A compound of thiol-containing cyclodextrin and spirulina, and its preparation method and application

Abstract

The present invention discloses a complex containing thiol cyclodextrin and spirulina, and its preparation method and application. The present invention first prepares a thiol cyclodextrin-active factor inclusion compound and a spirulina-active factor complex respectively, and then obtains the complex containing thiol cyclodextrin and spirulina through electrostatic interaction. The complex containing thiol cyclodextrin and spirulina provided by the present invention can not only enrich the types of microalgae complexes, but also can achieve stable and efficient delivery of active factors by being resistant to gastric acid and clearing gastrointestinal ROS. It can also achieve synergistic intestinal targeting through passive targeting by mutual entanglement with intestinal villi and active targeting by oxidation of intestinal mucus to form disulfide bonds, greatly improving the bioavailability of the active factors. In addition, the complex containing thiol cyclodextrin and spirulina described in the present invention can also be used for the preparation of intestinal-targeted foods or intestinal-targeted drugs.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a compound of thiol-containing cyclodextrin and spirulina, and a preparation method and application thereof. Background Art

[0002] The antioxidant properties of polyphenols can play a preventive role in chronic diseases such as Alzheimer's disease. However, after ingestion, they are easily degraded by gastric acid and enzymes in the stomach, making them ineffective in targeting the intestine. Furthermore, the abundant reactive oxygen species (ROS) in the intestine can oxidize polyphenols into quinones, rendering them ineffective, resulting in low bioavailability. To address these issues, research has developed delivery vehicles based on pH differences in the gastrointestinal tract to achieve intestinal-targeted delivery of polyphenols. However, these vehicles undergo a relatively slow pH response before targeted release, which can lead to delayed release. Alternatively, intestinal targeting can be achieved through non-covalent interactions (hydrogen bonding, electrostatic interactions, etc.) with electrolytes and ions in the intestinal fluid. However, the degree of response often varies with pH, ​​temperature, ion concentration, and other factors, making the system unstable. Some systems are even prone to cytotoxicity due to their positive charge. Therefore, the development of a stable, reliable, and efficient delivery vehicle is of great significance for the absorption of intestinal-targeted drugs and foods.

[0003] Spirulina is a spiral-shaped blue algae with an outer diameter of 3 to 5 μm and 14 to 16 nm micropores on its surface. Its main component is chlorophyll, which has green fluorescence. Due to its unique structure and performance characteristics, it has broad application prospects in the field of active factor delivery. For example, in [D. Zhong, D. Zhang, W. Chen, J. He, C. Ren, X. Zhang, N. Kong, W. Tao, M. Zhou. Orally deliverable strategy based on microalgal biomass for intestinal disease treatment. Sci. Adv., 2021, 7: eabi9265.], curcumin was loaded into the cavity of Spirulina to prepare a multi-purpose preparation based on Spirulina for the treatment of two intestinal diseases, colitis and colon cancer.

[0004] The existing technology only reports on loading nutrients into the cavity of Spirulina, and there is no relevant research on loading active factors on the complex of thiolcyclodextrin and spirulina. Whether loading active factors on the complex of thiolcyclodextrin and spirulina can achieve better delivery effect for intestinal targeted drugs has important practical significance for realizing efficient delivery of intestinal targeted drugs and food. Summary of the Invention

[0005] In order to solve the above problems, the present invention aims to construct a gastric acid-resistant, efficient and stable intestinal-targeted delivery carrier for active factors.

[0006] Based on the above objectives, the present invention provides a complex containing thiol cyclodextrin and spirulina. Based on electrostatic interaction, a thiol cyclodextrin-functionalized spirulina complex is constructed, which can synergize through active targeting by entanglement with intestinal villi and passive targeting by forming covalent disulfide bonds with intestinal mucus. The preparation raw materials include thiol cyclodextrin, spirulina and active factors.

[0007] Preferably, the active factor in the preparation raw material of the present invention is any one of fat-soluble polyphenols, drugs or probiotics.

[0008] More preferably, the active factor in the raw material for preparation of the present invention is resveratrol, and the total drug loading capacity of the complex of thiol-containing cyclodextrin and spirulina provided by the present invention to resveratrol is 63.4% to 78.3%.

[0009] Based on the above-mentioned preparation raw materials, the complex containing thiol cyclodextrin and spirulina provided by the present invention includes a thiol cyclodextrin-active factor inclusion complex (i.e., a thiol cyclodextrin@active factor inclusion complex) and a spirulina-active factor complex (i.e., a spirulina@active factor complex). By mass, the ratio of the spirulina@active factor complex to the thiol cyclodextrin@active factor inclusion complex is 1:0.8~100.

[0010] Preferably, the ratio of the spirulina@active factor complex to the mercaptocyclodextrin@active factor inclusion complex is 1:10 by mass.

[0011] Furthermore, in terms of molar ratio, the ratio of thiolcyclodextrin and active factor in the thiolcyclodextrin@active factor inclusion complex is 1:0.5-5; in terms of mass ratio, the ratio of spirulina and active factor in the spirulina@active factor complex is 1:0.005-0.02.

[0012] Preferably, the molar ratio of the thiol-cyclodextrin and the active factor in the thiol-cyclodextrin@active factor inclusion complex is 1:1; the mass ratio of the spirulina and the active factor in the spirulina@active factor complex is 1:0.01.

[0013] Furthermore, the present invention also provides a method for preparing the complex containing thiol cyclodextrin and spirulina, comprising: loading the active factor onto thiol cyclodextrin and spirulina to obtain a thiol cyclodextrin@active factor inclusion compound and a spirulina@active factor complex; dispersing the spirulina@active factor complex into water to form a concentration of 0.5 mg / mL -1 The dispersion of the thiol cyclodextrin @ active factor inclusion complex is dissolved in water to form a concentration of 10 mg mL-1 and then mixing the dispersion and the solution in a volume ratio of 1:0.5 to 100 at a temperature of 20 to 37 ° C and a rotation speed of 100 to 500 rpm for 1 to 48 hours to obtain the complex of the thiol-containing cyclodextrin and spirulina.

[0014] In addition, the present invention also provides the use of the complex of the thiol-containing cyclodextrin and spirulina in an intestinal-targeted functional food or an intestinal-targeted drug.

[0015] Through the above technical solution, combined with the embodiments, the complex of thiol-containing cyclodextrin and spirulina provided by the present invention has at least the following beneficial effects or advantages:

[0016] 1. The drug loading capacity of the complex of thiol-containing cyclodextrin and spirulina provided by the present invention for resveratrol is 63.4% to 78.3%, which is significantly higher than the drug loading capacity of thiol-containing cyclodextrin for resveratrol or the drug loading capacity of spirulina for resveratrol.

[0017] 2. The complex of thiol-containing cyclodextrin and Spirulina provided by the present invention exhibits significant adsorption capacity for mucin, with an adsorption capacity of 0.616±0.013 mg.

[0018] 3. The present invention simulates the release behavior of the complex containing thiol cyclodextrin and spirulina in gastric and intestinal fluids. The results show that the release of resveratrol within 2 hours is only 9.51% ± 1.19%. As the cyclodextrin and spirulina are gradually digested in the intestine, the structure of the complex is also destroyed, and resveratrol is rapidly released, reaching 91.12 ± 2.98% at 8 hours. This shows that the complex system of thiol cyclodextrin and spirulina constructed in the present invention can protect nutrients from being destroyed by gastric acid and achieve stable intestinal targeted release of active factors.

[0019] 4. After resveratrol is loaded into a complex of thiol-containing cyclodextrin and spirulina in the present invention, due to the complex's gastric acid resistance, synergistic intestinal targeting and intestinal ROS scavenging effects, the bioavailability of resveratrol after targeted delivery of resveratrol to the intestine reaches 84.98% ± 2.45%, which is 2.12 times that of free resveratrol, indicating that the complex of thiol-containing cyclodextrin and spirulina described in the present invention can significantly improve the bioavailability of resveratrol.

[0020] 5. The present invention compares the effects of the compound containing thiol cyclodextrin and spirulina and free resveratrol on ·OH, ·O2 before and after digestion. - ,·ABTS free radical scavenging effect found that before digestion, the two were respectively - There is no significant difference in the scavenging rate of ·ABTS free radicals; after digestion, the complex of the present invention containing thiol cyclodextrin and spirulina has no significant difference in the scavenging rate of ·OH, ·O2 -The scavenging rates of ABTS free radicals were significantly higher than those of resveratrol, indicating that the complex of thiol-containing cyclodextrin and spirulina prepared in the present invention can protect the loaded resveratrol from being destroyed and inactivated by gastric acid and excessive ROS in the intestine. It not only shows a generally applicable scavenging effect on ROS in the intestine, but also indicates that the resveratrol after intestinal targeted release is not inactivated and still has excellent free radical scavenging activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the infrared spectrum of mercaptocyclodextrin and resveratrol.

[0022] Figure 2 These are infrared spectra of Spirulina, Spirulina@resveratrol, thiol-cyclodextrin@resveratrol, and the complex of thiol-cyclodextrin and Spirulina.

[0023] Figure 3 The zeta potential diagrams of spirulina, resveratrol, thiol-cyclodextrin and the complex of thiol-cyclodextrin and spirulina are shown in FIG.

[0024] Figure 4 These are scanning electron microscope images of Spirulina, a complex of thiol-containing cyclodextrin and Spirulina, wherein (a) is a scanning electron microscope image of Spirulina, and (b) is a scanning electron microscope image of a complex of thiol-containing cyclodextrin and Spirulina.

[0025] Figure 5 For the digestion of resveratrol and the complex of thiol-containing cyclodextrin and spirulina to ·OH, ·O2 - ,· ABTS free radical scavenging efficiency diagram.

[0026] Figure 6 This figure shows the adhesion effects of Spirulina, thiol-cyclodextrin, and the complex of thiol-cyclodextrin and Spirulina on mucin.

[0027] Figure 7 This is the release curve of the complex of thiol-containing cyclodextrin and Spirulina in simulated gastric and intestinal fluids.

[0028] Figure 8 Bioavailability of a complex of resveratrol, thiol-containing cyclodextrin, and Spirulina.

[0029] Figure 9 The complex of resveratrol and thiol-containing cyclodextrin and spirulina after digestion is used to treat ·OH, ·O2 - ,· ABTS free radical scavenging efficiency diagram. DETAILED DESCRIPTION

[0030] The technical solutions of the present invention are described below with reference to the following embodiments; however, the present invention is not limited to the following embodiments.

[0031] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents and instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0032] Example 1

[0033] This embodiment provides methods for preparing a thiol-containing cyclodextrin@resveratrol inclusion complex, a spirulina@resveratrol complex, and a complex of thiol-containing cyclodextrin and spirulina.

[0034] 1. Preparation of Spirulina@Resveratrol Complex

[0035] The methods for culturing Spirulina and preparing the Spirulina@resveratrol complex in this example refer to the orally deliverable strategy based on microalgal biomass for intestinal disease treatment. Science Advances, 2021, 7, eabi9265.

[0036] 2. Preparation of Mercaptocyclodextrin@Resveratrol Inclusion Complex

[0037] 1) Preparation of mercaptocyclodextrin

[0038] Dissolve 95 mg of thiolated cysteine ​​in 10 mL of deionized water, then add 168 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 101 mg of N-hydroxysuccinimide, and stir at room temperature for 2 h to obtain activated N-acetylcysteine. Then add 5 mL of 60 mg mL -1 The mixture was stirred and reacted for 24 hours, and then freeze-dried to obtain a crude product. Acetone was added to the crude product to wash and remove unreacted N-acetylcysteine ​​to obtain thiol cyclodextrin.

[0039] 2) Preparation of mercaptocyclodextrin@resveratrol inclusion complex

[0040] Resveratrol and thiolcyclodextrin were weighed in a molar ratio of 1:1, 38 mg of resveratrol was dissolved in 10 mL of ethanol in the dark, 333 mg of thiolcyclodextrin was dissolved in 10 mL of deionized water and stirred continuously at 45°C, then the ethanol solution of resveratrol was added to the aqueous solution of thiolcyclodextrin, and stirred continuously at 45°C for 4 hours, followed by storage at 4°C for 12 hours. After 12 hours, the mixture was centrifuged at 4500 rpm for 5 minutes, and the resulting precipitate was washed three times with 50% ethanol to remove resveratrol adsorbed on the surface of thiolcyclodextrin. The ethanol on the precipitate was then removed by rotary evaporation and freeze-dried to obtain a thiolcyclodextrin@resveratrol inclusion complex.

[0041] 3. Preparation of the complex of thiol-containing cyclodextrin and Spirulina

[0042] The spirulina@resveratrol complex is dispersed in water to obtain a spirulina@resveratrol complex dispersion, the thiolcyclodextrin@resveratrol inclusion compound is dissolved in water to obtain a thiolcyclodextrin@resveratrol inclusion compound aqueous solution, the spirulina@resveratrol complex dispersion and the thiolcyclodextrin@resveratrol inclusion compound aqueous solution are mixed in a volume ratio of 1:0.5-100, and stirred at 20-37°C and a rotation speed of 100-500 rpm for 1-48 hours to obtain a complex containing thiolcyclodextrin and spirulina.

[0043] Example 2

[0044] In this example, infrared spectroscopy was performed on resveratrol, spirulina prepared in Example 1, spirulina@resveratrol, thiol-cyclodextrin, thiol-cyclodextrin@resveratrol inclusion complex, and the complex containing thiol-cyclodextrin and spirulina.

[0045] After analysis, the infrared spectra of resveratrol and mercaptocyclodextrin are shown in the attached figure. Figure 1 As shown, according to the attached Figure 1 , 1597, 1517 and 1433cm -1 The peaks at 3300-3500 cm are the characteristic peaks of the benzene ring skeleton of resveratrol. -1 The characteristic peak is the characteristic signal of phenolic hydroxyl group. In the infrared spectrum of mercaptocyclodextrin, 1709cm -1 The characteristic peak at 1562 cm is the stretching vibration of the newly formed ester -C=O, and the -NH bending vibration of the secondary amine of N-acetylcysteine ​​(amide II band) appears at 1562 cm -1 , 943cm -1 The skeletal vibration of the α-1,4 bond in hydroxypropyl-β-cyclodextrin is 756 cm -1 It is the skeleton vibration of hydroxypropyl-β-cyclodextrin, indicating that mercaptocyclodextrin has been successfully prepared.

[0046] The infrared spectra of Spirulina, Spirulina@resveratrol, mercaptocyclodextrin@resveratrol inclusion complex and the complex containing mercaptocyclodextrin and Spirulina are shown in the attached figure. Figure 2 As shown, according to the attached Figure 2 The main component of Spirulina is chlorophyll, and the characteristic vibration peak of the carboxylic acid carbonyl group in its structure appears at 1725-1700 cm -1 The stretching vibrations of -C=N- and -C=C- appear at 1690-1620 cm -1The infrared spectrum of Spirulina@Resveratrol shows characteristic peaks of both Spirulina and resveratrol. The infrared spectrum of Thiocyclodextrin@Resveratrol shows characteristic peaks of the resveratrol benzene ring skeleton in addition to the characteristic peaks of Thiocyclodextrin, indicating the successful preparation of the inclusion complex and the complex. The characteristic peaks of Thiocyclodextrin@Resveratrol and Spirulina@Resveratrol are both visible in the infrared spectrum of the complex containing Thiocyclodextrin and Spirulina, indicating that the complex containing Thiocyclodextrin and Spirulina according to the present invention has been successfully prepared.

[0047] Example 3

[0048] In this embodiment, resveratrol, the spirulina prepared in Example 1, thiol-cyclodextrin, and the complex containing thiol-cyclodextrin and spirulina were dispersed in a 50% ethanol aqueous solution at certain concentrations, and the zeta potential was measured.

[0049] The test results are as attached Figure 3 As shown in the results, the Zeta potential of Spirulina was -28.6±0.81mV, resveratrol was almost uncharged (2.25±0.03mV), and thiolcyclodextrin was positively charged due to its amino group (26.71±1.24mV), showing the potential to bind to negatively charged intestinal mucus and Spirulina based on electrostatic attraction. Therefore, the thiolcyclodextrin-functionalized Spirulina prepared by combining the positively charged thiolcyclodextrin@resveratrol and the negatively charged spirulina almost offset the opposite charges of the two (4.14±1.42mV), effectively eliminating the cytotoxicity of the positively charged thiolcyclodextrin, and also proving the successful combination of thiolcyclodextrin@resveratrol and spirulina@resveratrol.

[0050] Example 4

[0051] In this example, the loading amount and encapsulation efficiency of the mercaptocyclodextrin@resveratrol inclusion complex, the spirulina@resveratrol complex, and the complex containing mercaptocyclodextrin and spirulina prepared in Example 1 were determined.

[0052] First, prepare a standard solution of resveratrol, measure the UV absorbance of the standard solution at 308 nm, and fit the standard curve of resveratrol:

[0053] C (mg mL -1 )=(A-0.1226) / 0.0224(R2=0.9968)

[0054] Wherein, C and A represent the concentration of resveratrol standard solution and its corresponding UV absorbance, respectively.

[0055] A certain amount of thiol-cyclodextrin-resveratrol inclusion complex was ultrasonicated in ethanol for 5 minutes and then stirred for 2 hours. For the spirulina-resveratrol complex and the complex containing thiol-cyclodextrin and spirulina, the mixture was first ground and then dispersed in ethanol with ultrasound to extract the resveratrol. The supernatant was centrifuged at 4500 rpm and the absorbance of the supernatant was measured at 308 nm. The loading capacity and encapsulation efficiency of the thiol-cyclodextrin-resveratrol inclusion complex, the spirulina-resveratrol complex, and the complex containing thiol-cyclodextrin and spirulina were calculated according to formulas (1) and (2).

[0056]

[0057]

[0058] Based on this calculation, the drug loading capacity and encapsulation efficiency of thiol cyclodextrin for resveratrol were 18.3% to 24.1% and 60.3% to 88.4%, respectively; the drug loading capacity and encapsulation efficiency of spirulina for resveratrol were 36.1% to 40.7% and 80.7% to 93.4%, respectively; and the total drug loading capacity of the complex containing thiol cyclodextrin and spirulina for resveratrol was 63.4% to 78.3%.

[0059] Example 5

[0060] This example describes the microscopic morphology characterization of the complex of Spirulina, thiol-containing cyclodextrin and Spirulina prepared in Example 1.

[0061] Under a scanning electron microscope, Spirulina appears as a spiral structure with an outer diameter of 3 to 5 μm. Figure 4 As shown in (a), thiol cyclodextrin can generate intramolecular and intermolecular hydrogen bonds, and resveratrol is a crystal, so the complex containing thiol cyclodextrin and spirulina will attract each other through intramolecular hydrogen bonds to present a row-like rigid structure, as shown in the attached figure. Figure 4 As shown in (b), the surface-covered thiol cyclodextrin has a flexible structure, as shown in Figure 4 As shown in (b), the particles embedded in the structure are formed by the aggregation of hydrophobic resveratrol.

[0062] Example 6

[0063] This example compares the effects of free resveratrol and the complex of thiol-containing cyclodextrin and spirulina prepared in Example 1 on ·OH, ·O2 - ,·ABTS free radical scavenging efficiency.

[0064] 1. Evaluation of OH removal efficiency

[0065] The Fenton reagent method was used to examine the scavenging effect of a thiol-containing cyclodextrin-spirulina complex on ·OH. The thiol-containing cyclodextrin-spirulina complex was prepared into a sample solution with a concentration of 3 mg mL⁻¹. A 9 mM ferrous sulfate solution (50 μL), a 9 mM salicylic acid-ethanol solution (50 μL), and the sample solution (50 μL) were placed in a 96-well plate and mixed evenly. Then, 50 μL of 8.8 mM hydrogen peroxide solution was added to initiate the reaction. After incubation at 37°C for 0.5 h, the absorbance at a wavelength of 510 nm was measured. The positive control group for this experiment consisted of ascorbic acid, and the blank control group consisted of distilled water. The scavenging rate of the sample for ·OH was calculated according to formula (3).

[0066]

[0067] Where A0 is the absorbance of the distilled water blank control group, A S is the absorbance of the sample, A C An equal volume of salicylic acid-ethanol solution was used instead of ferrous sulfate solution as the reagent control group.

[0068] 2. Against O2 - Evaluation of removal efficiency

[0069] The compound of thiol-containing cyclodextrin and Spirulina was prepared into a sample solution with a concentration of 2 mg / mL. The pyrogallol method was used to determine the activity of the compound of thiol-containing cyclodextrin and Spirulina on·O2 - Scavenging effect. 5.0 mL of 50 mM Tris-HCl buffer (pH 8.2) and 0.5 mL of oligosaccharide solution were mixed evenly, preheated in a 37°C water bath for 10 minutes, and 1.0 mL of 3.5 mM pyrogallol solution was added. After 6 minutes of reaction, 0.5 mL of 8 mM HCl was quickly added to terminate the reaction. The absorbance was measured at a wavelength of 319 nm. The positive control group of this experiment was ascorbic acid, and the blank control group was distilled water. The scavenging rate of superoxide anions by the sample was calculated according to formula (4).

[0070]

[0071] Among them, A0 is the absorbance of the distilled water blank control group, A S is the absorbance of the sample.

[0072] 3. Ability to remove ABTS

[0073] Mix equal volumes of 7mM ABTS·water solution and 2.45mM potassium persulfate solution, store in the dark for more than 12 hours, and then dilute the ABTS-potassium persulfate mixture with ultrapure water to make its absorbance at 734nm around 0.2. Separately, prepare a complex of thiol-containing cyclodextrin and Spirulina at a concentration of 2mg mL -1The solution was placed in a 96-well plate with 200 μL of LABTS working solution. After 30 minutes of reaction in the dark, the absorbance at 734 nm was measured. The positive control group for this experiment was ascorbic acid, and the blank control group was distilled water. The scavenging rate of the sample for hydroxyl radicals was calculated according to formula (5).

[0074]

[0075] Among them, A0 is the absorbance of the distilled water blank control group, A S is the absorbance of the sample.

[0076] The free radical scavenging experiments were carried out using free resveratrol as a control. The experimental results are shown in the attached table. Figure 5 As shown, resveratrol has a significant effect on ·OH, ·O2 - The scavenging rates of ·ABTS were 63.45%±0.12%, 30.18%±0.41% and 69.29%±0.38% respectively. The scavenging rates of the complex of thiol-cyclodextrin and Spirulina for the above three free radicals were 70.22%±3.12%, 31.12%±0.81% and 62.18%±2.58% respectively.

[0077] Example 7

[0078] This example compares and tests the adsorption properties of Spirulina, thiol-cyclodextrin, and the complex containing thiol-cyclodextrin and Spirulina prepared in Example 1 on mucin.

[0079] Dissolve mucin in a concentration of

[0080] 0.5mg mL -1 In a pH 6.8 PBS, spirulina, thiol cyclodextrin, and the complex containing thiol cyclodextrin and spirulina were added at 0.3 mg mL -1 The mixture was dispersed in the mucus buffer at a concentration of 100 μg / ml. After incubation at 37°C for 2 hours, the mixture was centrifuged at 22,000 rpm for 10 minutes, and the free mucin in the supernatant was determined using the periodic acid-Schiff (PAS) colorimetric method. The amount of mucin bound to Spirulina, thiol-cyclodextrin, and the complex containing thiol-cyclodextrin and Spirulina was calculated as the difference between the total amount of mucin added and the free mucin content in the supernatant.

[0081] The test results are as attached Figure 6As shown in the results, Spirulina contains almost no thiol compounds and only relies on the weak hydrogen bonding between the polysaccharides and mucin to adsorb mucin, with an adsorption capacity of only 0.05±0.001 mg. Thiol cyclodextrin and the complex of thiol cyclodextrin and Spirulina showed significant mucus adhesion properties, with adhesion capacities of 0.412±0.023 mg and 0.616±0.013 mg, respectively. This is attributed to the following reasons: (1) the free hydroxyl groups contained in the complex form hydrogen bonds with the functional groups of mucin. (2) the resveratrol in the complex has a physical effect on mucin. (3) the thiol groups in the thiol cyclodextrin and the complex form disulfide bonds with the thiol groups in mucin. (4) chain entanglement, non-covalent interactions such as van der Waals forces and ionic interactions. Among them, the reason why the complex of thiol cyclodextrin and Spirulina has the strongest binding force to mucin is that, in addition to the synergistic effect of the above four factors, the hydrogen bonding between the polysaccharides on the surface of Spirulina and mucin also contributes to the adhesion. In fact, due to its unique structure, spirulina can entangle with the intestinal villi, so the actual intestinal targeting effect of the complex is even better. Based on this, it is inferred that the complex can prolong the retention time of resveratrol in the intestinal mucosa, thereby improving the bioavailability of resveratrol.

[0082] Example 8

[0083] This example simulates the release behavior of the complex of thiol-containing cyclodextrin and Spirulina prepared in Example 1 in gastric and intestinal fluids, and evaluates its release performance.

[0084] In this example, 40 mL of simulated gastric juice and intestinal fluid were used as the in vitro release medium. 6 mg of the compound containing thiol cyclodextrin and spirulina was dissolved in 0.5 mL of deionized water, which was then transferred into a dialysis bag and sealed. First, the dialysis bag was immersed in pH 2.0 PBS (artificial gastric juice) for 2 h; then the dialysis bag was removed and immersed in pH 6.8 PBS (artificial intestinal juice) for 3 h; finally, the bag was released in pH 7.4 PBS (artificial colonic fluid) for 3 h. During the test, samples were taken regularly, 1 mL of sample was taken each time, and an equal volume of fresh medium was added to the release system. After the release was completed, an equal volume of ethanol was added to the sample taken out, and the absorbance at 308 nm was measured by ultraviolet spectrophotometry. The cumulative release of resveratrol corresponding to each time point was calculated according to formula (6), and a release curve was drawn.

[0085]

[0086] Where m t and m0 represent the mass of resveratrol released at hour t and the total amount of resveratrol in the complex of thiol-cyclodextrin and Spirulina at the beginning of release, respectively.

[0087] Release curve as shown in the attached Figure 7As shown, hydroxypropyl-β-cyclodextrin is not digested in the gastrointestinal tract, but can be fermented by intestinal flora and supports the fermentation of probiotics such as bifidobacteria in the large intestine. After being fermented and utilized by intestinal microorganisms, it will produce short-chain fatty acids (such as acetic acid, butyric acid, etc.) that are beneficial to maintaining the health of the intestinal barrier; Spirulina is almost not digested in the stomach, so the complex of thiol-containing cyclodextrin and spirulina is stable in simulated gastric fluid, and the resveratrol release within 2 hours is only 9.51%±1.19%; as cyclodextrin and spirulina are gradually digested in the intestine, the structure of the complex is also destroyed, and the resveratrol therein is rapidly released, and the release amount reaches 91.12±2.98% in the 8th hour, indicating that the constructed system can protect nutrients from being destroyed by gastric acid and realize stable intestinal targeted release of active factors.

[0088] Example 9

[0089] This example evaluates the effect of the complex of thiol-containing cyclodextrin and spirulina prepared in Example 1 on improving the bioavailability of resveratrol.

[0090] In this example, an in vitro digestion method was used to study the in vitro bioavailability of the complex of thiol-containing cyclodextrin and Spirulina.

[0091] First, the complex of thiol-cyclodextrin and Spirulina was digested by simulated gastric and intestinal fluid in vitro: 3 mg·mL -1 The thiol-containing cyclodextrin-Spirulina complex was dissolved in simulated gastric fluid at 37°C and stirred for 2 hours. The pH was then adjusted to 7.4 with 1M NaOH. An equal proportion of trypsin was added and stirred for another 4 hours to obtain the thiol-containing cyclodextrin-Spirulina complex after gastrointestinal digestion. The digested solution was centrifuged at 10,000 g for 45 minutes at 4°C. The absorbance of the supernatant was measured using a microplate reader, and the in vitro bioavailability was calculated according to formula (7):

[0092]

[0093] The in vitro bioavailability of free resveratrol was evaluated after the same treatment with free resveratrol as control. The evaluation results are shown in the attached figure. Figure 8 As shown in the results, free resveratrol, without the protection of a carrier, has an in vitro bioavailability of only 40.08% ± 1.12% after simulated gastric and intestinal digestion. After resveratrol was loaded into the complex, the bioavailability of resveratrol reached 84.98% ± 2.45% after targeted delivery to the intestine, 2.12 times that of free resveratrol, due to the complex's gastric acid resistance, synergistic intestinal targeting, and intestinal ROS scavenging effects. This demonstrates that efficient intestinal targeted delivery using the complex is an effective way to improve the bioavailability of resveratrol.

[0094] Example 10

[0095] This example compares the effects of digested resveratrol and the complex of thiol-containing cyclodextrin and spirulina on ·OH, ·O2 - , ·The clearing effect of ABTS.

[0096] Resveratrol, thiol-containing cyclodextrin and spirulina complex were digested in simulated gastric and intestinal fluids to obtain digestive products. - , ABTS scavenging: the free radical scavenging efficiency was determined using the method of Example 6.

[0097] The evaluation results are as attached Figure 9 As shown, the digested resveratrol has an effect on ·OH, ·O2 - The clearance rates of ABTS were 52.51%±2.93%, 26.09%±1.83% and 63.46%±1.27%. The complex of thiol-cyclodextrin and spirulina released resveratrol after digestion in simulated gastric juice and simulated intestinal juice, and the clearance rates of the three free radicals were as high as 87.98%±0.06%, 34.20%±0.22% and 89.29%±0.01%, respectively. This indicated that the complex of thiol-cyclodextrin and spirulina could protect the loaded resveratrol from being destroyed and inactivated by gastric acid and excessive ROS in the intestine. It not only showed a generally applicable scavenging effect on ROS in the intestine, but also indicated that the resveratrol after intestinal targeted release was not inactivated and still had excellent free radical scavenging activity.

[0098] As described above, the present invention can be better implemented. The above embodiments only describe the preferred implementation methods of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various changes and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the protection scope determined by the present invention.

Claims

1. A complex of thiol-containing cyclodextrin and spirulina, characterized in that: The preparation raw materials include: mercaptocyclodextrin, spirulina and active factors; the active factor is resveratrol; The preparation method of the complex containing thiol cyclodextrin and spirulina comprises: loading the active factor onto thiol cyclodextrin and spirulina respectively to obtain a thiol cyclodextrin-active factor inclusion compound and a spirulina-active factor complex; dispersing the spirulina-active factor complex into water to form a dispersion, dissolving the thiol cyclodextrin-active factor inclusion compound in water to form a solution; and mixing the dispersion with the solution to obtain the complex containing thiol cyclodextrin and spirulina.

2. The complex of thiol-containing cyclodextrin and Spirulina according to claim 1, characterized in that The total drug loading capacity of the compound of the mercapto-containing cyclodextrin and spirulina to resveratrol is 63.4% to 78.3%.

3. The complex of thiol-containing cyclodextrin and Spirulina according to claim 1, characterized in that The concentration of the dispersion is 0.5 mg / mL, the concentration of the solution is 10 mg / mL, the volume ratio of the dispersion to the solution is 1:0.5-100, the mixing temperature is 20-37° C., the mixing speed is 100-500 rpm, and the mixing time is 1-48 h.

4. The complex of thiol-containing cyclodextrin and Spirulina according to claim 1, characterized in that In terms of molar ratio, the ratio of thiolcyclodextrin to active factor in the thiolcyclodextrin-active factor inclusion compound is 1:0.5-5, and in terms of mass ratio, the ratio of spirulina to active factor in the spirulina-active factor complex is 1:0.005-0.

02.

5. The complex of thiol-containing cyclodextrin and Spirulina according to claim 4, characterized in that In terms of molar ratio, the ratio of thiolcyclodextrin to active factor in the thiolcyclodextrin-active factor inclusion complex is 1:1; in terms of mass ratio, the ratio of spirulina to active factor in the spirulina-active factor complex is 1:0.01; in terms of mass ratio, the ratio of the spirulina-active factor complex to the thiolcyclodextrin-active factor inclusion complex is 1:0.8~100.

6. The complex of thiol-containing cyclodextrin and Spirulina according to claim 5, characterized in that Calculated by mass ratio, the ratio of the spirulina-active factor complex to the mercaptocyclodextrin-active factor inclusion compound is 1:

10.

7. Use of the complex of thiol-containing cyclodextrin and Spirulina according to claim 1 in the preparation of intestinal-targeted functional foods.

8. Use of the complex of thiol-containing cyclodextrin and Spirulina according to claim 1 in the preparation of intestinal targeted drugs.

Images