A composite emulsion for improving insomnia and preparation method thereof

Abstract

The present invention belongs to the technical field of functional preparations, and discloses a composite emulsion for improving insomnia and a preparation method thereof. The components of the composite emulsion are: 70-95% of a modified fucoidan solution with a concentration of 0.5-3 mg / mL, and the remainder is a medium-chain triglyceride containing melatonin, in terms of volume percentage; the content of melatonin in the composite emulsion is 0.02wt%. The composite emulsion has strong stability and high bioavailability, can effectively resist the acidic environment in the stomach, and protect melatonin from being delivered to the intestine. It has been verified that the average core sleep time of the subjects after taking the composite emulsion was increased by 21.05% compared with taking melatonin alone; the average wakefulness time was shortened by 40% compared with taking melatonin alone, and the effect was significant.

Description

Technical Field

[0001] The invention belongs to the technical field of functional preparations and relates to a composite emulsion for improving insomnia and a preparation method thereof. Background Art

[0002] Fucoidan (FUC) is non-toxic and highly biocompatible, exhibiting antiviral, anti-inflammatory, anti-tumor, and immunomodulatory properties. It has broad applications in therapy, pharmacy, diagnostics, and drug delivery. Using FUC as a delivery vehicle and combining it with other bioactive ingredients to form complex formulations holds great potential for the delivery of nutrients and drugs. For example, Chen Xing et al. prepared a binary complex system of whey protein isolate (WPI) and FUC, investigated the properties of the WPI-FUC complex under different conditions, and identified the optimal process.

[0003] Existing patent 202311498952.6 discloses a modified sulfated polysaccharide compound and a preparation method thereof. This patent uses concentrated sulfuric acid as a catalyst to carry out an esterification modification reaction to prepare a modified esterified polysaccharide compound, which has the ability to significantly improve the quality of fucoidan. However, the concentrated sulfuric acid used in this patent is highly corrosive, and there are safety issues in the production of the reagent, which has a certain adverse effect on the medicinal safety of the modified polysaccharide.

[0004] Existing patent 201810861854.7 discloses an oil-in-water emulsion of esterified modified soybean polysaccharide and its preparation method. The invention introduces starch octenyl succinate, leveraging the properties of modified starch to enhance the emulsifying activity of the polysaccharide. This invention effectively improves the emulsification stability of the polysaccharide. However, the starch octenyl succinate introduced in this application is a chemical macromolecular particle, which has certain implications for the safety of nanofunctional formulation production.

[0005] Therefore, how to improve the emulsification properties of natural polysaccharides and increase the bioavailability of functional ingredients remains an urgent industrial problem to be solved. Summary of the Invention

[0006] In order to improve the emulsification of natural polysaccharides and enhance the bioavailability of functional components, the present invention provides a composite emulsion for improving insomnia and a preparation method thereof.

[0007] First, the present invention provides a composite emulsion for improving insomnia, wherein the components of the composite emulsion are: 70-95% by volume of a modified fucoidan solution with a concentration of 0.5-3 mg / mL, and the remainder is a medium-chain triglyceride containing melatonin; the content of melatonin in the composite emulsion is 0.02 wt%.

[0008] Furthermore, for the above composite emulsion, the preparation method of the modified fucoidan is:

[0009] Disperse fucoidan in pure water to prepare a 20 wt% fucoidan solution, and adjust the pH of the fucoidan solution to 8.5 with a 0.5 mol / L NaOH solution;

[0010] Add 3-9% octenylsuccinic anhydride dilution and continue stirring the reaction at 45°C for 1 hour. During this time, use 0.5 mol / L NaOH solution to maintain the pH of the reaction solution at 8.5.

[0011] The reaction was terminated by adjusting the pH to 6.0 with 0.1 mol / L HCl solution;

[0012] The reaction solution was dialyzed for 48 h using a dialysis bag with a molecular weight cut-off of 3500 Da, evaporated and concentrated to remove water, and then freeze-dried to obtain modified fucoidan;

[0013] The octenyl succinic anhydride dilution solution is prepared by diluting octenyl succinic anhydride (OSA) with 5 times the volume of anhydrous ethanol.

[0014] On the other hand, the present invention provides a method for preparing the composite emulsion, comprising:

[0015] Dissolve the modified fucoidan in distilled water to prepare a modified fucoidan solution with a concentration of 0.5-3 mg / mL, heat it to 85°C and maintain it for 30 minutes, then cool it to 50°C;

[0016] Dissolving melatonin in medium-chain triglycerides so that the content of melatonin in the composite emulsion is 0.02 wt %, to obtain medium-chain triglycerides containing melatonin;

[0017] The modified fucoidan solution is mixed with the medium-chain triglyceride containing melatonin in a mixing ratio of 70-95% by volume of the modified fucoidan solution and the balance of the medium-chain triglyceride containing melatonin;

[0018] The mixture is homogenized in a shearing machine and then ultrasonically treated for 1 to 5 minutes to obtain the composite emulsion.

[0019] Furthermore, in the preparation method of the composite emulsion, the homogenization treatment conditions are: 10000 rpm, 3 min.

[0020] Finally, the present invention provides an application of the composite emulsion. Specifically, the composite emulsion is used to improve insomnia.

[0021] Compared with the prior art, the present invention "a composite emulsion for improving insomnia and its preparation method" has the following beneficial effects:

[0022] The present invention uses octenylsuccinic anhydride (OSA) to modify fucoidan, effectively improving its emulsification ability and forming a highly stable and bioavailable composite emulsion system. This helps effectively resist the acidic environment in the stomach and protects melatonin delivery to the intestines. The experimental group taking the composite emulsion demonstrated a 40.8% increase in average core sleep duration compared to the control group and a 21.05% increase compared to the melatonin group. The experimental group also experienced a 62.5% decrease in average wakefulness compared to the control group and a 40% decrease compared to the melatonin group. Furthermore, the number of patients experiencing significant effects and effective responses in the experimental group was 6 and 10, respectively, representing significant increases of 3 and 2, respectively, compared to the melatonin group. The overall effective rate was 25% higher than that of the melatonin group, demonstrating significant results. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The degree of substitution (DS) of fucoidan under different addition amounts of octenylsuccinic anhydride diluent.

[0024] Figure 2 Scanning electron micrographs of fucoidan (FUC) and modified fucoidan (OSA-FUC).

[0025] Figure 3 The particle size changes of the emulsion under different OSA-FUC concentration conditions.

[0026] Figure 4 The particle size changes of the emulsion under different MCT contents.

[0027] Figure 5 The particle size changes of the emulsion under different ultrasonic power conditions.

[0028] Figure 6 The particle size changes of the emulsion under different ultrasonic time conditions.

[0029] Figure 7 is the turbidity and whiteness of the emulsion under different MCT contents.

[0030] Figure 8 are the turbidity and whiteness of the emulsion under different OSA-FUC concentration conditions.

[0031] Figure 9 The turbidity and whiteness of the emulsion under different ultrasonic power conditions.

[0032] Figure 10 The turbidity and whiteness of the emulsion under different ultrasonic time conditions.

[0033] Figure 11 The following are the results of microstructure observation: A is the result of optical microscopy observation, B1 and B2 are the results of scanning electron microscopy observation, and C1 to C3 are the results of confocal laser scanning microscopy (CLSM) observation.

[0034] Figure 12 The particle size distribution trends under different pH conditions.

[0035] Figure 13 is the average particle size under different pH conditions.

[0036] Figure 14 The droplet morphology under different pH conditions.

[0037] Figure 15 Figure 3 is the particle size distribution trend under different NaCl concentration conditions.

[0038] Figure 16 is the average particle size under different NaCl concentration conditions.

[0039] Figure 17 The droplet morphology under different NaCl concentration conditions.

[0040] Figure 18 The cumulative release of melatonin from MCT and compound emulsion containing melatonin at different digestion stages.

[0041] Figure 19 The cumulative release of free fatty acids (FFA) from MCT and compound emulsions containing melatonin at different digestion stages.

[0042] Figure 20 This is the fitting curve of the cumulative release of FFA from the composite emulsion during digestion.

[0043] Figure 21 This is the fitting curve of the cumulative release of FFA during the digestion of MCT containing melatonin. DETAILED DESCRIPTION

[0044] The technical solutions of the present invention are described clearly and completely below with reference to the embodiments. It is obvious that the embodiments described are only a portion of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are also within the scope of protection of the present invention.

[0045] Example 1

[0046] This example describes the modification of fucoidan.

[0047] Disperse fucoidan in pure water to prepare a 20 wt% fucoidan (FUC) solution, and adjust the pH of the fucoidan solution to 8.5 with 0.5 mol / L NaOH solution;

[0048] Add 3-9% octenylsuccinic anhydride dilution and continue stirring the reaction at 45°C for 1 hour. During this time, use 0.5 mol / L NaOH solution to maintain the pH of the reaction solution at 8.5.

[0049] The reaction was terminated by adjusting the pH to 6.0 with 0.1 mol / L HCl solution;

[0050] The reaction solution was dialyzed for 48 h using a dialysis bag with a molecular weight cutoff of 3500 Da, evaporated and concentrated to remove water, and freeze-dried to obtain powdered modified fucoidan (OSA-FUC);

[0051] In the above steps, the octenyl succinic anhydride dilution solution is prepared by diluting octenyl succinic anhydride (OSA) with 5 times the volume of anhydrous ethanol.

[0052] Figure 1 The DS of fucoidan is determined by titration and is used to characterize the degree of esterification of fucoidan. Figure 1 It can be seen that when the OSA concentration is 3%, 6% and 9%, the DS values ​​are 0.020, 0.028 and 0.030, respectively, indicating that OSA-modified fucoidan (OSA-FUC) was prepared through the esterification reaction of FUC and OSA.

[0053] Figure 2 The following are scanning electron micrographs of fucoidan (FUC) and modified fucoidan (OSA-FUC). Figure 2 It can be seen that the surface morphology of fucoidan has changed significantly after being modified by OSA. The surface of the modified fucoidan has obvious wrinkles, a loose structure, and cavities, which effectively promotes emulsification properties.

[0054] Example 2

[0055] This example describes the preparation of the composite emulsion.

[0056] Dissolve OSA-FUC in distilled water to prepare an OSA-FUC solution with a concentration of 0.5-3 mg / mL. Heat to 85°C for 30 min and then cool to 50°C.

[0057] Prepare melatonin-containing MCT by dissolving 0.004 g of melatonin (the density of the composite emulsion is considered to be 1 g / mL) in 1-6 mL of medium-chain triglycerides (MCT).

[0058] Mix 14-19 mL of OSA-FUC solution with 1-6 mL of MCT containing melatonin to create a 20 mL mixture. The OSA-FUC solution and MCT containing melatonin should make up 70-95% and 5-30% of the mixture, respectively, with the melatonin content in each mixture at 0.02 wt%.

[0059] The mixed solution was stirred at 10,000 rpm for 3 min in a high-speed shearing machine (Shanghai Sample Model Factory, FJ200-SH) for homogenization, and then ultrasonically treated with an ultrasonic cell disruptor (Wuxi Xinyi, VOSHIN-1500C) at 0, 100, 200, 300, 400, and 500 W for 1 to 5 min to obtain composite emulsions of different concentrations.

[0060] Example 3

[0061] This example describes the effects of preparation parameters (OSA-FUC concentration, MCT content, ultrasonic power, and ultrasonic time) on the particle size, turbidity, and whiteness of the composite emulsion.

[0062] 1. Effect of preparation parameters on emulsion particle size

[0063] The stability of emulsions is affected by a variety of factors. According to Stokes' law, particle size is one of the factors that determine the success of emulsion preparation. The emulsions obtained above were measured and compared using a Malvern laser particle size analyzer. Parameters were: dispersed phase refractive index of 1.5, absorptivity of 0.001; continuous phase refractive index of 1.333; and test temperature of 25°C.

[0064] 1) Effect of OSA-FUC concentration on the particle size of composite emulsion

[0065] The effect of OSA-FUC concentration on the particle size of the composite emulsion was determined according to the composite emulsion preparation method described in Example 2. In this experiment, the other parameters were set as follows: MCT content was 5% v / v, ultrasonic power was 400W, and ultrasonic time was 3 minutes. The experimental results are shown in Figure 3 .

[0066] Figure 3 The figure shows the changes in particle size of the emulsion under different OSA-FUC concentrations (0.5, 1, 1.5, 2, 2.5, 3 mg / mL). Figure 3As shown, with increasing OSA-FUC concentration, the particle size first decreases and then increases. The initial emulsion particle size decreases from 5.15±0.14μm (0.5mg / mL) to 1.44±0.14μm (2.0mg / mL). When the OSA-FUC concentration exceeds 2.0mg / mL, the particle size increases to 2.16±0.03μm (3.0mg / mL). This trend may be attributed to the increased coverage of the composite emulsion droplets by the increased OSA-FUC concentration, resulting in a decrease in particle size. As the interfacial layer of each composite emulsion droplet gradually thickens, the emulsion particle size gradually increases. Based on these experimental results, the optimal OSA-FUC concentration is 2.0mg / mL.

[0067] 2) Effect of MCT content on particle size of composite emulsion

[0068] The effect of MCT content on the particle size of the composite emulsion was determined according to the composite emulsion preparation method described in Example 2. In this experiment, the setting conditions of other parameters were: OSA-FUC concentration was 2.0 mg / mL, ultrasonic power was 400 W, and ultrasonic time was 3 min. The experimental results are shown in Figure 4 .

[0069] Figure 4 The particle size changes of the emulsions with different MCT contents (5, 10, 15, 20, 25, 30% v / v) are shown. Figure 4 As shown, with increasing MCT content, the emulsion particle size gradually increased, from 5.25±0.13μm (5% v / v MCT) to 22.10±0.21μm (30% v / v MCT). The particle size differences between the groups were significant, with the smallest particle size occurring at 5% v / v MCT. This may be because OSA-FUC forms a network structure through non-covalent interactions, restricting the movement of the composite emulsion. However, at low MCT content, the distance between emulsion droplets in the composite emulsion system is greater, making it less likely to form a cross-linked network, resulting in smaller particle size. Based on these experimental results, the optimal MCT content is 5% v / v.

[0070] 3) Effect of ultrasonic power on particle size of composite emulsion

[0071] The effect of ultrasound power on the particle size of the composite emulsion was determined according to the composite emulsion preparation method described in Example 2. In this experiment, the other parameters were set as follows: OSA-FUC concentration was 2.0 mg / mL, MCT content was 5% v / v, and ultrasound time was 3 min. The experimental results are shown in Figure 5 .

[0072] Figure 5The figure shows the change of particle size of the emulsion under different ultrasonic powers (0W, 100W, 200W, 300W, 400W). Figure 5 As shown in the figure, when the ultrasonic power increases from 100W to 400W, the particle size decreases from 9.07±0.32μm to 2.84±0.06μm. This indicates that the cavitation and shearing effects of ultrasound break up and disperse the emulsion droplets, forming a uniform system. However, as the ultrasonic power increases, the heat energy generated promotes the aggregation of the droplets. More severe cavitation can strongly squeeze the droplets and cause them to recombine, disrupting the uniformity of the liquid and leading to the formation of more heterogeneous agglomerates. Based on these experimental results, the optimal power for ultrasonic treatment is 400W.

[0073] 4) Effect of ultrasonic time on particle size of composite emulsion

[0074] The effect of ultrasound time on the particle size of the composite emulsion was determined according to the composite emulsion preparation method described in Example 2. In this experiment, the other parameters were set as follows: OSA-FUC concentration was 2.0 mg / mL, MCT content was 5% v / v, and ultrasound power was 400 W. The experimental results are shown in Figure 6 .

[0075] Figure 6 The figure shows the change of particle size of the emulsion under different ultrasonic time conditions (0min, 1min, 2min, 3min, 4min, 5min). Figure 6 As shown, without ultrasonic treatment (i.e., 0 min), the particle size of the emulsion was 9.41 ± 0.69 μm. When the ultrasonic treatment time was 4 min, the particle size reached a minimum of 1.34 ± 0.03 μm. This indicates that ultrasonic treatment for 4 min significantly reduces the particle size. Based on these experimental results, the optimal ultrasonic treatment time is 4 min.

[0076] 2. Effect of preparation parameters on turbidity and whiteness

[0077] The composite emulsion was diluted 200 times in water and the absorbance of the sample was measured at 600 nm. The turbidity and whiteness values ​​of the emulsion were measured using a colorimeter. The turbidity and whiteness were calculated using the following formula:

[0078]

[0079]

[0080] Among them, A 600 is the absorbance at 600nm, 200 is the dilution factor, 0.01 is the optical path difference, L * is brightness, a * is redness / greenness, b* Yellowness / blueness.

[0081] Table 1 shows the brightness under different MCT content, OSA-FUC concentration, different ultrasound power and ultrasound time conditions ( L * )、Red and Green Value( a * )、Yellow-blue degree( b * ), color saturation ( c * ), hue angle (H), and plot the effects of the four preparation parameters on turbidity and whiteness based on the calculation formulas for turbidity and whiteness. When measuring the effects of the above parameters on turbidity and whiteness, the settings for the remaining parameters are the same as in "1. Effect of Preparation Parameters on Emulsion Particle Size."

[0082] Table 1. Effect of preparation parameters on turbidity and whiteness

[0083]

[0084] Figure 7 The effect of varying MCT content on turbidity and whiteness is shown. Turbidity and whiteness are important parameters reflecting the stability and uniformity of an emulsion system. Furthermore, the visual appearance of an emulsion is a crucial factor influencing consumer acceptance during product processing. Higher turbidity and whiteness values ​​indicate a milky white appearance. When the MCT content ranged from 5% to 15% v / v, the turbidity of the composite emulsion significantly increased from 32,734.44 ± 18.42 to 81,767.04 ± 64.46 (p < 0.05). When the MCT content exceeded 15% v / v, the turbidity of the composite emulsion significantly decreased (p < 0.05), due to the precipitation of the MCT due to the excessive MCT content.

[0085] Figure 8 The effect of different OSA-FUC concentrations on turbidity and whiteness is shown in Table 1. As the concentration of OSA-FUC increases, the turbidity of the emulsion increases significantly (p<0.05), while the whiteness does not change significantly. b * and c * The H gradually increases and approaches 90°, indicating that the increase in OSA-FUC concentration makes the composite emulsion more inclined to dark yellow.

[0086] Figure 9 、 10 The effects of different ultrasonic power and ultrasonic time on turbidity and whiteness are respectively Figure 9 、 10It can be seen that compared with the emulsion without ultrasonic treatment, the whiteness and turbidity of the emulsion treated with ultrasonic treatment increased significantly (p<0.05), and the stability and uniformity were improved.

[0087] In summary, the composite emulsion had the best stability and uniformity when using an OSA-FUC concentration of 2.0 mg / mL, an MCT content of 5% v / v, and ultrasonic treatment at 400 W for 4 min.

[0088] Example 4

[0089] This example describes the observation of the microstructure of the composite emulsion.

[0090] 10 μL of the composite emulsion (OSA-FUC at a concentration of 2.0 mg / mL and 5% v / v MCT) was dropped onto a glass slide, covered with a coverslip, and observed under a 100x upright microscope to assess droplet size and aggregation. MCT and OSA-FUC were stained with Nile red and Nile blue, respectively, and observed using confocal laser scanning microscopy (CLSM). 2 mL of the composite emulsion was stained with a mixture of Nile blue and Nile red-fixed fluorescent dyes for 5 minutes and then stored in the dark for 2 hours for morphology observation. The fluorescent dyes were activated using a 488 nm argon laser or a 633 nm helium-neon laser.

[0091] Figure 11 The microstructure observation results are given by Figure 11 As can be seen from optical microscopy (A), the composite emulsion system is uniform, with the formed droplets dispersed, effectively preventing aggregation during long-term storage. Scanning electron microscopy (B1 and B2) reveals that melatonin encapsulated in the composite emulsion appears as small, irregular particles, while the resulting emulsion is spherical with a smooth surface and no irregular structures. CLSM can be used to gain a deeper understanding of how OSA-FUC stabilizes droplets, thereby clarifying the emulsion stabilization mechanism. The green layer and red spheres in the CLSM results (C1–C3) represent OSA-FUC and MCT, respectively. It can be clearly seen that all MCT is encapsulated by OSA-FUC, indicating strong emulsion stability.

[0092] Example 5

[0093] This example describes the resistance of the composite emulsion to different environmental stresses.

[0094] 1. Effect of pH on emulsion stability

[0095] The pH of the emulsion was adjusted to 2.0, 4.0, 6.0, 8.0, and 10.0 using 1 mol / L HCl and 1 mol / L NaOH. The test tube was wrapped with tin foil and placed in a dark place for 6 h. The particle size distribution trend and average particle size under different pH conditions were analyzed using a Malvern 3000 laser particle size analyzer, and the changes in droplet morphology were observed using a microscope.

[0096] Figure 12 is the particle size distribution trend under different pH conditions. Figure 12 It can be seen that under different pH conditions, the 10μm particle size distribution accounts for the highest proportion. Figure 13 、 Figure 14 are the average particle size and droplet morphology under different pH conditions. Figure 13 、 Figure 14 As can be seen, under alkaline conditions, the emulsion particle size increased, the droplet morphology became larger, and the liquid aggregates increased, but no segregation occurred. Under neutral and acidic conditions, the emulsion droplets dispersed, the particle size was smaller, and the particle size change was not significant compared to the freshly prepared emulsion. This indicates that the composite emulsion is more stable under neutral and slightly acidic conditions, further demonstrating that the composite emulsion can effectively resist the acidic environment in the stomach and protect melatonin delivery to the intestine.

[0097] 2. Effect of NaCl concentration on emulsion stability

[0098] The NaCl concentration was adjusted (0-0.5 M), the test tube was wrapped with tin foil and placed in the dark for 6 h, and the particle size distribution trend and average particle size were analyzed using a Malvern 3000 laser particle size analyzer, and the droplet morphology changes were observed using a microscope.

[0099] Figure 15 is the particle size distribution trend under different NaCl concentration conditions. Figure 15 It can be seen that under different NaCl concentration conditions, the emulsion particle size distribution shows a bimodal morphology, with the particle size of 10 μm accounting for the highest proportion. Figure 16 、 Figure 17 are the average particle size and droplet morphology under different NaCl concentration conditions, respectively. Figure 16 、 Figure 17 It can be seen that the emulsion has good stability at low NaCl concentrations. As the NaCl concentration increases, the particle size tends to gradually increase and aggregate, but no segregation occurs, indicating that the composite emulsion can maintain system stability without being destroyed in a certain concentration of NaCl environment.

[0100] Example 6

[0101] This example describes the digestion of a composite emulsion, using MCT containing melatonin as a control.

[0102] 1. Experimental Design

[0103] (1) Simulated oral stage

[0104] Prepare simulated saliva fluid (SSF): a total volume of 100 mL with 0.55 g / L Na₂HPO₄, 0.27 g / L NaH₂PO₄, 1500 U / mL α-amylase, and 0.3 mol / L CaCl₂. Adjust the pH to 7.0 with 1 mol / L HCl. Preheat 5 mL of freshly prepared SSF in a 37°C water bath. Mix with an equal volume of the composite emulsion and shake in a 37°C water bath at 100 rpm for 2 minutes to obtain mixed solution a.

[0105] (2) Simulated gastric stage

[0106] Prepare simulated gastric fluid (SGF): a total volume of 50 mL, wherein the NaCl concentration is 0.342 M (2 g / L), the pepsin concentration is 3.2 g / L, and the pH is adjusted to 1.2 with 1 mol / L HCl. Take 5 mL of the mixed solution a obtained in step (1) and 15 mL of freshly prepared SGF, mix them, adjust the pH to 1.2 with 1 mol / L HCl, and shake in a constant temperature water bath at 100 rpm and 37°C for 2 h to obtain mixed solution b.

[0107] (3) Simulated small intestine stage

[0108] Prepare simulated small intestinal fluid (SIF): a total volume of 50 mL, containing a bile salt concentration of 48.5 mg / mL, pancreatic lipase of 12 mg / mL, CaCl2 of 0.75 mol / L, and a pH of 7.0. Take 10 mL of the mixture b obtained in step (2), add the same volume of freshly prepared SIF, mix thoroughly, adjust the pH to 7.0 with 0.1 mol / L NaOH, and shake in a constant temperature water bath at 100 rpm and 37°C for 2 h to simulate small intestinal digestion. During this period, use 0.2 mol / L NaOH to maintain the pH at 7.0. After digestion, obtain a mixture c. Record the volume of NaOH consumed during small intestinal digestion and calculate the free fatty acids (FFA).

[0109]

[0110] In the above formula, C NaOH : concentration of NaOH solution during titration (mol / L), V NaOH : The volume of NaOH solution consumed during 2 h of digestion (L), M oil : Average relative molecular weight of MCT (g / mol).

[0111] 2. Test results

[0112] (1) Melatonin release

[0113] The mixed solution c is used as the micelle component, and the release of melatonin is reflected by the change in the content of melatonin in the micelle component. Figure 18 Figure 2 shows the cumulative release of melatonin from MCT containing melatonin and the composite emulsion at different digestion stages. The figure shows that the cumulative release of melatonin from the composite emulsion (55.32±1.21%) is higher than that from MCT containing melatonin (24.20±1.11%), indicating that the in vitro bioaccessibility of melatonin in the composite emulsion is better than that in MCT containing melatonin.

[0114] Generally, the bioaccessibility of melatonin is positively correlated with the release of FFAs, and the presence of some FFAs is believed to increase the solubility of melatonin in micelles. Figure 18 The results presented may be due to the fact that the OSA-FUC binding layer of the composite emulsion effectively disperses the droplets to form smaller particles, which has a larger surface area exposed to pancreatic lipase, and the degree to which MCT is enzymatically hydrolyzed into free fatty acids (FFA) is higher, thereby releasing more melatonin; while for MCT containing melatonin, the bile salts and pancreatic lipase attached to the surface of MCT cause them to form micelles, delaying the release of melatonin.

[0115] (2) Free fatty acid (FFA) release

[0116] The release of FFA was determined by the pH-stat method, and the amount of NaOH required to maintain a neutral pH during digestion was recorded to track the extent of MCT digestion. Figure 19 is the cumulative release of free fatty acids (FFA) from MCT and compound emulsions containing melatonin at different digestion stages. Figure 19 As shown, the FFA release from both the melatonin-containing MCT and composite emulsions increased rapidly over time from 0 to 20 minutes. From 20 to 180 minutes, the FFA release from the composite emulsion reached 40% of the total release, exceeding the 25% from the melatonin-containing MCT, and the upward trend continued after 180 minutes. These results indicate that the composite emulsion system is more conducive to the sustained release of FFA.

[0117] Subsequently, five commonly used release kinetic models were used to evaluate the FFA release during the small intestinal digestion stage (see Table 2).

[0118] Table 2. FFA release kinetics during small intestinal digestion

[0119]

[0120] According to the release kinetics model in Table 2, the Figure 20 、 21The fitting curves of the cumulative release of FFA during the small intestine digestion of the composite emulsion and MCT containing melatonin. From the linear values, for MCT containing melatonin, the first-order model (M t / M ∞ =e （k1×t） +C1) has the highest fitting degree (R 2 =0.98); for the composite emulsion, the Peppas-Sahlin model has the highest fitting degree (R 2 =0.98), and the first-order model ranks second (R 2 =0.94). The results show that the main mechanism of MCT in small intestine digestion is direct diffusion. The rapid release of FFA within the first 20 minutes indicates that pancreatic lipase quickly attaches to the surface of MCT and decomposes it. After that, the release rate of FFA slows down, which may be due to the bile salts adsorbing layer by layer on the surface of MCT to form a new protective layer, blocking the contact between MCT and pancreatic lipase, and inhibiting the decomposition of MCT.

[0121] Both systems show high fitting degrees and correlations in the Peppas-Sahlin model. The difference in the m value (0.49 ± 0.05) in the model indicates that the two systems have different FFA release mechanisms: 0.43 < m < 0.85 corresponds to the Fick diffusion mechanism, which is a synergistic mechanism of substance release diffusion and particle dissolution. The process is roughly as follows: The composite emulsion droplets contact the complex enzyme system in the small intestine, the OSA-FUC shell structure is damaged, and lipase quickly enters the interior to decompose MCT, releasing free fatty acids. As the action time prolongs, MCT is gradually exhausted, and in addition, the droplets gradually form micelles, and the micelle layer prevents lipase from entering the interior of the droplets, and the release of FFA gradually slows down.

[0122] Example 7

[0123] This example describes the effect of the composite emulsion for improving insomnia.

[0124] Test subjects: Select subjects with at least two of the following characteristics: long sleep onset time, frequent awakenings, unstable sleep, inability to fall asleep again after waking up, premature morning awakening,多梦 (excessive dreaming), etc. The subjects are aged 17 - 65 years, including 30 females and 30 males. They are randomly divided into an experimental group, a melatonin group, and a control group, with 10 males and 10 females in each group. The subjects in the experimental group take 10 mL of the composite emulsion provided by the present invention (melatonin content 0.02 wt%) once in the morning and once in the evening, after meals; the melatonin group takes melatonin (MCT containing 0.02 wt% melatonin), with the same dosage and administration method as the experimental group; the control group does not take any medicine, and the usage effects of each group are evaluated (the results are shown in Table 3).

[0125] Evaluation Method: Using a self-controlled design, a smartwatch monitors various sleep data. Sensors record and collect data, accurately analyzing the various sleep stages, including deep sleep, core sleep, REM sleep, and wakefulness. This allows for the determination of the timing and proportion of each sleep stage, thereby characterizing sleep structure. Evaluation Criteria: An increase of more than 10% in core sleep time or a decrease in wakefulness time of more than 10% is considered significant; an increase of 5-10% or more in core sleep time or a decrease in wakefulness time of more than 5-10% is considered effective; and a fluctuation of less than 5% in core sleep time or wakefulness time is considered ineffective.

[0126] Table 3. Effects of improving insomnia

[0127]

[0128] Calculations from the data in Table 3 show that the experimental group experienced a 40.8% increase in average core sleep duration compared to the control group and a 21.05% increase compared to the melatonin group. Average wakefulness duration was shortened by 62.5% in the experimental group and 40% in the melatonin group. Furthermore, the number of patients experiencing significant effects and effective sleep in the experimental group was 6 and 10, respectively, an increase of 3 and 2, respectively, compared to the melatonin group, demonstrating significant improvement. The overall effective rate increased by 25% compared to the melatonin group, demonstrating a significant effect.

[0129] The embodiments described above are only some of the embodiments of the present invention, rather than all of them. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but merely represents selected embodiments of the present invention. All other embodiments obtained without creative effort and through deduction and substitution by a person of ordinary skill in the art based on the concept of the present invention are within the scope of protection of the present invention.

Claims

1. A composite emulsion for improving insomnia, characterized in that: The components of the composite emulsion are: 70-95% by volume of a modified fucoidan solution with a concentration of 0.5-3 mg / mL, and the remainder is a medium-chain triglyceride containing melatonin; The content of melatonin in the composite emulsion is 0.02wt%; The modified fucoidan preparation method comprises the following steps: dispersing fucoidan in pure water to prepare a 20 wt% fucoidan solution, and adjusting the pH of the fucoidan solution system to 8.5 using a 0.5 mol / L NaOH solution; Add 3-9% octenylsuccinic anhydride dilution and continue stirring the reaction at 45°C for 1 hour. During this time, use 0.5 mol / L NaOH solution to maintain the pH of the reaction solution at 8.

5. The reaction was terminated by adjusting the pH to 6.0 with 0.1 mol / L HCl solution; The reaction solution was dialyzed for 48 h using a dialysis bag with a molecular weight cut-off of 3500 Da, evaporated and concentrated to remove water, and then freeze-dried to obtain modified fucoidan; The octenyl succinic anhydride dilution solution is prepared by diluting octenyl succinic anhydride with 5 times the volume of anhydrous ethanol.

2. The method for preparing the composite emulsion according to claim 1, wherein include: Dissolve the modified fucoidan in distilled water to prepare a modified fucoidan solution with a concentration of 0.5-3 mg / mL, heat it to 85°C and maintain it for 30 minutes, then cool it to 50°C; Dissolving melatonin in medium-chain triglycerides so that the content of melatonin in the composite emulsion is 0.02 wt %, to obtain medium-chain triglycerides containing melatonin; The modified fucoidan solution is mixed with the medium-chain triglyceride containing melatonin in a mixing ratio of 70-95% by volume of the modified fucoidan solution and the balance of the medium-chain triglyceride containing melatonin; The mixture was homogenized in a shearing machine at 10,000 rpm for 3 minutes, and then ultrasonically treated for 1 to 5 minutes to obtain the composite emulsion.

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