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

Abstract

The invention 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-rosmarinic acid conjugate. According to the invention, beta-1, 3-glucan is extracted from microalgae Poterioochromonas malhamensis, and the beta-1, 3-glucan and rosmarinic acid are subjected to a simple esterification reaction to prepare the microalgae beta-1, 3-glucan-rosmarinic acid, so that the yield of the microalgae beta-1, 3-glucan-rosmarinic acid is increased, and the yield of the microalgae beta-1, 3-glucan-rosmarinic acid is increased. The microalgae beta-1, 3-glucan-rosmarinic acid conjugate can improve the antioxidant activity, anti-inflammatory activity and bioavailability of the rosmarinic acid, slow down the release speed of the rosmarinic acid in gastrointestinal tracts and improve the release and anti-cancer activity of the rosmarinic acid in colon.

Description

Technical Field

[0001] The present invention belongs to the technical field of the preparation of functional component delivery systems, and particularly relates to a preparation method and application of a microalgae β-1,3-glucan-rosmarinic acid conjugate. Background Art

[0002] Rosmarinic acid (RA) is a polyphenolic antioxidant with a wide range of health effects, including antibacterial, anticancer, anti-aging, antidiabetic, cardioprotective, hepatoprotective, and anti-inflammatory properties. However, RA is prone to degradation during gastrointestinal digestion, has poor stability, low absorption efficiency, and limited bioavailability due to the influence of first-pass metabolism, which limits its widespread 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 with controlled release in the colonic lesion area can enhance the therapeutic effect of acute colonic inflammation (ACS Nano, 2020, 14, 6887-6896), while 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 the cytotoxicity and apoptotic effect on AGS and CT26 cell lines (Arabian Journal of Chemistry, 2024, 17, 105402). However, these materials face challenges such as difficult biodegradation and possible systemic adverse effects.

[0003] Another approach 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 radical scavenging ability, photoprotection, antibacterial, and anti-inflammatory activities 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.) because their adhesion properties cause them to remain 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 the key to improving the bioavailability of RA and its application in the treatment of specific diseases. Summary of the Invention

[0004] The object of the present invention is to provide a preparation method and application of a microalgae β-1,3-glucan-rosmarinic acid conjugate, aiming to solve the problem that the existing RA delivery system cannot effectively exert the physiological activity of small molecules due to its limitations such as difficult biodegradation and low targeting effect on the colon.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] A microalgae β-1,3-glucan-rosmarinic acid conjugate, wherein the conjugate is a purified product obtained by an esterification reaction of rosmarinic acid and β-1,3-glucan in a mass ratio of 2-10:5;

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

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

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

[0010] (1) Dissolve rosmarinic acid in dimethyl sulfoxide to obtain a rosmarinic acid solution;

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

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

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

[0014] (5) Centrifuge the solution in the dialysis bag obtained in step (4), take the supernatant, and lyophilize to obtain the microalgae β-1,3-glucan-rosmarinic acid conjugate.

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

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

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

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

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

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

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

[0024] Use of the above-mentioned microalgal β-1,3-glucan-rosmarinic acid conjugate or the microalgal β-1,3-glucan-rosmarinic acid conjugate prepared by the above method in enhancing the physiological activity of rosmarinic acid, improving the bioavailability of rosmarinic acid, targeting the colon, and preparing anti-colorectal cancer drugs.

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

[0026] The β-1,3-glucan extracted from the microalgae Poterioochromonas malhamensis is used in the present invention to prepare microalgae β-1,3-glucan-rosmarinic acid through a simple esterification reaction with rosmarinic acid. The esterification reaction conditions are mild and the side effects of the raw materials are low. The β-1,3-glucan extracted from the microalgae Poterioochromonas malhamensis has a relatively high linearity and a low molecular weight, and it has good water solubility. The obtained microalgae β-1,3-glucan-rosmarinic acid conjugate can improve the stability of RA, enhance the physiological activities such as its own antioxidant activity and anti-inflammatory activity, effectively improve its cell absorption and bioavailability, effectively avoid degradation in the gastrointestinal physiological environment, achieve the targeted colon release of RA, improve the anti-colorectal cancer activity of RA, and has low systemic side effects and high therapeutic effects. Description of the Drawings

[0027] Figure 1 It is the NMR characterization of PβG used in the present invention.

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

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

[0030] Figure 4 It is the stability analysis of RA-PβG conjugate under different conditions in the present invention: (A) pH; (B) at different temperatures; (C) at different NaCl concentrations; (D) UV radiation; (E) under the storage condition of 4°C; (F) under the storage condition of 25°C.

[0031] Figure 5 It is the antioxidant activity (A-B), anti-inflammatory activity (C-F) and in vitro release curve (G) of RA-PβG conjugate in the present invention.

[0032] Figure 6 It is the antioxidant activity of free RA.

[0033] Figure 7 It is the cell transport map (A), transport efficiency (B), bioaccessibility (C), cell uptake (D) and bioavailability (E) of RA-PβG conjugate in the present invention.

[0034] Figure 8 It is the anti-colon cancer cell activity of RA-PβG conjugate in the present invention.

[0035] Figure 9Effect of RA-PβG conjugate on apoptosis of colon cancer cells in the present invention. Detailed implementation mode

[0036] The present invention will be further described below through specific examples. The following implementation cases should not be construed as limiting the protection scope of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention according to the above invention content and implement it specifically, which still belongs to the protection scope of the invention.

[0037] In the following examples, PβG is β-1,3-glucan, derived from the microalgae Poterioochromonas malhamensis. The extraction process is as follows: Take 1 g of freeze-dried Poterioochromonas malhamensis cell powder and add it to 10 mL of deionized water. Stir and extract at 50 °C in a water bath at a rotation speed of 500 rpm for 0.5 h, collect the supernatant, and precipitate it with 95% ethanol at -20 °C for 12 h. After precipitation, centrifuge at a centrifugal force of 4000 g at 4 °C for 20 min, discard the supernatant, collect the precipitate, wash the precipitate with 95% ethanol twice, then redissolve it with deionized water, and then treat it with DNase at 4 °C for 1 h to remove residual nucleic acids; then treat it with proteinase K at 4 °C for 2 h to remove residual proteins; the obtained solution is precipitated with 95% ethanol at -20 °C for 12 h, washed with 95% ethanol twice, then centrifuged at a centrifugal force of 4000 g at 4 °C for 10 min, discard the supernatant, collect the precipitate, wash the precipitate with 95% ethanol twice, add deionized water to redissolve it, then dialyze (MWCO = 3.5 kDa) at 4 °C for 24 h, and finally freeze-dry to obtain purified PβG for standby.

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

[0039] Through 1 HNMR, 13 C NMR, COSY, HSQC, and HMBC spectra were used to further characterize the structure of PβG, and the results are as Figure 1 shown. At 1 H NMR at approximately 2.50 ppm and 13 C NMR at approximately 39.5 ppm, strong peaks corresponding to the DMSO solvent were observed. This polysaccharide has a typical β-glycoside structure, and its 1 HNMR spectrum has chemical shifts of δ4.51, 3.68, 3.44, 3.26, and 3.20 in the head region. The 13The 13C nuclear magnetic resonance spectrum showed six significant signals at 60.93, 68.48, 72.91, 76.40, 86.27, and 103.12 ppm, respectively. The relatively pure nuclear magnetic resonance spectrum indicated that the polysaccharide was a linear glucan with fewer side chains and a purity of over 90%.

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

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

[0042] (1) Dissolve 50 mg of rosmarinic acid (RA) in 10 mL of dimethyl sulfoxide (DMSO), and stir at room temperature under nitrogen protection until completely dissolved to obtain an RA solution;

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

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

[0045] (4) Place the mixed solution obtained in step (3) in a dialysis bag (MWCO = 3.5 kDa), then immerse it in ultrapure water for dialysis for 24 h to remove residual DMSO and other small molecule substances, and change the water every 6 h;

[0046] (5) Centrifuge the solution in the dialysis bag in step (4) (8000 rpm, 10 min), take the supernatant, and freeze-dry at -80 °C to obtain the RA-PβG conjugate, denoted as 50-RA.

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

[0048] Take 400 μg each of 8-RA, 20-RA, and 50-RA and dissolve them in 1 mL of ultrapure water to obtain a 400 μg / mL RA-PβG aqueous solution. Mix 0.5 mL of the RA-PβG aqueous solution with 0.5 mL of Folin-Ciocalteu reagent and 1 mL of 10% Na 2 CO 3Mix the solutions, incubate in a water bath at 50 °C for 1 hour, cool to room temperature, let stand at room temperature for 15 minutes, and measure the absorbance at 725 nm with an enzyme-linked immunosorbent assay (ELISA) reader. Obtain the concentration of RA based on 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 the 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 were calculated to be 2.16%, 5.72%, and 16.56% respectively by the Folin-Ciocalteu method.

[0051] 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) according to the proportion of the drug on the polymer (grafting rate W%) and the following formula

[0052]

[0053] The degrees of substitution of the three RA-PβG conjugates of 8-RA, 20-RA, and 50-RA were calculated to be 0.010, 0.027, and 0.089 respectively from this formula.

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

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

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

[0057] Figure 2 For the NMR characterization of the RA-PβG conjugate. As Figure 2 of 1 1H NMR showed that 1H NMR analysis was performed on three RA-PβG conjugates with different degrees of substitution. The characteristic peak of DMSO solvent was observed at δ 2.50 ppm. After the reaction of PβG and RA, for 50-RA 1 1H NMR showed that 1H NMR analysis was performed on three RA-PβG conjugates with different degrees of substitution. The characteristic peak of DMSO solvent was observed at δ 2.50 ppm. After the reaction of PβG and RA, for 50-RA 1In the 1H NMR spectrum, the area ratio of the signal peaks at δ 5.20 ppm and δ 4.65 ppm is approximately 1:1, indicating that the hydroxyl proton signal at C6 of PβG disappears, suggesting that the hydroxyl group at C6 of PβG participates in the reaction. When the region of 8.1 - 6.0 ppm in the 1H NMR spectra of 8-RA, 20-RA, and 50-RA is magnified, aromatic ring hydrogen signals are observed at δ 7.06 - 6.49 ppm, indicating that RA reacts with PβG. The new characteristic spectra of the 50-RA conjugate appear in the range 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 the successful preparation of the conjugate.

[0058] (2) FITR Characterization of RA-PβG Conjugates

[0059] In the spectrum of RA ( Figure 3 in A), characteristic peaks of O-H stretching vibration are observed at 3370 cm-1 and 3232 cm-1 respectively, C=O stretching vibration at 1682 cm-1, aromatic ring C=C stretching vibration and phenolic C-O stretching vibration corresponding to 1605 cm-1 and 1525 cm-1 respectively, and phenolic C-O stretching vibration at 1278 cm-1. In addition, aromatic ring C-H bending vibration appears between 864 and 684 cm-1. For PβG, the broad bands at 3433 cm-1 and 2922 cm-1 are respectively attributed to the overlap of O-H stretching vibration and C-H stretching vibration, while the peaks at 1075 cm-1 and 1039 cm-1 correspond to C-O-C stretching vibration. 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 C-H stretching vibration of PβG. From 8-RA to 50-RA, with the increase of the substitution degree, the intensities of the O-H and C-H bands at 3433 cm-1 and 2922 cm-1 decrease. At the same time, the intensities of the ester-related peak at 1690 cm-1 and the RA-related peak at 1520 cm-1 increase, reflecting a higher level of substitution degree. The peak at 888 cm-1 becomes more obvious with the increase of the substitution degree, indicating enhanced C-H deformation vibration. These spectral changes together indicate the successful coupling of RA and PβG and highlight the structural modifications associated with the increase of the substitution degree.

[0060] (3) UV-vis Analysis of RA-PβG Conjugates

[0061] The coupling effect of RA and PβG was verified by UV-Vis spectral analysis ( Figure 3In B). RA has absorption peaks at approximately 326 nm and 288 nm. Characteristic absorption bands at 283 - 284 nm and 323 nm were observed in the spectra of 8 - RA, 20 - RA, and 50 - RA conjugates, while corresponding absorption peaks were not observed in the range of 200 to 400 nm for PβG, indicating that RA has been successfully conjugated to PβG. As the degree of substitution increases, the absorption intensity enhances.

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

[0063] The crystal structure of RA - PβG conjugates was further determined by XRD ( Figure 3 In C). RA shows sharp and distinct peaks at 2θ = 6°, 17°, 21°, 24°, 27°, and 28°, indicating its highly crystalline and ordered crystal structure. PβG shows two prominent diffraction peaks at approximately 2θ = 6° and 21°, indicating its semi - crystalline structure. In the RA - PβG conjugate, the peak at 2θ = 6° was observed, but with the increase in the degree of substitution, the intensity decreased. In addition, the peak at 21° shifted to a higher angle (22°) and the intensity also decreased, indicating that coupling occurred.

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

[0065] The thermal stability and degradation of RA - PβG conjugates were evaluated by TG - DSC analysis. In Figure 3 In D - E, the thermogravimetric curves show that the initial weight loss of PβG is 10.46% between 30°C and 152°C. A significant mass loss of 73.25% occurs between 285°C and 325°C, and the peak degradation temperature (Tp) is 301°C. After 400°C, the sample tends to be stable. For RA, the initial weight loss is 10.1% between 30°C and 125°C, and a further loss of 73.39% occurs between 288°C and 314°C, with Tp approximately 301.54°C. Similar to PβG, RA is also stable after 400°C. The three RA - PβG conjugates (8 - RA, 20 - RA, and 50 - RA) all show two - stage weight loss. The initial weight loss temperatures of 8 - RA, 20 - RA, and 50 - RA are delayed to 102°C, 195°C, and 227°C respectively. In the second stage, these conjugates lose 71.03%, 63.78%, and 43.27% of their mass respectively. The Tp values of 8 - RA, 20 - RA, and 50 - RA are delayed to 306.34°C, 306.98°C, and 309.83°C respectively. This indicates that the thermal stability increases correspondingly with the increase in the degree of substitution, following the order: 50 - RA > 20 - RA > 8 - RA > RA.

[0066] (6) SEM Characterization of RA-PβG Conjugates

[0067] The surface morphologies of PβG and RA-PβG conjugates were characterized by scanning electron microscopy (SEM) ( Figure 3 F-I in it are PβG, 8-RA, 20-RA, and 50-RA in sequence). PβG presented a rough and amorphous surface morphology. With the increase in the degree of substitution, the surface morphology of the RA-PβG conjugate gradually changed. The surface morphology of 8-RA was similar to that of PβG, while the amorphous structures of 20-RA and 50-RA gradually decreased and became smoother due to the gradual increase in the grafted RA fragments, indicating that a coupling reaction occurred 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 freeze-dried RA-PβG conjugate sample obtained in Example 1 was dissolved in ultrapure water at 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 with different pH values (1.0, 2.0, 3.0, 5.0, 7.0, 9.0, and 11.0) (the pH was adjusted with hydrochloric acid or sodium hydroxide), and left to stand at 25 °C for 3 hours. The treated samples were ultrafiltered with a 10 kDa ultrafiltration tube, and the Folin-Ciocalteu method was used to measure the retention rate of RA to evaluate the stability of the RA-PβG conjugate under different pH conditions. As Figure 4 shown in A in it, in a strong acid environment, free RA showed poor stability, and the retention rates at pH 1.0 and pH 2.0 were 41.81% and 43.28% respectively. After binding with PβG, the retention rate of RA in the conjugate increased. The retention rates of RA in 50-RA at pH 1.0 and pH 2.0 were 65.34% and 71.28% respectively. In the range of pH 3.0 - 7.0, there was no significant effect on the stability of RA. However, when the pH was higher than 7.0, the stability of 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 contrast, the retention rates of 8-RA, 20-RA, and 50-RA at pH 9.0 were 76.54%, 81.54%, and 89.58% respectively; at pH 11.0, the retention rates were 52.56%, 62.64%, and 88.80% respectively. The results showed that compared with free RA, RA in the RA-PβG conjugate showed better pH stability, and the higher the degree of substitution, the better the stability.

[0072] b. Thermal stability

[0073] Dissolve the freeze-dried RA-PβG conjugate sample obtained in Example 1 in ultrapure water at a concentration of 1 mg / mL. Incubate 1.0 mL of the freshly prepared RA-PβG conjugate sample solution in a water bath at 50, 60, 70, 80, and 90 °C for 60 minutes respectively. Ultrafilter the treated samples using a 10 kDa ultrafiltration tube, and adopt the Folin-Ciocalteu method to measure the retention rate of RA to evaluate the thermal stability of the RA-PβG conjugate. As Figure 4 shown in B below, as the temperature increases, the retention rate of free RA decreases, reaching the lowest level of 38.49% at 90 °C. At 90 °C, the RA retention rates of 8-RA, 20-RA, and 50-RA are 64.12%, 77.73%, and 84.47% respectively. All RA-PβG conjugates can effectively inhibit the thermal degradation of RA, showing a positive correlation with the degree of substitution.

[0074] c. Ionic stability

[0075] Dissolve the freeze-dried RA-PβG conjugate sample obtained in Example 1 in ultrapure water at a concentration of 1 mg / mL. Mix 1.0 mL of the freshly prepared RA-PβG conjugate sample solution with equal volumes of NaCl solutions at different concentrations (0, 50, 100, 250, 500, and 1000 mM) respectively, and let it stand for 24 hours. Ultrafilter the treated samples using a 10 kDa ultrafiltration tube, and adopt the Folin-Ciocalteu method to measure the retention rate of RA to evaluate the ionic strength stability of the RA-PβG conjugate. As Figure 4 shown in C below, as the ionic strength increases, the stabilities of free RA and RA-PβG conjugate decrease. Compared with the two conditions of pH and thermal stability, the influence of ionic strength on RA and RA-PβG conjugate seems to be smaller. In 1 M NaCl solution, the RA retention rates in free RA, 8-RA, 20-RA, and 50-RA are 81.01%, 86.15%, 87.78%, and 91.32% respectively. All RA-PβG conjugates can enhance the stability of RA in a wide range of ionic strengths.

[0076] d. Photostability

[0077] Dissolve the freeze-dried RA-PβG conjugate sample obtained in Example 1 in ultrapure water at a concentration of 1 mg / mL. Place 1.0 mL of the freshly prepared RA-PβG conjugate sample solution in a colorless glass tube respectively for the photostability test. At 25 °C, the distance is 30 cm, 254 nm, 17 μW / cm 2Irradiate under ultraviolet light for 6 h, ultrafilter the treated sample with a 10 kDa ultrafiltration tube, and use the Folin-Ciocalteu method to determine the retention rate of RA to evaluate the photostability of the RA-PβG conjugate. As Figure 4 shown in D of , as the ultraviolet light exposure time increases, the retention rate of free RA gradually decreases to 66.35% after 6 h, and the RA degradation rate of all RA-PβG conjugates is only 11.52% - 22.26%. Among them, the degradation rate of the 50-RA conjugate is the lowest, at 11.52%.

[0078] e. Storage stability

[0079] Dissolve the freeze-dried RA-PβG conjugate sample obtained in Example 1 in ultrapure water at a concentration of 1 mg / mL. Put 1.0 mL of the freshly prepared RA-PβG conjugate sample solution into glass tubes respectively, and explore the storage stability of the RA-PβG conjugate in ultrapure water under the conditions of 4 °C and 25 °C without light. Ultrafilter the treated sample with a 10 kDa ultrafiltration tube, and use the Folin-Ciocalteu method to determine the retention rate of RA to evaluate the storage stability of the RA-PβG conjugate. As Figure 4 shown in E - F of , during storage, the retention rates of both free RA and the RA-PβG conjugate decrease. After storing for 10 d at 4 °C and 25 °C, the retention rates of free RA are 63.41% and 47.77% respectively. In contrast, the RA-PβG conjugate shows a higher retention rate. At 4 °C, the retention rates of 8-RA, 20-RA, and 50-RA are 71.04%, 74.38%, and 84.10% respectively, which are 1.12 times, 1.17 times, and 1.33 times that of free RA respectively. At 25 °C, their retention rates are 54.44%, 63.85%, and 73.73% respectively, which are 1.14 times, 1.34 times, and 1.54 times that of free RA respectively. These results indicate that the RA-PβG conjugate can effectively improve the stability of RA.

[0080] (2) Antioxidant and anti-inflammatory activities of the RA-PβG conjugate.

[0081] a. In vitro antioxidant activity of the RA-PβG conjugate

[0082] Use an in vitro chemical model to explore the antioxidant activity of the RA-PβG conjugate. First, mix 1 mL of DPPH solution with 0.2 mL of samples with different concentrations (RA, PβG, and the RA-PβG conjugate). After incubating in the dark for 0.5 h, record the absorbance of the solution at 517 nm.

[0083]

[0084] A1 is the absorbance of the sample, A 0 is the absorbance of the blank group (without adding the sample).

[0085] Mix 2.45 mM potassium persulfate with 7 mM ABTS (1:1, v / v) to prepare the ABTS stock solution, and completely dissolve it in the dark for 16 h. Dilute it with 70% ethanol until the absorbance at 517 nm is maintained at about 0.70. Then, add 0.075 mL of various samples with different concentrations (RA, PβG, and RA-PβG conjugate) to the diluted ABTS solution (1 mL) and let it stand for 6 min. Measure the ABTS activity at the absorbance value of 517 nm.

[0086]

[0087] A 1 is the absorbance of the sample, A 0 is the absorbance of the blank group (without adding the sample).

[0088] The results are as Figure 5 shown in A - B. At the concentration of 166.7 μg / mL, the DPPH radical scavenging rates of PβG, 8 - RA, 20 - RA, and 50 - RA are 18.78%, 35.39%, 52.87%, and 82.90% respectively. At the concentration of 69.8 μg / mL, the ABTS radical scavenging rates of PβG, 8 - RA, 20 - RA, and 50 - RA are 5.32%, 14.69%, 20.36%, and 71.63% respectively. In addition, as Figure 6 shown, the effective half - inhibitory concentrations (IC 50 ) of free RA for scavenging DPPH and ABTS radicals are 5.27 μg / mL and 9.54 μg / mL respectively. The higher the degree of substitution of the RA - PβG conjugate, the stronger the antioxidant ability. The antioxidant activity of PβG is relatively low, and the DPPH scavenging ability is less than 20% at the highest test concentration, and there is almost no inhibitory effect on ABTS scavenging at 69.8 μg / mL. The IC 50 values of 50 - RA for scavenging DPPH and ABTS radicals are 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 are lower than those of free RA, showing enhanced antioxidant activity.

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

[0090] Use lipopolysaccharide (LPS) to stimulate RAW264.7 cells to produce an inflammatory response. At 5×10 5RAW264.7 cells were cultured overnight in a 24-well plate at a density of Figure 5 cells / well. The cells were incubated with 100 μL of LPS (1 μg / mL) with or without RA, PβG, and aqueous solutions of the RA-PβG conjugate for 24 h. NO and cytokines (TNF-α, IL-6, and IL-1β) in the cell supernatants were detected according to the kit detection procedure. Specifically, the concentrations of aqueous solutions of PβG and the RA-PβG conjugate (8-RA, 20-RA, and 50-RA) were 100 μg / mL. The RA concentrations in the 8-RA, 20-RA, and 50-RA conjugates corresponded to 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, the same as 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-fold compared with the control group ( Figure 5 in 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 by 4.19-fold after LPS exposure ( Figure 5 in D). Compared with the LPS group, the IL-1β levels 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 IL-6 level decreased after the cells were treated with the samples, and the inhibition rates were 9.15% (RA), 11.11% (PβG), 28.76% (8-RA), 42.48% (20-RA), and 76.91% (50-RA) ( 5 in E). In addition, the TNF-α level increased from 0.13 × 10 5 ± 0.07 ng / L to 4.80 × 10 Figure 5 ± 0.05 ng / L, an increase of 36.92-fold after LPS stimulation (

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

[0092] a. Transport efficiency

[0093] The transmembrane transport of RA-PβG was investigated using Caco-2 cell monolayers, and the schematic diagram is shown in Figure 7 A as shown. Caco-2 cells at a density of 1×10 2 cells / well were seeded on Transwell (12-well, 0.4 μm, 1.12 cm 5 surface area) to evaluate the cellular transport efficiency of RA-PβG conjugates (8-RA, 20-RA, 50-RA). During the incubation at 37 °C and 5% CO 2 2, the transepithelial electrical resistance (TEER) values were monitored. When the resistance value reached 500 Ω·cm 2 , the cell monolayer was ready for cell transport. In the apical-to-basolateral (AP-to-BL) experiment, 1.5 mL of blank HBSS was added to the BL side, and 0.5 mL of free RA (83 μg / mL), sample solutions (PβG and RA-PβG conjugates: both at a concentration of 500 μg / mL, and the RA contents in 8-RA, 20-RA, and 50-RA were 11, 29, and 83 μg / mL, respectively) were added to the AP side. To determine the transported amount, the basolateral solutions were taken at two different incubation times (2 h and 4 h). The following formula was used to calculate the transport efficiency of RA:

[0094]

[0095] where M BL is the amount of sample loaded on the BL side (μg), and M Inital is the amount of sample added on the AP side (μg).

[0096] The results are shown in Figure 7 B. For RA and RA-PβG conjugates, the transport efficiency was time-dependent. After 2 h 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 h, the transport efficiencies increased to 17.88% (RA), 37.83% (8-RA), 47.09% (20-RA), and 60.31% (50-RA). With the increase in the degree of substitution in RA-PβG conjugates, the transport efficiency increased. The results showed that RA-PβG conjugates significantly improved the transport efficiency of RA, with an increase rate of up to 373%.

[0097] b. Bioaccessibility

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

[0099] Prepare each digestive fluid - Oral digestive fluid SSF: 15.1 mL of 0.5 M KCl solution, 3.7 mL of 0.5 M KH 2 PO 4 solution, 6.8 mL of 1 M NaHCO 3 solution, 2.72 mL of 2 M NaCl solution, 0.5 mL of 0.15 M MgCl 2 (H 2 O) 6 solution, 0.06 mL of (NH 4 ) 2 CO 3 solution, and make up to 400 mL with ultrapure water. Dissolve 1.5 g of mucin in 400 mL of SSF to obtain the oral digestive fluid SSF. Gastric digestive fluid SGF: 6.9 mL of 0.5 M KCl solution, 0.9 mL of 0.5 M KH 2 PO 4 solution, 12.5 mL of 1 M NaHCO 3 solution, 18.05 mL of 2 M NaCl solution, 0.4 mL of 0.15 M MgCl 2 (H 2 O) 6 solution, 0.5 mL of 0.5 M (NH 4 ) 2 CO 3 solution, and make up to 400 mL with ultrapure water. Small intestine digestive fluid SIF: 6.8 mL of 0.5 M KCl solution, 0.8 mL of 0.5 M KH 2 PO 4 solution, 42.5 mL of 1 M NaHCO 3 solution, 30.85 mL of 2 M NaCl solution, 1.1 mL of 0.15 M MgCl 2 (H 2 O) 6 solution, 0.5 mL of (NH 4 ) 2 CO 3 solution, and make up to 400 mL with ultrapure water.

[0100] Oral digestion stage: Sequentially add the samples (RA, 8-RA, 20-RA, and 50-RA) at 10 mg / mL, 8 mL of SSF, 50 μL of 1.5 mM CaCl 2 、1 mL of 291.83 mg mL -1Mix α - amylase with 0.95 mL of ultrapure water. Incubate with continuous shaking at 37 °C and 150 rpm for 2 min. Adjust the pH to 7. Gastric phase: Sequentially add 20 mL of the above oral experimental solution and mix with 16 mL of SGF. Adjust the pH to 3 with 1 M HCl, and then add 2 mL of 19.32 mg mL -1 pepsin, 10 μL of 0.15 mM CaCl 2 , and make up the volume to 40 mL with ultrapure water. Incubate with continuous shaking at 37 °C and 150 rpm for 2 h. Intestinal phase: Sequentially add 20 mL of the gastric experimental solution, 8.5 mL of SIF, 5 mL of 57.54 mg mL -1 pancreatin, 2.5 mL of 171.38 mg mL -1 bile salts, 40 μL of 0.6 mM CaCl 2 , adjust the pH to 7 with 1 M NaOH, and make up the volume to 40 mL with ultrapure water. Incubate with continuous shaking at 37 °C and 150 rpm for 2 h. After the gastric digestion and intestinal digestion stages are completed, centrifuge the resulting mixture at 5000 rpm at 4 °C for 10 min, take the supernatant, and use the same procedure with a blank digestion solution without the sample as a reference. Test by the Folin - Ciocalteu method, subtract the background of the blank digestion solution, and calculate the bioaccessibility using the following formula:

[0101]

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

[0103] The RA in the digestion supernatant is considered the absorbable part and is defined as bioaccessibility. As Figure 7 shown by C in, the bioaccessibility of free RA is 32.71%, and the degradation rate is relatively fast. For the RA - PβG conjugate, the bioaccessibilities of 8 - RA, 20 - RA, and 50 - RA are 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. Cell uptake and bioavailability

[0105] Caco - 2 cells are cultured in DMEM medium supplemented with 20% FBS and cultured to the 21st passage in a humidified atmosphere of 37 °C and 5% CO 2 . The cultured cells are seeded in a 24 - well plate at a density of 1×10 5Cells / well. After 14 days, the cells were observed under a microscope to form a monolayer with tight fusion. They were washed 3 times with PBS buffer to remove impurities on the cell surface, and then a digestion sample diluted with HBSS buffer (free RA: 83 μg / mL, RA-PβG conjugate: 500 μg / mL) was added. After culturing for 4 h, pre-cooled HBSS buffer was added to terminate cell uptake. The cells were lysed on ice for 30 min with cell lysate (PBS containing 10% ethanol) and then sonicated (switch on for 10 s / 10 s, power 35%). After centrifuging the lysate (10,000 rpm, 2 min), the supernatant was taken, and the cellular uptake of RA was calculated according to the following formula:

[0106]

[0107] C Cell is the content of RA after cellular uptake, and C Total is the content of RA before cellular uptake.

[0108] The bioavailability was calculated according to the following formula:

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

[0110] F B is the bioaccessibility of the sample, and F A is the cellular uptake of the sample.

[0111] The results of cellular uptake efficiency are shown in Figure 7 D as shown below. After 4 h, there were significant differences in the cellular uptake of free RA and RA-PβG conjugate. Compared with free RA (19.20%), grafting PβG significantly improved the cellular uptake ability 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 could increase the cellular uptake rate of RA.

[0112] Based on the bioaccessibility and cellular uptake, the bioavailability of free RA was 6.24%, which was significantly lower than that of RA-PβG conjugates (12.17%, 24.33%, 31.56%), as shown in Figure 7 E as shown below. The bioavailabilities of 8-RA, 20-RA, and 50-RA were increased by 1.95-fold, 3.90-fold, and 5.06-fold, respectively, compared with free RA.

[0113] (4) Colon-targeted release performance

[0114] In vitro release tests of 50-RA and free RA were conducted as follows. The process was as follows: Simulated gastric fluid (SGF): 0.1M HCl (pH 1.2), 0 - 2h; Simulated intestinal fluid (SIF): Phosphate solution (pH 6.8), 2 - 6h; Simulated colon fluid (SCF): Phosphate solution (pH 7.4), 6 - 30 h. A dialysis bag (3500Da) was filled with 5 mL of 1.66 mg / mL RA solution or 10 mg / mL 50-RA solution (equivalent to 8.3 mg RA). The bag was immersed in a centrifuge tube containing 5 ml of release medium, placed in an incubator at a rotation speed of 150 rpm and a temperature of 37 ± 1°C. To maintain stable precipitation conditions, an equal amount of release medium was taken out at predetermined time intervals and replaced with fresh release medium.

[0115] The in vitro release curve of 50-RA was selected to evaluate the colon-targeting ability under simulated gastrointestinal conditions (SGF, SIF, and SCF) for 30 hours ( Figure 5 In these conditions, free RA was rapidly released within the first 2 hours, reaching 75.45%, due to the influence of the highly acidic environment, and was completely degraded during the subsequent 4-hour SIF digestion. This indicates that free RA is very sensitive to acidic conditions. In contrast, the RA-PβG conjugate (50-RA) released RA more slowly in these environments, and the cumulative release of RA in SGF and SIF was only 27.86%, confirming the improved stability. During this process, some of the released RA could pass through the dialysis membrane due to its low molecular weight. The RA in 50-RA reached complete release in SCF, and the release continued until 30 hours, indicating that the RA-PβG conjugate has good colon-targeted release ability.

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

[0117] To further evaluate the colon-targeting ability of the RA-PβG conjugate, human colorectal cancer cell lines CT26, HT29, and human hepatocellular carcinoma cell line HepG2 were cultured at a density of 1×10 4 cells / well and co-incubated with RA, PβG, and 50-RA for 24 h. AnnexinV-FITC / PI kits and flow cytometry were used to qualitatively and quantitatively test cell apoptosis.

[0118] The cytotoxicity of PβG, free RA, and 50-RA was as Figure 8 shown in A-I. PβG had certain cytotoxicity to CT26 and HT29 cells. At the highest concentration (500 μg / mL), the cell viabilities of CT26 and HT29 cells were 70.78% and 81.56% respectively (see Figure 8 A, D), while it had no obvious cytotoxicity to HepG2 cells (seeFigure 8 In G), it shows colon targeting ability. Similarly, free RA (see Figure 8 in B, E, H) and 50-RA (see Figure 8 in C, F, I) reduced the viability of CT26 and HT29 cells to a certain extent, but had no toxic effect on the viability of HepG2 cells. At the same concentration, 50-RA was significantly more toxic to CT26 and HT29 cells than PβG and free RA. Especially at 500 μg / mL, the survival rates of CT26 and HT29 cells in 50-RA were 37.67% and 52.84% respectively, much lower than the survival rates of CT26 and HT29 cells in free RA, which were 61.30% and 61.36% respectively.

[0119] Cultivate human colon cancer cell lines CT26, HT29 and human liver cancer cell line HepG2 at a density of 1×10 4 cells / well. Further, after AnnexinV-FITC / PI staining and co-incubating PβG, 50-RA and free RA (the concentrations of PβG and 50-RA are both 500 μg / mL, and the concentration of free RA is 83 μg / mL) with the stained cells for 24 h, the effects on cell apoptosis are shown in Figure 9 A. For the control group of CT26, HT29 and HepG2 cell lines, 97.20%, 96.20% and 97.10% of the normal cells survived and did not undergo apoptosis. After PβG and free RA acted on CT26 and HT29 cells, the apoptosis rates of CT26 and HT29 cells by PβG were 16.31% and 11.00% respectively, and the apoptosis rates of CT26 and HT29 cells by free RA were 19.40% and 12.62% respectively; the apoptosis rates of CT26 and HT29 cells by 50-RA were 29.18% and 23.96% respectively (see Figure 9 B, C). The apoptosis performances of HepG2 cells treated with PβG, RA and 50-RA were relatively low, which were 3.30%, 5.52% and 6.63% respectively (see Figure 9 D), which was consistent with the cytotoxicity results, indicating that the RA-PβG conjugate has colon targeting characteristics and promotes apoptosis of colon cancer cells to a greater extent than free RA.

Claims

1. A microalgae β-1,3-glucan-rosmarinic acid conjugate, characterized in that: The conjugate is a purified product of esterification reaction between rosmarinic acid and β-1,3-glucan at a mass ratio of 2-10:5; The β-1,3-glucan is extracted from the microalgae Poterioochromonas malhamensis, with a purity of more than 90%.

2. The microalgae β-1,3-glucan-rosmarinic acid conjugate according to claim 1, characterized in that: The molecular weight of the β-1,3-glucan is 15.5 kDa-17.0 kDa.

3. A method for preparing the microalgae β-1,3-glucan-rosmarinic acid conjugate according to any one of claims 1 to 2, characterized in that: The steps include: (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 period of time; (3) adding β-1,3-glucan to the solution obtained in step (2), and stirring for a certain period of time to carry out an esterification reaction; the β-1,3-glucan is obtained by water extraction and alcohol precipitation from the microalgae Poterioochromonas malhamensis, and has a purity of more than 90%; (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), taking the supernatant, and freeze-drying the supernatant to obtain a microalgae β-1,3-glucan-rosmarinic acid conjugate.

4. The preparation method according to claim 3, characterized in that: 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; and / or The molecular weight of the β-1,3-glucan is 15.5 kDa-17.0 kDa, preferably 16.1 kDa-16.5 kDa.

5. The preparation method according to claim 3, characterized in that: In step (2), stirring for 1-3 hours, preferably 2 hours; and / or In step (3), stirring is performed for 18-30 hours, preferably 24 hours.

6. The preparation method according to claim 3, characterized in that: In step (2), the molar ratio of rosmarinic acid, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine is 0.4-2:1:1, preferably, the molar ratio is 0.8-1.2:1:1, and more preferably, the molar ratio is 1:1:

1.

7. The preparation method according to claim 3, characterized in that: In step (4), the molecular weight cutoff MWCO of the dialysis bag is 3.5 kDa.

8. The preparation method according to claim 3, characterized in that: In step (1), the concentration of the rosmarinic acid solution is 0.5-10 mg / mL.

9. The preparation method according to claim 3, characterized in that: Steps (1) to (3) are all carried out under nitrogen protection.

10. Use of the microalgae β-1,3-glucan-rosmarinic acid conjugate according to any one of claims 1 to 2 or the microalgae β-1,3-glucan-rosmarinic acid conjugate prepared by the preparation method according to any one of claims 3 to 9 in improving the physiological activity of rosmarinic acid, improving the bioavailability of rosmarinic acid, targeting the colon and preparing anti-colon cancer drugs.

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