Rice-sourced vesicles as well as extraction method and application thereof

Abstract

According to the rice source vesicles and the extraction method and application thereof, the rice vesicles are extracted from rice through centrifugation and / or size exclusion chromatography, the extraction method is simple, and large-scale production is easy; the obtained rice vesicles are good in water solubility, excellent in permeability and easy to absorb, have the effects of regulating blood sugar and blood fat and reducing body weight, and have potential in the fields of functional food development, health management, medicine research and development and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological extraction, and particularly relates to a rice-derived vesicle and an extraction method and application thereof. BACKGROUND

[0002] Extracellular vesicles (EVs) are nanoscale lipid bilayer vesicles released by almost all cell types, including plant vesicles, microvesicles, and apoptotic bodies. EVs are widely present in various body fluids and cell supernatants, and stably carry important biomolecules such as nucleic acids, proteins, and lipids, and are involved in the regulation of pathological and physiological processes such as cell proliferation and differentiation, angiogenesis, and immune response, and have various biological activities, and are important carriers of information exchange. Plant vesicles are similar to mammalian EVs in morphology, composition, and function, but due to the presence of cell walls, they play an important role in a non-cell autonomous manner, and even can exchange biological information between species.

[0003] Plant exosome-like nanoparticles (PELNs) have a typical nanoscale vesicle structure with a diameter ranging from 30 to 300 nanometers. Under transmission electron microscopy (TEM), PELNs appear as flat spheres wrapped in a lipid bilayer, showing characteristic spherical, cup-shaped, or tea-tray-shaped shapes. This morphological feature is similar to animal-derived exosomes, indicating that they may have similar biogenesis mechanisms and physical and chemical properties.

[0004] PELNs have cross-border regulation ability and can be taken up by mammalian cells and regulate their physiological functions. This cross-border communication is mainly achieved in three ways: paracrine: PELNs bind to the target cell surface through receptor-ligand interaction, and then are endocytosed into the target cell, or are directly released into the target cell, thereby activating the target cell. Direct endocytosis: exosomes directly enter target cells through endocytosis. Membrane fusion: exosomes directly contact target cells for membrane fusion, causing non-selective transfer of nucleic acids and proteins in the exosomes to the target cells, causing a response in the target cells. Studies have shown that plant miRNAs (such as miR159) can cross-border regulate mammalian physiological functions, such as inhibiting tumor growth, improving energy metabolism, resisting oxidative stress, and regulating immune function of the body. This cross-border regulation function provides a theoretical basis for using PELNs for disease treatment.

[0005] PELNs show significant effects in regulating intestinal health. Studies have shown that exosomes from plants such as grapes, broccoli, ginger, and grapefruits play an important role in protecting intestinal inflammation and permeability, and are involved in shaping the intestinal microbiota. These exosomes interact with target cells such as intestinal epithelial cells, macrophages, and dendritic cells, promote E-cadherin expression and cell proliferation, and maintain intestinal barrier function.

[0006] Rice and its extracts have been proven to have multiple functions in many studies, but the active substances extracted from rice have certain limitations in water solubility, skin penetration and bioavailability, for example, certain phenolic / flavonoid components are not good in water solubility and are easy to be decomposed. Therefore, there is an urgent need in the art for a delivery carrier with good water solubility, good penetration and easy absorption to realize efficient use of active substances extracted from rice. SUMMARY

[0007] Embodiments of the present application provide a rice-derived vesicle and its extraction method and application to solve the problems in the related art, and the technical solutions are as follows: In a first aspect, embodiments of the present application provide an application of a rice-derived vesicle in the preparation of a functional product, which comprises: In a blood glucose and lipid control, blood glucose and lipid reducing, weight control or weight loss product.

[0008] In an embodiment, the product is a food, health product or drug.

[0009] In an embodiment, the preparation method of the rice-derived vesicle comprises the following steps: rice is pulverized in a buffer to obtain rice juice; the rice juice is purified by one or both of centrifugal separation and size exclusion chromatography to obtain the rice-derived vesicle.

[0010] In an embodiment, the rice is one or more than two combinations of indica rice, japonica rice, paddy, rice husk, brown rice or milled rice.

[0011] In an embodiment, the buffer is a phosphate buffer with a pH of 7.2-7.4; the mass ratio of rice to phosphate buffer is (0.8:1.2)-(2:1).

[0012] In an embodiment, the pulverization is performed by using a cell disruptor or grinding and crushing; the cell disruptor crushing time is 1-10 min.

[0013] In an embodiment, the centrifugation of the rice juice is as follows: The rice juice is centrifuged at 3000xg-7000xg for 10-20 min at 2-8℃, and the supernatant is collected; Centrifugation at 10000xg-14000xg for 20-30 min, and the supernatant is collected; Centrifugation at 100000xg-140000xg for 30-60 min, and the precipitate is collected and resuspended with PBS; The resuspension is centrifuged at 100000xg-140000xg in a sucrose density gradient for 30-90 min.

[0014] In an embodiment, the size exclusion chromatography column packing is agarose gel or agarose-dextran complex gel or agarose-polyacrylamide complex gel.

[0015] In an embodiment, the chromatography column is soaked for 8-12 hours after being washed with 0.1M aqueous sodium hydroxide solution until the pH of the effluent is 12.

[0016] Washed with pure water until the pH is 10; washed with PBS buffer until the effluent at the outlet is neutral.

[0017] In an embodiment, before the rice juice is subjected to size exclusion chromatography, the supernatant is collected by centrifugal separation; then filtered and concentrated by ultrafiltration to obtain the column chromatography liquid.

[0018] In an embodiment, the centrifugal separation is performed at 4-10°C at 7000xg-9000xg for 5-15 min. The ultrafiltration uses a membrane with a pore size range of 0.002-0.1 μm and a molecular weight cut-off of 1000-500000 Da; the concentration is to a particle concentration of greater than 1x10 11 6 / ml; then filtered through 10 um and 0.45 um.

[0019] In a second aspect, the embodiments of the present application provide a rice-derived vesicle, which is prepared by the preparation method of the rice-derived vesicle described above.

[0020] The advantages or beneficial effects of the above technical solutions at least include: The application of the rice-derived vesicle in the preparation of functional products, the rice is soaked in a buffer after being crushed, and the rice-derived vesicle is obtained after centrifugal separation or size exclusion chromatography purification; the preparation method is simple and easy to scale up. The rice-derived vesicle prepared by the application has the functions of regulating sugar metabolism and weight loss, and is expected to be used in the fields of blood sugar, blood lipid regulation, diabetes treatment and weight loss, etc. food, health products, and pharmaceuticals.

[0021] The above summary is merely intended to illustrate the present application and is not intended to limit in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of the present application will be readily apparent from a reading of the following detailed description of the application, which makes reference to the drawings described below. BRIEF DESCRIPTION OF DRAWINGS

[0022] In the drawings, like numerals refer to like elements throughout the various drawings. The drawings are not necessarily to scale, the emphasis instead being placed on the principles of the application. It should be understood that the drawings are merely depictions of some embodiments of the application and should not be construed as limiting the scope of the application.

[0023] Figure 1 TEM images of various rice vesicle samples prepared for this application; Figure 2 DSL detection particle size distribution graphs of various rice vesicle samples prepared for this application; Figure 3 Representative NTA detection graphs of rice vesicle samples prepared for this application; Figure 4 Representative HPLC purity detection graphs of rice vesicle samples prepared for this application; Figure 5 Inhibition of alpha-glucosidase activity by rice vesicle samples prepared for this application; Figure 6 Inhibition of protein tyrosine phosphatase (PTP-1B) activity by rice vesicle samples prepared for this application; Figure 7 Zebrafish blood glucose inhibition rate results for rice vesicle samples prepared for this application; Figure 8 Postprandial blood glucose level control results for mice for rice vesicle samples prepared for this application; Figure 9 Postprandial blood glucose AUC control results for mice for rice vesicle samples prepared for this application; Figure 10 Body weight control results for mice for rice vesicle samples prepared for this application; Figure 11 Fasting blood glucose control results for mice for rice vesicle samples prepared for this application; Figure 12 Serum total cholesterol control results for mice for rice vesicle samples prepared for this application. DETAILED DESCRIPTION

[0024] In the following, only certain exemplary embodiments are simply described. As those skilled in the art can recognize, the described embodiments can be modified in various different ways without departing from the spirit or scope of the present application. Therefore, the drawings and description are to be considered exemplary in nature - but not limiting.

[0025] Rice (Oryza sativa L.) is mainly divided into two subspecies: Indica and Japonica, in addition to special functional varieties such as black rice, red rice, glutinous rice, etc. It is one of the most important food crops in the world. Rice is rich in plant proteins such as glutelin and rice glutelin, and is rich in vitamins B, magnesium, zinc, selenium and other elements, which support the nervous system and metabolic function. Rice protein peptides have potential pharmacological activities in terms of blood pressure reduction, anti-fatigue, and immune regulation. Rice bran polyphenols, γ-oryzanol and other ingredients have free radical scavenging and inflammation regulation effects, and rice germ oil can be used for anti-fatigue and sports nutrition products. In summary, in addition to its basic energy supply function, rice also shows wide potential in the fields of functional food development, health management, and pharmaceutical research and development.

[0026] Rice and its extracts have been shown in many studies to have antioxidant, anti-inflammatory, melanin production inhibition, collagen synthesis promotion / inhibition of matrix metalloproteinase (MMP) regulation of apoptosis, and other functions. Natural active substances in rice that regulate sugar metabolism and affect body weight include: resistant starch (RS), γ-aminobutyric acid (GABA), phenolic and flavonoid compounds, dietary fiber, and bioactive proteins (such as α-amylase inhibitors. The active substances extracted from rice also have certain limitations in terms of water solubility, skin permeability, and bioavailability. For example, some phenolic / flavonoid ingredients are not well soluble in water and are easily decomposed. Therefore, there is an urgent need in the art for a delivery carrier that is well soluble in water, has good permeability, and is easily absorbed, and rice vesicles can perfectly achieve this function.

[0027] The processing of rice has a significant impact on the retention and activity of natural blood sugar-lowering substances. During processing, the natural nutrients in the skin layer, aleurone layer and germ of rice (such as vitamins, minerals, γ-aminobutyric acid, etc.) can be activated or retained under the action of water and heat, while the structure of starch changes, which can affect its digestion and absorption characteristics. For example, the processing technology of steamed rice is believed to be able to retain more nutrients such as B vitamins, dietary fiber and minerals, and has a lower glycemic index. However, processing can also result in the loss of certain nutrients, such as trace elements such as iron and zinc, which can be reduced during milling. In addition, the processing process can affect the activity of natural bioactive substances in rice (such as proteins that inhibit α-amylase activity), thereby affecting their potential for blood sugar reduction. The choice of processing method has an important impact on the retention and activity of natural blood sugar-lowering substances in rice. Therefore, the present application provides a rice-derived vesicle and its extraction method and application.

[0028] The present application provides an extraction method of a rice-derived vesicle, comprising the following steps: The rice is crushed in a buffer solution to obtain rice juice; the rice juice is purified by one or both of centrifugal separation and size exclusion chromatography to obtain the rice-derived vesicles.

[0029] Size exclusion chromatography (SEC) is an industrial liquid chromatography technique that separates molecules based on their size. The separation is based on the volume exclusion effect of solute molecules in the mobile phase using the pore size distribution of the porous gel stationary phase. The larger the molecular volume (i.e., the hydrodynamic volume in the solvent), the more difficult it is to enter the gel pores, and the earlier it is eluted from the column by the stationary phase "exclusion"; smaller molecules can penetrate deeper into the pores, have a longer retention time, and are eluted later.

[0030] Centrifugation is a method of separating different components in a mixture by centrifugal force according to density or mass differences. The centrifugation method used in the present application includes but is not limited to (1) sedimentation centrifugation: relying on centrifugal force to make particles settle; (2) filtration centrifugation: liquid is pressed out by centrifugal force, and solid forms a filter cake; (3) continuous centrifugation: material continuously enters and exits to achieve large-scale separation; (4) differential centrifugation: multiple rotors produce different centrifugal forces to achieve particle classification; (4) density gradient centrifugation: separating target vesicles in a gradient medium.

[0031] In the present application, the rice is crushed in a buffer solution and extracted, which can dissolve the active substances in the rice into the solution. Purification by centrifugation or size exclusion chromatography can extract high-quality nanoscale vesicles from rice.

[0032] As one embodiment, the rice is one or more combinations of indica rice, japonica rice, paddy, rice hull, brown rice, or milled rice.

[0033] In the present embodiment, the paddy is the mature fruiting body of Oryza sativa without removing the husk (rice hull), belonging to the Oryza sativa of the Poaceae family, including naturally propagated rice and artificially genetically modified rice. The rice hull is the outer hard protective structure removed during paddy processing, also known as chaff or rice bran. Brown rice is the whole grain rice obtained by removing the outermost hull of paddy, retaining the skin, aleurone layer, embryo, and endosperm.

[0034] As one embodiment, the buffer solution is a phosphate buffer solution with a pH of 7.2-7.4; the mass ratio of rice to phosphate buffer solution is (0.8:1.2) to (2:1).

[0035] The phosphate buffer can dissolve the vesicles and active substances in the rice cells and keep their activity, avoiding quality decline in subsequent purification process.

[0036] As one of the embodiments, the crushing is performed by using a wall-breaking machine or grinding crushing; the wall-breaking time of the crusher is 1-10 min. The crushing is to make the buffer fully contact with the rice cells, and thus the crushing mode can be any conventional mode, and in the present embodiment, grinding and wall-breaking machine are preferred.

[0037] As one of the embodiments, the process of centrifugation of the rice juice is as follows: The rice juice is centrifuged at 3000xg-7000xg at 2-8°C for 10-20 min, and the supernatant is collected; The supernatant is collected after centrifugation at 10000xg-14000xg for 20-30 min; The precipitate is collected after centrifugation at 100000xg-140000xg for 30-60 min, and the precipitate is resuspended with PBS; The resuspension is subjected to sucrose density gradient centrifugation at 100000xg-140000xg for 30-90 min.

[0038] The rice juice is subjected to preliminary separation by removing the precipitate residue under low-speed centrifugation, and then the vesicles are separated by precipitate separation under high-speed centrifugation, and finally the vesicles are further purified by sucrose density gradient centrifugation. In the present embodiment, the purification of the vesicles can be achieved only by centrifugation, which is simple and can be prepared by most laboratories or manufacturers.

[0039] As one of the embodiments, the size exclusion chromatography is performed, and the filler of the exclusion chromatography column is agarose gel or agarose-dextran composite gel or agarose-polyacrylamide composite gel; the chromatography column is soaked for 8-12 h after being washed with 0.1M sodium hydroxide aqueous solution until the pH of the effluent is 12. The chromatography column is washed with pure water until the pH is 10; and the chromatography column is washed with PBS buffer until the effluent is neutral.

[0040] As one of the embodiments, the rice juice is subjected to centrifugal separation to collect the supernatant before size exclusion chromatography, and then filtered and concentrated by ultrafiltration to obtain the column chromatography liquid.

[0041] As one of the embodiments, the centrifugal separation is performed at 4-10°C at 7000xg-9000xg for 5-15 min. As one of the embodiments, filtration is a process of separating solid particles and liquid from fluid using porous media. The materials used include, but are not limited to: fibrous materials, membrane materials, activated carbon, ceramic and microglass, filter paper (cellulose fiber), polypropylene fiber, polyester fiber, nylon, microporous membrane, ultrafiltration membrane, nanofiltration membrane, reverse osmosis membrane, PTFE (polytetrafluoroethylene), glass fiber, stainless steel mesh, ceramic membrane, diatomite, etc. Preferably, a multi-size ceramic membrane filter is used in the present embodiment to remove solid insoluble substances, and a filtered supernatant is obtained.

[0042] As one of the embodiments, ultrafiltration uses an ultrafiltration membrane with a pore size range of 0.002-0.1 μm and a molecular weight cutoff of 1000-500000 Da; the particle concentration is concentrated to more than 1×10 11 Individuals / ml; and then filtered through 10 um and 0.45 um.

[0043] The present application also provides a rice-derived vesicle prepared by the above-mentioned method for preparing a rice-derived vesicle.

[0044] The present application also provides the use of the above-mentioned rice-derived vesicle in the preparation of a blood glucose control, blood glucose lowering, weight control, weight loss product. The present application first found that rice vesicles have the effects of blood glucose control and blood glucose lowering; and also have the effect of weight loss.

[0045] As one of the embodiments, the product is a food, health product or drug.

[0046] The following is further illustrated with specific examples.

[0047] Example 1: Centrifugation method for extracting rice vesicles Take 1 kg of each of the following fresh rice materials: brown indica rice, milled indica rice, indica rice grain, indica rice husk, brown japonica rice, milled japonica rice, japonica rice grain, and japonica rice husk, and use an ultrasonic cleaning machine to repeatedly clean and disinfect the materials with purified water for 3-5 times to remove surface dirt; After cleaning, transfer the materials to a cell wall breaking machine, add 800 g of PBS buffer to immerse the surface of the plants, and then break the cell walls until they are completely pulverized; Centrifuge the obtained homogenate at 8000 x g at 4°C for 10 min, discard the precipitate, centrifuge the supernatant at 12000 x g for 30 min, discard the precipitate, and collect the precipitate layer in the centrifuge tube after centrifugation at 120000 x g for 60 min, resuspend with PBS, and take samples for particle count detection. If the particle concentration does not reach 1×10 11 Individuals / ml, concentrate with an ultrafiltration tube with a molecular weight cutoff of 100K, to a particle concentration of more than 1×10 11 Individuals / ml, and obtain a rice vesicle stock solution.

[0048] Example 2 Size exclusion chromatography extraction of rice vesicles Freshly harvested rice materials (1 kg each) of long-grain brown rice, long-grain milled rice, indica rice grain, indica rice hull, japonica brown rice, japonica milled rice, japonica rice grain, and japonica rice hull were obtained and repeatedly washed and sterilized with purified water using an ultrasonic washing machine for 3-5 times to remove surface dirt. The washed materials were transferred to a cell wall breaking machine and immersed in 2 kg of PBS buffer to immerse the surface of the plants, and then the cell wall was broken to completely crush the materials. The obtained homogenate was centrifuged at 8000 x g at 4°C for 10 min, and the precipitate was discarded. The filtrate was obtained by removing the solid insoluble substances through a large size ceramic membrane filter. The concentration of vesicle particles in the filtrate was detected, and the concentration ratio was calculated according to the detected concentration. The filtrate was pumped into an ultrafiltration concentration tank, and ultrafiltration concentration was performed using a 100K ultrafiltration membrane. The concentration was performed until the particle concentration was greater than 1 x 10 11 After filtration through 10 um and 0.45 um, the sample was stored in a cold storage. A chromatography system was prepared, and agarose gel was used as the filler. The chromatography column and pipeline were washed and sterilized with 0.1M sodium hydroxide aqueous solution. After the pH of the effluent was adjusted to 12, the pipeline was soaked for 8-12 hours. The pipeline was washed with purified water until the pH was about 10, and then washed with PBS buffer for 2-3 column volumes (CV) until the pH was neutral. The pipeline was connected for loading. Each batch of loading was performed according to 30% of the loading capacity of the molecular sieve volume of the chromatography column. Online detection was performed at a wavelength of 260 nm. The target rice vesicle chromatography peak was collected. After collection, the total vesicle solution was combined and filtered through a 0.45 um filter membrane. The sample was stored in a cold storage. The obtained sample was a rice vesicle stock solution.

[0049] Characterization and performance detection of vesicles (1) Transmission electron microscopy (TEM) detection of rice vesicle extracts 10 μL of rice vesicle samples from various sources (long-grain brown rice, long-grain milled rice, indica rice grain, indica rice hull, japonica brown rice, japonica milled rice, japonica rice grain, and japonica rice hull) were dropped onto a copper grid, absorbed for 5 min, and the floating liquid was absorbed with filter paper. The grid was stained with phosphotungstic acid solution for 1 min, and the floating liquid was absorbed with filter paper. The sample was dried at room temperature for 20 min. After drying, the sample was observed in a Tecnai G2 Spirit transmission electron microscope (TEM). The TEM images are shown in Figure 1 As can be seen from Figure 1 , the particles in the rice vesicle samples from fresh rice grain, rice hull, brown rice, and milled rice are spherical and relatively complete.

[0050] (2) Particle size distribution determination of vesicle extracts ​Turn on the Malvern Nano ZS instrument, select a clean sample cell, and after equilibrating the background with PBS buffer, take the rice vesicle samples of various sources (long round brown rice, long round milled rice, long round rice grain, long round rice husk, round brown rice, round milled rice, round rice grain, round rice husk) diluted 10 times, slowly inject into the detection sample cell to avoid bubbles, and detect 3 readings to take the average particle size to report the results; the particle size distribution curve is shown in Figure 2 , from left to right, respectively, are the particle size distribution curves of long round brown rice, long round milled rice, long round rice grain, long round rice husk, round brown rice, round milled rice, round rice grain, and round rice husk.

[0051] From the particle size distribution results of Figure 2 , it can be seen that the vesicles from fresh rice grain, rice husk, brown rice, and milled rice sources are distributed in the range of 50-1000 nm, with an average particle size in the range of 132.2-269.3 nm, and the PDI is less than 0.3, with good sample consistency.

[0052] (3) Vesicle extract content and purity detection Vesicle content determination: dilute the vesicle solutions of different sources of rice (long round brown rice, long round milled rice, long round rice grain, long round rice husk, round brown rice, round milled rice, round rice grain, and round rice husk) according to the concentration range of the ZetaView® NTA instrument, and load the detection solution to detect the vesicle particle content in the solution; Vesicle purity determination: use a Thermo UltiMate 3000 high-performance liquid chromatograph, 4.6 x 200 mm, agarose gel filler column, mobile phase: 10 mM PBS + 300 mM NaCl, pH 7.2-7.4, isocratic elution, flow rate 0.15 mL / min, detection wavelength UV 280 nm and UV 260 nm, elution time 35 min, injection volume 100 μL; the detection results are shown in Table 1; the NTA detection graph is shown in Figure 3 ; and the HPLC purity detection graph is shown in Figure 4 .

[0053] Table 1

[0054] The detection results of Table 1 show that vesicles can be extracted from rice grain, rice husk, brown rice, and milled rice of different sources, and the purity is more than 70%; the average particle size is consistent with the test results of the Malvern Nano ZS instrument.

[0055] (4) Stability evaluation of rice vesicle extract Storage stability: select long round brown rice vesicle extract as a representative sample for stability test, store the long round brown rice vesicle stock sample at 4°C for 30 days and at room temperature for 3 days, and measure the particle size and HPLC purity at 1, 3, 5, 7, 14, and 30 days.

[0056] Freeze-thaw stability: The raw solution of brown rice vesicles was stored at -20°C for 4 hours, then placed at room temperature until completely thawed, which was a freeze-thaw cycle. A total of 5 freeze-thaw cycles were performed, and the particle size and HPLC purity of the 1st, 3rd and 5th freeze-thaw samples were determined. The results are shown in Table 2.

[0057] Table 2

[0058] The data in Table 2 show that the average particle size of the raw solution of rice vesicles changed slightly after being stored at 4°C for 30 days, at room temperature for 3 days, and repeated freeze-thawing for 5 times, but the purity did not change much, indicating that the vesicles prepared in this application have good stability.

[0059] (5) Inhibition experiment of rice vesicle extract on α-glucosidase activity 50µL of α-glucosidase was mixed uniformly with 50µL of rice vesicles of different concentrations, and PBS buffer was added to maintain a total volume of 150µL. After incubation at 37°C for 15 min, 50µL of p-nitrophenyl glucoside (NPG, 10mM) was added to start the reaction. After incubation for 15 min, 60µL of Na2CO3 was added to terminate the reaction; the absorbance at 405nm was measured using an enzyme marker to quantify the release of p-nitrophenol (PNP); the inhibition rate I% was calculated according to the following formula, and the IC50 value was calculated; p I%=[1- (C-D) / (A-B)]×100% ; wherein: A is the negative group (PBS + enzyme liquid + NPG); B is the blank group (PBS + NPG); C is the sample group (sample + enzyme liquid + NPG); D is the sample control group (sample + PBS + NPG). p p p p

[0060] The inhibition rate curve is shown in Figure 5 Table 3.

[0061] Table 3

[0062] Each type of rice vesicle showed certain α-glucosidase inhibitory ability, among which the IC 50 value of brown rice vesicles was 0.30 E+10 Particles / mL, with the best inhibitory ability.

[0063] (6) Inhibition experiment of rice vesicle extract on protein tyrosine phosphatase (PTP-1B) activity ​​​​​10 μL PTP-1B enzyme was mixed with 10 μL of different concentrations of rice vesicles, 10 μL of PBS was added to keep the total volume at 100 μL; after incubation at 37°C for 10 min, 10 μL of disodium p-nitrophenyl phosphate (pNPP, 10 mM) was added to start the reaction; after incubation for 30 min, 20 μL of stop solution NaOH was added to stop the reaction; the release amount of p-nitrophenol (PNP) was quantified by measuring the absorbance at 405 nm using an enzyme label instrument. The inhibition rate I% was calculated according to the following formula, as shown in Figure 6 The IC50 value was calculated, and the results are shown in Table 4. I% = [1 - (C-D) / (A-B)] x 100%; Wherein: A is the negative group (PBS + enzyme solution + pNPP); B is the blank group (PBS + pNPP); C is the sample group (sample + enzyme solution + pNPP); D is the sample control group (sample + PBS + pNPP).

[0064] Table 4

[0065] As can be seen from Table 4, various types of rice vesicles all showed good PTP-1B enzyme inhibition ability, among which the IC50 value of brown rice vesicles was 2.63 E+10 Particles / mL, and the inhibition ability was the best.

[0066] (7) Zebrafish blood glucose inhibition rate test Reagent consumables: including glucose (modeling drug, Shanghai Biotech, item number: A600218-0500), acarbose (positive drug, MCE, item number: HY-B0089), 6 / 12 well plates, 1.5 mL / 10 mL / 50 mL centrifuge tubes, etc.

[0067] Instrument equipment: medical-grade blood glucose meter, body microscope, clean bench, constant temperature incubator, zebrafish breeding system, ultrapure water system, analytical balance (one ten thousandth), freeze grinding instrument.

[0068] Test procedure: 4% glucose solution was prepared by using zebra fish embryo culture solution, and zebra fish developed to 3dpf were treated with 4% glucose for 48h, and the solution was changed every 24h to construct high blood glucose model. The blank group, model group and test group were 1.0% rice vesicle products (long-grain brown rice, milled long-grain rice, long-grain rice, long-grain rice husk, long-grain brown rice, milled long-grain rice, long-grain rice, long-grain rice husk), and each group contained 40 zebra fish. Except the blank group, the working solution of each group was prepared by E3 culture water containing 4% glucose, and 5mL of solution was set in each well. Each test group was placed in a constant temperature incubator at 28.5±0.5℃ for 48h, and the solution was changed once during the incubation. After 5dfp, all fish in each group were collected, washed with E3 culture water for three times, centrifuged briefly, and the excess water was absorbed. Then, zirconium beads were added, and the zebra fish tissue was homogenized by high-throughput frozen grinder. 2uL of zebra fish tissue homogenate was taken, and the blood glucose content in zebra fish tissue was detected by blood glucose meter.

[0069] Result calculation: the blood glucose inhibition rate was calculated according to the following formula: Inhibition rate (%) = [(S model control group- S test group)] / S model control group]x100%; In the formula, S is the blood glucose value of zebra fish.

[0070] The blood glucose values of the experimental group and the model control group were subjected to two-tailed T test, and p<0.05 was significantly different. The results are shown in Table 5 and Figure 7 .

[0071] Table 5

[0072] Compared with the model control group, the rice vesicle product in the experimental group at a concentration of 1.0% could significantly inhibit the blood glucose content of zebra fish embryos (p<0.05), and had the effect of lowering blood glucose.

[0073] (8) Postprandial blood glucose control experiment of mice The effect of rice vesicles on delaying the increase of postprandial blood glucose level was evaluated by measuring the postprandial blood glucose level of mice. C57BL / 6J mice (54) were divided into 9 groups, 6 in each group. After fasting for 16h, the C57 mice were respectively given intragastrically: 1) starch; 2) starch and long-grain brown rice vesicles; 3) starch and milled long-grain rice vesicles; 4) starch and long-grain rice vesicles; 5) starch and long-grain rice husk vesicles; 6) starch and long-grain brown rice vesicles; 7) starch and milled long-grain rice vesicles; 8) starch and long-grain rice vesicles; 9) starch and long-grain rice husk vesicles. After the mice in each group were respectively given the same amount of sample, the postprandial blood glucose value was measured at different time points (0, 30, 60, 90, 120min), and the results are shown in Figure 8 ; and the area under the curve (AUC) was calculated by trapezoidal rule, and the results are shown in Figure 9 .

[0074] From Figure 8 It can be seen that the blood glucose values of mice after meals all rapidly increased within 0~60 min after meals, and gradually decreased within 60~120 min. The blood glucose values of the sample groups were significantly lower than those of the control group, indicating that rice vesicles can delay the increase of postprandial blood glucose, which shows that rice vesicles can improve the hyperglycemic state of mice by regulating sugar metabolism related pathways.

[0075] (9) Changes of body weight, blood glucose and blood lipids of high-sugar and high-fat model mice 7-week-old C57BL / 6J mice were randomly divided into groups, 10 mice in each group, and the test period was 6 weeks: normal diet control group; high-sugar and high-fat diet control group; high-sugar and high-fat diet + various rice vesicle groups (mixing rice vesicle stock solution 1:1 into daily drinking water for mice). During the test period, the body weight of mice was recorded every week, and the results are shown in Table 6 and Figure 10 ; the blood glucose meter was used to determine the fasting blood glucose value of mice every week, and the specific results are shown in Table 7 and Figure 11 ; the serum cholesterol value of mice was determined every other week (8 weeks of test period), and the specific results are shown in Table 8 and Figure 12 .

[0076] Table 6

[0077] Table 7

[0078] Table 8

[0079] The experimental results show that rice vesicles from various sources significantly delay the increasing trend of fasting blood glucose and serum cholesterol of high-sugar and high-fat diet mice, and significantly delay the increasing trend of body weight.

[0080] The application extracts a kind of nanometer size vesicle from rice by centrifugation or chromatography and the like method firstly, and carries out quality characterization and function research of the vesicle, and finds that the extract has the functions of regulating sugar and lipid metabolism and weight loss, and is expected to be used in the fields of blood sugar and blood lipid regulation, diabetes treatment and weight loss and the like food, health care product and drug.In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application.Furthermore, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples.In addition, the person skilled in the art can combine and combine the different embodiments or examples described in the specification and the features of the different embodiments or examples without contradiction.

[0081] In addition, the terms "first", "second" are only for the purpose of description, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features.Therefore, the features defined with "first", "second" can explicitly or implicitly include at least one of the features.In the description of the present application, the meaning of "a plurality of" is two or more, unless otherwise specifically limited.

[0082] The above is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can easily think of various changes or replacements within the technical range disclosed in the present application, which should be covered in the protection scope of the present application.Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. The application of rice-derived vesicles in the preparation of functional products, characterized in that, The applications include: Applications in products for controlling blood sugar and lipids, lowering blood sugar and lipids, controlling weight, or losing weight.

2. The application according to claim 1, characterized in that, The product in question is food, health supplement, or medicine.

3. The application according to claim 1 or 2, characterized in that, The method for preparing rice-derived vesicles includes: The following steps: Rice is pulverized in a buffer solution to obtain rice juice; the rice juice is purified by one or two of centrifugation and size exclusion chromatography to obtain the rice-derived vesicles.

4. The application according to claim 3, characterized in that, The rice is one or a combination of two or more of the following: indica rice, japonica rice, paddy rice, rice husk, brown rice, or polished rice.

5. The application according to claim 3, characterized in that, The buffer solution is a phosphate buffer with a pH of 7.2 to 7.4; the mass ratio of rice to phosphate buffer is (0.8:1.2) to (2:1). Crushing is performed using a cell wall breaker or grinding machine; the cell wall breaking time of the crusher is 1 to 10 minutes.

6. The application according to claim 3, characterized in that, The process of centrifuging rice water is as follows: Centrifuge the rice water at 3000×g~7000×g for 10~20 min at 2~8℃ and collect the supernatant; Centrifuge at 10000×g~14000×g for 20~30 min and collect the supernatant; Centrifuge at 100,000×g to 140,000×g for 30 to 60 min, collect the precipitate, and resuspend the precipitate in PBS; The resuspended solution was centrifuged at a sucrose density gradient of 100,000×g to 140,000×g for 30 to 90 minutes.

7. The application according to claim 3, characterized in that, Size exclusion chromatography was performed using agarose gel, agarose-dextran composite gel, or agarose-polyacrylamide composite gel as the column packing material. The column was rinsed with 0.1M sodium hydroxide aqueous solution until the pH of the outlet reached 12, and then soaked for 8-12 hours. Rinse with pure water until pH 10; rinse with PBS buffer until the outlet liquid is neutral.

8. The application according to claim 3, characterized in that, Before size exclusion chromatography, the rice juice is first centrifuged to collect the supernatant; then it is concentrated by filtration and ultrafiltration to obtain the column chromatography solution.

9. The application according to claim 8, characterized in that, The centrifugation conditions are: centrifugation at 7000×g to 9000×g for 5 to 15 minutes at 4 to 10°C; Ultrafiltration uses ultrafiltration membranes with a pore size range of 0.002~0.1μm and a molecular weight cutoff of 1000~500000 Da; concentration is achieved to a particle concentration greater than 1×10⁻⁶. 11 10 μL / ml; then filtered at 10 μm and 0.45 μm.

10. A rice-derived vesicle, characterized in that, It is prepared by the preparation method described in any one of claims 3-9.

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