Nanoparticles loaded with phycocyanin, preparation method and application thereof

Abstract

The present invention discloses a nanoparticle loaded with phycocyanin, a preparation method and application thereof, and belongs to the technical field of biological preparations. The nanoparticle loaded with phycocyanin of the present invention is a zein nanoparticle loaded with phycocyanin or a zein fucoidan nanoparticle loaded with phycocyanin. The present invention selects plant-derived zein as a carrier material to construct nanoparticles to deliver phycocyanin, and adds marine biopolysaccharide fucoidan to improve the stability of zein-based nanoparticles to prepare PC‑zein‑FU nanoparticles. The prepared PC‑zein‑FU nanoparticles have good anti-allergic activity, providing a theoretical basis for the development and practical application of phycocyanin production.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological preparations, and in particular relates to a nanoparticle loaded with phycocyanin, a preparation method and application thereof. Background Art

[0002] Phycocyanin exists in microalgae and has attracted more and more attention for its natural blue color and wide range of physiological functions. Its physiological functions such as anti-inflammatory, anti-allergic, anti-tumor, antioxidant, and immunomodulatory have been fully verified. However, purified phycocyanin is prone to precipitation or dissociation after being dissolved in water, and is highly sensitive to environmental factors such as temperature, pH value, and light. Direct oral administration can easily affect the functional activity of phycocyanin and reduce its bioavailability, thus limiting its application in actual product production. Summary of the invention

[0003] In view of the problems existing in the prior art, the object of the present invention is to provide a nanoparticle loaded with phycocyanin, a preparation method and application thereof.

[0004] In order to achieve the above object, the present invention adopts the following technical solution:

[0005] A phycocyanin-loaded nanoparticle, wherein the nanoparticle is a phycocyanin-loaded zein nanoparticle or a phycocyanin-loaded zein-fucoidan nanoparticle.

[0006] In a specific embodiment, the nanoparticles are zein nanoparticles loaded with phycocyanin, and the preparation method is as follows:

[0007] Phycocyanin and zein are dissolved in a 75% ethanol aqueous solution, ultrapure water is added, stirred, ethanol and insoluble matter are removed, and a zein nanoparticle dispersion system loaded with phycocyanin is obtained. After freeze drying, zein nanoparticles loaded with phycocyanin are obtained.

[0008] In a specific embodiment, the concentration ratio of phycocyanin to Zein is 1:5.

[0009] In a specific embodiment, the phycocyanin-loaded zein-fucoidan nanoparticles are prepared as follows:

[0010] Phycocyanin and zein are dissolved in a 75% ethanol aqueous solution, added to a fucoidan solution, stirred, and the ethanol and insoluble matter are removed to obtain a zein-fucoidan composite colloidal dispersion system. After freeze-drying, zein-fucoidan nanoparticles loaded with phycocyanin are obtained.

[0011] In a specific embodiment, the mass ratio of zein to fucoidan is 20:1 to 1:2, preferably 10:1 to 1:2, and more preferably 1:1.

[0012] In a specific embodiment, the concentration ratio of phycocyanin to Zein is 1:5.

[0013] Application of the above-mentioned phycocyanin-loaded nanoparticles in the preparation of drugs or preparations with anti-allergic activity.

[0014] Advantages of the technical solution of the present invention

[0015] The present invention uses a nanoscale carrier system to encapsulate and deliver phycocyanin, greatly improving the water dispersibility, chemical stability, release characteristics and biological efficacy of the compound. Specifically, plant-derived corn zein (zein) is selected as a carrier material to construct nanoparticles to deliver phycocyanin, and marine biopolysaccharide fucoidan is added to improve the stability of zein-based nanoparticles to prepare PC-zein-FU nanoparticles. The prepared PC-zein-FU nanoparticles have good anti-allergic activity, providing a theoretical basis for the development and practical application of phycocyanin production. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Scanning electron microscopy images of PC-zein and PC-zein-FU nanoparticles (A is PC-zein and B is PC-zein-FU);

[0017] Figure 2 Fluorescence detection results of composite nanoparticles;

[0018] Figure 3 Circular dichroism test results of composite nanoparticles;

[0019] Figure 4 Effects of phycocyanin on specific IgE, IgG, IgG1, histamine and mMCP-1 in mice;

[0020] Figure 5 Effects of phycocyanin on IL-4, IL-3, and IFN-γ levels in mouse spleen cells;

[0021] Figure 6 Mouse duodenum tissue sections (A: PBS group; B: TM group; C: phycocyanin group; D: PC-zein group; E: PC-zein-FU group). DETAILED DESCRIPTION

[0022] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art.

[0023] The present invention is further described in detail below in conjunction with specific examples and with reference to data. The following examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0024] In the following examples, phycocyanin was sourced from Xi'an Musen Bioengineering Co., Ltd.; zein was sourced from Shanghai Xingye Biotechnology Co., Ltd.; and fucoidan was sourced from Henan Sanhua Biotechnology Co., Ltd.

[0025] Example 1

[0026] A Zein nanoparticle loaded with phycocyanin, the preparation method is as follows:

[0027] Weigh a certain amount of phycocyanin (PC) and dissolve it in 75% ethanol aqueous solution to prepare a phycocyanin stock solution with a concentration of 1mmol / L. Weigh a certain amount of zein and dissolve it in 75% ethanol solution to prepare a zein stock solution with a concentration of 200mmol / L. Take 200μL of phycocyanin stock solution and mix it with 5mL zein stock solution, and dilute it to 100mL with 75% ethanol solution. The zein concentration is 10μmol / L and the phycocyanin concentration is 2μmol / L. Take 20mL of phycocyanin-zein (PC-zein) mixture and slowly add it to 60mL ultrapure water, and stir it continuously at a speed of 1000g / min for 30min. And use a rotary evaporator to remove ethanol in the system. Then centrifuge at low speed to remove insoluble matter, and obtain a zein nanoparticle dispersion system loaded with phycocyanin, which is stored at 4℃ for use. Another part of the dispersion system was freeze-dried to obtain zein nanoparticles loaded with phycocyanin (PC-zein).

[0028] Example 2

[0029] A zein-fucoidan nanoparticle loaded with phycocyanin, the preparation method is as follows:

[0030] 200 mg of phycocyanin and 1.0 g of zein were weighed and dissolved in 100 mL of 75% (v / v) ethanol solution. Different masses (5, 10, 50, 100, 200 mg) of fucoidan were weighed and dissolved in 40 mL of water. Under 1000 g magnetic stirring, 10 mL of the mixed solution of phycocyanin and zein was slowly added to 40 mL of fucoidan and stirred for 30 min. After that, the ethanol in the mixed system was removed by vacuum rotary evaporation. Finally, the sample was centrifuged at low speed (3000 g, 10 min) to remove possible large aggregates and undissolved substances to obtain a zein-fucoidan composite colloidal dispersion system. The final nanoparticle samples contained zein and fucoidan in mass ratios (zein / FU, w / w) of 20:1, 10:1, 2:1, 1:1, and 1:2, respectively, to obtain phycocyanin-loaded zein-fucoidan nanoparticles (PC-zein-FU).

[0031] Characterization of phycocyanin-loaded composite nanoparticles and determination of encapsulation efficiency

[0032] Characterization: The nanoparticle dispersions prepared in Example 1 and Example 2 were diluted 10 times with pure water, 1 mL of the sample was taken and placed in a sample cell, and the average particle size, particle size distribution, PDI and The parameters were set as follows: equilibrium time 45 s, detection angle 173°, and temperature 25°C.

[0033] Encapsulation efficiency: Take 0.5 mL of the nanoparticle dispersion prepared in Example 1 and Example 2, respectively, and mix them with 1.0 mL of ethyl acetate, vortex shake for 1 min, and then centrifuge to take the supernatant, and extract phycocyanin from the nanoparticles into ethyl acetate. Repeat this extraction step 3 times. Collect the supernatant, dry the collected supernatant with nitrogen, then dissolve it with acetonitrile, and use a high performance liquid chromatography system to detect the content of phycocyanin.

[0034] Encapsulation efficiency (%) = C1 / C2*100%

[0035] C1 is the phycocyanin content extracted and detected from the nanoparticle dispersion; C2 is the phycocyanin content added when preparing the sample.

[0036] The results are shown in Table 1.

[0037] Table 1 Average particle size, PDI, and Potential and encapsulation efficiency of phycocyanin

[0038]

[0039]

[0040] Note: The data in the table are mean ± standard deviation. Different superscript letters in the same column indicate statistically significant differences (p < 0.05)

[0041] As shown in Table 1, the average particle size of PC-zein nanoparticles is 126.3 nm, and the PDI is 0.28. The potential value is -53.3mV. Adding fucoidan (PC-zein-FU 20:1 ), the average particle size of the nanoparticles increased to 158.5nm. This is because less fucoidan was added, resulting in bridging flocculation and aggregation between particles. In addition, when the mass ratio of zein to fucoidan was 20:1, The potential value is relatively low, providing a small electrostatic repulsion, and zein and fucoidan form an insoluble electrostatic complex. As the fucoidan content continues to increase, the average particle size of PC-zein-FU nanoparticles decreases to 96.8 (PC-zein-FU 10:1 )、90.4(PC-zein-FU 2:1 )、83.6(PC-zein-FU 1:1 ) and 80.3(PC-zein-FU 1:2 ), which is because as the content of fucoidan increases, the surface charge of the complex increases, the electrostatic repulsion increases, and the particle size decreases. In addition, PDI increases from 0.23 (PC-zein-FU 10:1 ) is reduced to 0.19 (PC-zein-FU 1:2 ), PC-zein-FU nanoparticles Potential from -47.2mV (PC-zein-FU 10:1 ) to -22.5mV(PC-zein-FU 1:2 ). The above results show that with the increase of fucoidan, PC-zein-FU nanoparticles gradually become a more uniform colloidal system, and these nanoparticle systems have sufficient electrostatic repulsion to resist nanoparticle aggregation. In addition, without the addition of fucoidan, the encapsulation efficiency of phycocyanin in zein nanoparticles was 86.9%. When the mass ratio of zein and fucoidan was 20:1, the encapsulation efficiency of phycocyanin in the composite nanoparticles decreased to 69.6%. This phenomenon indicates that when the mass ratio of zein and fucoidan was 20:1, the composite nanoparticles tended to aggregate and precipitate during the preparation and encapsulation of phycocyanin. When the fucoidan content increased, the encapsulation efficiency of phycocyanin in the composite nanoparticles was significantly higher than that in zein nanoparticles, and PC-zein-FU 1:2The composite nanoparticles showed the highest phycocyanin encapsulation efficiency of 97.1%. The above results showed that the presence of fucoidan can significantly improve the encapsulation efficiency of phycocyanin in zein nanoparticles, and PC-zein-FU nanoparticles have excellent encapsulation properties.

[0042] Scanning electron microscopy observation of the microstructure of phycocyanin-loaded composite nanoparticles

[0043] 5 mL of fresh PC-Zein and PC-Zein-FU nanoparticle dispersion prepared in Example 1 and Example 2 was coated on a silicon wafer and dried naturally at room temperature. The silicon wafer was fixed on a sample plate with conductive glue, and the sample was sprayed with gold. The scanning electron microscope was used for observation and photographing at an accelerating voltage of 3.0 kV.

[0044] The surface morphology and microstructure of nanoparticles observed by scanning electron microscopy Figure 1 As shown. Both PC-zein and PC-zein-FU (1:1) nanoparticle samples present a typical spherical nanoparticle structure. However, the addition of fucoidan causes differences in the microscopic morphology, size and distribution uniformity of the particle surface. The particle size distribution of PC-zein nanoparticles is relatively uniform, but these nanoparticles are tightly connected to each other to form an adhesion state. This is mainly due to the strong hydrophobic attraction between zein nanoparticles, which leads to the phenomenon of particle water transport and aggregation during the drying process. However, the surface of PC-zein-FU nanoparticles is relatively smooth. The main reason is that the addition of fucoidan improves the surface hydrophobicity of zein particles, increases the surface hydrophilicity of nanoparticles, and thus reduces the aggregation of particles due to hydrophobic effects.

[0045] Mechanism of action of fucoidan and zein in composite nanoparticles

[0046] The fluorescence of the tyrosine group in the protein is very sensitive to its surrounding microscopic environment. The addition of polysaccharides can change the conformation of the protein and thus affect the fluorescence intensity. Therefore, the fluorescence value of the tyrosine group can better reflect the role of fucoidan and zein in the composite nanoparticles. 20:1 、PC-zein-FU 10:1 、PC-zein-FU 2:1 、PC-zein-FU 1:1 、PC-zein-FU 1:2 The fluorescence spectrum of Figure 2As shown: After excitation at 285nm, the fluorescence spectrum of zein nanoparticles is between 290-400nm, and the peak is around 333nm, which is mainly due to the presence of tyrosine in zein. After binding with fucoidan, the intrinsic fluorescence intensity of zein nanoparticles decreases. When the mass ratio of zein to fucoidan is 20:1, the fluorescence intensity is lower than that of 10:1. This may be due to the aggregation of composite nanoparticles when the mass ratio of zein to fucoidan is 20:1, which causes the tyrosine in zein to be shielded and the fluorescence intensity to decrease. In addition, as the content ratio of zein to fucoidan changes from 10:1 to 1:2, the intrinsic fluorescence intensity of zein nanoparticles gradually decreases. This indicates that fucoidan quenches the intrinsic fluorescence of zein, which may be because the negatively charged fucoidan binds to the positively charged tyrosine residues through electrostatic attraction. As the mass of fucoidan increases, more tyrosine residues in zein are consumed.

[0047] PC-zein, PC-zein-FU 20:1 、PC-zein-FU 10:1 、PC-zein-FU 2:1 、PC-zein-FU 1:1 、PC-zein-FU 1:2 The circular dichroism spectrum of Figure 3 As shown, it is used to study the conformation and secondary structure of proteins. The circular dichroism spectrum of zein nanoparticles shows two negative peaks at 208nm and 223nm. After adding fucoidan, the secondary structure of zein nanoparticles changes, which is due to the intermolecular interaction between zein and fucoidan. When the mass ratio of Zein to fucoidan is 20:1, the a-helical structure decreases, which may be due to the slight aggregation between the composite nanoparticles. As the fucoidan content continues to increase, the a-helical structure is enhanced, probably because the nanoparticles become compact when the ratio of zein to fucoidan is 10:1 to 1:2. The change in the secondary structure of Zein particles in the presence of fucoidan indicates that intermolecular interactions occur between zein and fucoidan.

[0048] Study on the anti-allergic effect of PC-zein and PC-zein-FU (1:1) nanoparticles

[0049] The following anti-allergic effect study was conducted using PC-zein-FU (1:1) nanoparticles constructed with a mass ratio of zein to fucoidan of 1:1, because when the amount of fucoidan was increased, the encapsulation rate of phycocyanin did not increase much.

[0050] Balb / c mice to establish food allergy model:

[0051] 6-8 weeks old SPF female Balb / c mice were used as the research subjects. The mice were sensitized with shrimp allergen tropomyosin to establish a mouse allergy model. The mice were divided into PBS negative control group, tropomyosin positive control group, phycocyanin group, zein+phycocyanin group, zein+fucoidan+phycocyanin group (PC-zein-FU 1:1 ), 8 mice in each group. Tropomyosin positive control group and each test group were injected with tropomyosin and alum adjuvant mixed solution (tropomyosin 10mg / ) on days 0, 7, 14, and 21, and PBS negative control group mice were injected with PBS and alum adjuvant mixed solution as negative control. Each test group was gavaged three times a week from the week before sensitization, phycocyanin, zein+phycocyanin, zein+fucoidan+phycocyanin, and gavage volume 10mg / . From the 28th day, the PBS negative control group, tropomyosin positive control group, phycocyanin group, zein+phycocyanin group, zein+fucoidan+phycocyanin group mice were gavaged. Tropomyosin positive control group, phycocyanin group, zein+phycocyanin group, zein+fucoidan+phycocyanin group were gavaged with 10mg tropomyosin (dissolved in 200mL PBS solution) every 2-3 days, and gavage was performed 10 times in total. The PBS negative control group was gavaged with 200 mL of PBS solution. On day 50, blood was collected from the eyeballs of mice, and duodenal tissue and spleen were obtained. The levels of tropomyosin-specific IgE, IgG1, IgG2, histamine, and mMCP-1 in the serum of mice in each group were detected, spleen cells were cultured and spleen cytokine levels were determined, and duodenal tissue was histologically evaluated.

[0052] The detection method of tropomyosin-specific IgE, IgG1, IgG2, histamine and mMCP-1 levels in mouse serum is as follows:

[0053] Serum samples were collected and the levels of IgE, IgG, IgG1 and IgG2 were determined by indirect ELISA. 100 mL of coating solution containing tropomyosin was added to each well of a 96-well plate and incubated at 4°C overnight. The plate was washed 3 times with PBST for 5 minutes each time. 200 mL of PBST containing 5% BSA was added to each well and incubated at 37°C for 2 hours. After washing the plate again, 100 mL of mouse serum from each group diluted by the same multiple (IgE diluted 5 times, IgG, IgG1 and IgG2 diluted 200 times) was added to each well and incubated at 37°C for 1 hour. After washing the plate again, HRP-labeled goat anti-mouse IgE (1000 times), IgG1 and IgG2 (5000 times) were added to each well and incubated at 37°C for 1 hour. After washing the plate again, 100 mL of TMB colorimetric solution was added to each well and incubated at 37°C for 15 minutes. Then add 50mL 2mol / L H2SO4 to terminate the reaction, and detect the absorbance at 450nm.

[0054] The content of histamine and mMCP-1 in mouse serum was determined using the corresponding detection kits, and the detection was carried out according to the kit detection instructions.

[0055] The results are as follows Figure 4 As shown: Compared with the tropomyosin (TM) group, phycocyanin (PC) group, phycocyanin-zein (PC-zein) group, and phycocyanin-zein-fucoidan (PC-zein-FU) group, the IgE / IgG / IgG1 antibody levels, histamine and mast cell protease-1 (mMCP-1) release were all decreased, but all higher than the blank group. Among them, the level of zein+fucoidan+phycocyanin group decreased the most. The decrease in histamine content indicates that phycocyanin nanoparticles can alleviate the allergic symptoms caused by tropomyosin. The decrease in mMCP-1 content indicates that the PC-zein-FU group reduces the degranulation reaction of mast cells.

[0056] The methods for spleen cell culture and cytokine analysis are as follows:

[0057] The spleens of mice in each experimental group (n=10) were collected and gently crushed with a syringe plunger, and the cell tissue was filtered through a 100 μm nylon filter to obtain a single cell suspension. The suspension was then transferred to a 15 mL tube and centrifuged at 1000 g for 5 minutes. The supernatant was then discarded, 5 mL of red blood cell lysis buffer was added to lyse the red blood cells, and 5 mL of sterile RPMI-1640 complete medium was added to stop the reaction after the reaction was completed. The reaction was centrifuged again, and the cells were washed twice with 10 mL of RPMI-1640 medium, suspended, and transferred to a 96-well plate at a density of 2×10 6 / well, and cultured at 37°C for 72 hours under (control group, TM, CO2). The levels of IFN-γ, IL-4, IL-5 and IL-13 were determined by ELISA kits.

[0058] The concentrations of Th2-related cytokines (IL-4 and IL-13) and Th1-related cytokines (IFN-γ) in the spleen of mice were detected to determine whether phycocyanin and the formed nanoparticles could affect the production of cytokines in the immune response. Figure 5 As shown. Compared with the TM group, the concentrations of IL-4 and IL-13 cytokines in the mouse epithelial cell supernatants of the PC, PC-zein, and PC-zein-FU groups decreased, while the concentration of IFN-γ increased. Interestingly, the concentrations of IL-4 and IL-13 cytokines in the mouse epithelial cell supernatants of the PC-zein-FU group decreased to the maximum, while the concentration of IFN-γ increased the most.

[0059] IFN-γ secreted by Th1-related cells can inhibit IgE production, while cytokines including IL-4 and IL-13 can stimulate Th2-related cells to produce IgE, which can induce systemic allergic reactions. The experimental results showed that the expression of Th2-related cytokines (IL-4 and IL-13) in mouse skin cells in the PC-zein-FU group was the most downregulated, and the expression of Th1-related cytokines (IFN-γ) was the most upregulated, indicating that compared with the phycocyanin group and the PC-zein group, PC-zein-FU nanoparticles can better maintain the stability of phycocyanin, achieve sustained-release delivery, and improve bioavailability, thereby more effectively regulating the Th1 / Th2 balance, inhibiting Th2 immune response and promoting Th1 immune response, thereby inhibiting shrimp tropomyosin allergic reactions.

[0060] The histological evaluation method of duodenal tissue was as follows: the duodenum was cut to approximately 4 cm in length, fixed with 10% formalin, processed with standard histological sectioning techniques (section thickness was 5 mm, stained with hematoxylin), and then observed and photographed under an optical microscope.

[0061] The results are as follows Figure 6 As shown, from Figure 6 It can be seen that the duodenal tissue of mice in the PBS group was normal without pathological phenomena; the duodenal tissue of the tropomyosin group showed obvious pathological phenomena; while the PC group, PC-zein group and PC-zein-FU group still had pathological phenomena, but the symptoms were significantly alleviated. Among them, the mice in the PC-zein-FU group had the mildest allergic symptoms.

[0062] In summary, the studies show that PC-zein-FU nanoparticles can maintain the stability of phycocyanin and improve the bioavailability of phycocyanin, thereby achieving good anti-allergic effects.

[0063] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. A phycocyanin-loaded nanoparticle, characterized in that: The nanoparticles are zein-fucoidan nanoparticles loaded with phycocyanin; The preparation method of the zein-fucoidan nanoparticles loaded with phycocyanin is as follows: Dissolving phycocyanin and zein in a 75% ethanol aqueous solution, adding the solution to the fucoidan, stirring, removing the ethanol and insoluble matter, obtaining a zein-fucoidan composite colloidal dispersion system, and freeze-drying to obtain zein-fucoidan nanoparticles loaded with phycocyanin; The mass ratio of zein to fucoidan is 1:1; The concentration ratio of phycocyanin to Zein is 1:

5.

2. Use of the phycocyanin-loaded nanoparticles according to claim 1 in the preparation of drugs or preparations with anti-allergic activity.

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