A porphyridium polysaccharide-astaxanthin non-covalent complex, and a preparation method and application thereof

Abstract

This invention provides a non-covalent complex of *Plasmodium purpureum* polysaccharide and astaxanthin, its preparation method, and its applications, relating to the fields of biomedicine and functional food technology. The complex is spontaneously formed by the non-covalent interaction between *Plasmodium purpureum* polysaccharide and astaxanthin; the non-covalent interaction consists of hydrogen bonds and hydrophobic interactions. This invention, for the first time, reveals the mechanism by which *Plasmodium purpureum* polysaccharide and astaxanthin form a non-covalent complex through hydrogen bonds and hydrophobic interactions using multiple techniques including infrared spectroscopy, ultraviolet spectroscopy, proton nuclear magnetic resonance spectroscopy, and transmission electron microscopy. Isothermal titration calorimetry results show that the two bind with high affinity at a near 1:1 stoichiometric ratio (K = 3.10 × 10⁻⁶). 5 M ‑1 The binding process is dominated by hydrogen bonds and can proceed spontaneously (ΔH<0, ΔS<0, ΔG<0). Cell experiments show that this complex can synergistically alleviate ox-LDL-induced oxidative stress and inflammatory responses in endothelial cells, and has broad application prospects in the preparation of anti-atherosclerotic drugs or functional foods.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and functional food technology, specifically to a non-covalent complex of *Plasmodium purpureum* polysaccharide and astaxanthin, its preparation method, and its application. Background Technology

[0002] Atherosclerosis is the main pathological basis of cardiovascular disease, and its occurrence and development are closely related to vascular endothelial damage, oxidative stress, and chronic inflammation. While commonly used statins have some efficacy, long-term use can lead to side effects such as liver damage and muscle toxicity. Therefore, finding safe and effective anti-atherosclerotic active substances from natural products has become a research hotspot.

[0003] Astaxanthin is a natural, fat-soluble carotenoid with extremely strong antioxidant activity, as well as various pharmacological activities such as anti-inflammatory, lipid-lowering, and anti-atherosclerotic effects. However, astaxanthin's extremely poor water solubility, low chemical stability, and low oral bioavailability severely limit its widespread application in the food and pharmaceutical fields.

[0004] *Porphyra yezoensis* is a marine single-celled red algae whose synthesized extracellular sulfated polysaccharides possess unique colloidal properties and various biological activities. *Porphyra yezoensis* polysaccharides contain a large number of sulfate and uronic acid groups, exhibiting excellent antiviral, antitumor, lipid-lowering, and hypoglycemic activities. While there are existing reports on the preparation methods and applications of *Porphyra yezoensis* polysaccharides, no research has been found on the formation of complexes between *Porphyra yezoensis* polysaccharides and astaxanthin through non-covalent interactions.

[0005] While there are reports of combining polysaccharides with astaxanthin in existing technologies, such as preparing astaxanthin microspheres using fucoidan, preparing astaxanthin formulations by encapsulating Haematococcus pluvialis powder with β-cyclodextrin, and using dextran and phenylboronic acid to modify astaxanthin to form nanocarriers for the treatment of atherosclerosis, these technologies mostly focus on dosage form development or chemical modification. They either require end-group modification of astaxanthin or encapsulation with encapsulation materials. Summary of the Invention

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution: a non-covalent complex of *Plasmodium purpureum* polysaccharide and astaxanthin, wherein the complex is spontaneously formed by the non-covalent interaction between *Plasmodium purpureum* polysaccharide and astaxanthin; The non-covalent interactions are hydrogen bonds and hydrophobic interactions.

[0007] Preferably, the number of binding sites n between the *Plasmodium purpureum* polysaccharide and astaxanthin is 1.16 ± 0.08, and the binding constant K is 3.10 × 10⁻⁶. 5 M -1The enthalpy change ΔH during the combination process is -36.23 kcal / mol, and the entropy change ΔS is -91.7 cal·mol. -1 ·K -1 The Gibbs free energy ΔG < 0 indicates that the bonding process is an exothermic reaction, dominated by hydrogen bonds, and can proceed spontaneously.

[0008] A method for preparing a non-covalent complex of *Plasmodium purpureum* polysaccharide and astaxanthin includes the following steps: (1) Dissolve the polysaccharide of *Porphyra yezoensis* in pure water or buffer solution to prepare a polysaccharide solution with a concentration of 0.5-5 mg / mL; (2) Dissolve astaxanthin in anhydrous ethanol to prepare astaxanthin stock solution; (3) Under stirring conditions, the astaxanthin mother liquor is added dropwise to the Porphyra yezoensis polysaccharide solution so that the final concentration of ethanol in the mixture is 1%-10% and the final concentration of astaxanthin is 0.01-2 mg / mL. The mixture is stirred at 15-30℃ in the dark for 10-60 minutes to obtain the Porphyra yezoensis polysaccharide-astaxanthin non-covalent complex solution.

[0009] Preferably, the concentration of the *Plasmodium purpureum* polysaccharide solution is 1-2 mg / mL.

[0010] Application of a non-covalent complex of *Plasmodium purpureum* polysaccharide and astaxanthin in anti-atherosclerotic drugs or functional foods.

[0011] This invention provides a non-covalent complex of *Plasmodium purpureus* polysaccharide and astaxanthin, its preparation method, and its application. It possesses the following beneficial effects: 1. The non-covalent binding mechanism between *Porphyra yezoensis* polysaccharides and astaxanthin was revealed for the first time. This was achieved through Fourier transform infrared spectroscopy (FTIR), ultraviolet-visible absorption spectroscopy (UV-Vis), and proton nuclear magnetic resonance spectroscopy (NMR). 1 Multiple techniques, including 1H NMR, transmission electron microscopy (TEM), pyrene fluorescence probe method, and isothermal titration calorimetry (ITC), have for the first time confirmed at the molecular level that the polysaccharide of *Porphyra yezoensis* and astaxanthin form a non-covalent complex through hydrogen bonding and hydrophobic interactions, rather than a simple physical mixture.

[0012] 2. The preparation method is simple and the conditions are mild. This invention only requires physical mixing of Porphyra yezoensis polysaccharide and astaxanthin under mild conditions. There is no need for chemical modification of astaxanthin, no need for high-energy equipment such as high-pressure homogenization, and no need for complicated encapsulation processes. The operation is simple, the cost is low, and it is easy to industrialize.

[0013] 3. The complex exhibits high affinity. ITC results show that the binding constant K between *Porphyra yezoensis* polysaccharide and astaxanthin reaches 3.10 × 10⁻⁶. 5 M -1 The number of binding sites is close to 1:1, indicating that the two can spontaneously form a stable complex.

[0014] 4. It has synergistic anti-atherosclerotic activity. The combined use of Porphyromonas auricula-judae polysaccharide and astaxanthin can synergistically reduce the level of reactive oxygen species (ROS) in ox-LDL-induced endothelial cells and increase the activity of superoxide dismutase (SOD). At the same time, it synergistically activates the PPARγ pathway and inhibits the TNF-α and IL-6-mediated inflammatory response, thus exhibiting a good synergistic effect in anti-atherosclerosis. Attached Figure Description

[0015] Figure 1 Fourier transform infrared (FTIR) spectra of Porphyra yezoensis polysaccharide, astaxanthin, and their complex. Figure 2 The 1H NMR spectra of *Plasmodium purpureum* polysaccharide, astaxanthin, and their complex (H NMR spectra) 1 HNMR spectrum; Figure 3 The image shows the UV-Vis absorption spectra of Porphyra yezoensis polysaccharide and its complex. Figure 4 Transmission electron microscopy (TEM) images of polysaccharides and astaxanthin from *Porphyra yezoensis*. Figure 5 The graph shows the variation curves of the I1 / I3 values ​​of the pyrene fluorescent probe in different concentrations of *Porphyra yezoensis* polysaccharide solutions and composite systems. Figure 6 Isothermal titration calorimetry (ITC) curves and thermodynamic parameter fitting plots for the titration of astaxanthin with polysaccharides from *Plasmodium purpureum*. Figure 7 Histogram showing the effect of different concentrations of *Porphyra yezoensis* polysaccharide solution on HUVECs cell viability; Figure 8 Histogram showing the effect of different concentrations of *Porphyra yezoensis* polysaccharide solution on the viability of HUVECs damaged by ox-LDL; Figure 9 Histogram showing the effect of different concentrations of astaxanthin stock solution on the viability of HUVECs; Figure 10 Histogram showing the effect of different concentrations of astaxanthin stock solution on the viability of HUVECs cells damaged by ox-LDL; Figure 11 Figure showing the synergistic protective effect of astaxanthin and Porphyra yezoensis polysaccharide against ox-LDL-damaged HUVECs; Figure 12 Figure showing the synergistic effect analysis of astaxanthin combined with Porphyromonas auricula-judae polysaccharide (HSA and Loewe model); Figure 13 Histogram showing the effect of different treatments on ROS levels in HUVEC cells with ox-LDL-induced damage; Figure 14Histogram showing the effect of different treatments on PPARγ mRNA expression levels in HUVEC cells with ox-LDL-induced damage; Figure 15 Histogram showing the effect of different treatments on TNF-α mRNA expression levels in HUVEC cells with ox-LDL-induced damage; Figure 16 Histogram showing the effect of different treatments on IL-6 mRNA expression levels in HUVEC cells with ox-LDL-induced damage. Detailed Implementation

[0016] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] This invention provides a non-covalent complex of *Porphyra yezoensis* polysaccharide and astaxanthin and its preparation method, the specific process of which is as follows: Preparation of polysaccharides from *Porphyra yezoensis*: A flat-plate photobioreactor was used to continuously cultivate *Porphyra yezoensis* under light. After the algal cells reached the stationary phase, they were collected and centrifuged. The supernatant was collected, concentrated by rotary evaporation, and four volumes of 90% ethanol were added. The mixture was then incubated at 4°C for 24 hours for alcohol precipitation. The precipitate was collected by centrifugation at 9000 rpm for 10 minutes, and the crude polysaccharide precipitate was reconstituted with an appropriate amount of pure water. The mixture was magnetically stirred until the polysaccharide solution was homogeneous. The homogeneous polysaccharide solution was placed in a constant-temperature water bath and preheated at 45°C for 10 minutes. Then, 1.5% (w / w) papain was added, and the mixture was enzymatically hydrolyzed for 2 hours. After hydrolysis, the hydrolysate was transferred to a boiling water bath and heated for 10 minutes to denature and inactivate the enzyme, thus terminating the reaction. The mixture was centrifuged at 9000 rpm for 15 minutes at 4°C, and the supernatant was collected. The supernatant was filtered, dialyzed, and freeze-dried to obtain *Porphyra yezoensis* polysaccharide.

[0018] Preparation of *Porphyra yezoensis* polysaccharide-astaxanthin complex Accurately weigh the polysaccharide from *Porphyra yezoensis* and dissolve it in pure water to prepare a 1 mg / mL solution. Accurately weigh the astaxanthin and dissolve it in anhydrous ethanol to prepare a 3 mg / mL stock solution. Under magnetic stirring, add the astaxanthin stock solution dropwise to the *Porphyra yezoensis* polysaccharide solution, so that the final concentrations of astaxanthin in the system are 0.025, 0.05, 0.1, 0.5, 1.0, and 2.0 mg / mL, respectively, and the final concentration of ethanol does not exceed 5%. Stir and react at 25°C in the dark for 30 minutes to obtain the *Porphyra yezoensis* polysaccharide-astaxanthin complex solution.

[0019] Physical characterization of the polysaccharide-astaxanthin complex from *Porphyra yezoensis* Fourier transform infrared spectroscopy (FTIR) analysis: In the infrared spectrum of the complex ( Figure 1 The OH stretching vibration peak of *Plasmodium purpureum* polysaccharide (approximately 3419 cm⁻¹) -1 When mixed with astaxanthin, the blue color shifted to 3425 cm⁻¹. -1 The C=O stretching vibration peak of astaxanthin (1651 cm⁻¹) -1 Redshifted to 1637cm -1 Astaxanthin at 3438cm -1 and 2920cm -1 The absorption peak at 972 cm⁻¹ has basically disappeared; astaxanthin at 972 cm⁻¹ -1 1197cm -1 1550cm -1 The characteristic peaks at [locations] almost completely disappeared; the characteristic peaks of *Porphyra yezoensis* polysaccharide at 1024 cm⁻¹... -1 1079cm -1 1154cm -1 The COC peaks at the locations were shifted to 1026 cm⁻¹. -1 1079cm -1 1153cm -1 Furthermore, the intensity decreases significantly. The blue shift of the OH peak and the red shift of the C=O peak are typical spectral features of hydrogen bond formation.

[0020] 1H NMR spectrum ( 1 HNMR analysis: Nuclear magnetic resonance spectroscopy shows ( Figure 2 The hydroxyl and methyl proton peaks of astaxanthin and the anomeric proton peaks of polysaccharides in the complex showed significant chemical shifts and broadening, further indicating the presence of hydrogen bonds and hydrophobic interactions between the two.

[0021] Ultraviolet-Vis absorption spectroscopy (UV-Vis) analysis: Ultraviolet spectroscopy shows ( Figure 3 When combined with astaxanthin, the characteristic absorption peak of *Porphyra yezoensis* polysaccharide shifted from 200 nm to 195 nm, proving that astaxanthin and *Porphyra yezoensis* polysaccharide interacted; no characteristic peak of astaxanthin was observed at 470-500 nm, because its signal was masked by the strong absorption of polysaccharide.

[0022] Transmission electron microscopy (TEM) analysis: Transmission electron microscopy images show ( Figure 4 The combination of *Plasmodium purpureum* polysaccharide and astaxanthin formed well-shaped complex particles, further confirming the effective binding between the two.

[0023] Pyrene fluorescence spectroscopy analysis: Using pyrene as a fluorescent probe, the I1 / I3 values ​​of different concentrations of *Porphyra yezoensis* polysaccharides were measured. The results showed that when the polysaccharide concentration increased to 1 mg / mL, the I1 / I3 value decreased significantly, indicating the formation of hydrophobic microregions by the polysaccharide. With the polysaccharide concentration fixed at 1 mg / mL, the I1 / I3 value showed a phased change after adding gradient concentrations of astaxanthin: at extremely low AST concentrations (0.025-0.05 mg / mL), I1 / I3 showed a slight rebound compared to the EPS group alone; as the AST concentration further increased (0.1-2 mg / mL), I1 / I3 showed a continuous decreasing trend. Figure 5 This indicates that astaxanthin and polysaccharides synergistically enhance the hydrophobicity of the system, confirming the existence of hydrophobic interactions.

[0024] Isothermal titration calorimetry (ITC) analysis: The thermodynamic parameters of the interaction between *Porphyra yezoensis* polysaccharide and astaxanthin were determined using an isothermal titration calorimeter. Titration of the astaxanthin solution into the *Porphyra yezoensis* polysaccharide solution yielded a series of exothermic peaks, with the peak heights gradually decreasing with increasing titration frequency, indicating that the binding sites on the *Porphyra yezoensis* polysaccharide gradually approach saturation. Thermodynamic parameters were obtained by fitting the data using a "onesetofsites" model. Figure 6 The number of binding sites, n = 1.16 ± 0.08, indicates that approximately 1.16 astaxanthin molecules bind to 1 *Plasmodium purpureum* polysaccharide molecule, with a stoichiometric ratio close to 1:1; the binding constant K = 3.10 × 10⁻⁶. 5 M -1 The corresponding dissociation constant K_D≈3.23μM indicates that the two have a high affinity; the enthalpy change ΔH=-36.23kcal / mol and the entropy change ΔS=-91.7cal·mol -1 ·K -1 ΔG < 0. The above thermodynamic parameters indicate that the bonding process is an exothermic reaction, driven by enthalpy, with hydrogen bonding being the main driving force, accompanied by contributions from hydrophobic interactions, and the bonding process can proceed spontaneously.

[0025] Evaluation of the anti-atherosclerotic activity of the *Plasma gondii* polysaccharide-astaxanthin complex at the cellular level Human umbilical vein endothelial cells (HUVECs) induced by oxidized low-density lipoprotein (ox-LDL, 20 μg / mL) were used as a model.

[0026] Cytotoxicity evaluation of *Porphyra yezoensis* polysaccharides and astaxanthin: The CCK-8 assay was used to detect the effects of different concentrations of *Porphyra yezoensis* polysaccharides (12.5, 25, 50, 100, 200, 400 μg / mL) on the activity of HUVECs. The results showed that within the concentration range of 12.5-400 μg / mL ( Figure 7The cell viability of HUVECs remained above 95% in all groups treated with different concentrations of *Porphyra yezoensis* polysaccharide, indicating that *Porphyra yezoensis* polysaccharide had no significant cytotoxicity to HUVECs. The effects of different concentrations of astaxanthin (1.25, 2.5, 5, 10, 20, 40 μg / mL) on HUVEC activity were also investigated. Figure 9 The results showed that within the concentration range of 1.25-20 μg / mL, the cell viability of each treatment group remained above 100%, indicating that astaxanthin had no significant toxicity to HUVECs within this concentration range; when the astaxanthin concentration increased to 40 μg / mL, the cell viability decreased significantly (P<0.001), showing obvious cytotoxicity.

[0027] CCK-8 assay for cell viability: HUVECs were seeded in 96-well plates, and blank control group, model group (20 μg / mL ox-LDL), positive control group (10 μg / mL simvastatin), and EPS single drug group (50, 100, 200 μg / mL) were set up. Figure 8 ), AST monotherapy group (1.25-20 μg / mL) ( Figure 10 ) and combination therapy (EPS+AST) Figure 11 After 24 hours of treatment, CCK-8 solution was added to each well, and the cells were incubated for 30 minutes. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated. Results showed that after ox-LDL stimulation, cell viability in the model group decreased to approximately 70%, indicating successful establishment of the damage model. After intervention with EPS single-drug therapy (25, 50 μg / mL), cell viability recovered to approximately 85% (P<0.01). Figure 8 Cell viability recovered in a dose-dependent manner after intervention with AST monotherapy (1.25-20 μg / mL), with the 5-10 μg / mL concentration group showing the best recovery effect. Figure 10 In the combined experiment () Figure 11 Cell viability was significantly restored in all groups treated with EPS at concentrations of 50 μg / mL and 100 μg / mL combined with AST at concentrations of 5-20 μg / mL (P<0.01), with the combinations of EPS at 50 μg / mL + AST at 10-20 μg / mL and EPS at 100 μg / mL + AST at 5-20 μg / mL showing the best recovery effects. The combined effect weakened when the EPS concentration was increased to 200 μg / mL.

[0028] Synergistic effect analysis: To further quantitatively evaluate the combined efficacy of EPS and AST, synergistic effect analysis was performed on CCK-8 data using SynergyFinder software. Synergistic scores were calculated using the HSA (Highest Single Agent) model and the Loewe model as reference models. Figure 12The results showed that the HSA synergy score was -0.493, indicating that the overall combined effect was close to the optimal level of the single drug, with no obvious antagonism; the Loewe synergy score was 5.408, showing a significant synergistic effect. The red areas in the heatmap represent concentration combinations with strong synergistic effects, mainly concentrated in the EPS 50-100 μg / mL and AST 5-20 μg / mL ranges. Combining the two models, a synergy score (δ) > 10 indicates a significant synergistic effect, with the EPS 50 μg / mL and AST 5 μg / mL combination showing the highest δ value of 10.85. Therefore, EPS 50 μg / mL and AST 5 μg / mL were selected as the synergistic combination group for further mechanistic studies.

[0029] Oxidative stress marker detection: Intracellular reactive oxygen species (ROS) levels were detected using the DCFH-DA fluorescent probe. Figure 13 The results showed that, compared with the model group, the treatment group with the polysaccharide-astaxanthin complex of *Plasmodium purpureum* significantly reduced ROS levels, and the effect was better than that of astaxanthin or polysaccharide alone, indicating that the two have a synergistic effect in anti-oxidative stress.

[0030] RT-qPCR detection: PPARγ was detected using RT-qPCR. Figure 14 ), TNF-α ( Figure 15 ) and IL-6 ( Figure 16 The expression levels of PPARγ mRNA were measured. Results showed that, compared to the control group, PPARγ expression was significantly downregulated, while TNF-α and IL-6 expression were significantly upregulated. Compared to the model group, the EPS monotherapy group showed the most significant upregulation of PPARγ, while the AST monotherapy group significantly inhibited TNF-α and IL-6 expression. The combination group (EPS+AST) showed the best effect in inhibiting TNF-α, and also exhibited good effects in promoting PPARγ expression and inhibiting IL-6. These results indicate that the combination of EPS and AST can exert a synergistic endothelial protective effect by synergistically activating the PPARγ pathway and inhibiting the TNF-α and IL-6-mediated inflammatory response.

[0031] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A non-covalent complex of *Porphyra yezoensis* polysaccharide and astaxanthin, characterized in that: The complex is spontaneously formed by the non-covalent interaction between Porphyra yezoensis polysaccharide and astaxanthin. The non-covalent interactions are hydrogen bonds and hydrophobic interactions.

2. The non-covalent complex of *Plasma gondii* polysaccharide-astaxanthin according to claim 1, its preparation method, and its application, characterized in that: The number of binding sites n for the *Porphyra yezoensis* polysaccharide and astaxanthin was 1.16 ± 0.08, and the binding constant K was 3.10 × 10⁻⁶. 5 M -1 The enthalpy change ΔH during the combination process is -36.23 kcal / mol, and the entropy change ΔS is -91.7 cal·mol. -1 ·K -1 .

3. A method for preparing a non-covalent complex of *Plasmodium purpureum* polysaccharide and astaxanthin, used to prepare the non-covalent complex of *Plasmodium purpureum* polysaccharide and astaxanthin as described in claim 1 or 2, characterized in that: Includes the following steps: (1) Dissolve the polysaccharide of *Porphyra yezoensis* in pure water or buffer solution to prepare a polysaccharide solution with a concentration of 0.5-5 mg / mL; (2) Dissolve astaxanthin in anhydrous ethanol to prepare astaxanthin stock solution; (3) Under stirring conditions, the astaxanthin mother liquor is added dropwise to the Porphyra yezoensis polysaccharide solution so that the final concentration of ethanol in the mixture is 1%-10% and the final concentration of astaxanthin is 0.01-2 mg / mL. The mixture is stirred at 15-30℃ in the dark for 10-60 minutes to obtain the Porphyra yezoensis polysaccharide-astaxanthin non-covalent complex solution.

4. The method for preparing a non-covalent complex of *Plasma gondii* polysaccharide and astaxanthin according to claim 3, characterized in that: The concentration of the *Porphyra yezoensis* polysaccharide solution is 1-2 mg / mL.

5. The application of the non-covalent complex of *Porphyra yezoensis* polysaccharide-astaxanthin according to claim 1 or 2, characterized in that: It can be used in anti-atherosclerotic drugs or functional foods.

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