Preparation method and application of microalgae beta-1, 3-glucan-rosemary acid conjugate

Abstract

The application belongs to the technical field of functional component delivery system preparation, and particularly discloses a preparation method and application of a microalgae beta-1,3-glucan-rosemary acid conjugate. The beta-1,3-glucan extracted from microalgae Poterioochromonas malhamensis is esterified with rosemary acid to obtain the microalgae beta-1,3-glucan-rosemary acid. The microalgae beta-1,3-glucan-rosemary acid conjugate can improve the antioxidant activity, anti-inflammatory activity, bioavailability of rosemary acid, slow down the release speed of rosemary acid in the gastrointestinal tract, improve the release of rosemary acid in the colon and the anti-cancer activity.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of functional component delivery system preparation, and particularly relates to a preparation method and application of a microalgae beta-1,3-glucan-rosemary acid conjugate. BACKGROUND

[0002] Rosmarinic acid (RA) is a polyphenolic antioxidant with a wide range of health effects, including antibacterial, anticancer, anti-aging, anti-diabetic, heart-protective, liver-protective, and anti-inflammatory properties. However, RA is easily degraded in the gastrointestinal tract during digestion, has poor stability, low absorption efficiency, and is affected by first-pass metabolism, resulting in limited bioavailability, which limits its wide application. To address these issues, various delivery systems have been developed to improve the stability and efficacy of RA. For example, polyethylene glycolated RA-derived nanoparticles can enhance the treatment of acute colonic inflammation by controlled release in the colon (ACS Nano, 2020, 14, 6887-6896), and RA loaded in solid lipid nanoparticles exhibits good stability during storage (Colloids and Surfaces B: Biointerfaces, 2014, 115, 109-117). In addition, RA encapsulated in organic silica nanoparticles enhances cytotoxicity and apoptosis effects on AGS and CT26 cell lines (Arabian Journal of Chemistry, 2024, 17, 105402). However, these materials face challenges such as difficulty in biodegradation and possible systemic adverse effects.

[0003] Another method to address the limited bioavailability of RA and reduce side effects is to combine RA with natural carbohydrate polymers to form conjugates. For example, grafting RA onto chitosan can form chitosan-RA conjugates, which exhibit stronger free radical scavenging ability, photoprotection, antibacterial, and anti-inflammatory activity compared to free RA (Carbohydrate Polymers, 2021, 273, 118619; Carbohydrate Polymers, 2022, 296, 119943). However, chitosan-RA conjugates have limitations in targeted delivery for colon diseases such as ulcerative colitis, colon cancer, etc., as their adhesive properties cause them to be retained in the upper gastrointestinal tract and fail to effectively reach the colon. Therefore, developing an RA delivery system that can effectively target the colon is key to improving the bioavailability of RA and its application in the treatment of specific diseases. SUMMARY

[0004] The present application aims to provide a preparation method and application of a microalgae beta-1,3-glucan-rosemary acid conjugate, and aims to solve the problem that the existing RA delivery system cannot effectively exert physiological activity of small molecules due to its limitations such as difficulty in biodegradation and low colon targeting effect.

[0005] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0006] A microalgae beta-1,3-glucan-rosemary acid conjugate, the conjugate being a purified product of an esterification reaction of rosemary acid and beta-1,3-glucan in a mass ratio of 2-10:5;

[0007] The beta-1,3-glucan is extracted from microalgae Poterioochromonas malhamensis and has a purity of more than 90%.

[0008] The molecular weight of the beta-1,3-glucan is 15.5 kDa-17.0 kDa.

[0009] A preparation method of a microalgae beta-1,3-glucan-rosemary acid conjugate, comprising the following steps:

[0010] (1) dissolving rosemary acid in dimethyl sulfoxide to obtain a rosemary acid solution;

[0011] (2) adding N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine to the rosemary acid solution, and stirring for a certain time to activate the rosemary acid;

[0012] (3) adding beta-1,3-glucan to the solution obtained in step (2) and stirring for a certain time to perform esterification reaction; the beta-1,3-glucan is extracted from microalgae Poterioochromonas malhamensis by water extraction and alcohol precipitation, and has a purity of more than 90%;

[0013] (4) placing the solution obtained in step (3) in a dialysis bag and dialyzing with ultrapure water to remove solvents and small molecules;

[0014] (5) centrifuging the solution in the dialysis bag in step (4) to obtain a supernatant, and freeze-drying to obtain a microalgae beta-1,3-glucan-rosemary acid conjugate.

[0015] Preferably, steps (1)-(3) are all performed under nitrogen protection.

[0016] Further, in step (1), the concentration of the rosemary acid solution is 0.5-10 mg / mL.

[0017] Further, in step (2), the mass ratio of rosmarinic acid, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine is 0.4-2:1:1, preferably, the mass ratio is 0.8-1.2:1:1, more preferably, the mass ratio is 1:1:1.

[0018] Further, in step (3), the mass ratio of rosmarinic acid to β-1,3-glucan is 2-10:5, preferably, the mass ratio of rosmarinic acid to β-1,3-glucan is 4-6:5, more preferably, the mass ratio of rosmarinic acid to β-1,3-glucan is 1:1.

[0019] Further, in step (2), stirring is performed for 1-3 hours, preferably 2 hours.

[0020] Further, in step (3), stirring is performed for 18-30 hours, preferably 24 hours.

[0021] Further, in step (3), the molecular weight of the β-1,3-glucan is 15.5kDa-17.0kDa, preferably 16.1kDa-16.5kDa.

[0022] Further, in step (4), the molecular weight cut-off (MWCO) of the dialysis bag is 3.5kDa.

[0023] Further, in step (5), the centrifugal speed is 8000-12000rpm, and the centrifugal time is 8-10min.

[0024] The microalgae β-1,3-glucan-rosmarinic acid conjugate or the microalgae β-1,3-glucan-rosmarinic acid conjugate prepared by the above method can be used for improving the physiological activity of rosmarinic acid, improving the bioavailability of rosmarinic acid, targeting the colon, and preparing anti-colon cancer drugs.

[0025] Compared with the prior art, the advantages and beneficial effects of the present application are as follows:

[0026] The present application utilizes the beta-1,3-glucan extracted from microalgae Poterioochromonas malhamensis to prepare microalgae beta-1,3-glucan-rosemary acid through a simple esterification reaction with rosemary acid. The esterification reaction condition is mild, and the side effect of raw materials is low. The beta-1,3-glucan extracted from microalgae Poterioochromonas malhamensis has high linearity and low molecular weight, and has good water solubility. The microalgae beta-1,3-glucan-rosemary acid conjugate obtained can improve the stability of RA, improve the antioxidant activity, anti-inflammatory activity and other physiological activities of RA itself, effectively improve the cell absorption and bioavailability of RA, effectively avoid degradation in the physiological environment of the gastrointestinal tract, realize the targeted colon release of RA, improve the anti-colon cancer activity of RA, and has low systemic side effects and high treatment effect. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 NMR characterization of PβG used in the present application.

[0028] Figure 2 NMR characterization of different RA-PβG conjugates in the present application.

[0029] Figure 3 Performance and morphology characterization of different RA-PβG conjugates in the present application: (A) FITR; (B) UV-vis; (C) XRD; (D-E) thermodynamics; (F-I) SEM.

[0030] Figure 4 Stability analysis of RA-PβG conjugates in the present application under different conditions: (A) pH; (B) under different temperatures; (C) different NaCl concentrations; (D) UV radiation; (E) under 4℃ storage conditions; (F) under 25℃ storage conditions.

[0031] Figure 5 Antioxidant activity (A-B), anti-inflammatory activity (C-F) and in vitro release curve (G) of RA-PβG conjugates in the present application.

[0032] Figure 6 Antioxidant activity of free RA.

[0033] Figure 7 Cell transport diagram (A), transport efficiency (B), bioavailability (C), cell uptake (D) and bioavailability (E) of RA-PβG conjugates in the present application.

[0034] Figure 8 Anti-colon cancer cell activity of RA-PβG conjugates in the present application.

[0035] Figure 9This invention relates to the effect of RA-PβG conjugate on apoptosis of colon cancer cells. Detailed Implementation

[0036] The present invention will be further illustrated below with specific embodiments. The following embodiments should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above-described invention will still fall within the scope of protection of the invention.

[0037] The PβG used in the following examples is β-1,3-glucan derived from the microalga Poterioochromonas malhamensis. The extraction process is as follows: 1 g of freeze-dried Poterioochromonas malhamensis cell powder was added to 10 mL of deionized water and extracted by stirring at 500 rpm in a 50°C water bath for 0.5 h. The supernatant was collected and precipitated with 95% ethanol at -20°C for 12 h. After precipitation, the precipitate was centrifuged at 4000g for 20 min at 4°C. The supernatant was discarded, and the precipitate was collected. The precipitate was washed twice with 95% ethanol and then redissolved in deionized water. DNase was then used to treat the precipitate at 4°C for 1 h to remove residual nucleic acids. Proteinase K was then used to treat the precipitate at 4°C for 2 h to remove residual proteins. The resulting solution was precipitated with 95% ethanol at -20°C for 12 h. The precipitate was washed twice with 95% ethanol and then centrifuged at 4000g for 10 min at 4°C. The supernatant was discarded, and the precipitate was collected. The precipitate was washed twice with 95% ethanol and then redissolved in deionized water. The solution was then dialyzed at 4°C (MWCO = 3.5 kDa) for 24 h. Finally, the purified PβG was lyophilized for later use.

[0038] The molecular weight of PβG, determined by gel permeation chromatography, is approximately 16.3 kDa. Based on the molecular weight of glucose (180 Da), the theoretical degree of polymerization of PβG is approximately 90.6%, and the total sugar content, determined by the phenol-sulfuric acid method, is 90.94%.

[0039] pass 1 HNMR, 13 The structure of PβG was further characterized by C NMR, COSY, HSQC, and HMBC spectra, and the results are as follows: Figure 1 As shown. In 1 H NMR approximately 2.50 ppm and 13 A strong peak corresponding to DMSO solvent was observed at approximately 39.5 ppm using C NMR. This polysaccharide exhibits a typical β-glycoside structure. 1 The HNMR spectra show chemical shifts of δ 4.51, 3.68, 3.44, 3.26, and 3.20 in the head region. C6, C4, C2, C5, C3, and C1... 13C NMR spectrum showed 6 significant signals at 60.93, 68.48, 72.91, 76.40, 86.27 and 103.12 ppm, respectively. The relatively pure NMR spectrum indicated that the polysaccharide was a linear glucan with few side chains, and the purity was as high as more than 90%.

[0040] Example 1: Preparation of RA-PβG conjugate

[0041] The preparation of the RA-PβG conjugate comprises the following steps:

[0042] (1) 50 mg of rosmarinic acid (RA) was dissolved in 10 mL of dimethyl sulfoxide (DMSO) and stirred at room temperature under nitrogen protection until complete dissolution to obtain a RA solution;

[0043] (2) 27 mg (0.14 mmol) of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC·HCl) and 17 mg (0.14 mmol) of 4-dimethylaminopyridine (DMAP) were added to the RA solution obtained in step (1) to activate the carboxyl group of RA, and stirred at room temperature under nitrogen protection for 2 hours to obtain a mixed solution;

[0044] (3) 50 mg of PβG powder derived from microalgae Poterioochromonas malhamensis was added to the mixed solution obtained in step (2), and stirred at room temperature under nitrogen protection for 24 hours to obtain a mixed solution;

[0045] (4) The mixed solution obtained in step (3) was placed in a dialysis bag (MWCO = 3.5 kDa) and then immersed in ultrapure water for dialysis for 24 hours to remove residual DMSO and other small molecular substances, and the water was replaced every 6 hours;

[0046] (5) The solution in the dialysis bag in step (4) was centrifuged (8000 rpm, 10 min) to take the supernatant, and then freeze-dried at -80°C to obtain the RA-PβG conjugate, which was recorded as 50-RA.

[0047] Similarly, steps (1)-(5) were repeated, only adjusting the mass of RA to be 8 mg and 20 mg, respectively, to finally obtain two RA-PβG conjugates, which were recorded as 8-RA and 20-RA, respectively.

[0048] Dissolve 400 μg of 8-RA, 20-RA and 50-RA in 1 mL of ultrapure water to obtain 400 μg / mL of RA-PβG aqueous solution. Mix 0.5 mL of RA-PβG aqueous solution with 0.5 mL of Folin-phenol reagent and 1 mL of 10% Na2CO3 solution, incubate in a 50°C water bath for 1 hour, cool to room temperature, stand at room temperature for 15 minutes, and measure the absorbance at 725 nm using an enzyme marker. Calculate the concentration of RA according to the absorbance, and then calculate the grafting rate (W%, the content of RA in the RA-PβG conjugate) according to the following formula.

[0049] W% = content of RA in sample / sample amount * 100% (1-1)

[0050] The grafting rates of RA in the three RA-PβG conjugates of 8-RA, 20-RA and 50-RA are 2.16%, 5.72% and 16.56% respectively, calculated by the Folin-phenol method.

[0051] According to the ratio of the drug on the polymer (grafting rate W%) and the following formula, calculate the degree of substitution (DS, which refers to the average degree of substitution of hydroxyl groups on each polymer unit by other chemical groups)

[0052]

[0053] According to this formula, the degrees of substitution of the three RA-PβG conjugates of 8-RA, 20-RA and 50-RA are 0.010, 0.027 and 0.089 respectively.

[0054] Example 2: Characterization of RA-PβG conjugate

[0055] Characterize the RA-PβG conjugate prepared in Example 1.

[0056] (1) NMR characterization of RA-PβG conjugate

[0057] Figure 2 Characterize the RA-PβG conjugate by NMR. As Figure 2 1 HNMR shows that the RA-PβG conjugates with three different degrees of substitution are characterized by 1 HNMR analysis. The characteristic peak of DMSO solvent is observed at δ 2.50 ppm, and the 1 ​In the 1H NMR spectra, the area ratio of the signal peaks at δ5.20 ppm and δ4.65 ppm was approximately 1:1, indicating the disappearance of the hydroxyl proton signal at C6 of PβG, suggesting that the hydroxyl group at C6 of PβG participated in the reaction. When the 1H NMR spectra of 8-RA, 20-RA, and 50-RA were magnified in the 8.1–6.0 ppm region, aromatic ring hydrogen signals were observed at δ7.06–6.49 ppm, indicating that RA reacted with PβG. New characteristic spectra of the 50-RA conjugate appeared in the ranges of 7.38–7.46 ppm, 6.47–7.09 ppm, and 6.14–6.25 ppm. These peaks correspond to the characteristic absorption of RA, indicating that the conjugate was successfully prepared.

[0058] (2) FITR characterization of RA-PβG conjugates

[0059] In the spectrum of RA ( Figure 3 In RA-PβG conjugates, characteristic peaks of OH stretching vibration were observed at 3370 cm⁻¹ and 3232 cm⁻¹, C=O stretching vibration at 1682 cm⁻¹, aromatic ring C=C stretching vibration and phenolic CO stretching vibration at 1605 cm⁻¹ and 1525 cm⁻¹, respectively, and phenolic CO stretching vibration at 1278 cm⁻¹. Furthermore, aromatic ring CH bending vibrations appeared between 864 and 684 cm⁻¹. For PβG, the broadband at 3433 cm⁻¹ and 2922 cm⁻¹ is attributed to the overlap of OH and CH stretching vibrations, respectively, while the peaks at 1075 cm⁻¹ and 1039 cm⁻¹ correspond to COC stretching vibrations. In the RA-PβG conjugate, the peak at 1690 cm⁻¹ represents the C=O stretching vibration of the ester bond, indicating successful esterification, while the peak at 2922 cm⁻¹ indicates the CH stretching vibration of PβG. From 8-RA to 50-RA, the intensities of the OH and CH bands at 3433 cm⁻¹ and 2922 cm⁻¹ decreased with increasing substitution degree. Simultaneously, the intensities of the ester-related peak at 1690 cm⁻¹ and the RA-related peak at 1520 cm⁻¹ increased, reflecting a higher level of substitution. The peak at 888 cm⁻¹ became more prominent with increasing substitution degree, indicating enhanced CH deformation vibrations. These spectral changes collectively demonstrate successful coupling of RA with PβG and highlight the structural modifications associated with increased substitution degree.

[0060] (3) UV-vis analysis of RA-PβG conjugates

[0061] The coupling effect between RA and PβG was verified by UV-Vis spectroscopy analysis. Figure 3(B) RA exhibits absorption peaks at approximately 326 nm and 288 nm. Characteristic absorption bands at 283–284 nm and 323 nm are observed in the spectra of the 8-RA, 20-RA, and 50-RA conjugates, while no corresponding absorption peaks are observed in the 200–400 nm range of PβG, indicating that RA has been successfully conjugated to PβG. The absorption intensity increases with increasing degree of substitution.

[0062] (4) XRD analysis of RA-PβG conjugate

[0063] The crystal structure of the RA-PβG conjugate was further determined by XRD. Figure 3 (C). RA exhibits sharp and clear peaks at 2θ = 6°, 17°, 21°, 24°, 27°, and 28°, indicating its highly crystalline and ordered crystal structure. PβG shows two significant diffraction peaks at approximately 2θ = 6° and 21°, indicating a semi-crystalline structure. In the RA-PβG conjugate, the peak at 2θ = 6° is observed, but its intensity decreases with increasing degree of substitution. Furthermore, the peak at 21° shifts to a higher angle (22°) and its intensity also decreases, indicating coupling.

[0064] (5) Thermodynamic TG-DSC analysis of RA-PβG conjugate

[0065] The thermal stability and degradation of the RA-PβG conjugate were evaluated using TG-DSC analysis. Figure 3 In the DE assay, the thermal degradation curves showed that PβG experienced an initial weight loss of 10.46% between 30℃ and 152℃. A significant weight reduction of 73.25% occurred between 285℃ and 325℃, with a peak degradation temperature (Tp) of 301℃. The sample tended to stabilize after 400℃. For RA, the initial weight loss was 10.1% between 30℃ and 125℃, with a further loss of 73.39% between 288℃ and 314℃, and a Tp of approximately 301.54℃. Similar to PβG, RA also stabilized after 400℃. All three RA-PβG conjugates (8-RA, 20-RA, and 50-RA) showed two stages of weight loss. The initial weight loss temperatures for 8-RA, 20-RA, and 50-RA were delayed to 102℃, 195℃, and 227℃, respectively. In the second stage, these conjugates lost 71.03%, 63.78%, and 43.27% of their mass, respectively. The thermal stability (Tp) of 8-RA, 20-RA, and 50-RA was delayed to 306.34 °C, 306.98 °C, and 309.83 °C, respectively. This indicates that thermal stability increases with increasing degree of substitution, following the order: 50-RA > 20-RA > 8-RA > RA.

[0066] (6) SEM characterization of RA-PβG conjugate

[0067] The surface morphology of PβG and RA-PβG conjugates was characterized using scanning electron microscopy (SEM). Figure 3 In the middle FI (in order, PβG, 8-RA, 20-RA, and 50-RA), PβG exhibits a rough and amorphous surface morphology. With increasing degree of substitution, the surface morphology of the RA-PβG conjugates gradually changes. The surface morphology of 8-RA is similar to that of PβG, while the amorphous structure of 20-RA and 50-RA gradually decreases and becomes smoother due to the increasing number of grafted RA fragments, indicating a coupling reaction between RA and PβG.

[0068] Example 3: Performance testing of RA-PβG conjugates

[0069] (1) Stability analysis of RA-PβG conjugates

[0070] a. pH stability

[0071] The lyophilized RA-PβG conjugate sample obtained in Example 1 was dissolved in ultrapure water to a concentration of 1 mg / mL. 1.0 mL of the freshly prepared RA-PβG conjugate sample solution was mixed with 1.0 mL of aqueous solutions at different pH values ​​(1.0, 2.0, 3.0, 5.0, 7.0, 9.0, and 11.0) (pH adjusted with hydrochloric acid or sodium hydroxide). The mixture was allowed to stand at 25°C for 3 hours. The treated sample was then ultrafiltered using a 10 kDa ultrafiltration tube. The retention rate of RA was determined using the Folin-Ciocalteu method to assess the stability of the RA-PβG conjugate under different pH conditions. Figure 4 As shown in Figure A, free RA exhibits poor stability under strongly acidic conditions, with retention rates of 41.81% and 43.28% at pH 1.0 and pH 2.0, respectively. The retention rate of RA in the conjugates improved after binding with PβG. Specifically, the retention rates of RA in 50-RA at pH 1.0 and pH 2.0 were 65.34% and 71.28%, respectively. Within the pH range of 3.0–7.0, there was no significant effect on the stability of RA. However, above pH 7.0, the stability of both free RA and all conjugates decreased significantly. At pH 9.0 and 11.0, the retention rates of RA decreased to 65.93% and 39.48%, respectively. In comparison, the retention rates of 8-RA, 20-RA, and 50-RA at pH 9.0 were 76.54%, 81.54%, and 89.58%, respectively; and at pH 11.0, they were 52.56%, 62.64%, and 88.80%, respectively. These results indicate that RA in the RA-PβG conjugate exhibits better pH stability compared to free RA, and the higher the degree of substitution, the better the stability.

[0072] b. Thermal stability

[0073] The lyophilized RA-PβG conjugate samples obtained from Example 1 were dissolved in ultrapure water at a concentration of 1 mg / mL. 1.0 mL of freshly prepared RA-PβG conjugate sample solution was incubated in a water bath at 50, 60, 70, 80 and 90 °C for 60 min, respectively. The treated samples were ultrafiltered with 10 kDa ultrafiltration tubes. The retention rate of RA was determined by Folin-Ciocalteu method to evaluate the thermal stability of RA-PβG conjugates. As shown in Fig. B, the retention rate of free RA decreased with the increase of temperature, reaching the lowest level of 38.49% at 90 °C. While the RA retention rates of 8-RA, 20-RA and 50-RA were 64.12%, 77.73% and 84.47% at 90 °C, respectively. All RA-PβG conjugates could effectively inhibit the thermal degradation of RA, which was positively correlated with the degree of substitution. Figure 4

[0074] c. Ionic stability

[0075] The lyophilized RA-PβG conjugate samples obtained from Example 1 were dissolved in ultrapure water at a concentration of 1 mg / mL. 1.0 mL of freshly prepared RA-PβG conjugate sample solution was mixed with different concentrations of NaCl solution (0, 50, 100, 250, 500 and 1000 mM) at equal volume, respectively, and placed for 24 h. The treated samples were ultrafiltered with 10 kDa ultrafiltration tubes. The retention rate of RA was determined by Folin-Ciocalteu method to evaluate the ionic strength stability of RA-PβG conjugates. As shown in Fig. C, the stability of free RA and RA-PβG conjugates decreased with the increase of ionic strength. Compared with pH and thermal stability, the effect of ionic strength on RA and RA-PβG conjugates seemed to be smaller. The RA retention rates in free RA, 8-RA, 20-RA and 50-RA were 81.01%, 86.15%, 87.78% and 91.32% in 1 M NaCl solution, respectively. All RA-PβG conjugates could enhance the stability of RA in a wide range of ionic strength. Figure 4

[0076] d. Light stability

[0077] The lyophilized RA-PβG conjugate samples obtained from Example 1 were dissolved in ultrapure water at a concentration of 1 mg / mL. 1.0 mL of freshly prepared RA-PβG conjugate sample solution was placed in a colorless glass tube for light stability test. The light stability test was performed at 25 °C with a distance of 30 cm, 254 nm, 17 μW / cm 2 ​​The samples were irradiated under UV light for 6 h, and the treated samples were ultrafiltered using 10 kDa ultrafiltration tubes. The retention rate of RA was determined using the Folin-Ciocalteu method to evaluate the photostability of the RA-PBG conjugates. As shown in Table 1, the retention rate of free RA gradually decreased to 66.35% after 6 h of UV light exposure, while the degradation rate of RA in all RA-PBG conjugates was only 11.52% to 22.26%, with the 50-RA conjugate having the lowest degradation rate of 11.52%. Figure 4

[0078] e. Storage stability

[0079] The freeze-dried RA-PBG conjugate samples obtained in Example 1 were dissolved in ultrapure water at a concentration of 1 mg / mL. 1.0 mL of freshly prepared RA-PBG conjugate sample solution was placed in a glass tube, and the storage stability of the RA-PBG conjugate in ultrapure water at 4°C and 25°C without light was investigated, respectively. The treated samples were ultrafiltered using 10 kDa ultrafiltration tubes. The retention rate of RA was determined using the Folin-Ciocalteu method to evaluate the storage stability of the RA-PBG conjugates. As shown in Table 2, the retention rates of both free RA and RA-PBG conjugates decreased during storage. After 10 days of storage at 4°C and 25°C, the retention rate of free RA was 63.41% and 47.77%, respectively. In contrast, the RA-PBG conjugates exhibited higher retention rates. At 4°C, the retention rates of 8-RA, 20-RA, and 50-RA were 71.04%, 74.38%, and 84.10%, respectively, which were 1.12 times, 1.17 times, and 1.33 times that of free RA. At 25°C, the retention rates were 54.44%, 63.85%, and 73.73%, respectively, which were 1.14 times, 1.34 times, and 1.54 times that of free RA. These results indicate that the RA-PBG conjugates can effectively improve the stability of RA. Figure 4

[0080] (2) Antioxidant and anti-inflammatory activities of RA-PBG conjugates

[0081] a. In vitro antioxidant activity of RA-PBG conjugates

[0082] The antioxidant activity of RA-PBG conjugates was investigated using an in vitro chemical model. First, 1 mL of DPPH solution was mixed with 0.2 mL of different concentrations of samples (RA, PBG, and RA-PBG conjugates). After incubation in the dark for 0.5 h, the absorbance of the solution at 517 nm was recorded.

[0083]

[0084] ​​A1 is the absorbance of the sample, and A0 is the absorbance of the blank group (without sample).

[0085] ABTS stock solution was prepared by mixing 2.45 mM potassium persulfate with 7 mM ABTS (1:1, v / v) and allowing it to dissolve completely in the dark for 16 h. The solution was then diluted with 70% ethanol until the absorbance at 517 nm was maintained at approximately 0.70. Then, 0.075 mL of various concentrations of samples (RA, PβG, and RA-PβG conjugate) were added to the diluted ABTS solution (1 mL) and allowed to stand for 6 min. ABTS activity was measured at 517 nm.

[0086]

[0087] A1 is the absorbance of the sample, and A0 is the absorbance of the blank group (without sample).

[0088] The results are as follows Figure 5 As shown in Figure AB, at a concentration of 166.7 μg / mL, the DPPH radical scavenging rates of PβG, 8-RA, 20-RA, and 50-RA were 18.78%, 35.39%, 52.87%, and 82.90%, respectively. At a concentration of 69.8 μg / mL, the ABTS radical scavenging rates of PβG, 8-RA, 20-RA, and 50-RA were 5.32%, 14.69%, 20.36%, and 71.63%, respectively. Furthermore, as... Figure 6 As shown, the effective half-inhibitory concentration (IC50) of free RA for scavenging DPPH and ABTS radicals is... 50 The concentrations were 5.27 μg / mL and 9.54 μg / mL, respectively. The higher the degree of substitution of the RA-PβG conjugate, the stronger its antioxidant capacity. PβG had low antioxidant activity; its DPPH scavenging capacity was less than 20% at the highest tested concentration, and it had almost no inhibitory effect on ABTS scavenging at 69.8 μg / mL. The IC50 of 50-RA for scavenging DPPH and ABTS free radicals was... 50 The values ​​were 29.89 μg / mL and 42.39 μg / mL, respectively, corresponding to 4.95 μg / mL and 7.0 μg / mL of RA. These values ​​were lower than those of free RA, indicating enhanced antioxidant activity.

[0089] b. Anti-inflammatory activity of RA-PβG conjugates

[0090] RAW264.7 cells were stimulated to produce an inflammatory response using lipopolysaccharide (LPS). The concentration was 5 × 10⁻⁶ cells / cells. 5RAW264.7 cells were cultured overnight in 24-well plates at a cell / well density. Cells were incubated for 24 h with 100 μL LPS (1 μg / mL) with or without RA, PβG, and RA-PβG conjugates. NO and cytokines (TNF-α, IL-6, and IL-1β) in the cell supernatant were collected and analyzed according to the kit's detection procedure. Specifically, the concentration of PβG and RA-PβG conjugates (8-RA, 20-RA, and 50-RA) in the aqueous solution was 100 μg / mL. The RA concentrations in the 8-RA, 20-RA, and 50-RA conjugates were 2.2 μg / mL, 5.7 μg / mL, and 16.6 μg / mL, respectively. For direct comparison, the concentration of free RA was set at 16.6 μg / mL, consistent with the RA concentration in the 50-RA conjugate. The results showed that after RAW264.7 cells were exposed to 1 μg / mL LPS, the NO level in the cells increased significantly by 5.17 times compared with the control group. Figure 5 (C). The inhibitory effects of RA, PβG, 8-RA, 20-RA, and 50-RA on NO production were 11.63%, 18.26%, 24.50%, 31.66%, and 37.37%, respectively. The IL-1β level in the control group was 5.27 ± 0.20 ng / L, and the IL-1β level increased 4.19-fold after LPS exposure. Figure 5 (D). Compared with the LPS group, the levels of IL-1β in the free RA, PβG, and RA-PβG conjugate groups decreased by 28.03% (PβG), 18.05% (RA), 31.47% (8-RA), 45.94% (20-RA), and 65.90% (50-RA), respectively. Similarly, the levels of IL-6 in cells decreased after sample treatment, with inhibition rates of 9.15% (RA), 11.11% (PβG), 28.76% (8-RA), 42.48% (20-RA), and 76.91% (50-RA), respectively. Figure 5 (E). Furthermore, TNF-α levels decreased from 0.13 × 10⁻⁶. 5 The concentration increased from ±0.07 ng / L to 4.80 × 10⁻⁶. 5 ±0.05 ng / L, increased 36.92-fold after LPS stimulation ( Figure 5 (Middle F). Compared with the LPS group, the inhibition rates of RA and PβG were 10.42% and 17.29%, respectively, while the inhibition rates of the RA-PβG conjugate were 17.50% (8-RA), 27.71% (20-RA), and 46.25% (50-RA), respectively. The results indicate that the RA-PβG conjugate exhibits strong anti-inflammatory activity, and its activity is stronger than that of free RA.

[0091] (3) Bioavailability of RA-PβG conjugate.

[0092] a. Transfer efficiency

[0093] The transmembrane transport of RA-PβG was investigated using Caco-2 cell monolayers, as shown in the schematic diagram. Figure 7 As shown in Figure A. In a Transwell (12 wells, 0.4 μm, 1.12 cm²), 2 Inoculate 1×10 on the surface area) 5 Caco-2 cells per well were used to assess the cell transport efficiency of RA-PβG conjugates (8-RA, 20-RA, 50-RA). Transcutaneous electrical resistance (TEER) values ​​were monitored during culture at 37°C and 5% CO2. A TEER was established when the resistance reached 500 Ω·cm. 2 Cell transport can occur even within a single cell monolayer. In experiments involving apical (AP) to basal (BL) cell transport, 1.5 mL of blank HBSS was added to BL, and 0.5 mL of free RA (83 μg / mL) and sample solutions (PβG and RA-PβG conjugates: both at 500 μg / mL, with RA contents of 11, 29, and 83 μg / mL in 8-RA, 20-RA, and 50-RA, respectively) were added to AP. To determine the transported amount, basal-side solutions were taken at two different culture times (2 hours and 4 hours). The following formula was used to calculate the RA transport efficiency:

[0094]

[0095] Where M BL M represents the sample volume delivered to the BL side (μg). Inital The sample volume added to the AP side is (μg).

[0096] The results are as follows Figure 7 As shown in Figure B, the transport efficiency for RA and RA-PβG conjugates is time-dependent. After 2 hours of incubation, their transport efficiencies were 9.05% (RA), 22.54% (8-RA), 23.97% (20-RA), and 32.22% (50-RA), respectively. When the incubation time reached 4 hours, the transport efficiency increased to 17.88% (RA), 37.83% (8-RA), 47.09% (20-RA), and 60.31% (50-RA). The transport efficiency increased with increasing degree of substitution in the RA-PβG conjugate. The results show that the RA-PβG conjugate significantly improved the transport efficiency of RA, with an increase rate as high as 373%.

[0097] b. Biological accessibility

[0098] Based on the INFOGEST in vitro digestion model, the bioaccessibility of RA-PβG conjugates and RA was studied by simulating gastrointestinal digestion.

[0099] SSF: 15.1 mL of 0.5 M KCl solution, 3.7 mL of 0.5 M KH2PO4 solution, 6.8 mL of 1 M NaHCO3 solution, 2.72 mL of 2 M NaCl solution, 0.5 mL of 0.15 M MgCl2(H2O)6solution, 0.06 mL of (NH4)2CO3 solution, and made up to 400 mL with ultrapure water. 1.5 g of mucin was dissolved in 400 mL of SSF as the oral digestion solution SSF. SGF: 6.9 mL of 0.5 M KCl solution, 0.9 mL of 0.5 M KH2PO4 solution, 12.5 mL of 1 M NaHCO3 solution, 18.05 mL of 2 M NaCl solution, 0.4 mL of 0.15 M MgCl2(H2O)6solution, 0.5 mL of 0.5 M (NH4)2CO3 solution, and made up to 400 mL with ultrapure water. SIF: 6.8 mL of 0.5 M KCl solution, 0.8 mL of 0.5 M KH2PO4 solution, 42.5 mL of 1 M NaHCO3 solution, 30.85 mL of 2 M NaCl solution, 1.1 mL of 0.15 M MgCl2(H2O)6solution, 0.5 mL of (NH4)2CO3 solution, and made up to 400 mL with ultrapure water.

[0100] Oral phase: 10 mg / mL of sample (RA, 8-RA, 20-RA and 50-RA) was added sequentially to 8 mL of SSF, 50 μL of 1.5 mM CaCl2, 1 mL of 291.83 mg / mL -1 α-amylase, 0.95 mL of ultrapure water. And incubated at 37 °C for 2 min with continuous shaking at 150 rpm. The pH was adjusted to 7. Gastric phase: 20 mL of the above oral experimental solution was mixed with 16 mL of SGF, the pH was adjusted to 3 with 1 M HCl, and 2 mL of 19.32 mg / mL -1 pepsin, 10 μL of 0.15 mM CaCl2, and made up to 40 mL with ultrapure water. This was incubated at 37 °C for 2 h with continuous shaking at 150 rpm. Intestinal phase: 20 mL of the gastric experimental solution was added sequentially to 8.5 mL of SIF, 5 mL of 57.54 mg / mL -1 pancreatin, 2.5 mL of 171.38 mg / mL -1Bile salts and 40 μL of 0.6 mM CaCl2 were added, and the pH was adjusted to 7 with 1 M NaOH. The volume was then brought to 40 mL with ultrapure water. The mixture was incubated on a shaker at 37 °C and 150 rpm for 2 h with continuous shaking. After the gastric and intestinal digestion stages were completed, the resulting mixture was centrifuged at 5000 rpm at 4 °C for 10 min. The supernatant was collected, and a blank digest without sample was prepared using the same procedure as a reference. Bioaccessibility was determined using the Folin-Ciocalteu method, after subtracting the background from the blank digest, and calculated using the following formula:

[0101]

[0102] C RA The content of RA after digestion, C Total The content of RA in the total system.

[0103] The reabsorbable portion of RA in the digestion supernatant is considered absorbable and is defined as bioaccessibility. For example... Figure 7 As shown in Figure C, the bioavailability of free RA was 32.71% through simulated digestion, indicating a relatively rapid degradation rate. For RA-PβG conjugates, the bioavailability of 8-RA, 20-RA, and 50-RA were 44.14% (1.35 times that of free RA), 74.58% (2.28 times that of free RA), and 89.85% (2.74 times that of free RA), respectively.

[0104] c. Cellular uptake and bioavailability

[0105] Caco-2 cells were cultured in DMEM medium supplemented with 20% FBS at 37°C under a humidified atmosphere of 5% CO2 until passage 21. The cultured cells were then seeded into 24-well plates at a density of 1 × 10⁻⁶ cells / well. 5 Cells / well. After 14 days, cells were observed under a microscope to form a tightly confluent monolayer. The cells were washed three times with PBS buffer to remove surface impurities. Diluted digested sample (free RA: 83 μg / mL, RA-PβG conjugate: 500 μg / mL) was added with HBSS buffer and cultured for 4 h. Pre-chilled HBSS buffer was then added to stop cell uptake. Cells were lysed on ice for 30 min with cell lysis buffer (PBS containing 10% ethanol) and sonicated (10 s / 10 s, 35% power). After centrifugation (10000 rpm, 2 min), the supernatant was collected. RA cell uptake was calculated using the following formula:

[0106]

[0107] C Cell The content of RA after cellular uptake, C Total This represents the RA content before cellular uptake.

[0108] Bioavailability is calculated using the following formula:

[0109] Bioavailability (%) = F B ×F A ×100% (7)

[0110] F B It refers to the bioaccessibility of the sample, F A It refers to the cellular uptake of the sample.

[0111] Cellular uptake efficiency results are as follows Figure 7 As shown in Figure D, after 4 hours, there were significant differences in cellular uptake of free RA and RA-PβG conjugates. Compared with free RA (19.20%), grafted PβG significantly increased the cellular uptake capacity of RA: 8-RA (27.64%, 1.44-fold), 20-RA (32.55%, 1.70-fold), and 50-RA (35.17%, 1.83-fold). All RA-PβG conjugates increased RA cellular uptake.

[0112] Based on bioavailability and cellular uptake, the bioavailability of free RA was found to be 6.24%, significantly lower than that of the RA-PβG conjugate (12.17%, 24.33%, 31.56%). Figure 7 As shown in Figure E, the bioavailability of 8-RA, 20-RA, and 50-RA was 1.95 times, 3.90 times, and 5.06 times higher than that of free RA, respectively.

[0113] (4) Colon-targeted release performance

[0114] The in vitro release assays of 50-RA and free RA were performed as follows: Simulated gastric fluid (SGF): 0.1M HCl (pH 1.2), 0–2 h; Simulated intestinal fluid (SIF): phosphate solution (pH 6.8), 2–6 h; Simulated colonic fluid (SCF): phosphate solution (pH 7.4), 6–30 h. 5 mL of 1.66 mg / mL RA solution or 10 mg / mL 50-RA solution (equivalent to 8.3 mg RA) was filled into a dialysis bag (3500 Da). The bag was immersed in a centrifuge tube containing 5 mL of release medium and placed in an incubator at 150 rpm and 37 ± 1 °C. To maintain stable precipitation conditions, equal amounts of release medium were removed at predetermined time intervals and replaced with fresh release medium.

[0115] 50-RA was selected for colon-targeting ability to be evaluated by in vitro release curve analysis over 30 hours under simulated gastrointestinal conditions (SGF, SIF, and SCF). Figure 5In the presence of SGF and SIF, the cumulative release of RA from 50-RA was only 27.86%, which confirmed the improved stability of RA-PβG conjugate. During this process, part of the released RA could pass through the dialysis membrane due to its low molecular weight. RA in 50-RA reached complete release in SCF, and the release continued to 30 h, which indicated that the RA-PβG conjugate had good colon-targeting release ability.

[0116] (5) Anti-colon cancer activity of RA-PβG conjugate

[0117] To further evaluate the colon-targeting ability of RA-PβG conjugate, 1 x 10 4 Human colon cancer cell lines CT26 and HT29 and human hepatoma cell line HepG2 were cultured at a density of 1 x 10

[0118] Cytotoxicity of PβG, free RA and 50-RA was tested by MTT assay as described in Example 1. Figure 8 In the presence of SGF and SIF, the cumulative release of RA from 50-RA was only 27.86%, which confirmed the improved stability of RA-PβG conjugate. During this process, part of the released RA could pass through the dialysis membrane due to its low molecular weight. RA in 50-RA reached complete release in SCF, and the release continued to 30 h, which indicated that the RA-PβG conjugate had good colon-targeting release ability. Figure 8 In the presence of SGF and SIF, the cumulative release of RA from 50-RA was only 27.86%, which confirmed the improved stability of RA-PβG conjugate. During this process, part of the released RA could pass through the dialysis membrane due to its low molecular weight. RA in 50-RA reached complete release in SCF, and the release continued to 30 h, which indicated that the RA-PβG conjugate had good colon-targeting release ability. Figure 8 In the presence of SGF and SIF, the cumulative release of RA from 50-RA was only 27.86%, which confirmed the improved stability of RA-PβG conjugate. During this process, part of the released RA could pass through the dialysis membrane due to its low molecular weight. RA in 50-RA reached complete release in SCF, and the release continued to 30 h, which indicated that the RA-PβG conjugate had good colon-targeting release ability. Figure 8 In the presence of SGF and SIF, the cumulative release of RA from 50-RA was only 27.86%, which confirmed the improved stability of RA-PβG conjugate. During this process, part of the released RA could pass through the dialysis membrane due to its low molecular weight. RA in 50-RA reached complete release in SCF, and the release continued to 30 h, which indicated that the RA-PβG conjugate had good colon-targeting release ability. Figure 8 In the presence of SGF and SIF, the cumulative release of RA from 50-RA was only 27.86%, which confirmed the improved stability of RA-PβG conjugate. During this process, part of the released RA could pass through the dialysis membrane due to its low molecular weight. RA in 50-RA reached complete release in SCF, and the release continued to 30 h, which indicated that the RA-PβG conjugate had good colon-targeting release ability.

[0119] In the presence of SGF and SIF, the cumulative release of RA from 50-RA was only 27.86%, which confirmed the improved stability of RA-PβG conjugate. During this process, part of the released RA could pass through the dialysis membrane due to its low molecular weight. RA in 50-RA reached complete release in SCF, and the release continued to 30 h, which indicated that the RA-PβG conjugate had good colon-targeting release ability. 4The human colon cancer cell lines CT26, HT29 and human hepatoma cell line HepG2 were cultured at a density of cells per well, and further stained with Annexin V-FITC / PI. The effects of PβG, 50-RA and free RA (PβG and 50-RA at a concentration of 500 μg / mL, and free RA at a concentration of 83 μg / mL) on apoptosis were observed after incubation with the stained cells for 24 h. Figure 9 In Table A, for the control groups of CT26, HT29 and HepG2 cell lines, 97.20%, 96.20% and 97.10% of normal cells survived and did not undergo apoptosis. After PβG and free RA were applied to CT26 and HT29 cells, the apoptosis rates of PβG for CT26 and HT29 cells were 16.31% and 11.00%, respectively, and the apoptosis rates of free RA for CT26 and HT29 cells were 19.40% and 12.62%, respectively; the apoptosis rates of 50-RA for CT26 and HT29 cells were 29.18% and 23.96%, respectively (see Table B and Table C). Figure 9 In Table A, for the control groups of CT26, HT29 and HepG2 cell lines, 97.20%, 96.20% and 97.10% of normal cells survived and did not undergo apoptosis. After PβG and free RA were applied to CT26 and HT29 cells, the apoptosis rates of PβG for CT26 and HT29 cells were 16.31% and 11.00%, respectively, and the apoptosis rates of free RA for CT26 and HT29 cells were 19.40% and 12.62%, respectively; the apoptosis rates of 50-RA for CT26 and HT29 cells were 29.18% and 23.96%, respectively (see Table B and Table C). Figure 9 In Table D, the apoptosis rates of PβG, RA and 50-RA for HepG2 cells were 3.30%, 5.52% and 6.63%, respectively, which were consistent with the cytotoxicity results, indicating that the RA-PβG conjugate had a colon targeting property and promoted a greater degree of apoptosis of colon cancer cells than free RA.

Claims

1. A method for the preparation of a microalgal β-1,3-glucan- rosmarinic acid conjugate, characterized in that, Comprising the following steps: (1) dissolving rosmarinic acid in dimethyl sulfoxide to obtain a rosmarinic acid solution; (2) adding N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine to the rosmarinic acid solution and stirring for a certain time; (3) adding β-1,3-glucan into the solution obtained in step (2), the mass ratio of rosmarinic acid to β-1,3-glucan being 4-6:5, and stirring for a certain time to perform esterification reaction; the β-1,3-glucan is extracted from microalgae by water extraction and alcohol precipitation, and the purity is more than 90%; Poterioochromonas malhamensis ​ (4) placing the solution obtained in step (3) in a dialysis bag and dialyzing with ultrapure water; (5) centrifuging the solution in the dialysis bag in step (4) to obtain the supernatant, freeze-drying, and obtaining the microalgal β-1,3-glucan-rosmarinic acid conjugate.

2. The production method according to claim 1, characterized by, In step (3), the mass ratio of rosmarinic acid to β-1,3-glucan is 1:1; and / or The molecular weight of the β-1,3-glucan is 15.5 kDa-17.0 kDa.

3. The preparation method according to claim 2, characterized in that, The molecular weight of the β-1,3-glucan is 16.1 kDa-16.5 kDa.

4. The method of claim 1, wherein, In step (2), the stirring time is 1-3 hours; and / or in step (3), the stirring time is 18-30 hours.

5. The preparation method according to claim 4, characterized in that, In step (2), the stirring time is 2 hours; and / or in step (3), the stirring time is 24 hours.

6. The method of claim 1, wherein, In step (2), the molar ratio of rosmarinic acid, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine is 0.4-2:1:

1.

7. The preparation method according to claim 6, characterized in that, The molar ratio of rosmarinic acid, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine is 0.8-1.2:1:

1.

8. The preparation method according to claim 7, characterized in that, The molar ratio of rosmarinic acid, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine is 1:1:

1.

9. The preparation method according to claim 1, characterized in that, In step (4), the molecular weight cut-off (MWCO) of the dialysis bag is 3.5 kDa.

10. The method of claim 1, wherein, In step (1), the concentration of the rosmarinic acid solution is 0.5-10 mg / mL.

11. The method of claim 1, wherein, Steps (1)-(3) are all carried out under nitrogen protection.

12. The microalgal β-1,3-glucan-rosmarinic acid conjugate prepared by the preparation method of any one of claims 1-11.

13. The use of the microalgal β-1,3-glucan-rosmarinic acid conjugate of claim 12 in the preparation of an anti-colon cancer drug.

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