Exosome-like nano-vesicle dual-carrier for regulating and controlling glucose metabolism, method and application of exosome-like nano-vesicle dual-carrier

Abstract

The invention discloses an exosome-like nano-vesicle dual-carrier for regulating glucose metabolism, a method and application, and belongs to the field of biotechnology and bioengineering. According to the tartary buckwheat exosome-like nano-vesicle dual-carrier, the structural advantage of a tartary buckwheat exosome-like nano-vesicle is taken as an entry point, and chlorogenic acid with an alpha-amylase activity inhibition effect is loaded into an inner core cavity of the tartary buckwheat exosome-like nano-vesicle through ultrasonic loading; selenium nanoparticles with an anti-oxidation effect are combined to an exosome-like nano-vesicle lipid membrane of the tartary buckwheat through in-situ reduction, and the tartary buckwheat exosome-like nano-vesicle dual-carrier with a multi-element synergistic regulation effect is constructed. Cell and animal test results show that the prepared dual-carrier is safe and free of toxic and side effects in an effective dose range, can cooperatively regulate and control blood glucose rise caused by high-fat diet in a multi-angle and multi-channel mode, and has good development and application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and bioengineering, and specifically belongs to an exosome-like nanovesicle dual carrier, method and application for regulating sugar metabolism. Background Art

[0002] Diabetes is a chronic metabolic disease characterized by high blood sugar. In recent years, the number of diabetes patients worldwide has generally been on the rise and younger, and has become the second type of chronic non-communicable disease that seriously affects human health after cardiovascular and cerebrovascular diseases. Diabetes is difficult to cure. Once you are sick, you need to take medication for life. Long-term use of hypoglycemic drugs can cause various side effects such as flatulence and digestive system disorders. Sugar metabolism disorders are closely related to the body's blood sugar level and the occurrence of diabetes, and are typical symptoms of diabetes. Therefore, research and development of functional foods, beverages or health products that have the ability to regulate sugar metabolism are of great significance for preventing and intervening in the occurrence of diabetes and maintaining the health of the body.

[0003] Active ingredients in food, such as polyphenols, flavonoids and nutrients, are commonly used nutritional ingredients for regulating sugar metabolism. They have attracted close attention due to their wide sources, safety and non-toxic side effects. However, polyphenols and flavonoids are easily degraded by light, acid and alkaline environments due to the presence of multiple hydroxyl groups in their structures. Nutrients such as selenium often exist in the form of nanoparticles, which are prone to aggregation and precipitation due to their high specific surface area, resulting in low oral bioavailability, which seriously limits the efficiency of regulating sugar metabolism. Chlorogenic acid (CGA) and selenium (Se) are commonly used active ingredients for regulating sugar metabolism. CGA is a hydrophilic phenolic substance that can inhibit α-amylase activity, delay starch digestion rate and increase blood sugar levels after meals, but its polyhydroxy structure is easily degraded by acid and alkaline environments. At the same time, as a water-soluble phenolic substance, its low absorption and transport efficiency is another important factor limiting its efficiency in regulating sugar metabolism. In addition, Se is an essential nutrient for the human body, and its nanoparticles SeNPs can effectively increase the activity of GSH-Px, relieve oxidative stress, and regulate the body's sugar metabolism. However, due to its high specific surface area, it is easy to aggregate and precipitate, which limits its application. SeNPs can effectively avoid their aggregation and precipitation by binding to the surface of polymer materials such as proteins and polysaccharides, thereby improving their stability. In addition, the regulation mechanism of sugar metabolism is complex, and the regulation mechanism of a single food active component is single, and the regulation effect is not ideal, resulting in a high incidence of diabetes. Therefore, the research and development of a multi-component synergistic sugar metabolism regulation system that can improve the oral bioavailability of food active components is of great significance for the prevention and treatment of the occurrence and development of diabetes. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the present invention provides an exosome-like nanovesicle dual carrier, method and application for regulating glucose metabolism. The idea of ​​multi-element coordinated regulation is adopted, and buckwheat exosome-like nanovesicles (ELNs) are used as carriers. Chlorogenic acid CGA is loaded into the inner core of buckwheat ELNs by ultrasonic loading, and selenium nanoparticles SeNPs are combined with the outer lipid membrane of buckwheat ELNs by in situ reduction to construct the buckwheat ELNs dual carrier, which provides a theoretical basis for the development of functional foods, beverages or health products for regulating glucose metabolism.

[0005] To achieve the above objectives, the present invention provides the following technical solutions: an exosome-like nanovesicle dual carrier for regulating sugar metabolism, comprising tartary buckwheat exosome-like nanovesicles, chlorogenic acid loaded in the inner core of the tartary buckwheat exosome-like nanovesicles, and selenium nanoparticles bound to the outer lipid membrane of the tartary buckwheat exosome-like nanovesicles.

[0006] Furthermore, chlorogenic acid was loaded into the inner core of buckwheat exosome-like nanovesicles using ultrasound.

[0007] Furthermore, selenium nanoparticles were bound to the outer lipid membrane of buckwheat exosome-like nanovesicles by in situ reduction.

[0008] The present invention also provides a method for preparing an exosome-like nanovesicle dual carrier for regulating sugar metabolism, and the specific steps are as follows:

[0009] The tartary buckwheat exosome-like nanovesicles and chlorogenic acid solution were slowly mixed in equal volumes and subjected to ice bath sonication to obtain a single carrier of the tartary buckwheat exosome-like nanovesicles;

[0010] The tartary buckwheat exosome-like nanovesicle single carrier, sodium selenite solution and ascorbic acid solution were mixed, an in situ reduction reaction was carried out, the pH of the solution was adjusted, and the tartary buckwheat exosome-like nanovesicle dual carrier was obtained by centrifugation.

[0011] Further, in the step of slowly mixing equal volumes of tartary buckwheat exosome-like nanovesicles and chlorogenic acid solution and performing ice bath sonication to obtain a single carrier of tartary buckwheat exosome-like nanovesicles:

[0012] An equal volume of 10 mg / ml tartary buckwheat exosome-like nanovesicle suspension and 75 mg / ml chlorogenic acid solution were mixed, ultrasonicated in an ice bath, allowed to stand, and centrifuged to obtain a single tartary buckwheat exosome-like nanovesicle carrier.

[0013] Furthermore, the ultrasonic condition is 20% to 30% amplitude, each ultrasonication is 120s, interval is 60s, and cycled 5 times; static incubation is 37°C for 60min; centrifugation is 8000g to 10,000g at 4°C for 10min.

[0014] Further, in the step of mixing the tartary buckwheat exosome-like nanovesicle single carrier, sodium selenite solution and ascorbic acid solution, and performing an in-situ reduction reaction to obtain the tartary buckwheat exosome-like nanovesicle dual carrier:

[0015] The single carrier of tartary buckwheat exosome-like nanovesicles was mixed with a sodium selenite solution with a concentration of 0.1M to 0.2M, stirred at room temperature at 1000 r / min for 3 hours, and then an ascorbic acid solution with a concentration of 0.1M to 0.2M was added for in situ reduction reaction;

[0016] After the in situ reduction reaction, the pH value was adjusted to 6.8, and the supernatant was obtained by centrifugation at 3000g-4000g for 10 min at 4°C; the supernatant was ultracentrifuged at 100,000g-120,000g for 2 h, and the precipitate was collected to obtain the buckwheat exosome-like nanovesicle dual carrier.

[0017] Furthermore, the tartary buckwheat exosome-like nanovesicles are prepared by differential centrifugation-sucrose density gradient centrifugation, wherein the mass / volume ratio of tartary buckwheat to phosphate buffer is 1:3.

[0018] The present invention also provides the use of the above-mentioned exosome-like nanovesicle dual carrier for regulating sugar metabolism in the preparation of sugar metabolism regulating functional food or beverage.

[0019] The present invention also provides the use of the above-mentioned exosome-like nanovesicle dual carrier for regulating glucose metabolism in the preparation of an oral health product for regulating glucose metabolism.

[0020] Compared with the prior art, the present invention has at least the following beneficial effects:

[0021] Plant ELNs have a phospholipid bilayer membrane structure and do not carry human or animal pathogens. They have significant advantages such as safety, non-toxicity, good biocompatibility, and low immunogenicity, and can carry hydrophilic / hydrophobic active components at the same time. In addition, in addition to lipids, proteins, and RNA carrying genetic information, plant ELNs also contain hydrophilic and lipophilic secondary metabolites derived from plants, making them have biological activities similar to those of the source plants. Based on this, the present invention provides an exosome-like nanovesicle dual carrier for regulating glucose metabolism, which takes the structural advantages of tartary buckwheat ELNs as the starting point, adopts a multi-element coordinated regulation approach, utilizes the unique lipid membrane structure of tartary buckwheat ELNs combined with the characteristics of chlorogenic acid and selenium nanoparticles in regulating glucose metabolism, loads chlorogenic acid that can inhibit α-amylase activity into the inner core of ELNs, and combines selenium nanoparticles with oxidative stress regulation into the outer lipid membrane of ELNs to construct a tartary buckwheat exosome-like nanovesicle dual carrier (SeNPs-ELNs-CGA), so as to improve the deficiencies of chlorogenic acid and selenium nanoparticles in the glucose metabolism regulation process, solve the problems of low oral bioavailability of CGA and SeNPs and limitations of glucose metabolism regulation, and provide a theoretical basis for the development of oral functional foods, beverages or health products for regulating glucose metabolism.

[0022] The bitter buckwheat exosome-like nanovesicle dual carrier (SeNPs-ELNs-CGA) prepared by the present invention can effectively reduce the gastrointestinal degradation rate of CGA and significantly improve the stability of SeNPs. At the same time, SeNPs are bound to the outer lipid membrane of ELNs, which can significantly improve the rigidity of the ELNs membrane, increase the gastrointestinal stability of the dual carrier, and thus improve the oral bioavailability of the carrier component. In addition, both carrier components of this dual carrier can regulate the body's sugar metabolism through different modes of action, and bitter buckwheat ELNs have biological activities similar to bitter buckwheat, and can be used as a biologically active carrier that can regulate sugar metabolism. Therefore, the bitter buckwheat ELNs dual carrier of the present invention can significantly improve the problems of low oral bioavailability of the carrier components chlorogenic acid and selenium nanoparticles and limited sugar metabolism regulation through multi-component synergistic action.

[0023] The buckwheat exosome-like nanovesicle dual carrier of the present invention loads chlorogenic acid with α-amylase activity inhibition into the inner core of the buckwheat exosome-like nanovesicle by ultrasonic loading, and uses in situ reduction to bind selenium nanoparticles with antioxidant effects to the outer lipid membrane of the buckwheat exosome-like nanovesicle to construct a buckwheat exosome-like nanovesicle dual carrier with multi-element synergistic regulation. The results of cell and animal experiments show that the dual carrier prepared by the present invention is safe and has no toxic side effects within the effective dose range, can synergistically regulate the increase in blood sugar caused by a high-fat diet from multiple angles and multiple pathways, and has good development and application prospects.

[0024] In vitro simulated gastrointestinal digestion experiments show that the buckwheat exosome-like nanovesicle dual carrier of the present invention has good gastrointestinal stability, which provides the possibility for its application in functional drinks or health products; and the buckwheat exosome-like nanovesicle dual carrier of the present invention can significantly delay the increase in postprandial blood glucose in mice, improve the glucose tolerance and insulin tolerance of mice fed a high-fat diet, promote the growth of probiotics related to glucose metabolism, activate the expression of multiple glucose metabolism-related signal pathways, and effectively regulate the glucose metabolism disorders caused by a high-fat diet. It will provide innovative ideas for the construction of multiple carriers of food active components based on the coordinated regulation of glucose metabolism, and has important guiding significance for the multi-dimensional coordinated regulation of glucose metabolism. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a characterization diagram of the morphology and particle size of buckwheat ELNs.

[0026] Figure 2 This is a characterization diagram of the buckwheat exosome-like nanovesicle dual carrier.

[0027] Figure 3 This is the gastrointestinal and storage stability diagram of the buckwheat exosome-like nanovesicle dual carrier.

[0028] Figure 4 This is a diagram showing the structural characteristics of the buckwheat exosome-like nanovesicle dual carrier.

[0029] Figure 5 The effect of buckwheat exosome-like nanovesicle dual carriers on cell activity.

[0030] Figure 6 To evaluate the safety of buckwheat exosome-like nanovesicle dual carriers in animals.

[0031] Figure 7 The effect of buckwheat exosome-like nanovesicle dual carriers on postprandial blood glucose levels in mice.

[0032] Figure 8 The present study is about the regulatory effect of buckwheat exosome-like nanovesicle dual carriers on the changes in body weight and fasting blood glucose in mice fed a high-fat diet.

[0033] Fig. 9 Changes in oral glucose tolerance and insulin tolerance in high-fat diet mice intervened by dual carriers of tartary buckwheat exosome-like nanovesicles.

[0034] Fig.10 Colon tissue analysis of high-fat diet mice intervened by dual carriers of Tartary Buckwheat exosome-like nanovesicles.

[0035] Fig.11 Analysis of intestinal flora in high-fat diet mice intervened by dual carriers of tartary buckwheat exosome-like nanovesicles.

[0036] Fig.12Proteomic analysis of liver tissue in high-fat diet mice treated with dual carriers of tartary buckwheat exosome-like nanovesicles. DETAILED DESCRIPTION

[0037] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0038] The present invention uses food active component delivery technology to provide a buckwheat exosome-like nanovesicle dual carrier, which can carry multiple components at the same time, has a size of about 500nm, has good gastrointestinal stability and high sugar metabolism regulation efficiency. The specific steps of its preparation are as follows:

[0039] Step 1: Extract buckwheat ELNs using differential centrifugation-sucrose density gradient centrifugation:

[0040] The crushed buckwheat seeds were mixed with a phosphate buffered saline solution, wherein the mass / volume ratio of buckwheat to PBS was 1:3, and the mixture was placed at 4°C overnight. The supernatant was subjected to differential centrifugation at 4°C, 1200g-1500g for 20 min, 3000g-4000g for 30 min, 8000g-10,000g for 60 min, and 100,000g-120,000g for 120 min, and the precipitate was taken.

[0041] Subsequently, the ELNs were purified by 8%, 30%, 45% and 60% sucrose solutions, and the ELNs were collected after centrifugation at 100,000g-120,000g for 120 min and resuspended in PBS, i.e., the tartary buckwheat ELNs suspension.

[0042] Step 2: Chlorogenic acid CGA was loaded into the inner core of buckwheat ELNs by ultrasonic loading to reduce the gastrointestinal degradation rate of CGA and improve its oral bioavailability.

[0043] The buckwheat ELNs suspension (10 mg / ml) and CGA solution (75 mg / ml) dissolved in anhydrous ethanol were mixed in equal volumes and then subjected to ice-bath sonication at 4°C to complete one-level loading. The excess unloaded CGA was removed to obtain buckwheat exosome-like nanovesicle single carriers (buckwheat ELNs-CGA).

[0044] Furthermore, the ultrasonic condition was 20% to 30% amplitude, each ultrasonication was 120 s, with an interval of 60 s, and the cycle was repeated 5 times.

[0045] Furthermore, after the end of ultrasound in one layer of loading, the cells were incubated at 37°C for 60 min to allow the plasma membrane of buckwheat ELNs to recover.

[0046] Furthermore, after the plasma membrane of tartary buckwheat ELNs was restored, the precipitate was discarded by centrifugation at 8000g-10,000g for 10 min at 4°C to remove the excess unloaded CGA.

[0047] Step 3: Selenium nanoparticles SeNPs are bound to the outer lipid membrane of buckwheat ELNs by in situ reduction. On the one hand, the stability of SeNPs can be improved, and on the other hand, the membrane rigidity of the buckwheat ELNs nano dual carrier can be enhanced, thereby improving its gastrointestinal stability and the oral bioavailability of the carried components.

[0048] After mixing and stirring the buckwheat exosome-like nanovesicle single carrier (buckwheat ELNs-CGA) and sodium selenite (Na2SeO3) solution, ascorbic acid solution was added to carry out in situ reduction reaction to achieve double loading, thus obtaining the buckwheat exosome-like nanovesicle dual carrier (SeNPs-buckwheat ELNs-CGA).

[0049] Furthermore, the tartary buckwheat ELNs-CGA was mixed with a Na2SeO3 solution having a concentration of 0.1M to 0.2M, and after stirring at room temperature at 1000 r / min for 3 h, an ascorbic acid solution having a concentration of 0.1M to 0.2M was added, and stirring was continued for 40 min.

[0050] Furthermore, after double loading, the pH value was adjusted to 6.8, and the supernatant was obtained by centrifugation at 3000g-4000g for 10 min at 4°C. The supernatant was then ultracentrifuged at 100,000g-120,000g for 2h, and the precipitate was collected and suspended in PBS to obtain the buckwheat exosome-like nanovesicle dual carrier.

[0051] The tartary buckwheat exosome-like nanovesicle dual carrier of the present invention is stored at 4° C. and can achieve multi-factor coordinated regulation of sugar metabolism, and thus can be added as a functional substance to oral functional foods, beverages or health products for regulating sugar metabolism.

[0052] Example 1

[0053] The present invention provides an exosome-like nanovesicle dual carrier for regulating sugar metabolism, which is prepared by the following steps:

[0054] 1. Preparation of Tartary Buckwheat ELNs:

[0055] (1) After the buckwheat seeds were broken by a wall breaking machine, they were mixed with phosphate buffered saline in a ratio of 1:3 and placed at 4°C overnight.

[0056] (2) Take the supernatant and perform differential centrifugation at 4°C. After centrifugation at 1200g for 20 min, take supernatant ①; after centrifugation at 3000g for 30 min, take supernatant ②; after centrifugation at 10,000g for 60 min, take supernatant ③; after centrifugation at 100,000g for 120 min, take precipitate ④;

[0057] (3) Subsequently, sucrose density gradient centrifugation was used for purification and precipitation④. Sucrose solutions with mass fractions of 60%, 45%, 30%, and 8% were added in sequence, and the mixture was centrifuged at 120,000 g for 120 min. The ELNs were collected and resuspended in PBS, i.e., the buckwheat ELN suspension.

[0058] Figure 1 This is a characterization diagram of the morphology and particle size of tartary buckwheat ELNs (TB-ELNs). TB-ELNs are spherical or ellipsoidal in shape, with a particle size of about 128nm and a Zeta potential of -9.2mV.

[0059] 2. Preparation of Tartary Buckwheat Exosome-like Nanovesicle Single Carrier (Tartary Buckwheat ELNs-CGA):

[0060] (1) The protein of buckwheat ELNs was quantified using a BCA protein quantification kit. 10 mg / ml of buckwheat ELNs was taken and mixed with an equal volume of 75 mg / ml CGA solution dissolved in anhydrous ethanol.

[0061] (2) Ultrasonic method was used to load CGA once.

[0062] Ice bath ultrasound was performed at 4°C with an amplitude of 20%, and the ultrasound was repeated for 5 times after 120s, followed by a 60s interval. After ultrasound, the cells were incubated at 37°C for 60min to restore the plasma membrane of buckwheat ELNs. The excess unloaded CGA was removed by centrifugation at 8000g for 10min at 4°C to obtain the supernatant.

[0063] (3) The supernatant after centrifugation in step (2) was mixed with 0.1 M Na2SeO3, and stirred at room temperature at 1000 r / min for 3 h. Then, 0.1 M ascorbic acid solution was added to the reaction solution, and stirring was continued at 1000 r / min for 40 min to perform an in situ reduction reaction to achieve double loading.

[0064] (4) After the reaction was completed, the pH value of the reaction solution was adjusted to 6.8, and the supernatant was obtained by centrifugation at 3000 g for 10 min at 4°C. The supernatant was centrifuged at 120,000 g for 2 h, and the precipitate was collected and suspended in PBS to obtain the buckwheat ELNs nano dual carrier suspension.

[0065] Figure 2 This is a characterization diagram of the buckwheat exosome-like nanovesicle dual carrier. SeNPs are combined with the outer lipid membrane of ELNs, making the dual carrier appear as a flower cluster with a particle size of about 500nm.

[0066] Figure 3 The figure shows the gastrointestinal and storage stability of the buckwheat exosome-like nanovesicle dual carrier. Figure 3As shown in a, compared with the free CGA and SeNPs groups, the tartary buckwheat ELNs dual carrier has good gastrointestinal stability, which can effectively reduce the degradation of CGA and SeNPs in the gastrointestinal tract and improve their bioavailability. GP value is often used to measure the phospholipid membrane rigidity of substances, such as Figure 3 As shown in b, the GP value of the buckwheat ELNs nano dual carrier did not change significantly under extremely acidic and neutral pH conditions, and the GP value was relatively high, indicating that the buckwheat ELNs dual carrier has certain advantages in overcoming the gastrointestinal environment of the oral route.

[0067] Figure 4 This is a diagram showing the structural characteristics of the buckwheat exosome-like nanovesicle dual carrier. Figure 4 a is the infrared spectra of tartary buckwheat ELNs and their carriers. Compared with the ELNs group, no new characteristic peaks appeared in the ELNs-CGA and ELNs-SeNPs groups, indicating that no new chemical bonds or functional groups were formed between ELNs and CGA and between ELNs and SeNPs, and they were mainly connected through non-covalent interactions. -1 To 3500cm -1 The characteristic peak between is mainly caused by the stretching vibration of hydroxyl groups, which is closely related to the intramolecular and intermolecular hydrogen bonds, reflecting the strength of hydrogen bonds. When CGA is added, the width of this peak becomes wider and the hydrogen bond strength increases, indicating that CGA and ELNs are mainly bound by hydrogen bonds. In addition, Figure 4 It can be seen in b that the buckwheat ELNs-SeNPs suspension is light orange, while after sodium dodecyl sulfate (SDS) is first added to the buckwheat ELNs and then reacted with SeNPs, the reaction solution becomes colorless, that is, SDS can prevent the binding of ELNs and SeNPs, indicating that buckwheat ELNs-SeNPs are mainly connected through hydrophobic interactions.

[0068] The buckwheat exosome-like nanovesicle dual carrier obtained in Example 1 was used as a test sample, and the effect of the present invention on regulating sugar metabolism was verified in the following steps:

[0069] (1) Biosafety evaluation of buckwheat ELNs and their dual vectors

[0070] To ensure the safety of buckwheat ELNs and their dual carriers, different concentrations of buckwheat ELNs and their nanocarriers were co-incubated with Caco-2 cells and HepG2 cells, and the MTT method was used to determine their effects on cell viability. Figure 5 As shown, within the sample concentration range of 20 μg / mL, the viability of Caco-2 cells (left) and HepG2 cells (right) were both greater than 80%, that is, each group of samples had no significant effect on the viability of Caco-2 cells and HepG2 cells within the concentration range of 20 μg / mL.

[0071] Based on the above experiments, the biosafety of buckwheat ELNs and their nanocarriers was further evaluated using C57 mice. After mice were gavaged with buckwheat ELNs and their nanocarriers daily for six weeks, Figure 6 As shown in the figure, the sections of the mouse heart, liver, spleen, lung and kidney showed no significant difference in the tissue sections of the four groups of samples compared with the normal group. The myocardial cell morphology was intact without obvious deformation; the hepatocytes, spleen cells, lung cells and kidney cells were uniform in size and regularly arranged, and no pathological tissue appeared. In addition, compared with the normal control group, there were no significant differences in the red blood cell, white blood cell, hemoglobin and platelet contents of the tartary buckwheat ELNs and its nano dual carrier groups. The contents of AST, ALT, BUN and CREA in the sample group were also not significantly different from those in the control group. It can be seen that long-term intragastric administration of the tartary buckwheat ELNs dual carrier did not cause inflammation and tissue lesions in mice, and had good biosafety, which could be used for the normal conduct of subsequent experiments.

[0072] (2) Changes in blood glucose levels in mice after meals

[0073] The effect of buckwheat ELNs-CGA nanocarriers in delaying the increase of postprandial blood glucose level was evaluated by measuring the postprandial blood glucose level of mice. C57 mice (30) were divided into 5 groups, 6 in each group. After fasting for 16 hours, the mice in each group were gavaged with: ① starch; ② starch and ELNs; ③ starch and CGA; ④ starch and ELNs+CGA (mix ELNs with CGA, ready for use); ⑤ starch and ELNs-CGA (load an equal amount of CGA on ELNs) After gavage of equal amounts of samples to each group of mice, the postprandial blood glucose values ​​were measured at different time points (0, 30, 60, 90, 120 min), and the area under the curve (AUC) was calculated by the trapezoidal rule.

[0074] like Figure 7 As shown, the postprandial blood glucose levels of mice increased rapidly within 0-60 minutes, and gradually decreased within 60-120 minutes. The blood glucose levels of the sample group were significantly lower than those of the control group, indicating that both buckwheat ELNs and CGA have the effect of delaying the increase in postprandial blood glucose levels, and the effect of ELNs-CGA in delaying the increase in postprandial blood glucose levels is significantly better than ELNs+CGA.

[0075] (3) Changes in body weight and fasting blood glucose in mice

[0076] C57 mice (72 mice) were randomly divided into 9 groups, 8 mice in each group, and the experimental period was 6 weeks: ① normal control group; ② high fat diet (HFD) model control group; ③ ELNs intervention group; ④ CGA intervention group; ⑤ ELNs-CGA intervention group (equivalent CGA was loaded on ELNs); ⑥ SeNPs intervention group; ⑦ ELNs-SeNPs intervention group (equivalent SeNPs were loaded on ELNs); ⑧ SeNPs+ELNs+CGA intervention group (ELNs were mixed with CGA and SeNPs and used immediately after preparation). ⑨ SeNPs-ELNs-CGA intervention group (tartary buckwheat ELNs dual carrier of the present invention).

[0077] Group ① was fed a normal diet; Group ② was given a high-fat diet (60% fat); Groups ③-⑨ were given a high-fat diet and gavaged with samples from the corresponding groups once a day. During the experiment, the weight of the mice was recorded weekly, and the fasting blood glucose level of the mice was measured by a blood glucose meter.

[0078] The results are as follows Figure 8 As shown, after 6 weeks of intervention, compared with the HFD group, the dual-carrier of buckwheat ELNs could significantly reduce the weight gain of mice fed a high-fat diet and reduce the increase in fasting blood glucose levels in mice.

[0079] (4) Oral glucose tolerance and insulin tolerance test

[0080] Mice fed a high-fat diet for 6 weeks were fasted for 12 hours and gavaged with glucose solution (2g / kg). The blood glucose level of the mice was measured at different time points (0, 30, 60, 90, 120min) after gavage using a blood glucose meter, and the oral glucose tolerance test (OGTT) and AUC of the mice were calculated. Mice were intraperitoneally injected with insulin (0.4U / kg), and the blood glucose level was measured at different time points (0, 30, 60, 90, 120min) after injection, and the insulin tolerance test (ITT), AUC and insulin resistance index (HOMA-IR) were calculated.

[0081] OGTT and ITT are closely related to the body's insulin sensitivity and blood glucose level. Fig. 9 As shown. Compared with the HFD group, the blood glucose levels of the buckwheat ELNs dual carriers at 0-120 min were lower than those of the HFD group, indicating that oral administration of the buckwheat ELNs dual carriers can significantly improve the insulin sensitivity of mice. From the AUC, it can be seen that CGA and SeNPs can significantly improve insulin sensitivity and glucose tolerance after being transported by buckwheat ELNs, and the dual carriers are significantly better than the mixed group in reducing blood glucose levels and improving insulin resistance.

[0082] (5) Colon tissue repair

[0083] Long-term high-fat diet can damage the integrity of the intestinal barrier, which prevents harmful substances from entering the blood and protects the health of the host. Fig.10 As shown in the results, the bitter buckwheat ELNs dual carrier can significantly delay the shortening of the colon caused by a high-fat diet, repair the damage to the colon epithelial barrier, and promote colon mucus secretion and the expression of colon tight junction proteins. In addition, the bitter buckwheat ELNs dual carrier can promote the growth of microvilli on the surface of the colon epithelium, and the length of microvilli is positively correlated with the tight junction of the colon epithelium. Therefore, oral administration of bitter buckwheat ELNs dual carrier can significantly improve the damage to the intestinal barrier function caused by a high-fat diet by promoting the secretion of goblet cell mucus and the arrangement of colon epithelial cell microvilli.

[0084] (6) Intestinal flora analysis

[0085] The intestinal microbiota is closely related to the regulation of glucose metabolism and the development of type 2 diabetes. Therefore, the intestinal microbiota of HFD mice after 6 weeks of intervention with tartary buckwheat exosome-like nanovesicles dual carriers was sequenced and analyzed using 16s RNA sequencing technology.

[0086] like Fig.11 As shown in the figure, the Venn diagram showed that the high-fat diet significantly affected the types and composition of the intestinal microbiota of mice. The bitter buckwheat exosome-like nanovesicle dual carrier can promote the proliferation of actinomycetes and Campylobacter. Actinomycetes are Gram-positive bacteria represented by Bifidobacterium, which plays an important role in maintaining the intestinal health of the host. Heat map analysis found that after the intervention of bitter buckwheat exosome-like nanovesicle dual carrier, the relative abundance of related pathogens was significantly reduced, ELNs-CGA significantly increased the abundance of Faecalibaculum, and ELNs-SeNPs showed a significant proliferation effect on Lachnospiraceae bacteria. The bitter buckwheat exosome-like nanovesicle dual carrier simultaneously increased the beneficial bacteria of Faecalibaculum and Lachnospiraceae. Faecalibaculum and Lachnospiraceae can promote the production of short-chain fatty acids (SCFAs). Long-term oral administration of tartary buckwheat ELNs dual carriers can inhibit the proliferation of pathogens, promote the growth of probiotics related to glucose metabolism, effectively regulate the body's glucose metabolism, and alleviate the hyperglycemia symptoms of HFD mice.

[0087] (7) Proteomic analysis of mouse liver tissue

[0088] Proteomic analysis was performed on liver tissues of high-fat diet mice after oral administration of buckwheat ELNs dual vectors. The data were analyzed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.

[0089] like Fig.12 As shown in the figure, the heat map and cluster analysis of non-phosphorylated and phosphorylated proteins related to glucose metabolism in liver tissues by KEGG analysis revealed that both phosphorylated and non-phosphorylated proteins were involved in the biological process of glucose metabolism. Compared with the HFD and HFD-(SeNPs+ELNs+CGA) mixed groups, the buckwheat ELNs dual carrier significantly increased the levels of proteins related to inflammation, oxidative stress and tight junctions, such as SOD, GSH, H2O2, TNF and ZO, and increased the phosphorylated proteins closely related to the regulation of hyperglycemia and the development of diabetes, such as PI3K, AKT, AMPK, MAPK and GSK3β. These research results indicate that the delivery of CGA and SeNPs by buckwheat ELNs can significantly improve their bioavailability. The nano dual carrier based on buckwheat ELNs can effectively regulate the expression of key proteins related to the glucose metabolism process in mice fed a high-fat diet through the synergistic effect of multiple components.

[0090] Example 2

[0091] The present invention provides an exosome-like nanovesicle dual carrier for regulating sugar metabolism, which is prepared by the following steps:

[0092] 1. Preparation of Tartary Buckwheat ELNs:

[0093] (1) After the buckwheat seeds were broken by a wall breaking machine, they were mixed with phosphate buffered saline in a ratio of 1:3 and placed at 4°C overnight.

[0094] (2) Take the supernatant and perform differential centrifugation at 4°C. After centrifugation at 1500g for 20 min, take supernatant ①; after centrifugation at 4000g for 30 min, take supernatant ②; after centrifugation at 8000g for 60 min, take supernatant ③; after centrifugation at 120,000g for 120 min, take precipitate ④;

[0095] (3) Subsequently, sucrose density gradient centrifugation was used for purification and precipitation④. Sucrose solutions with mass fractions of 60%, 45%, 30%, and 8% were added in sequence, and the mixture was centrifuged at 100,000 g for 120 min. The ELNs were collected and resuspended in PBS, i.e., the buckwheat ELN suspension.

[0096] 2. Preparation of Tartary Buckwheat Exosome-like Nanovesicle Single Carrier (Tartary Buckwheat ELNs-CGA):

[0097] (1) The protein of buckwheat ELNs was quantified using a BCA protein quantification kit. 10 mg / ml of buckwheat ELNs was taken and mixed with an equal volume of 75 mg / ml CGA solution dissolved in anhydrous ethanol.

[0098] (2) Ultrasonic method was used to load CGA.

[0099] Ice bath ultrasound was performed at 4°C with an amplitude of 30%, and the ultrasound was repeated for 5 times with an interval of 60 seconds after each ultrasound for 120 seconds. After the ultrasound, the cells were incubated at 37°C for 60 minutes to restore the plasma membrane of buckwheat ELNs. The excess unloaded CGA was removed by centrifugation at 10,000 g for 10 minutes at 4°C to obtain the supernatant.

[0100] (3) The supernatant after centrifugation in step (2) was mixed with 0.2 M Na2SeO3, and stirred at room temperature at 1000 r / min for 3 h. Then, 0.2 M ascorbic acid solution was added to the reaction solution, and stirring was continued at 1000 r / min for 40 min to perform an in situ reduction reaction to achieve double loading.

[0101] (4) After the reaction was completed, the pH value of the reaction solution was adjusted to 6.8, and the supernatant was obtained by centrifugation at 4000 g for 10 min at 4°C. The supernatant was centrifuged at 100,000 g for 2 h, and the precipitate was collected and suspended in PBS to obtain the buckwheat ELNs nano dual carrier suspension.

[0102] Example 3

[0103] The present invention provides an exosome-like nanovesicle dual carrier for regulating sugar metabolism, which is prepared by the following steps:

[0104] 1. Preparation of Tartary Buckwheat ELNs:

[0105] (1) After the buckwheat seeds were broken by a wall breaking machine, they were mixed with phosphate buffered saline in a ratio of 1:3 and placed at 4°C overnight.

[0106] (2) Take the supernatant and perform differential centrifugation at 4°C. After centrifugation at 1300g for 20 min, take supernatant ①; after centrifugation at 3500g for 30 min, take supernatant ②; after centrifugation at 9,000g for 60 min, take supernatant ③; after centrifugation at 110,000g for 120 min, take precipitate ④;

[0107] (3) Subsequently, sucrose density gradient centrifugation was used for purification and precipitation④. Sucrose solutions with mass fractions of 60%, 45%, 30%, and 8% were added in sequence, and the mixture was centrifuged at 110,000 g for 120 min. The ELNs were collected and resuspended in PBS, i.e., the buckwheat ELN suspension.

[0108] 2. Preparation of Tartary Buckwheat Exosome-like Nanovesicle Single Carrier (Tartary Buckwheat ELNs-CGA):

[0109] (1) The protein of buckwheat ELNs was quantified using a BCA protein quantification kit. 10 mg / ml of buckwheat ELNs was taken and mixed with an equal volume of 75 mg / ml CGA solution dissolved in anhydrous ethanol.

[0110] (2) Ultrasonic method was used to load CGA.

[0111] Ice bath ultrasound was performed at 4°C with an amplitude of 20%, and the ultrasound was repeated for 5 times after 120s, followed by a 60s interval. After ultrasound, the cells were incubated at 37°C for 60min to restore the plasma membrane of buckwheat ELNs. The excess unloaded CGA was removed by centrifugation at 9000g for 10min at 4°C to obtain the supernatant.

[0112] (3) The supernatant after centrifugation in step (2) was mixed with 0.15 M Na2SeO3, and stirred at room temperature at 1000 r / min for 3 h. Then, 0.15 M ascorbic acid solution was added to the reaction solution, and stirring was continued at 1000 r / min for 40 min to perform an in situ reduction reaction to achieve double loading.

[0113] (4) After the reaction was completed, the pH value of the reaction solution was adjusted to 6.8, and the supernatant was obtained by centrifugation at 3500 g for 10 min at 4 °C. The supernatant was centrifuged at 110,000 g for 2 h, and the precipitate was collected and suspended in PBS to obtain the buckwheat ELNs nano dual carrier suspension.

[0114] In summary, buckwheat ELNs can be used as a nanocarrier with hypoglycemic activity to carry food active ingredients, effectively improve the bioavailability of active ingredients, and improve their biological activity. Buckwheat ELNs dual carrier has significant beneficial effects in regulating sugar metabolism, its preparation method is simple and stable, the product has good biocompatibility and health benefits, and has broad market application prospects.

Claims

1. An exosome-like nanovesicle dual carrier for regulating sugar metabolism, characterized in that: It includes buckwheat exosome-like nanovesicles, chlorogenic acid loaded in the inner core of the buckwheat exosome-like nanovesicles, and selenium nanoparticles bound to the outer lipid membrane of the buckwheat exosome-like nanovesicles.

2. The exosome-like nanovesicle dual carrier for regulating sugar metabolism according to claim 1, characterized in that: Chlorogenic acid was loaded into the inner core of buckwheat exosome-like nanovesicles by ultrasound.

3. The exosome-like nanovesicle dual carrier for regulating sugar metabolism according to claim 1, characterized in that: Selenium nanoparticles were bound to the outer lipid membrane of buckwheat exosome-like nanovesicles by in situ reduction.

4. A method for preparing an exosome-like nanovesicle dual carrier for regulating sugar metabolism according to any one of claims 1 to 3, characterized in that: The specific steps are as follows: The tartary buckwheat exosome-like nanovesicles and chlorogenic acid solution were slowly mixed in equal volumes and subjected to ice bath sonication to obtain a single carrier of the tartary buckwheat exosome-like nanovesicles; The tartary buckwheat exosome-like nanovesicle single carrier, sodium selenite solution and ascorbic acid solution were mixed, an in situ reduction reaction was carried out, the pH of the solution was adjusted, and the tartary buckwheat exosome-like nanovesicle dual carrier was obtained by centrifugation.

5. The method for preparing an exosome-like nanovesicle dual carrier for regulating sugar metabolism according to claim 4, characterized in that: The step of slowly mixing equal volumes of tartary buckwheat exosome-like nanovesicles and chlorogenic acid solution and performing ice bath sonication to obtain a single carrier of tartary buckwheat exosome-like nanovesicles is as follows: An equal volume of 10 mg / ml tartary buckwheat exosome-like nanovesicle suspension and 75 mg / ml chlorogenic acid solution were mixed, ultrasonicated in an ice bath, allowed to stand, and centrifuged to obtain a single tartary buckwheat exosome-like nanovesicle carrier.

6. The method for preparing an exosome-like nanovesicle dual carrier for regulating sugar metabolism according to claim 5, characterized in that: The ultrasonic condition is 20% to 30% amplitude, each ultrasonication is 120s, interval is 60s, and cycled 5 times; the static incubation is 37°C for 60min; the centrifugation is 8000g to 10,000g for 10min at 4°C.

7. The method for preparing an exosome-like nanovesicle dual carrier for regulating sugar metabolism according to claim 4, characterized in that: The step of mixing a single carrier of tartary buckwheat exosome-like nanovesicles, a sodium selenite solution and an ascorbic acid solution, and performing an in-situ reduction reaction to obtain a dual carrier of tartary buckwheat exosome-like nanovesicles: The single carrier of tartary buckwheat exosome-like nanovesicles was mixed with a sodium selenite solution with a concentration of 0.1M to 0.2M, stirred at room temperature at 1000 r / min for 3 hours, and then an ascorbic acid solution with a concentration of 0.1M to 0.2M was added for in situ reduction reaction; After the in situ reduction reaction, the pH value was adjusted to 6.8, and the supernatant was obtained by centrifugation at 3000g-4000g for 10 min at 4°C; the supernatant was ultracentrifuged at 100,000g-120,000g for 2 h, and the precipitate was collected to obtain the buckwheat exosome-like nanovesicle dual carrier.

8. The method for preparing the exosome-like nanovesicle dual carrier for regulating sugar metabolism according to claim 4, characterized in that: The tartary buckwheat exosome-like nanovesicles are prepared by differential centrifugation-sucrose density gradient centrifugation, wherein the mass / volume ratio of tartary buckwheat to phosphate buffer is 1:

3.

9. Use of an exosome-like nanovesicle dual carrier for regulating glucose metabolism according to any one of claims 1 to 3 in the preparation of glucose metabolism regulating functional foods or drinks.

10. Use of the exosome-like nanovesicle dual carrier for regulating glucose metabolism according to any one of claims 1 to 3 in the preparation of an oral health product for regulating glucose metabolism.

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