A stabilized delivery system of phlorotannins for oral use, and a preparation method and application thereof

Abstract

The application discloses a kind of oral steady-state delivery phlorotannin complex system, and its preparation method and application, specifically include: using ɑ-amylase and starch glucosidase complex is handled to natural corn starch to obtain the porous corn starch with outer hole-inner channel structure, through self-adsorption effect loading phlorotannin after combination basic mineral ions and porous starch realizes intelligent gate control closed, and in the process of basic metal coordination, simultaneously using terahertz wave is assisted, finally after freeze drying, porous starch-phlorotannin powder is obtained.The preparation process of the method is simple, the obtained powder has pH intelligent response, the product is green and natural, can significantly improve the loading rate of phlorotannin, acid stability and oral availability.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, and in particular to an oral steady-state delivery system of brown algae polyphenols, its preparation method and application. Background Technology

[0002] Brown algae are large algae distributed in oceans worldwide. Their main pigment is fucoxanthin, which gives them their dark brown color. Common brown algae include kelp, wakame, Sargassum, Sargassum fusiforme, and nori. Brown algae contain abundant bioactive secondary metabolites, such as polysaccharides (fucose gum, alginic acid, fucoidan, brown starch, etc.), polyphenols (algal polyphenols, tannins, flavonoids), carotenoids, fucoxanthin, and some bioactive peptides. Algal polyphenols are complex structures polymerized using phloroglucinol units as monomers, with a degree of polymerization between 126 Da and 650 kDa. Containing numerous phenolic hydroxyl groups, algal polyphenols exhibit high hydrophilicity and strong bioactivity, such as antioxidant, anti-inflammatory, antibacterial, anti-obesity, and anti-photooxidation effects, making them widely applicable in functional foods, nutritional products, cosmetics, and pharmaceuticals. When phenolic substances are stimulated by external environmental factors such as temperature, pH, and light, brown algae polyphenols will degrade and lose their biological activity, which limits the oral application of brown algae polyphenols. Therefore, it is essential to improve and maintain the long-term stability and functionality of brown algae polyphenols.

[0003] Currently, various forms of phenolic stabilization delivery systems exist, such as liposomes, emulsions, nanoparticles, nanocapsules, and porous materials. For example, Lei Dai et al. proposed using zein-rhamnolipid composite nanoparticles to encapsulate curcumin (a flavonoid polyphenol) to improve its stability. However, these nanoparticles are highly unstable in strongly acidic environments and are only suitable for delivery in neutral environments, limiting their application. Weiming Chen et al. used sugarcane leaf polyphenols-zein to prepare Pickering emulsions for curcumin delivery, which showed good protection for curcumin under different salt concentrations, temperatures, and UV stimulation. However, the Pickering emulsion delivery system is prone to flocculation and aggregation, thus affecting the polyphenol encapsulation efficiency. Li Anping et al. used bamboo shoot dietary fiber to combine with polyphenols to improve the bioavailability of polyphenols. However, the combination with polyphenols requires high-voltage electrostatics and high-speed vacuum stirring, making the preparation complex and unsuitable for industrial production. Maryam Wahab et al. prepared porous corn starch loaded with curcumin and resveratrol using an enzymatic hydrolysis method. The encapsulation efficiency of curcumin reached 80.16%, and that of resveratrol reached 88.33%. Although the encapsulation efficiency of curcumin and resveratrol was high, significant leakage of active substances into the stomach still occurred in subsequent in vitro digestion experiments. Therefore, there is an urgent need for a method to protect the stable delivery of brown algae polyphenols in the gastrointestinal tract, preserve the physical stability of brown algae polyphenols in the gastrointestinal tract, reduce the significant leakage of active substances into the stomach, and thus improve the oral bioavailability of brown algae polyphenols and the high-value utilization of brown algae. Among the various stable delivery systems reported, porous materials have attracted widespread attention due to their advantages such as large specific surface area, large pore volume, and ability to physically adsorb active substances. Porous materials are widely available and are mainly divided into two categories: organic materials and inorganic materials. Organic materials such as natural polysaccharides (including starch, cyclodextrin, pectin, chitosan, cellulose, etc.) have attracted much attention due to their excellent biocompatibility, safety, non-toxicity, and environmental friendliness. Among them, starch, with its wide availability, low cost, high biocompatibility, and status as a primary energy source for humans, has become a research hotspot. Currently, the preparation methods for starch-based porous materials mainly include physical methods, chemical methods, and enzymatic hydrolysis. Physical methods, such as microwaves and ultrasound, use external forces to disrupt the starch structure, creating a porous structure. Chemical methods utilize solvent exchange with ethanol or acids to obtain a porous structure. Enzymatic hydrolysis uses enzymes such as amylase to act on the glycosidic bonds of starch to form a porous structure. Enzymatic hydrolysis has become one of the most popular methods for preparing porous starch due to its high efficiency, short processing time, simple process, mild reaction conditions, and environmental friendliness. However, despite its significant advantages in preparing porous starch, enzymatic hydrolysis still has some limitations in applications such as maintaining the stability of active substances. For example, when using a single porous starch to load active substances for in vivo targeted / steady-state delivery, there are many leakage issues, resulting in unsatisfactory loading rates and release performance of the active substances.When active substances are released in large quantities in the stomach, some acid-labile active substances will be degraded in large quantities in the stomach and thus lose their functional activity. Summary of the Invention

[0004] To address the shortcomings of existing bio-enzymatic hydrolysis methods, this invention proposes a novel solution. Based on the significantly increased hydroxyl group exposure in the porous starch formed after bio-enzymatic hydrolysis, an alkaline metal coordination technique is introduced to intelligently gate and seal the external pores of the porous starch. However, alkaline metal coordination suffers from drawbacks such as long processing time and low efficiency. Therefore, terahertz waves are combined to assist alkaline metal coordination, thereby achieving highly efficient conversion. This technology can reduce the degree of leakage of active substances, decrease the release rate in the stomach, thereby reducing the loss rate of active substances, significantly improving the stability and bioavailability of active substances, and constructing a pH-responsive gated release brown algal polyphenol transport system.

[0005] The purpose of this invention is to provide an orally administered, stabilized delivery system for brown algae polyphenols, its preparation method, and its application. This invention uses corn starch as raw material, obtaining porous corn starch through a green enzymatic process. The porous corn starch is loaded with brown algae polyphenols via self-adsorption, with the two primarily bound by hydrogen bonds and other forces. The porous structure maintains the original structure of the brown algae polyphenols while better retaining them. Furthermore, alkaline metal coordination technology (assisted by terahertz wave technology) is used to seal the external pores of the porous starch, achieving intelligent sealing of the external pores under acidic conditions. The porous starch-brown algae polyphenol system prepared by this invention exhibits intelligent pH responsiveness and acid-shielding release, mitigating leakage in the stomach, thus providing an orally administered, stabilized delivery system for brown algae polyphenols.

[0006] The specific technical solution of the present invention to solve the above problems is as follows:

[0007] This invention provides a method for preparing a porous corn starch-brown algae polyphenol composite system, comprising the following steps:

[0008] (1) Dissolve corn starch in phosphate buffer solution, then add α-amylase and amyloglucosidase, incubate for a period of time, add alkaline solution to adjust pH to 10.0-11.0 to terminate the enzyme reaction, centrifuge, collect the precipitate, wash, and obtain porous corn starch.

[0009] (2) Dissolve brown algae polyphenols in water to prepare an aqueous solution of brown algae polyphenols; dissolve the obtained porous corn starch in an aqueous solution to prepare an aqueous solution of porous corn starch; then mix the two, add alkaline metal salts, and incubate with terahertz waves for a period of time, then centrifuge, collect the precipitate, and dry to obtain a porous corn starch-brown algae polyphenol composite system.

[0010] In one embodiment of the present invention, in step (1), the ratio of starch mass (g) to the total enzyme activity (U) of α-amylase and amyloglucosidase is 1:(300-450), g:U.

[0011] In one embodiment of the present invention, in step (1), the enzyme activity ratio of α-amylase to amyloglucosidase is 1:(3-8).

[0012] In one embodiment of the present invention, in step (1), the pH of the phosphate buffer solution is 4.5-5.5.

[0013] In one embodiment of the present invention, in step (1), the mass-to-volume ratio of corn starch to phosphate buffer solution is 1:(8-10)(m / V, g / mL).

[0014] In one embodiment of the present invention, in step (1), the incubation reaction is carried out at 45-50°C and 150-200 rpm for 2-6 hours with shaking.

[0015] In one embodiment of the present invention, in step (1), the alkaline solution is selected from calcium chloride solution, sodium chloride solution, sodium bicarbonate solution, potassium chloride solution, etc.

[0016] In one embodiment of the present invention, in step (1), the concentration of sodium bicarbonate solution is 0.1-1 mol / L.

[0017] In one embodiment of the present invention, in step (2), the concentration of the brown algae polyphenol aqueous solution is 0.8-1 mg / mL.

[0018] In one embodiment of the present invention, in step (2), the concentration of the porous corn starch aqueous solution is 4-5 mg / mL.

[0019] In one embodiment of the present invention, in step (2), the mass ratio of brown algae polyphenols to porous corn starch is (0.4-1):25.

[0020] In one embodiment of the present invention, in step (2), the alkaline metal salt is selected from calcium chloride, sodium chloride, sodium bicarbonate, potassium chloride, etc.

[0021] In one embodiment of the present invention, in step (2), the concentration of the alkaline metal salt relative to the brown algae polyphenol-porous starch system is 100-500 mM. Preferably, it is 200-400 mM.

[0022] In one embodiment of the present invention, in step (2), an alkaline metal salt is prepared into an alkaline metal salt solution and then added to the brown algae polyphenol-porous starch system. The volume ratio of the brown algae polyphenol-porous starch system to the added alkaline metal salt solution is (5-2):1. Specifically, 4:1 can be selected.

[0023] In one embodiment of the present invention, in step (2), the concentration of the alkaline metal salt solution is 50-100 mM.

[0024] In one embodiment of the present invention, in step (2), the conditions for the terahertz wave are: frequency of 0.1-0.5THz and power of 80-120mW.

[0025] In one embodiment of the present invention, in step (2), the incubation time is 2-4 hours.

[0026] In one embodiment of the present invention, the preparation method specifically includes the following steps:

[0027] S1. Dissolve corn starch in phosphate buffer solution, with a mass-to-volume ratio of starch to phosphate buffer solution of 1:(8-10)(m / V, g / mL);

[0028] S2. Prepare a compound enzyme by taking α-amylase and amyloglucosidase and add it to the system described in S1. Incubate at 45-50℃ and 150-200 rpm for 1-8 hours with shaking.

[0029] S3. Add sodium bicarbonate solution to adjust the pH of the reaction system to 10.0-11.0 to terminate the enzyme reaction. Then, centrifuge to remove the supernatant, wash repeatedly with water, centrifuge again to obtain porous starch precipitate.

[0030] S4. The precipitate described in S3 is dried overnight in an oven at 55-60℃ and then ground to obtain porous corn starch with multiple pore sizes.

[0031] S5. Dissolve brown algae polyphenols in water to prepare an aqueous solution of brown algae polyphenols with a concentration of 0.8-1 mg / mL;

[0032] S6. Dissolve the porous corn starch obtained in S4 in an aqueous solution to obtain a porous corn starch aqueous solution with a concentration of 4-5 mg / mL;

[0033] S7. Add the solution described in S5 to the solution described in S6, and shake at 35-38℃ and 150-200rpm for 2-6 hours;

[0034] S8. Add a calcium chloride solution with a concentration of 40-80mM to the solution system described in S7, and incubate with terahertz wave assistance at 0-4℃ for 1-4h;

[0035] S9. Centrifuge the solution system described in S8, remove the supernatant to obtain the precipitate, and freeze-dry it to obtain a porous corn starch-brown algae polyphenol composite system.

[0036] Preferably, when adding the complex enzyme in step S2, the system described in S1 should be preheated at 45-50°C.

[0037] The present invention provides a porous corn starch-brown algae polyphenol composite system based on the above method.

[0038] The present invention also provides a formulation product comprising the above-mentioned porous corn starch-brown algae polyphenol composite system.

[0039] In one embodiment of the present invention, the preparation product is a pharmaceutical, health food, special medical food, special diet, or ordinary food.

[0040] In one embodiment of the present invention, the dosage form of the pharmaceutical product includes oral solid dosage form and oral liquid dosage form.

[0041] In one embodiment of the present invention, the oral solid dosage form includes: powder, tablet, capsule, granule, etc.

[0042] The beneficial effects of this invention are as follows:

[0043] (1) Porous starch is prepared by combining corn starch as raw material with compound enzyme method. It has the characteristics of low price, daily consumption, high efficiency, green and biodegradable. Furthermore, the pore size can be obtained between 0.8-1.3μm through process control, so that the pore size formed by porous starch is controllable.

[0044] (2) Porous corn starch and brown algae polyphenols are mainly bonded together through hydrogen bonds, electrostatic adsorption, and van der Waals forces, preserving the original structure of the brown algae polyphenols. Enzymatic hydrolysis exposes more hydroxyl groups in the porous starch, thereby enhancing intermolecular forces and significantly increasing the retention capacity of brown algae polyphenols. Furthermore, by utilizing alkaline mineral ions to form alkaline-responsive coordination bonds, the external pores of the porous starch are gated and sealed. Compared to porous starch, the addition of alkaline metal coordination technology can increase the loading capacity by up to 43%.

[0045] (3) After porous starch and brown algae polyphenols are combined, the metal coordination bonds formed seal the external pores of the porous starch in an acidic environment, providing acid shielding properties that prevent leakage of contents and protect the bioactivity of brown algae polyphenols. Compared with porous starch, the alkaline metal coordination technology reduces the initial release rate of brown algae polyphenols in the stomach by 45%, while maintaining the antioxidant capacity of brown algae polyphenols at over 98%.

[0046] (4) Using terahertz wave technology to assist the coordination process of alkaline metals can effectively improve the interaction between alkaline mineral ions and porous starch. Terahertz waves reduce the energy barrier of coordination reaction by precisely controlling molecular motion, enabling alkaline mineral ions and porous starch to complete coordination in a short time, thus improving the efficiency of traditional coordination reaction.

[0047] (5) This invention expands the development and utilization of corn starch and brown algae polyphenols in functional foods, improves the economic value of agricultural by-products, and promotes the high-value utilization of marine resources. Detailed Implementation

[0048] The following detailed description, in conjunction with specific embodiments, further illustrates the specific implementation of the present invention. Many specific details are set forth in the following description to provide a thorough understanding of the invention; however, the invention may also be implemented in other ways different from those described herein. Those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0049] Corn starch (amylose content 27%) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; α-amylase (35 U / mg) and amyloglucosidase (100,000 U / mL) were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; and brown algae polyphenols were purchased from Shaanxi Panier Biotechnology Co., Ltd.

[0050] This invention discloses a method for preparing a reconstituteable, orally stable, brown algae polyphenol powder, which specifically includes the following steps:

[0051] S1. Dissolve corn starch in phosphate buffer solution, with the ratio of starch to phosphate buffer solution being 1:(8-10)(m / V, g / mL);

[0052] S2. Prepare a compound enzyme by taking α-amylase and amyloglucosidase and add it to the system described in S1. Incubate at 45-50℃ and 150-200 rpm for 1-8 hours with shaking.

[0053] S3. Add sodium bicarbonate solution to adjust the pH of the reaction system to 10.0-11.0 to terminate the enzyme reaction. Then, centrifuge to remove the supernatant, wash repeatedly with water, centrifuge again to obtain porous starch precipitate.

[0054] S4. The precipitate described in S3 is dried overnight in an oven at 55-60℃ and then ground to obtain porous corn starch with multiple pore sizes.

[0055] S5. Dissolve brown algae polyphenols in water to prepare an aqueous solution of brown algae polyphenols with a concentration of 0.8-1 mg / mL;

[0056] S6. Dissolve the porous corn starch obtained in S4 in an aqueous solution to obtain a porous corn starch aqueous solution with a concentration of 4-5 mg / mL;

[0057] S7. Add the solution described in S5 to the solution described in S6, and incubate with shaking at 35-38℃ and 150-200 rpm for 2-6 hours;

[0058] S8. Add a calcium chloride solution with a concentration of 40-80mM to the solution system described in S7, and incubate with terahertz wave assistance at 0-4℃ for 1-4h;

[0059] S9. Centrifuge the solution system described in S8, remove the supernatant to obtain the precipitate, and freeze-dry it to obtain porous corn starch-brown algae polyphenol powder.

[0060] Example 1

[0061] S1. Dissolve corn starch in phosphate buffer solution at a ratio of starch to phosphate buffer solution of 1:10 (m / V, g / mL);

[0062] S2. Prepare a compound enzyme by mixing α-amylase and amyloglucosidase (α-amylase to amyloglucosidase enzyme activity ratio (U) = 1:5) and add it to the system described in S1, wherein the starch mass (g) to total enzyme activity ratio (U) = 1:360, and incubate at 50℃ and 150rpm for 2h with shaking.

[0063] S3. Add sodium bicarbonate solution to adjust the pH of the reaction system to 10.0 to terminate the enzyme reaction. Then centrifuge to remove the supernatant, wash repeatedly with water, centrifuge again to obtain the precipitate.

[0064] S4. The precipitate described in S3 is dried in an oven at 55°C overnight, and then ground to obtain porous corn starch;

[0065] S5. Dissolve brown algae polyphenols in water to prepare an aqueous solution of brown algae polyphenols with a concentration of 1 mg / mL;

[0066] S6. Dissolve the porous corn starch obtained in S4 in an aqueous solution to obtain a porous corn starch aqueous solution with a concentration of 5 mg / mL;

[0067] S7. Add the solution described in S5 to the solution described in S6 (V / V = 1:5), and shake at 37°C and 150 rpm for 4 hours;

[0068] S8. Add a 50 mM calcium chloride solution to the solution system described in S7 (the volume ratio of the brown algae polyphenol-porous starch system to the added calcium chloride solution is 4:1, and the amount of calcium chloride added relative to the brown algae polyphenol-porous starch system is 200 mmol / L). Incubate with terahertz wave assistance at 4℃ for 2 h, with a terahertz wave frequency of 0.4 THz and a power of 100 mW.

[0069] S9. Centrifuge the solution system described in S8, remove the supernatant to obtain the precipitate, and freeze-dry it to obtain porous corn starch-brown algae polyphenol powder.

[0070] Example 2

[0071] S1. Dissolve corn starch in phosphate buffer solution at a ratio of starch to phosphate buffer solution of 1:10 (m / V, g / mL);

[0072] S2. Prepare a compound enzyme by mixing α-amylase and amyloglucosidase (α-amylase to amyloglucosidase enzyme activity ratio (U) = 1:5) and add it to the system described in S1, wherein the starch mass (g) to total enzyme activity ratio (U) = 1:360. Incubate at 50℃ and 150rpm for 4 hours with shaking.

[0073] S3. Add sodium bicarbonate solution to adjust the pH of the reaction system to 10.0 to terminate the enzyme reaction. Then centrifuge to remove the supernatant, wash repeatedly with water, centrifuge again to obtain the precipitate.

[0074] S4. The precipitate described in S3 is dried in an oven at 55°C overnight, and then ground to obtain porous corn starch;

[0075] S5. Dissolve brown algae polyphenols in water to prepare an aqueous solution of brown algae polyphenols with a concentration of 1 mg / mL;

[0076] S6. Dissolve the porous corn starch obtained in S4 in an aqueous solution to obtain a porous corn starch aqueous solution with a concentration of 5 mg / mL;

[0077] S7. Add the solution described in S5 to the solution described in S6 (V / V = 1:5), and shake at 37°C and 150 rpm for 4 hours;

[0078] S8. Add a 50 mM calcium chloride solution to the solution system described in S7 (the volume ratio of the brown algae polyphenol-porous starch system to the added calcium chloride solution is 4:1), and incubate with terahertz wave assisted at 4°C for 2 h. The terahertz wave frequency is 0.4 THz and the power is 100 mW.

[0079] S9. Centrifuge the solution system described in S8, remove the supernatant to obtain the precipitate, and freeze-dry it to obtain porous corn starch-brown algae polyphenol powder.

[0080] Example 3

[0081] S1. Dissolve corn starch in phosphate buffer solution at a ratio of starch to phosphate buffer solution of 1:10 (m / V, g / mL);

[0082] S2. Prepare a compound enzyme by mixing α-amylase and amyloglucosidase (α-amylase to amyloglucosidase enzyme activity ratio (U) = 1:5) and add it to the system described in S1, wherein the starch mass (g) to total enzyme activity ratio (U) = 1:360, and incubate at 50℃ and 150rpm for 6h with shaking.

[0083] S3. Add sodium bicarbonate solution to adjust the pH of the reaction system to 10.0 to terminate the enzyme reaction. Then, centrifuge to remove the supernatant, wash repeatedly with water, centrifuge again to obtain the precipitate.

[0084] S4. The precipitate described in S3 is dried in an oven at 55°C overnight, and then ground to obtain porous corn starch;

[0085] S5. Dissolve brown algae polyphenols in water to prepare an aqueous solution of brown algae polyphenols with a concentration of 1 mg / mL;

[0086] S6. Dissolve the porous corn starch obtained in S4 in an aqueous solution to obtain a porous corn starch aqueous solution with a concentration of 5 mg / mL;

[0087] S7. Add the solution described in S5 to the solution described in S6 (V / V = 1:5), and shake at 37°C and 150 rpm for 4 hours;

[0088] S8. Add a 50 mM calcium chloride solution to the solution system described in S7 (the volume ratio of the brown algae polyphenol-porous starch system to the added calcium chloride solution is 4:1), and incubate with terahertz wave assisted at 4°C for 2 h. The terahertz wave frequency is 0.4 THz and the power is 100 mW.

[0089] S9. Centrifuge the solution system described in S8, remove the supernatant to obtain the precipitate, and freeze-dry it to obtain porous corn starch-brown algae polyphenol powder.

[0090] Comparative Example 1

[0091] S1. Dissolve brown algae polyphenols in water to prepare an aqueous solution of brown algae polyphenols with a concentration of 1 mg / mL;

[0092] S2. Dissolve corn starch in an aqueous solution to obtain a corn starch aqueous solution with a concentration of 5 mg / mL;

[0093] S3. Add the solution described in S1 to the solution described in S2 (V / V = 1:5), shake at 37°C and 150 rpm for 4 hours, centrifuge to obtain the precipitate, and freeze-dry the precipitate to obtain corn starch-brown algae polyphenol powder.

[0094] Comparative Example 2

[0095] S1. Dissolve corn starch in phosphate buffer solution at a ratio of starch to phosphate buffer solution of 1:10 (m / V, g / mL);

[0096] S2. Prepare a compound enzyme by mixing α-amylase and amyloglucosidase (α-amylase to amyloglucosidase enzyme activity ratio (U) = 1:5) and add it to the system described in S1, wherein the starch mass (g) to total enzyme activity ratio (U) = 1:360, and incubate at 50℃ and 150rpm for 2h with shaking.

[0097] S3. Add sodium bicarbonate solution to adjust the pH of the reaction system to 10.0 to terminate the enzyme reaction. Then, centrifuge to remove the supernatant, wash repeatedly with water, centrifuge again to obtain the precipitate.

[0098] S4. The precipitate described in S3 is dried in an oven at 55°C overnight, and then ground to obtain porous corn starch;

[0099] S5. Dissolve brown algae polyphenols in water to prepare an aqueous solution of brown algae polyphenols with a concentration of 1 mg / mL;

[0100] S6. Dissolve the porous corn starch obtained in S4 in an aqueous solution to obtain a porous corn starch aqueous solution with a concentration of 5 mg / mL;

[0101] S7. Add the solution described in S5 to the solution described in S6 (V / V = 1:5), and shake at 37°C and 150 rpm for 4 hours; centrifuge to remove the supernatant, and freeze-dry the resulting precipitate to obtain porous corn starch-brown algae polyphenol powder.

[0102] Comparative Example 3

[0103] S1. Dissolve corn starch in phosphate buffer solution at a ratio of starch to phosphate buffer solution of 1:10 (m / V, g / mL);

[0104] S2. Prepare a compound enzyme by mixing α-amylase and amyloglucosidase (α-amylase to amyloglucosidase enzyme activity ratio (U) = 1:5) and add it to the system described in S1, wherein the starch mass (g) to total enzyme activity ratio (U) = 1:360. Incubate at 50℃ and 150 rpm for 2 hours with shaking.

[0105] S3. Add sodium bicarbonate solution to adjust the pH of the reaction system to 10.0 to terminate the enzyme reaction. Then, centrifuge to remove the supernatant, wash repeatedly with water, centrifuge again to obtain the precipitate.

[0106] S4. The precipitate described in S3 is dried in an oven at 55°C overnight, and then ground to obtain porous corn starch;

[0107] S5. Dissolve brown algae polyphenols in water to prepare an aqueous solution of brown algae polyphenols with a concentration of 1 mg / mL;

[0108] S6. Dissolve the porous corn starch obtained in S4 in an aqueous solution to obtain a porous corn starch aqueous solution with a concentration of 5 mg / mL;

[0109] S7. Add the solution described in S5 to the solution described in S6 (V / V = 1:5), and shake at 37°C and 150 rpm for 4 hours;

[0110] S8. Add a 50 mM calcium chloride solution to the solution system described in S7 (the volume ratio of the brown algae polyphenol-porous starch system to the added calcium chloride solution is 4:1), and incubate at 4°C for 2 h.

[0111] S9. Centrifuge the solution system described in S8, remove the supernatant to obtain the precipitate, and freeze-dry it to obtain porous corn starch-brown algae polyphenol powder.

[0112] Comparative Example 4

[0113] S1. Dissolve corn starch in phosphate buffer solution at a ratio of starch to phosphate buffer solution of 1:10 (m / V, g / mL);

[0114] S2. Prepare a compound enzyme by mixing α-amylase and amyloglucosidase (α-amylase to amyloglucosidase enzyme activity ratio (U) = 1:5) and add it to the system described in S1, wherein the starch mass (g) to total enzyme activity ratio (U) = 1:360, and incubate at 50℃ and 150rpm for 4h with shaking.

[0115] S3. Add sodium bicarbonate solution to adjust the pH of the reaction system to 10.0 to terminate the enzyme reaction. Then, centrifuge to remove the supernatant, wash repeatedly with water, centrifuge again to obtain the precipitate.

[0116] S4. The precipitate described in S3 is dried in an oven at 55°C overnight, and then ground to obtain porous corn starch;

[0117] S5. Dissolve brown algae polyphenols in water to prepare an aqueous solution of brown algae polyphenols with a concentration of 1 mg / mL;

[0118] S6. Dissolve the porous corn starch obtained in S4 in an aqueous solution to obtain a porous corn starch aqueous solution with a concentration of 5 mg / mL;

[0119] S7. Add the solution described in S5 to the solution described in S6 (V / V = 1:5), and shake at 37°C and 150 rpm for 4 hours; centrifuge to remove the supernatant, and freeze-dry the resulting precipitate to obtain porous corn starch-brown algae polyphenol powder.

[0120] Comparative Example 5

[0121] S1. Dissolve corn starch in phosphate buffer solution at a ratio of starch to phosphate buffer solution of 1:10 (m / Vg / mL);

[0122] S2. Prepare a compound enzyme by mixing α-amylase and amyloglucosidase (α-amylase to amyloglucosidase enzyme activity ratio (U) = 1:5) and add it to the system described in S1, wherein the starch mass (g) to total enzyme activity ratio (U) = 1:360, and incubate at 50℃ and 150rpm for 6h with shaking.

[0123] S3. Add sodium bicarbonate solution to adjust the pH of the reaction system to 10.0 to terminate the enzyme reaction. Then, centrifuge to remove the supernatant, wash repeatedly with water, centrifuge again to obtain the precipitate.

[0124] S4. The precipitate described in S3 is dried in an oven at 55°C overnight, and then ground to obtain porous corn starch;

[0125] S5. Dissolve brown algae polyphenols in water to prepare an aqueous solution of brown algae polyphenols with a concentration of 1 mg / mL;

[0126] S6. Dissolve the porous corn starch obtained in S4 in an aqueous solution to obtain a porous corn starch aqueous solution with a concentration of 5 mg / mL;

[0127] S7. Add the solution described in S5 to the solution described in S6 (V / V = 1:5), and shake at 37°C and 150 rpm for 4 hours; centrifuge to remove the supernatant, and freeze-dry the resulting precipitate to obtain porous corn starch-brown algae polyphenol powder.

[0128] Indicator Measurement:

[0129] (1) Determination of polyphenol loading in brown algae

[0130] A 4% (w / w) mixed solution system was prepared by combining an aqueous solution of brown algae polyphenols (1 mg / mL) and a suspension of porous corn starch (5 mg / mL). This system was incubated in a shaker (37℃, 150 rpm) for 4 h, and then centrifuged (6000 × g, 15 min). 500 μL of the supernatant was diluted by half and placed in a 10 mL centrifuge tube. 500 μL of Folin-Ciocalteu (1 mol / L) reagent was added, and the mixture was allowed to stand in the dark for 5 min. Then, 5 mL of 10% (m / v) Na₂CO₃ solution was added, shaken well, and allowed to react in the dark at room temperature for 60 min. The absorbance of the solution was measured at a wavelength of 765 nm, and the loading was calculated according to the following formula (1):

[0131]

[0132] Where: M0 is the total mass of added brown algal polyphenols, mg; M1 is the mass of polyphenols in the supernatant calculated based on the gallic acid standard curve, mg; M ps The mass, in g, is the dry starch sample obtained after freeze-drying following loading.

[0133] (2) DPPH free radical scavenging rate

[0134] A solution of 1 mg / mL brown algae polyphenols and a solution of 20 mg / mL porous corn starch (corn starch)-brown algae polyphenol powder were incubated in 0, 0.1, and 0.2 mol / L glacial acetic acid for 1 h, respectively. After treatment, the DPPH free radical scavenging ability of the samples was measured. A DPPH solution with a concentration of 0.04 mg / mL was prepared using anhydrous ethanol. 100 μL of DPPH solution was added to 100 μL of water, denoted as A. control Take 100 μL of the solution obtained after the above treatment and add 100 μL of DPPH solution, denoted as A. sample Take 100 μL of the sample solution to be tested and add 100 μL of anhydrous ethanol, denoted as A. blank Incubate at room temperature in the dark for 30 min, and measure the absorbance at 517 nm. Calculate the DPPH free radical scavenging rate according to the following formula (2);

[0135]

[0136] (3) Analysis of the in vitro release performance of starch-brown algae polyphenols

[0137] 250 mg of porous corn starch (corn starch)-brown algae polyphenol powder was placed in 25 mL of artificial gastric juice and incubated in a shaker (150 rpm, 37 °C). The mixture was centrifuged (6000 × g, 5 min) at time intervals of 0 min, 30 min, 60 min, 90 min, and 120 min, and 4 mL of supernatant was collected. An equal volume of fresh artificial gastric juice was added to maintain a constant volume of 25 mL. The content of brown algae polyphenols in the collected supernatant was determined. The brown algae polyphenol content was calculated using the gallic acid standard curve, and the cumulative release rate was calculated. A data graph was plotted with time on the x-axis and the cumulative release rate on the y-axis. The cumulative release rate was calculated according to the following formula (3):

[0138]

[0139] In the formula: V1 refers to the volume of the released medium (25 mL), V2 refers to the volume of the removed medium (4 mL), C n It refers to the content of brown algal polyphenols in the releasing medium, m i This refers to the mass of brown algae polyphenols in starch.

[0140] Tables 1 and 2 summarize the effects of different preparation methods on the polyphenol loading, DPPH free radical scavenging rate, and cumulative release rate of brown algae.

[0141] Table 1. Effects of different preparation methods on polyphenol loading and DPPH free radical scavenging rate of brown algae.

[0142]

[0143] Table 2. Effects of different preparation methods on the cumulative release rate of brown algae polyphenols

[0144]

[0145] The loading capacity determination reflects the adsorption capacity and binding degree of porous starch for BAP, and measures whether porous starch can more effectively load BAP. Table 1 shows that Example 1 (the optimal example) has a higher loading capacity, increasing by 76%, 43%, and 21% compared to Comparative Examples 1, 2, and 3, respectively, indicating that the combination of enzymatic hydrolysis, basic metal coordination, and terahertz wave technology can significantly improve the loading rate of brown algae polyphenols. The DPPH free radical scavenging capacity was measured after treating different samples with different concentrations (0.0, 0.1, and 0.2 mol / L) of glacial acetic acid for 1 h, measuring the protective ability of different treatment methods on the activity of brown algae polyphenols under strong acid conditions. All examples and comparative examples (except Comparative Example 1) maintained a high DPPH free radical scavenging rate at different glacial acetic acid concentrations, all higher than Comparative Example 1. Example 1 (the optimal example) showed the highest DPPH free radical scavenging rate at glacial acetic acid concentrations of 0.1 mol / L and 0.2 mol / L, at 98.65% and 97.34%, respectively. This indicates that the combination of porous starch with alkaline metal coordination and terahertz wave technology can protect the bioactivity of brown algal polyphenols in a strongly acidic environment.

[0146] The cumulative release rate in the stomach was measured among different groups to reflect whether the product could exert an acid-shielded release effect on brown algae polyphenols in the stomach. Table 2 shows that at 0 min and 120 min in gastric juice, the cumulative release rate of brown algae polyphenols in Example 1 (the optimal example) was 21.64% and 26.74%, respectively, both significantly lower than the other examples and the comparative example. This indicates that the porous starch prepared by enzymatic hydrolysis, combined with basic metal coordination and terahertz wave assistance, can better retain brown algae polyphenols within the pores of the porous starch, reducing the likelihood of significant leakage of the porous transport system after entering the digestive system.

[0147] Example 4

[0148] Referring to Example 1, the concentration of calcium chloride solution in S8 was adjusted while other parameters remained unchanged to obtain the corresponding complex system. The loading of brown algae polyphenols and the cumulative release rate of brown algae polyphenols of the corresponding products were tested, and the results are shown in Table 3.

[0149] Table 3. Effects of different calcium chloride solution concentrations on the loading and cumulative release rate of brown algae polyphenols.

[0150]

[0151] Based on Example 1 (the optimal example), while keeping other experimental conditions constant, the concentration of calcium chloride solution was varied to explore the optimal concentration. The effect of calcium chloride solution concentration in the range of 30-100 mmol / L on the loading and cumulative release rate of brown algae polyphenols was determined, and the results are shown in Table 3. With the increase of calcium chloride concentration, the loading of brown algae polyphenols showed an upward trend, while the cumulative release rate of brown algae polyphenols in the stomach showed a downward trend. This indicates that the higher the ion concentration, the better the encapsulation effect of alkaline metal coordination technology on the external pores of porous starch. However, when the metal salt concentration was below 50 mmol / L, the loading of brown algae polyphenols was low and the cumulative release rate was high, failing to achieve the purpose of preparing an orally administered, stable delivery system of brown algae polyphenols. When the concentration of metal salt reaches 50 mmol / L (i.e., the amount of alkaline metal salt added relative to the brown algae polyphenol-porous starch system is 200 mM), compared with porous starch, the loading of brown algae polyphenols is significantly increased by 43%, the initial release rate of brown algae polyphenols in the stomach is reduced by 45%, and the antioxidant capacity of brown algae polyphenols is maintained at over 98%.

[0152] The above results indicate that the implementation of the method of the present invention can significantly improve the loading rate of brown algae polyphenols, has excellent protective effect on brown algae polyphenols in acidic environment and has a controlled release effect in the stomach, alleviating the occurrence of leakage, providing a new method and data support for the subsequent steady-state transport of active substances, and providing theoretical support for the development of functional foods.

[0153] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A method for preparing an orally administered, stabilized delivery system of brown algae polyphenols, comprising the following steps: (1) Dissolve corn starch in phosphate buffer solution, then add α-amylase and amyloglucosidase, incubate for a period of time, add alkaline solution to adjust pH to 10.0-11.0 to terminate the enzyme reaction, centrifuge, collect the precipitate, wash, and obtain porous corn starch. The incubation conditions are 45-50 °C and 150-200 rpm with shaking incubation for 2 h; The ratio of the mass of starch to the total enzyme activity of α-amylase and amyloglucosidase is 1:(300-450), g:U; (2) Dissolve brown algae polyphenols in water to prepare an aqueous solution of brown algae polyphenols; dissolve the obtained porous corn starch in the aqueous solution to prepare an aqueous solution of porous corn starch; then mix the two, add alkaline metal salts, and incubate with terahertz waves for a period of time, then centrifuge, collect the precipitate, dry, and obtain a porous corn starch-brown algae polyphenol composite system. The mass ratio of the brown algae polyphenols to the porous corn starch is (0.4-1):25; The alkaline metal salt is selected from any one of calcium chloride, sodium chloride, sodium bicarbonate, and potassium chloride; The concentration of the alkaline metal salt relative to the brown algae polyphenol-porous starch system is 200-500 mM; The terahertz wave frequency is 0.1-0.5 THz, the power is 80-120 mW, and the incubation time is 2-4 h.

2. The preparation method according to claim 1, characterized in that, In step (1), the enzyme activity ratio of α-amylase to amyloglucosidase is 1:(3-8).

3. A porous corn starch-brown algae polyphenol composite system prepared by the method described in any one of claims 1 or 2.

4. A pharmaceutical preparation, characterized in that, The formulation product comprises the porous corn starch-brown algae polyphenol complex system as described in claim 3.

5. The formulation product according to claim 4, characterized in that, The dosage forms of the pharmaceutical products include oral solid dosage forms and oral liquid dosage forms.

Images