A flavonoid compound-epipremnum pinnatum binary complex nanomicelle and a preparation method thereof
By using the self-assembly of physalis protein to form flavonoid-physalis protein binary composite nanomicelles, the problems of poor water solubility and low stability of flavonoids are solved, achieving highly efficient solubility and improved stability, making it suitable for the food and pharmaceutical fields.
Patent Information
- Application Number
- CN202311057302.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-08-22
- Publication Date
- 2026-03-10
- Estimated Expiration
- 2043-08-22
AI Technical Summary
Flavonoids suffer from poor water solubility, easy degradation, and low stability, which limits their efficient use in the food and pharmaceutical fields.
Using phytoesophageal protein as the encapsulating wall material, a binary composite nanomicelle of flavonoids and phytoesophageal protein was formed through a self-assembly method. The flavonoids were then encapsulated in the hydrophobic cavity of the phytoesophageal protein micelles by hydrogen bonding, van der Waals forces, hydrophobic interactions, and electrostatic interactions, forming a core-shell structured composite nanomicelle.
It improves the solubility, stability, and bioavailability of flavonoids, and the preparation process is simple and suitable for industrial production.
Smart Images
Figure CN116966146B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of protein deep processing, and particularly relates to a flavonoid compound-leafy grass protein binary compound nanomicelle and a preparation method thereof. BACKGROUND
[0002] The flavonoid compound structure is often connected with phenolic hydroxyl, methoxyl, methyl, isopentenyl and other functional groups. In addition, it is often combined with sugar to form glycosides. At present, more than 10,000 kinds of flavonoid compounds are known, which can be divided into six categories of flavones, dihydroflavones, isoflavones, flavonols, flavanols and anthocyanins according to the different structures. Flavonoids are the main effective components of many medicinal plants, for example, baicalin and baicalein in Scutellaria baicalensis Georgi are flavone and flavonol compounds, and catechin in Camellia sinensis is a flavanol compound. In plants, flavones are mostly combined with sugar to exist in the form of flavonoid glycosides, and a small part exists in the free state. Modern pharmacological studies have shown that flavonoids have many pharmacological activities such as anticancer, antioxidant, anti-inflammatory, anti-atherosclerosis and antitumor, and have wide application prospects in the fields of drug development and food health care.
[0003] Moreover, with the continuous enhancement of people's nutrition and health consciousness, the demand for food-derived functional factors is increasing. Flavonoids are attracting more and more attention due to their antioxidant, antibacterial, anti-inflammatory, cardiovascular protection and other health benefits.
[0004] However, most flavonoids have problems such as poor water solubility, easy degradation and low stability, which seriously limit their efficient use in the fields of food and medicine. SUMMARY
[0005] The purpose of the present application is to provide a flavonoid compound-leafy grass protein binary compound nanomicelle and a preparation method thereof. The flavonoid compound-leafy grass protein binary compound nanomicelle prepared by the present application has small particle size, good dispersibility, good biocompatibility, high stability and good encapsulation effect, and can improve the solubility, stability and bioavailability of flavonoids.
[0006] In order to achieve the above purpose, the present application provides the following technical scheme:
[0007] The present application provides a preparation method of a flavonoid compound-leafy grass protein binary compound nanomicelle, comprising the following steps:
[0008] The flavonoid compound-leafy grass protein binary compound nanomicelle is obtained by mixing the leafy grass protein solution and the ethanol solution of the flavonoid compound for self-assembly.
[0009] Preferably, the leafwort protein solution comprises leafwort protein and a solvent; the molecular weight of the leafwort protein is 44-46 kDa; and the purity of the leafwort protein is ≥80%.
[0010] Preferably, the solvent is water or a phosphate buffer solution, and the pH value of the phosphate buffer solution is 7.8 to 8.2;
[0011] The mass concentration of phytoestrogen in the phytoestrogen solution is 2.5–5 mg / mL.
[0012] Preferably, the flavonoids include one of galangin, kaempferol, quercetin, and myricetin.
[0013] Preferably, the mass concentration of the flavonoids in the ethanol solution of the flavonoids is 6.9–10.5 mg / mL.
[0014] Preferably, the mass ratio of physalis protein to flavonoids in the physalis protein solution is (8-10):1.
[0015] Preferably, the self-assembly temperature is 20-25°C and the time is 30-60 min; the self-assembly is carried out under stirring conditions, and the stirring speed is 300-500 rpm.
[0016] Preferably, the self-assembly yields a nanomicelle base solution; the self-assembly further includes: centrifuging the nanomicelle base solution to obtain a flavonoid-leafwort protein binary composite nanomicelle; the centrifugation speed is 3000-5000 r / min; the time is 5-10 min; and the temperature is 0-4℃.
[0017] The present invention provides a flavonoid-leafwort protein binary composite nanomicelle prepared by the preparation method described above, comprising leafwort protein nanomicelles and flavonoid compounds loaded in the leafwort protein nanomicelles.
[0018] Preferably, the particle size of the flavonoid-grass protein binary composite nanomicelles is 320-480 nm; the loading of flavonoids is 5.5-11.2%.
[0019] This invention provides a method for preparing flavonoid-leafwort protein binary composite nanomicelles, comprising the following steps: mixing a leafwort protein solution and an alcoholic solution of flavonoids for self-assembly to obtain flavonoid-leafwort protein binary composite nanomicelles. The preparation method provided by this invention uses leafwort protein as the encapsulating wall material. During the self-assembly of leafwort protein in solution to form leafwort protein micelles, flavonoids are encapsulated in the hydrophobic cavities of the leafwort protein micelles through hydrogen bonds, van der Waals forces, hydrophobic interactions, and electrostatic interactions. The loaded nanomicelles formed by leafwort protein have small particle sizes and high stability, while exhibiting high encapsulation efficiency and loading capacity for flavonoids. This invention, by loading flavonoids into leafwort protein nanomicelles, forms a binary composite with a core-shell structure. The resulting composite micelles have small particle sizes, relatively stable solutions, and high loading efficiency, which can significantly improve the solubility, stability, and bioavailability of flavonoids. Meanwhile, the preparation method provided by this invention is simple and suitable for industrial production.
[0020] This invention provides flavonoid-leafwort protein binary composite nanomicelles prepared by the preparation method described above, comprising leafwort protein nanomicelles and flavonoid compounds loaded within the leafwort protein nanomicelles. The flavonoid-leafwort protein binary composite nanomicelles prepared by this invention have small particle size, good dispersibility, good biocompatibility, high stability, and good encapsulation effect, which can significantly improve the solubility, stability, and bioavailability of flavonoid compounds. Attached Figure Description
[0021] Figure 1 Transmission electron microscopy images of the flavonoid-leafwort protein binary composite nanomicelles prepared in Examples 1-4;
[0022] Figure 2 Comparative images of the flavonoid-leafwort protein binary composite nanomicelles prepared in Examples 1-4 and the aqueous solution of the flavonoid compounds.
[0023] Figure 3 The encapsulation efficiency and loading of the flavonoid-leafwort protein binary composite nanomicelles prepared in Examples 1-4;
[0024] Figure 4 The figures show a comparison of the particle sizes of the flavonoid-leafwort protein binary composite nanomicelles prepared in Examples 1-4. Detailed Implementation
[0025] This invention provides a method for preparing binary composite nanomicelles of flavonoids and physalis protein, comprising the following steps:
[0026] By mixing a solution of physalis protein and an ethanol solution of flavonoids, self-assembly was carried out to obtain binary composite nanomicelles of flavonoids and physalis protein.
[0027] In this invention, unless otherwise specified, all raw materials / components used in the preparation are commercially available products well known to those skilled in the art.
[0028] In this invention, the leafwort protein solution comprises leafwort protein and a solvent. Leafwort protein is used as the coating material in this invention. The leafwort protein is a plant protein, specifically a hydrophilic plant protein. Plant proteins have the advantages of being low in sugar, low in fat, low in cholesterol, and high in fiber. The leafwort protein is derived from leafwort. Leafwort has the advantages of being easy to cultivate, widely distributed, high-yielding, and highly adaptable to the environment, and its protein content is as high as 36%. In this invention, the molecular weight of the leafwort protein is preferably 44–46 kDa, more preferably 44.53 kDa; the isoelectric point is preferably 4.0; and the purity of the leafwort protein is preferably ≥80% (by mass), more preferably 82–85%. Using leafwort protein as the coating material in this invention enables the preparation of stable, small-particle-size nanomicelles. In this invention, when no flavonoids are loaded, the particle size of the leafwort protein nanomicelles is preferably 220–240 nm. The difference between the maximum and minimum particle sizes of the phytoesophageal protein nanomicelles is ≤10 nm. This invention utilizes phytoesophageal protein micelles as a carrier to prepare supported phytoesophageal protein nanomicelles that encapsulate flavonoids. The resulting supported nanomicelles have small particle sizes, good stability, and are non-toxic.
[0029] In this invention, the solvent is preferably water or a phosphate buffer solution, more preferably a phosphate buffer solution. The water is preferably ultrapure water. The pH value of the phosphate buffer solution is preferably 7.8–8.2, more preferably 8.
[0030] In this invention, the mass concentration of phytoestrogen in the phytoestrogen solution is preferably 2.5 to 5 mg / mL, and more preferably 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL or 5 mg / mL.
[0031] In this invention, the ethanol solution of the flavonoids preferably comprises flavonoids and ethanol. The flavonoids preferably include one of galangin, kaempferol, quercetin, and myricetin. The number of hydroxyl groups on the B ring of the flavonoids provided by this invention shows a gradually increasing trend, and the encapsulation efficiency and loading of the resulting flavonoid-leafwort protein binary composite nanomicelles also increase with the increase of the number of hydroxyl groups. In this invention, the ethanol is preferably anhydrous ethanol. The mass concentration of the flavonoids in the ethanol solution is preferably 6.9–10.5 mg / mL, specifically preferably 6.9 mg / mL, 7 mg / mL, 7.3 mg / mL, 10 mg / mL, or 10.5 mg / mL.
[0032] In this invention, the mass ratio of phytoestrogen and flavonoids in the phytoestrogen protein solution is preferably (8-10):1, more preferably 10:1.
[0033] In this invention, the self-assembly temperature is preferably 20–25°C, and the time is preferably 30–60 min, more preferably 30–50 min, and even more preferably 30–40 min. The self-assembly is carried out under stirring conditions, and the stirring speed is preferably 300–500 rpm, more preferably 300 rpm. The stirring is preferably carried out using magnetic stirring. In this invention, the self-assembly process is as follows: the phytoesophageal protein molecules self-assemble in the mixed solution to form phytoesophageal protein micelles. During this process, the phytoesophageal protein molecules encapsulate flavonoid compounds within the hydrophobic cavities of the phytoesophageal protein micelles through hydrogen bonds, van der Waals forces, hydrophobic interactions, and electrostatic interactions, forming supported nanomicelles.
[0034] In this invention, the self-assembly yields a nanomicelle base solution; preferably, the self-assembly process further includes: centrifuging the nanomicelle base solution to obtain a flavonoid-leafwort protein binary composite nanomicelle. The centrifugation speed is preferably 3000–5000 r / min, more preferably 3000 r / min; the centrifugation time is preferably 5–10 min, more preferably 5 min; and the temperature is preferably 0–4 °C, more preferably 4 °C.
[0035] The preparation method provided by this invention uses physalis protein and flavonoids as raw materials and employs a self-assembly method to prepare a physalis protein-flavonoid compound nanomicelle. The obtained nanomicelles are uniformly dispersed, have small particle size, high stability, and high loading efficiency; moreover, the preparation process is simple and has good biocompatibility.
[0036] The present invention provides a flavonoid-leafwort protein binary composite nanomicelle prepared by the preparation method described above, comprising leafwort protein nanomicelles and flavonoid compounds loaded in the leafwort protein nanomicelles.
[0037] In this invention, the particle size of the flavonoid-leafwort protein binary composite nanomicelles is 320–480 nm, preferably 320–470 nm. The encapsulation efficiency of the flavonoids by the leafwort protein micelles is preferably 49.5–99%, specifically 49.54%, 59.21%, 85.36%, or 98.95%. The loading (mass percentage) of the flavonoids in the flavonoid-leafwort protein binary composite nanomicelles is preferably 5.5–11.2%, more preferably 5.5–11%, specifically 5.55%, 6.63%, 9.56%, or 10.92%.
[0038] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0039] The measurement methods used in the following examples include:
[0040] Determination of the particle size of phytoesophageal protein-flavonoid nanomicelles: The particle size of phytoesophageal protein-flavonoid nanomicelles was determined using a dynamic light scattering instrument (ZSU3100, Malvern Instruments Ltd., UK).
[0041] Morphological observation of phytoesophageal protein-flavonoid nanomicelles: The morphology of phytoesophageal protein-flavonoid nanomicelles was observed using a transmission electron microscope (JEM-1400Plus, Nippon Electron Co., Ltd.).
[0042] Determination of loading rate of phytoesophageal protein-flavonoid nanomicelles: The loading rate of phytoesophageal protein-flavonoid nanomicelles was determined using a fully automated multi-functional microplate reader (HH3500, Pontyclum, UK).
[0043] Example 1
[0044] 1) Dissolve galangin in anhydrous ethanol to make the concentration of galangin 7 mg / mL;
[0045] 2) Accurately weigh 12.5 mg of galangin and dissolve it in 5 mL of pH 8.0 phosphate buffer. Quickly add 0.2 mL of galangin / anhydrous ethanol solution, stir magnetically for 30 min, and centrifuge at 3000 r / min for 5 min to remove unencapsulated galangin, thus obtaining galangin-galangin micelles (denoted as Gal-EDP). Figure 3As shown, the encapsulation efficiency of galangin by the leafwort protein nanomicelles was 49.54%, the loading was 5.55% (w / w), and the particle size was 350.6 nm.
[0046] Example 2
[0047] 1) Dissolve kaempferol in anhydrous ethanol to make the concentration of kaempferol 7 mg / mL;
[0048] 2) Accurately weigh 12.5 mg of kaempferol protein and dissolve it in 5 mL of pH 8.0 phosphate buffer. Quickly add 0.2 mL of kaempferol / anhydrous ethanol solution, stir magnetically for 30 min, and centrifuge at 3000 r / min for 5 min to remove unencapsulated kaempferol, thus obtaining kaempferol protein-kaempferol micelles (denoted as Kae-EDP); Figure 3 As shown, the encapsulation efficiency of kaempferol by the leafy grass protein nanomicelles was 59.21%, the loading was 6.63% (w / w), and the particle size was 445.2 nm.
[0049] Example 3
[0050] 1) Dissolve quercetin in anhydrous ethanol to make the quercetin concentration 7 mg / mL;
[0051] 2) Accurately weigh 12.5 mg of phytoesin and dissolve it in 5 mL of pH 8.0 phosphate buffer. Quickly add 0.2 mL of quercetin / anhydrous ethanol solution, stir magnetically for 30 min, and centrifuge at 3000 r / min for 5 min to remove unencapsulated quercetin, thus obtaining phytoesin-quercetin micelles (denoted as Que-EDP); Figure 3 As shown, the encapsulation efficiency of quercetin in the leafy green protein nanomicelles was 85.36%, the loading was 9.56% (w / w), and the particle size was 320.6 nm.
[0052] Example 4
[0053] 1) Dissolve myricetin in anhydrous ethanol to make the myricetin concentration 7 mg / mL;
[0054] 2) Accurately weigh 12.5 mg of myricetin and dissolve it in 5 mL of pH 8.0 phosphate buffer. Quickly add 0.2 mL of myricetin / anhydrous ethanol solution, stir magnetically for 30 min, and centrifuge at 3000 r / min for 5 min to remove unencapsulated myricetin, thus obtaining myricetin-myricetin micelles (denoted as Myr-EDP). Figure 3 As shown, the encapsulation efficiency of myricetin nanomicelles was 98.95%, the loading was 10.92% (w / w), and the particle size was 328.5 nm.
[0055] Comparative Example 1
[0056] 1) Dissolve quercetin in anhydrous ethanol to make the quercetin concentration 7 mg / mL;
[0057] 2) Accurately weigh 12.5 mg of phytoesin and dissolve it in 5 mL of pH 8.0 phosphate buffer. Quickly add 0.2 mL of quercetin / anhydrous ethanol solution, stir magnetically for 6 h, and centrifuge at 3000 r / min for 5 min to remove unencapsulated quercetin, thus obtaining phytoesin-quercetin micelles. The encapsulation rate of quercetin in the phytoesin nanomicelles was 39.74%, and the loading was 4.45% (mass).
[0058] Comparative Example 2
[0059] 50 mg each of whey protein isolate (WPI), mealworm protein (TMP), and yeast protein (YP) were dissolved in pure water to prepare protein solutions with a mass concentration of 2 mg / mL. After magnetic stirring for 2 hours, the solutions were placed in a 4°C refrigerator for overnight hydration. After returning to room temperature, the pH was adjusted to 7.0 with NaOH, and the solutions were heated to 85°C and sonicated at 40 kHz for 30 minutes to obtain denatured protein solutions. A 6 mg / mL myricetin solution was prepared using anhydrous ethanol as the solvent. The 6 mg / mL myricetin solution was added to the protein solution at a volume ratio of 1:20 to ensure thorough complexation between the protein and myricetin. The solutions were magnetically stirred in the dark for 6 hours to obtain WPI-myr, TMP-Myr, and YP-Myr hydrogels. The embedding efficiency and loading of the hydrogels prepared in Comparative Example 2 are listed in Table 1.
[0060] Table 1. Encapsulation efficiency and loading of WPI-myr, TMP-Myr, and YP-Myr hydrogels.
[0061] Product name Embedding rate (% w / w) Loading capacity (% w / w) WPI-myr 74.71 8.96 TMP-Myr 72.73 8.73 YP-Myr 62.44 7.49
[0062] As shown in Table 1, compared with whey protein isolate (WPI), yellow mealworm protein (TMP), and yeast protein (YP), the leafy grass protein (EDP) used in this invention has a higher encapsulation rate and loading of myricetin.
[0063] Figure 1 Transmission electron microscopy (TEM) images of the flavonoid-leafwort protein binary composite nanomicelles prepared in Examples 1-4, wherein... Figure 1 The top left image is a transmission electron microscope (TEM) image of the flavonoid-leafwort protein binary composite nanomicelles prepared in Example 1. Figure 1 The upper right image is a transmission electron microscope (TEM) image of the flavonoid-leafwort protein binary composite nanomicelles prepared in Example 2. Figure 1 The lower left image shows a transmission electron microscope (TEM) image of the flavonoid-leafwort protein binary composite nanomicelles prepared in Example 3. Figure 1The lower right image is a transmission electron microscope (TEM) image of the flavonoid-leafwort protein binary composite nanomicelles prepared in Example 4. Figure 1 It can be seen that the flavonoid-leafwort protein binary composite nanomicelles prepared in Examples 1-4 of the present invention have a spherical particle morphology and the particle size distribution of the nanoparticles in the micelles is 320-470 nm.
[0064] Figure 2 Comparative images of the flavonoid-leafwort protein binary composite nanomicelles prepared in Examples 1-4 and the aqueous solution of the flavonoid compounds. Figure 2 In the upper left figure, 1 is an aqueous solution of galangin, and 2 is an aqueous solution of galangin-loaded leafwort protein nanomicelles. Figure 2 In the upper right figure, 1 is an aqueous solution of kaempferol, and 2 is an aqueous solution of kaempferol-encapsulated loaded leafwort protein nanomicelles. Figure 2 In the lower left figure, 1 is an aqueous solution of quercetin, and 2 is an aqueous solution of quercetin-loaded leafy green protein nanomicelles. Figure 2 In the lower right figure, 1 represents an aqueous solution of myricetin, and 2 represents an aqueous solution of supported leafwort protein nanomicelles encapsulated with myricetin. Figure 2 It can be seen that flavonoids (galangin, kaempferol, quercetin, myricetin) are completely insoluble in aqueous solution, forming a large amount of precipitate; after being encapsulated by physalis protein nanomicelles, flavonoids are encapsulated in loaded physalis protein nanomicelles, which can be soluble in aqueous solvent systems. This proves that the physalis protein-flavonoid nanomicelles prepared in this invention can improve the solubility of flavonoids.
[0065] Figure 3 This is a bar chart comparing the encapsulation efficiency and loading of flavonoids in the binary composite nanomicelles of flavonoids and fern protein prepared in Examples 1-4. Figure 3 It can be seen that the flavonoids used in Examples 1 to 4 range from galangin, kaempferol, quercetin to myricetin, and the number of hydroxyl groups on the B ring shows a gradually increasing trend. The encapsulation efficiency and loading of the resulting flavonoid-leafwort protein binary composite nanomicelles also show an increasing trend.
[0066] Figure 4 The images show a size comparison of the flavonoid-leafwort protein binary composite nanomicelles prepared in Examples 1-4, and the leafwort protein nanomicelles. Figure 4 It can be seen that the particle size of the composite nanomicelles obtained after encapsulating flavonoids in Examples 1 to 4 is larger than that of the leafwort protein nanomicelles, with the Kae-EDP prepared in Example 3 having the largest particle size.
[0067] The analytical results of the physalis protein-flavonoid nanomicelles in Examples 1-4 above show that the average particle size of the physalis protein-flavonoid nanomicelles prepared in this invention is between 320 and 470 nm, and they exhibit good dispersibility. Furthermore, the solubility of flavonoids can be significantly improved after being encapsulated by physalis protein nanomicelles.
[0068] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.
Claims
1. A preparation method of flavonoids-phytochelatin binary complex nanomicelles, characterized in that, The method comprises the following steps: mixing a protein solution of the Malva sylvestris L and an ethanol solution of the flavonoids to perform self-assembly, to obtain a flavonoids-Malva sylvestris L protein binary compound nanomicelle; the flavonoids are myricetin; the self-assembly is performed for 30-60 min at 20-25℃; the mass ratio of the Malva sylvestris L protein to the flavonoids in the protein solution of the Malva sylvestris L is (8-10):1; the protein solution of the Malva sylvestris L comprises the Malva sylvestris L protein and a solvent, the solvent is a phosphate buffer solution, and the pH value of the phosphate buffer solution is 8.
0.
2. The production method according to claim 1, characterized by, the molecular weight of the Malva sylvestris L protein is 44-46 KDa, and the purity of the Malva sylvestris L protein is ≥80%.
3. The preparation method according to claim 2, characterized in that, the mass concentration of the Malva sylvestris L protein in the protein solution of the Malva sylvestris L is 2.5-5 mg / mL.
4. The method of claim 1, wherein, the mass concentration of the flavonoids in the ethanol solution of the flavonoids is 6.9-10.5 mg / mL.
5. The preparation method according to claim 1, characterized in that, the self-assembly is performed under stirring, and the stirring speed is 300-500 rpm.
6. The production method according to claim 1 or 5, characterized by, the self-assembly obtains a nanomicelle base solution; after the self-assembly, the nanomicelle base solution is further centrifuged to obtain the flavonoids-Malva sylvestris L protein binary compound nanomicelle, the centrifugation speed is 3000-5000 r / min, the centrifugation time is 5-10 min, and the centrifugation temperature is 0-4℃.
7. The flavonoid-epigallocatechin binary complex nanomicelle prepared by the method of any one of claims 1-6, characterized in that, the method comprises the following steps: 8.The flavonoids-phytomenadione binary complex nanomicelle according to claim 7, characterized in that, the flavonoids-Malva sylvestris L protein binary compound nanomicelle has a particle size of 320-480 nm, and the loading amount of the flavonoids is 5.5-11.2%.