Liver-targeted lycopene plastid globules and application thereof

Abstract

The invention relates to liver-targeted lycopene plastid pellets and application thereof, the problems that lycopene is poor in water solubility, easy to oxidize and degrade and the like can be solved through the nanoscale structure of plastid membranes wrapping lycopene of the plastid pellets, and the liver-targeted lycopene plastid pellets serve as a natural delivery system. The plastid pellets can improve the water solubility of lycopene and the resistance to ions, acid, heat and other conditions, have good gastrointestinal stability and intestinal permeability, can effectively deliver lycopene to blood and liver and improve the bioavailability of lycopene, and can be applied to the fields of biological medicine, functional food and the like. In addition, the plastid pellets are derived from natural edible plant tissues, can meet the pursuit of consumers on health without side effects, and have wide development prospects.

Description

Technical Field

[0001] The present invention belongs to the fields of functional foods and biotechnology, and particularly relates to a liver-targeted lycopene plastid microsphere and its application. Background Art

[0002] Lycopene is an isoprenoid compound derived from plant-based foods and has strong antioxidant activity. Its antioxidant activity is 100 times that of vitamin E and 2 times that of β-carotene. It can delay the oxidation of proteins, lipids, and DNA, regulate the cell cycle and cell gap junctions, and has significant effects in preventing and treating non-alcoholic fatty liver, inhibiting the proliferation of tumor cells, and preventing cardiovascular and cerebrovascular diseases. However, lycopene has extremely poor water solubility and stability. It is insoluble in water and is easily degraded by environmental factors such as light and heat, as well as internal factors such as the extreme pH of the stomach, gastrointestinal digestive enzymes, and physical peristalsis, which destroys the structure of lycopene and results in extremely low in vivo bioavailability, thus seriously affecting its application. Therefore, improving the bioavailability of lycopene has become a key issue in the efficient utilization of lycopene. Since lycopene is almost insoluble in solvents such as water and ethanol and has a relatively high solubility in oily substances. Therefore, to address the issue of improving the bioavailability of lycopene, methods for enhancing the water solubility of lycopene are mainly adopted at present. However, increasing the water solubility of lycopene often requires various composite materials to assist lycopene in dissolving in water. In essence, it is to add a water-soluble outer shell, which not only increases the cumbersome steps of industrial production but also raises the cost of industrial production. On the other hand, the composite materials used are often chemical materials that cannot be degraded by organisms, which may cause allergic reactions and chemical residues during consumer contact, posing a potentially great impact on the physical health of consumers and endangering their physical health. At the same time, the in-vivo stability of this way of increasing the water solubility of lycopene remains questionable. For example, Patent CN109419775B authorizes a water-soluble lycopene and its preparation method and application. Its formula is 1 part by weight of lycopene oil, 1 - 5 parts by weight of a solid dispersion carrier material mixture, and 1 - 5 parts by weight of cyclodextrin, where the solid dispersion carrier material mixture is composed of polyethylene glycol 6000 and polyvinylpyrrolidone k30 mixed in a mass ratio of 3:2. The prepared lycopene system significantly improves the water solubility of lycopene, but the process method used in its formula requires strict and precise control of the proportion of materials such as the solid dispersion carrier material and lycopene oil, which requires huge costs and poses great risks in the actual production process. Therefore, developing a new type of water-soluble lycopene system with high stability and low safety risk is of great significance for promoting the application of lycopene in various fields. Plastoglobules are a special organelle structure derived from the chloroplasts of higher plants. In mature plant fruits, plastoglobules are the main metabolic sites of carotenoids. They are mostly spherical in shape, with a particle size between 30 - 500 nm, wrapped by a single-layer plasma membrane, and stably exist in the plastid matrix. Therefore, in plants such as tomatoes and watermelons, plastoglobules are rich in lycopene and exist in the form of spherical nanoparticles. Lycopene is encapsulated inside by the plasma membrane, which is a natural lycopene encapsulation system. The nanoscale size of plastoglobules enables them to easily penetrate the intestinal mucosa and intestinal wall, thus being taken up by the intestine and further entering the systemic circulation to achieve effective delivery of lycopene. In addition, the plasma membrane of plastoglobules has amphiphilicity, which can embed lycopene in it, significantly improving its water solubility and making it more easily absorbed by the human liver. These characteristics make plastoglobules perform well in enhancing the stability, water solubility, and biodegradability of lycopene and are expected to be used as a potential natural nanodelivery system for liver-targeted delivery applications. Currently, in the publicly available or authorized patents, there are almost no precedents involving natural nanosystems loaded with carotenoids for liver-targeted delivery applications. Therefore, such inventions have broad application prospects. Summary of the Invention

[0003] Technical problem to be solved: Aiming at the above technical problems, the object of the present invention is to provide a liver-targeted natural lycopene plastid sphere, which is green and safe, can improve the water solubility of lycopene and its resistance to external adverse factors, has good gastrointestinal stability and intestinal permeability, can effectively deliver lycopene to the blood and liver, improve the in vivo bioavailability of lycopene, and can be applied to the fields of biomedicine and functional foods, etc.

[0004] Technical solution: A liver-targeted lycopene plastid sphere, the structure of the plastid sphere is a plasma membrane wrapping lycopene. Furthermore, the plastid sphere is derived from plant fruits rich in lycopene. Furthermore, the plant fruits rich in lycopene include tomatoes, watermelons, peppers, grapefruits and guavas. A preparation method of a liver-targeted lycopene plastid sphere, comprising the following steps: S1: After rinsing the cut plant fruits rich in lycopene with a pre-cooled extraction buffer, add the pre-cooled extraction buffer according to a mass-volume ratio of 1:(3-7), and homogenize and stir to obtain crude juice; S2: After filtering the crude juice, collect the filtrate, centrifuge the filtrate and collect the precipitate, add the extraction buffer to the precipitate according to a mass-volume ratio of 1:(1-4) for re-dissolution, and perform ultrasonic treatment after re-dissolution to obtain a suspension; S3: Add the suspension, 35-38 wt.% sucrose solution, 18-20 wt.% sucrose solution, 12-15 wt.% sucrose solution and 3-5 wt.% sucrose solution to a centrifuge tube for centrifugation, and collect the topmost substance, which is the lycopene plastid sphere. Furthermore, the extraction buffer in step S1 includes 20 mM Tricine-KOH, 450 mM sorbitol, 10 mM EDTA-Na2, 10 mM NaHCO3 and 0.5 wt.% bovine serum albumin. Furthermore, the homogenization and stirring in step S1 is to stir for 1-3 seconds every 1-3 seconds, and stir a total of 1-5 times. Furthermore, the filtering material in step S2 is 2-5 layers of degreased cotton gauze and a 60-80 μm filter screen, the centrifugation conditions are 1200 g, 4 °C, 10-15 min, and the ultrasonic treatment frequency is 60-80 Hz, 1-3 min. Furthermore, the ratio of the suspension, 35-38 wt.% sucrose solution, 18-20 wt.% sucrose solution, 12-15 wt.% sucrose solution and 3-5 wt.% sucrose solution in step S3 is 3:3:3:2:4. Further, the centrifugation conditions in step S3 are 80,000-120,000 g, 4 °C, and 2-4 h. The present invention provides an application of liver-targeted lycopene plastid nanoparticles in biomedicine and functional foods. Beneficial effects: 1. The present invention uses a gentle pulping speed to crush the fruit dices, maximally retaining the integrity of the plastid nanoparticles, and uses ultrasonic treatment and density gradient centrifugation methods for extraction. The prepared plastid nanoparticles have a complete structure and contain rich carotenoids, especially lycopene, improving the retention rate and stability of lycopene in environments with different salt ion concentrations, pH values, and temperatures. 2. As a delivery system for lycopene, the plastid nanoparticles of the present invention have good gastrointestinal stability and good intestinal permeability. The permeability coefficients in the duodenum, jejunum, and ileum are 32.00 times, 39.00 times, and 143.00 times that of free lycopene, respectively. 3. The lycopene plastid nanoparticles of the present invention can increase the in vivo bioavailability of lycopene. Compared with free lycopene, the peak plasma concentration time of lycopene in the plastid nanoparticles is delayed by 2 h, the peak blood drug concentration is 1.96 times higher, and the bioavailability is increased by 1.71 times. 4. The delivery of lycopene by the plastid nanoparticles of the present invention can significantly increase the enrichment of lycopene in the liver. Compared with free lycopene, the peak liver concentration time of lycopene in the plastid nanoparticles is delayed by 7 h, the peak liver drug concentration is 4.90 times higher, and the bioavailability is increased by 7.28 times. The average particle size of the plastid nanoparticles is about 200 nm, and they can be passively enriched in the liver through the hepatic sinusoidal space, realizing the liver-targeted delivery of lycopene, providing technical theoretical support for the research of liver-targeted delivery of biomedicines and foods. 5. Directly using the extracted plastid nanoparticles in plants is simpler and lower in cost than extracting lycopene and then encapsulating or loading it. Moreover, the plastid nanoparticles are derived from natural edible plant tissues, are green and environmentally friendly, and are safer than other delivery systems, meeting consumers' pursuit of health without side effects. Description of the drawings Figure 1 It is a diagram of the separation of the crude extract at different density gradients after density gradient centrifugation in Example 1; Figure 2 It is a Western-blot transfer membrane color development diagram of the top layer and the second layer substances collected after density gradient centrifugation in Example 2; Figure 3 It is a TEM transmission electron microscope diagram of the top layer substance collected after density gradient centrifugation in Example 3; Figure 4It is the CLSM laser confocal fluorescence microscopy image of plastid globules excited by 488nm single channel in Example 3; Figure 5 It is the CLSM laser confocal fluorescence microscopy image of plastid globules excited by 488 / 543nm dual channels in Example 3; Figure 6 It is the particle size distribution diagram of plastid globules in Example 3; Figure 7 It is the result diagram of lycopene retention rate of plastid globules under different salt ions, pH, and temperature in Example 4; Figure 8 It is the result diagram of lycopene retention rate during in vitro simulated digestion in Example 4; Figure 9 It is the Transwell permeability diagram of intestinal mucus in Example 5; Figure 10 It is the intestinal permeability coefficient diagram in Example 5; Figure 11 It is the CLSM laser confocal fluorescence microscopy image of intestinal permeability in Example 5. A is free lycopene, and B is plastid globules; Figure 12 It is the mucus layer thickness diagram under the maximum fluorescence intensity at different treatment times in Example 5; Figure 13 It is the change diagram of lycopene concentration in blood over time in Example 6; Figure 14 It is the change diagram of lycopene concentration in liver over time in Example 6. Detailed implementation mode In order to more clearly and thoroughly understand the technical solution of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and embodiments. The following embodiments are explanations of the present invention and the present invention is not limited to the following embodiments: Example 1 Extraction of plastid globules: Preparation of pre-cooled extraction buffer: The extraction buffer contains: 20mM Tricine-KOH (pH 8.4), 450mM sorbitol, 10mM EDTA-Na2, 10mM NaHCO3, 0.5% wt.% bovine serum protein component. After washing fresh, mature tomato fruits, remove the seeds inside the tomato fruits and then dice them. Rinse the diced tomato fruits with pre-cooled extraction buffer. After adding the pre-cooled extraction buffer at a ratio of 1:5 (w / v), use a multi-functional juicer to homogenize and stir, intermittently pressing the button 2 times every 2 seconds at a low gear, for 2 seconds each time, for a total of 4 times. After filtering through 4 layers of degreased cotton gauze and a 60 μm filter, collect the filtrate. Centrifuge the filtrate at 1200 g and 4 °C for 10 min, collect the precipitate, and resuspend the above precipitate by adding extraction buffer at a ratio of 1:4 (w / v). And perform extraction by ultrasonic treatment and density gradient centrifugation. Ultrasonically treat the resuspended suspension at a frequency of 60 Hz for 3 min. Sequentially add 6 mL of the suspension, 6 mL of 38 wt.% sucrose solution, 6 mL of 20 wt.% sucrose solution, 4 mL of 15 wt.% sucrose solution, and 8 mL of 5 wt.% sucrose solution to the bottom of a 30 mL transparent ultracentrifuge tube. After preparing the density gradient centrifuge tube, centrifuge it at 100000 g and 4 °C for 2 h. The results after density gradient centrifugation are as Figure 1 shown. Use a pipette to separately collect the topmost layer and the second layer containing plastoglobules. Example 2 Identification of plastoglobules: The top layer and the second layer substances are used for western-blot determination. Vortex the extracted top layer and second layer substances in the extraction buffer for 3 min. The composition of the extraction buffer is as follows: 200 mM Tris (pH 8.0), 150 mM NaCl, add 2.5 mL of protease inhibitor mixture, 1 mM PMSF, and 0.125% (v / v) Triton X-100 to each 1 mL of the extraction buffer. Then centrifuge at 13000 g at 4 °C for 15 min and collect the supernatant. After 12% SDS-PAGE, transfer the proteins to a polyvinylidene difluoride (PVDF) membrane. Incubate the transferred membrane with antibodies against Arabidopsis thaliana AtPGL35 protein (a specific protein on the surface of plastoglobules) and Arabidopsis thaliana AtToC75 protein (a specific protein on the surface of chromoplast membranes) diluted 1:2500, and then incubate with anti-rabbit IgG alkaline phosphatase conjugate (diluted 1:80000). Finally, use the chromogenic substrate of alkaline phosphatase to develop the color of the transferred membrane. The substrate includes 66 mL of nitroblue tetrazolium (50 mg / mL) and 33 mL of 5-bromo-4-chloro-3-indolyl phosphate (50 mg / mL). Dissolve the above substrates in 10 mL of buffer. The composition of the buffer is: 100 mM Tris-HCl, 100 mM NaCl, and 5 mM MgCl2, pH 9.5. Finally, perform chemiluminescent color development of the treated transferred membrane in a chemiluminescence imager. The results are as Figure 2As shown, the AtPGL35 antibody is an antibody against the characteristic membrane protein of plastoglobules, and the ToC75 antibody is an antibody against the characteristic membrane protein of chromoplasts. After imaging with a chemiluminescence imager, different bands appeared on the transfer membranes of the top and sub-layer substances. Looking at the top-layer substance, the position of the band on the transfer membrane incubated with the PGL35 antibody was around 35KDa, and it can be determined that this is the characteristic protein of plastoglobules, that is, plastoglobules exist in the top layer; no band appeared on the transfer membrane incubated with the ToC75 antibody, indicating that there is no chromoplast membrane in the top layer. On the other hand, a band around 35KDa also appeared on the transfer membrane of the sub-layer substance incubated with the PGL35 antibody, indicating that plastoglobules also exist in the sub-layer; an obvious band appeared on the transfer membrane incubated with the ToC75 antibody, indicating that there is a chromoplast membrane in the sub-layer substance. Through comprehensive analysis, after density gradient centrifugation, plastoglobules were collected in the top-layer substance, and there were no residues of substances such as chromoplast membranes in the top layer, that is, plastoglobules were successfully extracted in this extraction process and had a high purity. Example 3 Characterization of plastoglobules: (1) Morphological structure of plastoglobules Take a drop of the extracted plastoglobule sample suspension and adsorb it on a copper grid for 1 min, then stain the sample on the copper grid with 2 wt.% uranyl acetate solution for 30 seconds, and air-dry it naturally. Observe the morphology of the extracted plastoglobules using a TEM transmission electron microscope. As Figure 3 shown, the extracted plastoglobules were observed to be spherical in TEM, and the particle size was about 100 nm. (2) Observation by CLSM confocal laser scanning fluorescence microscope To further observe by CLSM, since plastoglobules potentially contain lycopene and lycopene has autofluorescence, the CLSM experiment can be designed to observe through the autofluorescence of lycopene. And since plastoglobules have a lipid membrane and lipid substances do not have autofluorescence, it is necessary to stain the plasma membrane with a lipophilic dye before observation. Add Nile red dye to the extracted plastoglobule sample at a ratio of 1:100, incubate for 20 min, then fix the sample on a microscope slide, and observe by CLSM after single-channel excitation at 488 nm wavelength and dual-channel excitation at 488 nm and 543 nm wavelengths, and collect 1,024×1,024 pixel images. It can be seen from Figure 4 that CLSM successfully captured the structure of the autofluorescence of lycopene in single-channel excitation at 488 nm wavelength, and its morphology was spherical at the nanoscale, which was consistent with the spherical structure of plastoglobules. Therefore, lycopene may be wrapped inside the plastoglobules; through Figure 5It can be seen that in the plastoglobule samples treated with Nile red dye, dual-channel co-localization results can be captured. The red color represents Nile red dye excited at a wavelength of 543 nm, while the green color represents lycopene excited at a wavelength of 488 nm. Through co-localization, it can be seen that the Nile red dye channel and the lycopene channel overlap, indicating that plastoglobules encapsulate lycopene through the plasma membrane and can serve as a potential natural nano-delivery system. (3) Particle size analysis The particle size of the plastoglobules was determined using a laser particle size analyzer. Slowly drip 200 μL of the prepared plastoglobule suspension into the sample inlet of the particle size analyzer until the light shielding rate reaches 8 - 15% and then start the measurement. Each sample was scanned three times and the average value was taken. The refractive index of the sample was 1.476, the dispersion medium was deionized water with a refractive index of 1.333. It can be seen from Figure 6 that the average particle size of the plastoglobules is 204.4 ± 2.05 nm, the particle size shows a single-peak distribution, and its PDI is 0.475, indicating that the particle sizes are uniform and the stability of the plastoglobules is good. (4) Lycopene content in plastoglobules Sample treatment: 1 mL of the plastoglobule suspension was thoroughly mixed with 3 mL of an extraction solvent (methanol / ethyl acetate / petroleum ether, 1:1:1, v / v / v) containing 0.1 g / L BHT, extracted with an ultrasonic machine at an amplitude of 70% for 30 min, and centrifuged at 1320 g and 4 °C for 3 min. The upper organic phase was collected, and the residue was extracted 2 more times under the same conditions with 2 mL of the above extraction solvent. The combined upper organic phases were blown to dry under nitrogen, then dissolved in 1 mL of dichloromethane, filtered through a 0.22 μm PTFE membrane, and the filtrate was determined by high-performance liquid chromatography. The lycopene content was calculated using a standard curve. High-performance liquid chromatography detection conditions: The detection wavelength was 472 nm, the chromatographic column was C18, the mobile phase was a mixed solution of methanol, acetonitrile and dichloromethane (20:75:5), the flow rate was 1.0 mL / min, and the injection volume was 20 μL. The results show that the main substance in the plastoglobules is lycopene, and the lycopene concentration is 50.45 μg / mL, indicating that the plastoglobules are loaded with lycopene inside. Example 4 Lycopene stability test: (1) Salt ion stability analysis: The plastid vesicles suspension was mixed with 0 mM, 50 mM, 100 mM, 150 mM, 200 mM NaCl solutions at a ratio of 1:1 (v / v), and then stirred continuously at 300 rpm for 1 h at room temperature (25 °C). 1 mL of the mixed solution was taken from each sample, centrifuged at 5000 g and 25 °C for 10 min, and the supernatant was taken. The free lycopene suspension of the same mass was used as a control. (2) pH stability analysis: The plastid vesicles suspension was mixed with solutions of pH 3.0, pH 6.0, pH 9.0, and pH 12.0 at a ratio of 1:1 (v / v), and then stirred continuously at 300 rpm for 1 h at room temperature (25 °C). 1 mL of the mixed solution was taken from each sample, centrifuged at 5000 g and 25 °C for 10 min, and the supernatant was taken. The free lycopene suspension of the same mass was used as a control. (3) Temperature stability analysis: The plastid vesicles suspension was mixed with distilled water at a ratio of 1:1 (v / v), and then stirred continuously at 300 rpm for 1 h at 27 °C, 37 °C, 47 °C, 57 °C, 67 °C, 77 °C, 87 °C, and 100 °C respectively. 1 mL of the mixed solution was taken from each sample, centrifuged at 5000 g and 25 °C for 10 min, and the supernatant was taken. The free lycopene suspension of the same mass was used as a control. (4) In vitro simulated digestion stability analysis The in vitro simulated digestion stability analysis of plastid vesicles was carried out by the INFOGEST 2.0 method. Briefly, first, simulated gastric juice containing 2000 U / mL pepsin and 60 U / mL gastric lipase was prepared and incubated at 37 °C. Then, the plastid vesicles sample was added to the simulated gastric juice at a ratio of 1:1 (v / v), the pH was adjusted to 3.0, and the mixture was stirred for 2 h. Subsequently, an equal volume of simulated intestinal juice containing 200 U / mL trypsin was added. The mixture was stirred continuously at pH 7.0 and 37 °C for another 2 h. Initial lycopene content determination: 100 μL of the sample solution was taken at 0 min and mixed with 900 μL of ethyl acetate. After centrifugation at 10000 g and 4 °C for 10 min, the supernatant was taken for analysis. Lycopene retention determination: 200 μL of the sample solution was collected at 30, 60, 90, 120, 150, 180, 210, and 240 min respectively and centrifuged. 100 μL of the supernatant was taken, and the extraction was repeated according to the steps of sample treatment in (5) HPLC high-performance liquid chromatography. The content of lycopene was determined by an enzyme-labeled instrument, and the retention rate was calculated according to the following formula. Free lycopene was used to replace plastid vesicles at the same concentration for the above experiments. The extracted lycopene was analyzed for its lycopene content by an enzyme-labeled instrument at a wavelength of 472 nm. The lycopene retention rate was calculated according to the following formula: It can be seen from Figure 7 that among different salt ion concentrations, pH values, and temperatures, plastid globules significantly improve the water solubility of lycopene. The retention rate of lycopene is increased by loading with plastid globules. The retention rate of plastid globules is higher than 70% under different salt ion concentrations, and also has a relatively high retention rate in alkaline environments and at high temperatures. The above results indicate that plastid globules maintain good stability with environmental changes (salt ions, pH, temperature), which further confirms the potential application of plastid globules as a natural delivery system to resist harsh environments. Through Figure 8 it can be seen that during the simulated gastrointestinal digestion process, plastid globules can significantly resist the harsh gastrointestinal environment. The retention rate in the stomach is higher than 60%, and the retention rate in intestinal digestion is higher than 40%. The stability results of plastid globules in the gastrointestinal tract indicate that it can become a potential natural delivery system. Example 5 Intestinal permeability behavior test: (1) Transwell intestinal mucus permeability evaluation The permeability of plastid globules and free lycopene in intestinal mucus was compared through Transwell intestinal mucus permeability experiments. Weigh 50 mg of porcine small intestine mucus and spread it evenly on the upper chamber of the Transwell plate. Pre-add 600 μL of 0.01 M PBS to the lower chamber of the Transwell plate in advance. Then, after putting the upper chamber back on the Transwell plate, add 200 μL of the plastid globule sample and free lycopene sample with a lycopene concentration of 0.1 mg / mL to the upper chamber, and incubate at 37 °C for 0.5, 1, 2, and 4 h respectively. When sampling at each time point, take out 200 μL of the sample from the lower chamber of the Transwell plate. Extract lycopene and measure the lycopene content using an enzyme-linked immunosorbent assay (ELISA) at a wavelength of 472 nm. The lycopene permeability is calculated according to the following formula. The experimental results are as Figure 9 shown. It can be seen from the figure that with the change of incubation time, the permeability of free lycopene is extremely low, and there is no significant change among incubation times. However, the permeability of plastid globules gradually increases, and the permeability reaches 37% at 4 h, which is significantly higher than that of free lycopene. This is because the natural system of plastid globules can enhance the water solubility of lycopene and its particle size is nanoscale, which can penetrate through the intestinal mucus layer mainly composed of mucin network, preliminarily proving that plastid globules can be used as a natural nano-delivery system. (2) Ex vivo intestinal permeability evaluation The duodenum, jejunum, and ileum were removed from 7-week-old Sprague Dawley rats and quickly placed in Krebs-Ringer buffer. 4 cm of each intestinal segment was taken and rinsed with Krebs-Ringer buffer for standby. 0.4 mL of liposome samples and free lycopene samples with a lycopene concentration of 0.1 mg / mL were added to each intestinal segment. After closing the intestinal orifice, they were placed in 3 mL of 0.01 M PBS and incubated in a shaker at 37°C for 2 h. 200 μL of the external intestinal PBS was taken, and the samples were extracted. The content of lycopene was determined by an enzyme-linked immunosorbent assay (ELISA) reader, and the permeability coefficient was calculated according to the following formula. Among them, P app represents the permeability coefficient, dQ / dt represents the lycopene permeation rate (mg / s), A represents the intestinal surface area (cm 2 ), and C represents the initial lycopene concentration (mg / cm 3 ). It can be seen from Figure 10 that for the duodenum, jejunum, and ileum, the intestinal permeability coefficients of liposomes were 32.00-fold, 39.00-fold, and 143.00-fold higher than those of free lycopene, respectively, which further confirmed the results of the Transwell intestinal mucus permeability evaluation. This indicates that in the real system, liposomes are still more likely to penetrate the intestine than free lycopene, which also further proves the ability of liposomes as a natural delivery system. (3) Observation by CLSM laser confocal fluorescence microscopy To further observe the permeability of liposomes and free lycopene in intestinal mucus, CLSM laser confocal fluorescence microscopy was used for observation. 20 μL of 10 μg / mL Alexa Fluor 555-WGA dye solution was added to the intestinal mucus, and 200 μL of free lycopene samples and extracted liposome samples were added respectively, and they were incubated at 37°C for 0, 10, 30, and 60 min. Finally, observation was carried out by CLSM after dual-channel excitation at wavelengths of 488 nm and 553 nm. Images with 20 μm in the z-axis and 1024×1024 pixels were collected. It can be seen through Figure 11 that both the lycopene in free lycopene and liposomes was excited and showed green light, while the mucin in the intestinal mucus was excited to red light after being stained with Alexa Fluor 555-WGA dye. And it can be seen from Figure 11 A that free lycopene did not show excessive permeation with the prolongation of the incubation time. For Figure 11For the plastid globule sample, with the extension of the incubation time, the plastid globules significantly penetrated into the inner part of the mucus layer. And the maximum fluorescence image in the following figure represents the mucus thickness where the maximum fluorescence intensity of the two samples is located with the penetration over time. As Figure 12 shown, for free lycopene, with the extension of time, the maximum fluorescence was always located at the position where the mucus thickness was 2 μm, that is, free lycopene did not show obvious penetration over time; while for the plastid globules, with the extension of time, the maximum fluorescence intensity ranged from 2 μm to 14 μm, indicating that the plastid globules continuously penetrated in the mucus layer over time. This further shows that the plastid globules can pass through the intestine and enter the body for the next step of lycopene delivery, and also proves that the plastid globules can be used as a natural delivery system. Example 6 Metabolic kinetics test: (1) Blood metabolic kinetics test Sample preparation: To determine the metabolic state and delivery state of free lycopene and plastid globule samples in a real system and measure the pharmacokinetics. Briefly, 7-week-old C57BL / 6J mice were used. Free lycopene and plastid globule samples with the same dose (0.6 mg / kg) were used to perform gavage on the mice respectively. The mice were sacrificed at 0, 0.5, 1, 2, 4, 8, 12, and 24 h respectively, and their sera were collected into 1.5 mL anticoagulant tubes. Immediately, they were centrifuged at 5000 g for 5 min. After collecting the supernatant, 100 μL was taken, 100 μL of ethanol was added, and after mixing, 200 μL of n-hexane was added. After mixing, it was centrifuged at 3000 g at 4 °C for 10 min. The upper layer liquid was collected, and 200 μL of n-hexane was added again and the above steps were repeated three times. The collected upper layer liquid was blown to dry with nitrogen, then 200 μL of dichloromethane was added to dissolve it, and after filtering with a 0.22 μm PTFE membrane, the filtrate was measured by liquid chromatography-tandem mass spectrometry (LC-MS / MS). The results were calculated through the lycopene standard curve. LC-MS / MS determination: Column C18, column temperature 40 °C, flow rate 0.2 mL / min, injection volume 3 μL. Mobile phase A was a mixture of methanol, acetonitrile, and dichloromethane in a ratio of 25:60:15, and mobile phase B was methanol. The liquid phase conditions are shown in Table 1. The mass spectrometry conditions were m / z 536.3—>444.4, ion source ESI, positive ion mode, Dwell was set to 0.015 s, Cone voltage was 50 V, and Collision voltage was 10 V. Table 1 Liquid phase conditions Time Mobile phase (A%:B%) 0 min 60:40 10 min 80:20 As Figure 13 shown, the maximum plasma drug concentration (C max ) and the time to reach the maximum plasma drug concentration (T max) and use the area under the curve (AUC 0-t ) to represent the relative bioavailability in blood (Table 2). The plastid globules significantly improved the oral utilization of lycopene in mice, increased its blood drug concentration by 1.96 times, delayed the time to reach the maximum blood drug concentration by 2 h, and increased the relative bioavailability in blood by 1.71 times, indicating that the plastid globules can not only improve the bioavailability of lycopene in mice but also have a sustained-release effect, proving that the plastid globules can be used as a natural delivery system. Table 2 Pharmacokinetic characteristics of free lycopene and plastid globules in mouse serum (2) Hepatic metabolic kinetics test Sample preparation: To determine the hepatic delivery of free lycopene and plastid globule samples and measure the hepatic pharmacokinetics. Briefly, 7-week-old C57BL / 6J mice were used. Free lycopene and plastid globule samples at the same dose (0.6 mg / kg) were used to gavage the mice respectively. The mice were sacrificed at 0, 0.5, 1, 2, 4, 8, 12, and 24 h respectively, and their livers were collected in cryotubes, freeze-dried, ground into powder, 40 mg was weighed, 100 μL of 300 mM Vc solution was added, after mixing, 200 μL of n-hexane was added and ultrasonically treated for 5 min, then centrifuged at 10000 g at 4 °C for 10 min, the upper layer liquid was collected, and 200 μL of n-hexane was added again and the above steps were repeated three times. The collected upper layer liquid was blown to dry with nitrogen, then 200 μL of dichloromethane was added to dissolve it, and after filtering with a 0.22 μm PTFE membrane, the filtrate was also measured by liquid chromatography-tandem mass spectrometry (LC-MS / MS), and the results were calculated through the lycopene standard curve. As Figure 14 shown, the maximum hepatic drug concentration (C max ), the time to reach the maximum hepatic drug concentration (T max ) of free lycopene and plastid globules were statistically analyzed, and the area under the curve (AUC 0-t ) was used to represent the relative bioavailability in the liver (Table 3). The plastid globules significantly increased the enrichment degree of lycopene in the mouse liver, increased its hepatic drug concentration by 4.90 times, delayed the time to reach the maximum hepatic drug concentration by 7 h, and increased the relative bioavailability in the liver by 7.28 times, indicating that the plastid globules can not only improve the bioavailability of lycopene in the mouse liver but also have a sustained-release effect, proving that the plastid globules can be used as a natural delivery system for hepatic targeted delivery. Table 3 Pharmacokinetic characteristics of free lycopene and plastid globules in mouse liver Group <![CDATA[C max (ng / mL)]]> <![CDATA[T max (h)]]> <![CDATA[AUC 0-t (ng·h / mL)]]> Free lycopene 3.43±0.28 1 37.10±1.89 Plastoglobule 16.80±2.29 8 270.18±5.66 The above are only the preferred embodiments of the present invention, and do not impose any form of limitation on the present invention. Any person skilled in the art can, without departing from the spirit and technical solution of the present invention, make many possible changes and modifications to the technical solution of the present invention by using the methods and technical contents disclosed above, or modify it into equivalent embodiments with equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the technical solution of the present invention still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A liver-targeted lycopene plastid microsphere, characterized in that, The plastoglobule structure is a plasma membrane encapsulating lycopene.

2. The liver-targeted lycopene plastid microsphere according to claim 1, wherein The plastoglobules are derived from plant fruits rich in lycopene.

3. The liver-targeted lycopene plastid microsphere according to claim 2, wherein, The plant fruits rich in lycopene include tomatoes, watermelons, peppers, grapefruits, and guavas.

4. The preparation method of a liver-targeted lycopene plastid microsphere according to claim 1, characterized in that, It includes the following steps: S1: After rinsing the cut plant fruits rich in lycopene with a pre-cooled extraction buffer, add the pre-cooled extraction buffer at a mass-to-volume ratio of 1:(3 - 7), and homogenize and stir to obtain crude juice; S2: After filtering the crude juice, collect the filtrate, centrifuge the filtrate and collect the precipitate, add the extraction buffer to the precipitate at a mass-to-volume ratio of 1:(1 - 4) for reconstitution, and perform ultrasonic treatment after reconstitution to obtain a suspension; S3: Add the suspension, 35 - 38 wt.% sucrose solution, 18 - 20 wt.% sucrose solution, 12 - 15 wt.% sucrose solution, and 3 - 5 wt.% sucrose solution to a centrifuge tube for centrifugation, and collect the topmost substance, which is the lycopene plastoglobule.

5. The preparation method of a liver-targeted lycopene plastid microsphere according to claim 4, characterized in that: The extraction buffer in step S1 includes 20 mM Tricine-KOH, 450 mM sorbitol, 10 mM EDTA-Na2, 10 mM NaHCO3, and 0.5 wt.% bovine serum albumin.

6. The preparation method of a liver-targeted lycopene plastid microsphere according to claim 4, characterized in that: The homogenization and stirring in step S1 is to stir for 1 - 3 seconds every 1 - 3 seconds, with a total of 1 - 5 stirrings.

7. The preparation method of a liver-targeted lycopene plastid microsphere according to claim 4, characterized in that: The filtering material in step S2 is 2 - 5 layers of degreased cotton gauze and a 60 - 80 μm filter screen. The centrifugation conditions are 1200 g, 4°C, 10 - 15 min, and the ultrasonic treatment frequency is 60 - 80 Hz for 1 - 3 min.

8. The preparation method of a liver-targeted lycopene plastid microsphere according to claim 4, characterized in that: The ratio of the suspension, 35 - 38 wt.% sucrose solution, 18 - 20 wt.% sucrose solution, 12 - 15 wt.% sucrose solution, and 3 - 5 wt.% sucrose solution in step S3 is 3:3:3:2:

4.

9. The preparation method of a liver-targeted lycopene plastid microsphere according to claim 4, characterized in that: The centrifugation conditions in step S3 are 80000 - 120000 g, 4°C, 2 - 4 h.

10. Use of a liver-targeted lycopene plastoglobule according to claim 1 in biomedicine and functional foods.

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