A preparation method of a fucoidan steady-state anthocyanin controllable release oral dissolving film regulated by a pulse electric field

Abstract

The present application relates to a kind of fucoidan steady-state anthocyanin controllable release oral dissolving film preparation method for pulse electric field regulation, belong to food dietary supplement film preparation field.The method utilizes high field intensity pulse electric field to process fucoidan aqueous solution, then after fucoidan aqueous solution after pulse electric field processing is mixed with blueberry anthocyanin extract solution evenly, after low field intensity pulse electric field processing, obtain fucoidan-anthocyanin mixed solution;Hydroxypropyl methyl cellulose solution is added to fucoidan-anthocyanin mixed solution and is magnetically stirred to obtain fucoidan-anthocyanin-hydroxypropyl methyl cellulose mixed solution;Glycerol is added to the obtained mixed solution, and after adjusting the solution pH to 2-4, film forming liquid is obtained;Film forming liquid is dried to form film, and it is obtained.The present application is based on the high-low intensity transformation regulation technology of pulse electric field, realizes the high steady-state combination of fucoidan and anthocyanin, reaches the steady-state effect and controllable release effect of anthocyanin in oral environment, and lays the foundation for its efficient absorption and utilization.

Description

TECHNICAL FIELD

[0001] The application relates to a preparation method of a fucoidan steady-state anthocyanin controllable release oral dissolving film regulated by a pulsed electric field, and belongs to the field of food dietary supplement film preparations. BACKGROUND

[0002] Anthocyanin is a phenolic compound and one of main pigments constituting the color diversity of plants, and has multiple physiological functions such as antioxidant, regulation of sugar and lipid metabolism, immune regulation and relief of visual fatigue. Anthocyanin mainly exists in the form of yellow ionized cations, which is easy to degrade under the stress of external factors (temperature and light, etc.) and internal environment (intestinal first-pass effect), resulting in poor stability and low oral bioavailability. At present, the existing researches on the steady state of anthocyanin mainly focus on the structural modification, microcapsule embedding and interaction of macromolecular components, and the product types are mostly solution systems. However, in order to break the single product type and expand the universality of the population, it is necessary to develop a new steady-state delivery system loaded with anthocyanin.

[0003] The oral dissolving film is a new type of drug and dietary supplement system, which is light and thin in texture, convenient to transport and carry, and is welcomed by children and the elderly, and has high compliance for patients with difficulty in swallowing, and has good application prospect. In addition, the oral dissolving film preparation can be absorbed into the blood circulation through the oral mucosa, avoiding the intestinal first-pass effect and improving the bioavailability of active ingredients. The application pre-develops a carrier form loaded with anthocyanin based on the oral dissolving film, so as to broaden the steady-state delivery form of anthocyanin, and make the anthocyanin be efficiently absorbed through the oral mucosa and improve the bioavailability. However, the high steady state of anthocyanin in the oral dissolving film can cope with the stress of the oral environment, which is an important prerequisite for efficient absorption and utilization of anthocyanin. SUMMARY

[0004] The application aims to provide a preparation method of a fucoidan steady-state anthocyanin controllable release oral dissolving film regulated by a pulsed electric field, which can be used as a dietary nutrition supplement film loaded with anthocyanin to meet the dietary nutrition needs of the human body.

[0005] A preparation method of a fucoidan steady-state anthocyanin controllable release oral dissolving film regulated by a pulsed electric field, comprising the following process steps:

[0006] The fucoidan water solution is treated by high field intensity pulsed electric field, then the fucoidan water solution treated by pulsed electric field is mixed with blueberry anthocyanin extract solution uniformly, and then the mixed solution is treated by low field intensity pulsed electric field to obtain a fucoidan-anthocyanin mixed solution; the hydroxypropyl methyl cellulose solution is added into the fucoidan-anthocyanin mixed solution to obtain a fucoidan-anthocyanin-hydroxypropyl methyl cellulose mixed solution by magnetic stirring; a certain amount of food-grade glycerol is added into the obtained mixed solution, and the pH of the solution is adjusted to 2-4, and then the solution is continuously stirred by magnetic force for a period of time to obtain a film-forming solution; and the film-forming solution is dried to form a film.

[0007] As a non-thermal processing technology, pulsed electric field (PEF) can regulate the structure and properties of biological macromolecules through high-voltage electric field environment, and is suitable for the interaction between temperature-sensitive molecules. The present application is based on the regulation of biological macromolecules by pulsed electric field to develop a mouth-dissolving film preparation.

[0008] In the present application, the "high field intensity" and "low field intensity" in the "high field intensity pulsed electric field" and "low field intensity pulsed electric field" are only used to distinguish the two times of pulsed treatment in the present application.

[0009] Further, the high field intensity pulsed electric field has an electric field intensity of 15-25 kV / cm, a pulse frequency of 1000-1200 Hz, and a pulse time of 10-100 μs.

[0010] Further, the low field intensity pulsed electric field has an electric field intensity of 10-15 kV / cm, a pulse frequency of 800-1000 Hz, and a pulse time of 10-100 μs.

[0011] In the present application, the concentration of the fucoidan water solution is 2-8 mg / mL; the concentration of the blueberry anthocyanin extract solution is 1-10 mg / mL; and the mass ratio of the blueberry anthocyanin extract to fucoidan is 1:20-60.

[0012] Further, the solvent of the blueberry anthocyanin extract solution is composed of 1 mol / L citric acid and 1 mol / L sodium citrate buffer solution with pH=3.

[0013] In the present application, the hydroxypropyl methyl cellulose water solution is prepared by the following method: hydroxypropyl methyl cellulose is dissolved in 80% of total volume of 100℃ water, and then 20% of total volume of 25℃ water is added, and after magnetic stirring, a hydroxypropyl methyl cellulose solution with a concentration of 20-60 mg / mL is obtained, wherein the magnetic stirring rate is 500-800 r / min, and the stirring time is 1-2 h.

[0014] In the present application, the mass ratio of hydroxypropyl methyl cellulose to fucoidan is 10-30:1.

[0015] In the present application, the magnetic stirring rate is 500-800 r / min, and the stirring time is 20-40 min.

[0016] In the present application, the glycerol is added in an amount of 1-3% of the total volume of the film-forming solution.

[0017] In the present application, the fucoidan-anthocyanin-hydroxypropyl methyl cellulose solution is adjusted to a pH of 2-4 using 0.1 mol / L HCl solution or 0.1 mol / L NaOH solution.

[0018] In the present application, the obtained film-forming solution is cast onto a film-forming plate and placed in a blast drying oven for drying to form a film, with a drying temperature of 38-42 DEG C and a drying time of 8-16 h.

[0019] The present application has the following beneficial effects: the present application regulates the fucoidan-anthocyanin steady-state system by pulse electric field technology, uses hydroxypropyl methyl cellulose as a film-forming matrix, and constructs an anthocyanin-loaded oral dissolving film. First, based on high-intensity pulse electric field regulation, the spatial structure of fucoidan is changed, the molecular chain is stretched, and more groups are exposed. Then, low-intensity pulse electric field treatment promotes intermolecular motion, thereby realizing high-steady-state combination of fucoidan and anthocyanin, achieving the steady-state effect and controllable release effect of anthocyanin in the oral environment, and laying a foundation for efficient absorption and utilization. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 (A) is the retention rate of anthocyanin in the film-forming solution of the present application, Comparative Example 1, Comparative Example 2 and Example 1 after 8 days of light storage; Figure 1 (B) is the retention rate of anthocyanin in the film-forming solution of the present application, Comparative Example 1, Comparative Example 2 and Example 1 after 30 min under simulated oral environment pH 5-8. * represents a significant difference p<0.05, ** represents a significant difference p<0.01, and different lowercase letters represent a significant difference within the group p<0.05.

[0021] Figure 2 (A-C) are the thickness, tensile strength and elongation at break, and disintegration time of the oral dissolving film of the present application, Comparative Example 1, Comparative Example 2 and Example 1, respectively. Different lowercase letters represent a significant difference between groups p<0.05.

[0022] Figure 3 (A) and (B) are the retention rates of anthocyanin in the oral dissolving film of the present application, Comparative Example 1, Comparative Example 2 and Example 1 after 8 days of light storage. * represents a significant difference p<0.05, ** represents a significant difference p<0.01.

[0023] Figure 4(A~C) are the antioxidant activity evaluation of DPPH, ABTS and CAA of the mouth dissolving film of the present application comparative example 1, comparative example 2 and example 1 respectively. Different lowercase letters represent significant differences between groups p < 0.05.

[0024] Figure 5 (A) and (B) are the release amount and slope coefficient of the mouth dissolving film of the present application comparative example 1, comparative example 2 and example 1 respectively; Figure 5 (C~E) are the antioxidant activity evaluation of DPPH, ABTS and CAA of the mouth dissolving film of the present application comparative example 1, comparative example 2 and example 1 respectively after 30 min of release. Different lowercase letters represent significant differences between groups p < 0.05. DETAILED DESCRIPTION

[0025] The following non-limiting examples can enable those of ordinary skill in the art to more fully understand the present application, but in no way limit the present application.

[0026] The test methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0027] A preparation method of a pulse electric field regulated fucoidan steady-state anthocyanin controllable release mouth dissolving film, comprising the following process steps:

[0028] (1) Dissolve fucoidan in water, and obtain a PEF-fucoidan aqueous solution of a certain concentration after high-field pulse electric field regulation;

[0029] (2) Dissolve blueberry anthocyanin extract in a citric acid-sodium citrate solution to prepare an anthocyanin extract solution of a certain concentration;

[0030] (3) Add the anthocyanin extract solution obtained in step (2) to the PEF-fucoidan aqueous solution obtained in step (1), and then perform high-field pulse electric field treatment and magnetic stirring to obtain a PEF-fucoidan-anthocyanin complex solution;

[0031] (4) Slowly add a hydroxypropyl methylcellulose solution to the PEF-fucoidan-anthocyanin / hydroxypropyl methylcellulose complex solution obtained by magnetic stirring in step (3);

[0032] (5) Add a certain volume fraction of food-grade glycerol dropwise to the PEF-fucoidan-anthocyanin / hydroxypropyl methylcellulose complex solution obtained in step (4), adjust the final pH to 2~4, and continue magnetic stirring for 20~40 min;

[0033] (6) Cast the solution obtained in step (5) onto a film forming plate, place it in a blast drying oven, dry it into a film, and then take it out after equilibrating in a desiccator for a period of time.

[0034] Preferably, in step (1), the fucoidan concentration is 2-8 mg / mL, the high-field strength pulse electric field intensity is 15-25 kV / cm, the pulse frequency is 1000-1200 Hz, and the pulse time is 10-100 μs.

[0035] Preferably, in step (2), the blueberry anthocyanin extract concentration is 1-10 mg / mL; the solvent of the blueberry anthocyanin extract solution is composed of 1 mol / L citric acid and 1 mol / L sodium citrate buffer solution with pH=3.

[0036] Preferably, in step (3), the mass ratio of the blueberry anthocyanin extract to fucoidan is 1:20-60; the low-field strength pulse electric field intensity is 10-15 kV / cm, the pulse frequency is 800-1000 Hz, and the pulse time is 10-100 μs.

[0037] Preferably, in step (4), the hydroxypropyl methyl cellulose aqueous solution is prepared by the following method: hydroxypropyl methyl cellulose is dissolved in 80% of the total volume of 100°C water, and then 20% of the total volume of 25°C water is added, and after magnetic stirring, a hydroxypropyl methyl cellulose solution with a concentration of 20-60 mg / mL is obtained, the magnetic stirring rate is 500-800 r / min, and the stirring time is 1-2 h; the mass ratio of hydroxypropyl methyl cellulose to fucoidan is 10-30:1.

[0038] Preferably, in step (5), the final volume fraction of food-grade glycerol added is 1%-3%, the final pH is adjusted to 2-4, the stirring rate is 500-800 r / min, and the stirring time is 20-40 min.

[0039] Preferably, in step (6), the drying temperature is 38-42°C, and the drying time is 8-16 h.

[0040] Example 1

[0041] A preparation method of a pulse electric field regulated fucoidan steady-state anthocyanin controllable release oral dissolving film:

[0042] The fucoidan was dissolved in distilled water to prepare a fucoidan solution with a concentration of 4 mg / mL, which was subjected to pulsed electric field treatment at an electric field strength of 20 kV / cm, a pulse frequency of 1000 Hz, and a pulse time of 30 μs to obtain a PEF-fucoidan solution. The blueberry anthocyanin extract was dissolved in a citric acid-sodium citrate buffer solution with a pH of 3 to prepare an anthocyanin extract solution with a concentration of 10 mg / mL. 0.8 mL of the blueberry anthocyanin extract solution was added to 39.2 mL of the PEF-fucoidan solution with a concentration of 4 mg / mL, and the mixture was subjected to magnetic stirring at a stirring rate of 250 r / min for 10 min, followed by pulsed electric field treatment at an electric field strength of 10 kV / cm, a pulse frequency of 800 Hz, and a pulse time of 30 μs to obtain a PEF-fucoidan-anthocyanin mixed solution. A hydroxypropyl methylcellulose solution with a concentration of 40 mg / mL was prepared by a cold-hot method (hydroxypropyl methylcellulose was dissolved in 80% of the total volume of water at 100°C, and then 20% of the total volume of water at 25°C was added). The PEF-fucoidan-anthocyanin mixed solution was mixed with 40 mL of the hydroxypropyl methylcellulose solution with a concentration of 40 mg / mL, and the mixture was subjected to magnetic stirring at a stirring rate of 600 r / min for 30 min to obtain a PEF-fucoidan-anthocyanin / hydroxypropyl methylcellulose mixed solution. Food-grade glycerol was added to the PEF-fucoidan-anthocyanin / hydroxypropyl methylcellulose mixed solution in an amount of 2.5% by volume, and the final pH of the solution was adjusted to 3.0. The mixture was subjected to magnetic stirring at a stirring rate of 600 r / min for 30 min to obtain a film-forming solution with a final volume of about 80 mL. The film-forming solution was poured into a film-forming plate, and the plate was placed in an oven for drying at 40°C for 10 h to obtain a film. After the film was balanced in a desiccator for 6 h, it was removed to obtain a pulsed electric field-regulated fucoidan steady-state anthocyanin controllable release oral dissolving film, which was named as PEF-FU-BA / HPMC.

[0043] Comparative Example 1

[0044] A method for preparing an anthocyanin oral dissolving film:

[0045] Blueberry anthocyanin extract was dissolved in citric acid-sodium citrate buffer solution with pH = 3 to prepare an anthocyanin extract solution with a concentration of 10 mg / mL. 0.8 mL of the blueberry anthocyanin extract solution was added to 39.2 mL of distilled water, and the mixture was stirred magnetically for 10 min at a stirring rate of 250 r / min to obtain an anthocyanin solution as a control. A hydroxypropyl methylcellulose solution with a concentration of 40 mg / mL was prepared by a cold-hot method (hydroxypropyl methylcellulose was dissolved in 80% of the total volume of water at 100°C, and then 20% of the total volume of water at 25°C was added). The anthocyanin solution was mixed with 40 mL of the hydroxypropyl methylcellulose solution with a concentration of 40 mg / mL, and the mixture was stirred magnetically for 30 min at a stirring rate of 600 r / min to obtain an anthocyanin / hydroxypropyl methylcellulose mixed solution. Food-grade glycerol was added to the anthocyanin / hydroxypropyl methylcellulose mixed solution in an amount of 2.5% by volume, and the final pH of the solution was adjusted to 3.0. The mixture was stirred magnetically for 30 min at a stirring rate of 600 r / min to obtain a film-forming solution, and the final volume of the film-forming solution was about 80 mL. The film-forming solution was poured into a film-forming plate and dried in an oven at 40°C for 10 h to obtain a film, which was balanced in a desiccator for 6 h, and then the film was removed to obtain an anthocyanin oral dissolving film, which was named BA / HPMC.

[0046] Comparative Example 2

[0047] A method for preparing a fucoidan steady-state anthocyanin controlled-release oral dissolving film

[0048] Fucoidan solution was prepared by dissolving fucoidan in distilled water to obtain a fucoidan solution with a concentration of 4 mg / mL. Blueberry anthocyanin extract was dissolved in a citric acid-sodium citrate buffer solution with a pH of 3 to obtain an anthocyanin extract solution with a concentration of 10 mg / mL. 0.8 mL of the blueberry anthocyanin extract solution was added to 39.2 mL of the fucoidan solution with a concentration of 4 mg / mL, and the mixture was stirred magnetically for 10 min at a stirring rate of 250 r / min to obtain a fucoidan-anthocyanin mixed solution. A hydroxypropyl methylcellulose solution with a concentration of 40 mg / mL was prepared by the cold-hot method (hydroxypropyl methylcellulose was dissolved in 80% of the total volume of water at 100°C, and then 20% of the total volume of water at 25°C was added). The fucoidan-anthocyanin mixed solution was mixed with 40 mL of the hydroxypropyl methylcellulose solution with a concentration of 40 mg / mL, and the mixture was stirred magnetically for 30 min at a stirring rate of 600 r / min to obtain a fucoidan-anthocyanin / hydroxypropyl methylcellulose mixed solution. Food-grade glycerol was added to the fucoidan-anthocyanin / hydroxypropyl methylcellulose mixed solution in an amount of 2.5% by volume, and the final pH of the solution was adjusted to 3.0. The mixture was stirred magnetically for 30 min at a stirring rate of 600 r / min to obtain a film-forming solution with a final volume of about 80 mL. The film-forming solution was poured into a film-forming plate and dried in an oven at 40°C for 10 h to obtain a film. After the film was equilibrated in a desiccator for 6 h, it was removed from the plate to obtain a fucoidan-based anthocyanin controlled-release oral film, which was named FU-BA / HPMC.

[0049] The evaluation results of Comparative Example 1, Comparative Example 2 and Example 1 are as follows:

[0050] I. Evaluation of the stability of the fucoidan-based anthocyanin controlled-release oral film solution system regulated by pulsed electric fields

[0051] The retention rates of anthocyanin in the BA / HPMC, FU-BA / HPMC and PEF-FU-BA / HPMC composite solutions were determined under simulated light irradiation and a wide pH range (5-8) in the oral cavity to evaluate the steady-state effect of fucoidan on anthocyanin regulated by pulsed electric fields. The content of anthocyanin was determined by pH differential spectrophotometry, and the retention rate was calculated according to the following formula:

[0052] Retention rate of anthocyanin (%) = C t / C0×100[1]

[0053] wherein C t and C0are the concentrations of anthocyanin at time t and the initial time, respectively, mg / mL.

[0054] The retention rates of anthocyanin are shown in Table 1. Figure 1As shown in Fig. 4A, the anthocyanin retention rate gradually decreased with the extension of storage time. It is worth noting that the addition of fucoidan and the PEF-regulated fucoidan significantly improved the retention rate of anthocyanin during storage. After 8 days of light storage, the retention rate of anthocyanin in FU-BA / HPMC and PEF-FU-BA / HPMC was 1.76 and 2.76 times higher than that in BA / HPMC, respectively. In order to further evaluate the adaptability of anthocyanin in the oral environment, the retention rate of anthocyanin in BA / HPMC, FU-BA / HPMC and PEF-FU-BA / HPMC complex solutions was measured at pH 5-8. As shown in Fig. 4B, the retention rate of anthocyanin in FU-BA / HPMC and PEF-FU-BA / HPMC complex solutions was higher than that in BA / HPMC at pH 5-8 after 30 min, and the retention rate of anthocyanin after PEF regulation was the best. In summary, fucoidan and PEF-regulated fucoidan have a high steady-state effect on anthocyanin in the film-forming solution, and the steady-state effect of anthocyanin after PEF regulation is the best. Figure 1 B, the retention rate of anthocyanin in FU-BA / HPMC and PEF-FU-BA / HPMC complex solutions was higher than that in BA / HPMC at pH 5-8 after 30 min, and the retention rate of anthocyanin after PEF regulation was the best. In summary, fucoidan and PEF-regulated fucoidan have a high steady-state effect on anthocyanin in the film-forming solution, and the steady-state effect of anthocyanin after PEF regulation is the best.

[0055] II. Evaluation of the physical properties of PEF-regulated fucoidan steady-state anthocyanin oral dissolving film

[0056] The thickness and mechanical properties of the oral dissolving film were evaluated to determine the product forming ability and application stability. The thickness of the oral dissolving film was measured using a digital micrometer, and the mechanical properties of the oral dissolving film were measured according to the method of GB / T 1040.3-2006 "Determination of tensile properties of plastics". The film was cut into a long strip of 100 mm x 15 mm and fixed on an electronic tensile testing machine to measure the tensile strength (TS) and elongation at break (EAB) of the film. The results are shown in Fig. 5A and Fig. 5B. Figure 2 As shown in Fig. 5A and Fig. 5B, the addition of fucoidan increased the thickness of the oral dissolving film and improved the tensile strength and elongation at break of the oral dissolving film.

[0057] The disintegration properties of the oral dissolving film were further evaluated, which was crucial for the effective release of the active ingredients loaded. The disintegration time of the oral dissolving film was determined by fixing the film sample (3 cm x 2 cm) on a support glass frame and placing it in a petri dish. One drop of water (200 μL) was titrated on the surface of the oral dissolving film. The time required for the disintegration and pore formation of each film was quantified as the disintegration time (s). Each sample was measured in parallel for 10 times to ensure the accuracy of the test. The results of the disintegration time are shown in Fig. 6A and Fig. 6B. Figure 2As shown in Fig. 5, the disintegration time of BA / HPMC group was the shortest (47.48 s), and the addition of fucoidan prolonged the disintegration time of the oral film. The disintegration time of FU-BA / HPMC group was 52.08 s, which was related to the thickness of the film itself. In addition, due to the increase of intermolecular interaction force between the components of the oral film after PEF regulation, the binding tightness was enhanced, which led to the prolongation of the disintegration time of the oral film. The disintegration time of PEF-FU-BA / HPMC group was 58.11 s.

[0058] Three, content and color stability of fucoidan-stabilized anthocyanin oral film regulated by pulsed electric field

[0059] The BA / HPMC, FU-BA / HPMC and PEF-FU-BA / HPMC oral films were subjected to simulated light accelerated test to evaluate the content and color stability of anthocyanin under light stress. The retention rate was calculated according to formula [1], and the color difference value (ΔE) of color change was calculated according to the following formula:

[0060]

[0061] The results are shown in Figure 3 As shown in Fig. 6, within 8 days of light treatment, the retention rate of anthocyanin decreased with increasing storage time, and the ΔE value of the oral film gradually increased. After 8 days of light treatment, the retention rate of anthocyanin in BA / HPMC oral film was 7.06%, while the retention rates of anthocyanin in FU-BA / HPMC and PEF-FU-BA / HPMC groups were increased by 3.99 and 5.50 times, respectively. At the same time, the ΔE value of BA / HPMC oral film reached 5.34, which was significantly higher than that of FU-BA / HPMC (2.45) and PEF-FU-BA / HPMC (2.03) oral film groups. In summary, the results of retention rate and color change showed that fucoidan complexed anthocyanin oral film improved the color and content stability of anthocyanin, and the effect of fucoidan stabilized anthocyanin oral film regulated by pulsed electric field was the most significant.

[0062] Four, evaluation of antioxidant activity of fucoidan-stabilized anthocyanin oral film regulated by pulsed electric field

[0063] To prove that the oral film has good biological activity after formation, we first evaluated its antioxidant activity by DPPH and ABTS free radical scavenging ability. As shown in Fig. 7, the DPPH and ABTS free radical scavenging rates of BA / HPMC, FU-BA / HPMC and PEF-FU-BA / HPMC oral films were 48.62%, 59.62% and 63.62%, respectively, and 48.62%, 59.62% and 63.62%, respectively. Figure 4A, B, compared with BA / HPMC, the antioxidant activity of fucoidan complexed oral dissolving film was significantly enhanced, and the antioxidant activity of anthocyanin alone in the carrier group was low. The results showed that after adding fucoidan, the antioxidant performance of the oral dissolving film was significantly improved, indicating that the presence of fucoidan could reduce the loss of antioxidant activity of anthocyanin caused by temperature stress during drying. Notably, the antioxidant capacity of the oral dissolving film after pulse electric field regulation was the strongest, indicating that the pulse electric field treatment promoted the ability of fucoidan to stabilize anthocyanin, thereby retaining the activity of anthocyanin and improving its antioxidant capacity.

[0064] CAA assay is a cell-based method for evaluating antioxidant capacity, which indirectly assesses the bioavailability and cellular uptake of active ingredients. Therefore, the present application further verifies the antioxidant activity of the oral dissolving film at the cellular level. As shown in Figure 4 C, the CAA value of BA / HPMC was 7.12 μmol QE equiv. / 100 mg BA, which was significantly lower than that of FU-BA / HPMC (9.89 μmol QE equiv. / 100 mg BA) and PEF-FU-BA / HPMC (12.40 μmol QE equiv. / 100 mg BA) (p<0.05). In summary, the DPPH, ABTS and cellular antioxidant results all showed that the presence of fucoidan could enhance the antioxidant activity of anthocyanin, and the effect was more significant after pulse electric field regulation, proving that the oral dissolving film formed based on the pulse electric field regulated fucoidan stabilization technology had a significant effect on the retention of anthocyanin activity.

[0065] V. Evaluation of the in vitro release effect of pulse electric field regulated fucoidan stabilized anthocyanin oral dissolving film

[0066] For oral dissolving film preparations, whether the effective ingredients can be released controllably in the oral cavity is the key to achieving their functional effects, therefore, it is necessary to evaluate the release effect and content change of anthocyanin in the oral dissolving film. The release behavior of anthocyanin in the oral dissolving film was evaluated by simulating the pH value of the oral cavity. 6 cm 2 The oral dissolving film was placed in 20 mL pH 6.8 buffer solution and shaken in a water bath at 37°C. Samples (1 mL) were taken at 2, 4, 6, 8, 10, 15, 20, 25 and 30 minutes, and an equal volume of fresh buffer solution was added. The pH differential method was used to determine the content of anthocyanin at different time points. At the same time, the antioxidant activity of anthocyanin was determined after 30 minutes of release to evaluate the activity retention ability of the oral dissolving film after release.

[0067] The release results are shown in Figure 5As shown in A, with the extension of release time, the release rate of anthocyanins in BA / HPMC reached a peak at 4 min, which was 64.21%. Due to the weak binding strength of anthocyanins in the binary system, the free anthocyanins were rapidly released after the disintegration of the oral film. It is worth noting that the release peak of FU-BA / HPMC and PEF-FU-BA / HPMC in the oral film appeared at 10 min, which was due to the strong binding strength of the ternary system, which prolonged the disintegration time of the oral film, resulting in the delay of the release peak of anthocyanins, showing the slow-release and controlled-release effect. From Figure 5 As shown in the change of release slope coefficient in B, the slope of BA / HPMC was significantly higher than that of the other two groups, further indicating that the presence of fucoidan had a slow-release effect on the oral film. Among them, the release amount of anthocyanins in the PEF-FU-BA / HPMC group was the highest, which was 75.72%, which may be due to the better dispersion of the ternary system after the regulation of the pulse electric field. After the disintegration of the oral film, anthocyanins were more easily released, and the anthocyanins released by the protection of fucoidan were not easily degraded by the stress of the oral pH environment. However, after the release peak, with the extension of time, the residence time of anthocyanins in the oral environment (alkaline pH) was longer, resulting in a degradation rate greater than the release rate, so the content of anthocyanins showed a downward trend. It is worth noting that when the oral film gradually decomposed, FU-BA / HPMC and PEF-FU-BA / HPMC still had a stabilizing effect on anthocyanins, which showed that with the increase of release time, fucoidan delayed the loss of anthocyanins in the oral environment, and the content of anthocyanins was significantly higher than that of the control group (p<0.05) after 30 min of release. Further evaluation of the antioxidant activity of BA / HPMC, FU-BA / HPMC and PEF-FU-BA / HPMC after 30 min of release. The results are shown in Figure 5 As shown in C-E, the antioxidant capacity of FU-BA / HPMC and PEF-FU-BA / HPMC groups was significantly better than that of the control group, and the antioxidant activity of the oral film after the regulation of the pulse electric field was the highest. In summary, under the regulation of the pulse electric field, the formed oral film has a controlled-release effect, and the release amount of anthocyanins is the largest, and its antioxidant activity is the most significant after release, which proves that the steady-state effect of the pulse electric field regulated fucoidan on the formation of the anthocyanin controlled-release oral film has a significant technical effect.

Claims

1. A method for preparing a fucoidan steady-state anthocyanin controllable release oral dissolving film regulated by pulsed electric field, characterized in that: a fucoidan water solution is treated by high-field pulsed electric field, then the fucoidan water solution treated by pulsed electric field is mixed with a blueberry anthocyanin extract solution, and the mixture is treated by low-field pulsed electric field to obtain a fucoidan-anthocyanin mixed solution; a hydroxypropyl methylcellulose solution is added to the fucoidan-anthocyanin mixed solution and magnetically stirred to obtain a fucoidan-anthocyanin-hydroxypropyl methylcellulose mixed solution; a certain amount of food-grade glycerol is added to the mixed solution, the pH of the solution is adjusted to 2-4, and the solution is continuously magnetically stirred for a period of time to obtain a film-forming solution; and the film-forming solution is dried to form a film, thereby obtaining the fucoidan steady-state anthocyanin controllable release oral dissolving film regulated by pulsed electric field, wherein, the high-field pulsed electric field has an electric field strength of 15-25 kV / cm, a pulse frequency of 1000-1200 Hz, and a pulse time of 10-100 μs; the low-field pulsed electric field has an electric field strength of 10-15 kV / cm, a pulse frequency of 800-1000 Hz, and a pulse time of 10-100 μs; the concentration of the fucoidan water solution is 2-8 mg / mL; the concentration of the blueberry anthocyanin extract solution is 1-10 mg / mL; the mass ratio of the blueberry anthocyanin extract to fucoidan is 1:20-60; the hydroxypropyl methylcellulose water solution is prepared by adding hydroxypropyl methylcellulose to 80% of a total volume of water at 100°C to dissolve, then adding 20% of a total volume of water at 25°C, magnetically stirring at a speed of 500-800 r / min for 1-2 h to obtain a hydroxypropyl methylcellulose solution with a concentration of 20-60 mg / mL; and the mass ratio of the hydroxypropyl methylcellulose to fucoidan is 10-30:

1. The magnetic stirring speed is 500-800 r / min, and the stirring time is 20-40 min. The amount of glycerol added is 1%-3% of the total volume of the film-forming solution. The fucoidan-anthocyanin-hydroxypropyl methylcellulose solution is adjusted to a pH of 2-4 using 0.1 mol / L HCl solution or 0.1 mol / L NaOH solution. The obtained film-forming solution is cast onto a film-forming plate and placed in a blast drying oven to dry the film, with a drying temperature of 38°C-42°C and a drying time of 8-16 h. ​ ​ 2. The method of claim 1, wherein: ​ 3. The method of claim 1, wherein: ​ 4. The method of claim 1, wherein: ​ 5. The method of claim 1, wherein: ​

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