in a shaker at 37 ℃. Thereafter, the pH of the system was adjusted to 7.4, mixed with 50mL of simulated intestinal fluid, and incubated for another 4 hours. During the whole incubation process, at a certain time point, a proper amount of sample solution is taken out, and the same volume of simulated gastric fluid or simulated intestinal fluid is supplemented. The release rate was calculated by measuring the quercetin concentration in the samples at different time points.
The biological availability is calculated according to the following formula:
C represents the quercetin content in the intestinal digestive juice after simulated gastrointestinal digestion, and C o represents the quercetin content in the nanoparticle dispersion (or free quercetin sample) before simulated gastrointestinal digestion.
In FIG. 3, que is quercetin ethanol solution and Zein-Que is Zein-loaded Zein nanoparticle. It can be seen from fig. 3 that after nanoparticle encapsulation, the content of quercetin in gastrointestinal tract digestive juice continuously increases, the ternary nanoparticle systems of example 1 and example 2 release higher amounts than zein-loaded nanoparticles, while the quercetin ethanol solution release is very low, probably due to poor solubility of quercetin Pi Sushui, precipitate formation, difficulty in detection in digestive juice, and possible decomposition damage due to poor stability of quercetin in simulated gastrointestinal tract digestive juice, resulting in a lower content of quercetin in simulated digestive juice. The nanoparticle coating can well protect the quercetin and increase the water solubility of the quercetin, so that the effect of improving the bioavailability is achieved. The bioavailability was calculated based on the release in simulated gastrointestinal tract, examples 1 and 2 were 57.1% and 52.1% for the quercetin zein loaded nanoparticles, 42.8% for the quercetin ethanol solution, and only 24.8%.
Example 11
To the quercetin-loaded gelatin-carboxymethyl starch double-modified zein nanoparticle dispersion obtained in example 1, 0.2mL of a calcium chloride solution of a certain concentration was added under stirring, so that the final calcium ion concentration of the mixed system was 0.5mM, and stirring was continued for 1 hour. Centrifuging at 3000rpm for 10min to remove large particles, and obtaining the calcium ion modified drug-loaded composite nano particles.
The analysis and detection show that the particle size is 264nm, and the encapsulation rate of quercetin is 92%.
Stability investigation
Thermal stability: samples prepared in this example were taken, incubated in a water bath at 80℃for 0.5 hours, and then cooled to room temperature. The particle size of the sample and the retention of quercetin were measured. The particle size is kept unchanged, and the retention rate of quercetin reaches 99%.
Light stability: the sample prepared in this example was taken and placed under an ultraviolet lamp (363 nm, 30W) and irradiated for 2 hours. The retention rate of quercetin reaches 97%.
Storage stability: taking the sample prepared in the embodiment, storing for one month at 4 ℃, sampling and detecting the particle size and the content of quercetin in the sample, and calculating the quercetin retention rate. The particle size is 272nm, and the retention rate of quercetin is 94%.
Salt stability: different amounts of NaCl solids were added directly to the samples prepared in this example, magnetically stirred (500 rpm) for 5 minutes to give NaCl concentrations in the solution of 0-40mM. After all samples were left at 25℃for 24 hours, the change in particle size of the nanoparticles in the solution was determined.
When the NaCl concentration is from 10mM to 80mM, the calcium ion modified drug-loaded composite nanoparticle system prepared in the embodiment can keep good stability, no precipitation occurs, and the particle size is only slightly increased. At a NaCl concentration of 80mM, the particle size was 283nm. In contrast, the quercetin-loaded gelatin-carboxymethyl starch double-modified zein nanoparticle dispersion obtained in example 1 had a 50% increase in particle size at 30mM NaCl concentration; at a NaCl concentration of 50mM, the system became very turbid and precipitation occurred, probably due to NaCl breaking electrostatic repulsion between nanoparticles, resulting in instability of the solution. The results show that the introduction of Ca 2+ improves the ionic stability of the composite nanoparticle.
Sustained release experiment:
An in vitro simulated gastric fluid and intestinal fluid were prepared as in example 10, 25mL of freshly prepared dispersion was mixed with 25mL of simulated gastric fluid and the pH of the mixed solution was adjusted to 1.5. The mixed solution was then incubated in a shaker at 37℃and shaken at 120rpm for 120 minutes. The above solution was mixed with 50mL of simulated intestinal fluid, the pH was adjusted to 7.4 and incubation continued for 240 minutes. Throughout the simulated digestion experiments, samples were taken every 30 minutes and supplemented with the corresponding simulated digests. The sample solution thus taken out was centrifuged at 12000rpm for 10 minutes, and then filtered with a microporous membrane of 0.45 μm to obtain a supernatant, and the released amount of quercetin was determined as described in example 1.
As shown in fig. 4, the nanoparticle dispersion prepared by adding Ca 2+ can slow the release rate of quercetin in the simulated stomach and intestine, which indicates that the addition of Ca 2+ may change the structure of the composite nanoparticle, and form a gel-like structure on the surface, thereby enhancing the resistance to simulated gastrointestinal fluid and improving the slow release effect.
Claims (8)
1. A quercetin-loaded protein-polysaccharide ternary composite nanoparticle is characterized in that: the composite nanoparticle with a core-shell structure for delivering the quercetin is constructed by taking the zein-coated quercetin as a core and assembling gelatin and carboxymethyl starch into a shell.
2. A preparation method of a quercetin-loaded protein-polysaccharide ternary composite nanoparticle is characterized by comprising the following steps of: the method specifically comprises the following steps:
(1) Preparation of ethanol solution of quercetin-zein
0.6 G zein was dissolved in 30 mL 80% aqueous ethanol, then pressed against quercetin: adding quercetin into zein the mass ratio of 1:5-1:30, and stirring thoroughly to dissolve to obtain quercetin-zein ethanol solution;
(2) Preparation of gelatin solution
Dissolving a certain amount of gelatin in 20mL of deionized water, stirring at 40 ℃ for 0.5 h, and then stirring at room temperature for 0.5 h to prepare gelatin solution with a certain concentration, wherein the mass of gelatin is 12.5mg-100mg;
(3) Preparation of quercetin-loaded gelatin-modified zein nanoparticle dispersion liquid
Taking 5mL of the ethanol solution of quercetin-zein obtained in the step (1), slowly dripping the solution into 20mL of the gelatin solution obtained in the step (2), and stirring at a speed of 400-1000 rpm for 10-60min;
(4) Preparation of carboxymethyl starch solution
Dissolving a certain amount of carboxymethyl starch in 25mL of deionized water, and stirring overnight to prepare carboxymethyl starch solution with a certain concentration, wherein the mass of carboxymethyl starch is 25mg-200mg;
(5) Preparation of quercetin-loaded gelatin-carboxymethyl starch double-modified zein nanoparticle dispersion liquid
Slowly dripping the quercetin-loaded gelatin-modified zein nanoparticle dispersion liquid obtained in the step (3) into the carboxymethyl starch solution obtained in the step (4), stirring at a speed of 400-1000 rpm for 30-120min, removing ethanol under reduced pressure on a rotary evaporator, and supplementing the volume to 50mL by deionized water to obtain the quercetin-loaded gelatin-carboxymethyl starch dual-modified zein nanoparticle dispersion liquid.
3. The method for preparing the quercetin-loaded protein-polysaccharide ternary composite nanoparticle, according to claim 2, is characterized in that: and (3) carrying out freeze drying on the dispersion liquid of the quercetin-loaded gelatin-carboxymethyl starch double-modified zein nano particles obtained in the step (5) to obtain the quercetin-loaded protein-polysaccharide ternary composite nano particles.
4. The method for preparing the quercetin-loaded protein-polysaccharide ternary composite nanoparticle, according to claim 2, is characterized in that: the mass ratio of the quercetin to the zein in the step (1) is 1:5-1:20; the mass of the gelatin in the step (2) is 20mg-50mg; the mass of the carboxymethyl starch in the step (4) is 50mg-150mg.
5. The method for preparing the quercetin-loaded protein-polysaccharide ternary composite nanoparticle, according to claim 2, is characterized in that: the stirring speed in the step (3) is 500-600 rpm, and the stirring time is 10-30min; the stirring speed in the step (5) is 500-800 rpm, and the stirring time is 30-60min.
6. A quercetin-loaded protein-polysaccharide ternary composite nanoparticle is used for preparing antioxidant drugs and functional foods.
7. A preparation method of a calcium ion modified drug-loaded composite nanoparticle is characterized by comprising the following steps: stirring the quercetin-loaded gelatin-carboxymethyl starch double-modified zein nanoparticle dispersion liquid, adding 0.1-1 mL of calcium chloride solution while stirring, so that the final calcium ion concentration of the mixed system is 0.15 mM-1.5 mM, and continuing stirring for 0.5-2 hours; centrifuging at 3000 rpm for 10 min to remove large particles, and obtaining the calcium ion modified drug-loaded composite nanoparticle.
8. The application of the calcium ion modified drug-loaded composite nano-particles in preparing antioxidant drugs and functional foods.
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