Ginkgo flavone-hedysarum polybotrys polysaccharide dual-delivery liposome as well as preparation method and application thereof

Abstract

The invention relates to the technical field of food engineering, and discloses ginkgo flavone-hedysarum polybotrys polysaccharide dual-delivery liposome as well as a preparation method and application thereof. The liposome disclosed by the invention consists of a core liposome and an outer-layer modifier, ginkgo flavone and hedysarum polybotrys polysaccharide are embedded in the core liposome at the same time, and the outer modifier is a food-borne polysaccharide modification material grafted with vitamin B12. According to the invention, the defect of poor solubility of the two nutrients is overcome through the application of the liposome, and conditions are provided for the two nutrients to pass through the intestinal barrier and enrich the brain through the surface modification of the liposome. The invention also verifies the synergistic effect of the two nutrients in the aspect of improving the learning and memory ability through an animal model, and provides an effective path for improving the bioavailability of the two nutrients through surface modification, so that the dual-delivery liposome disclosed by the invention has a good application prospect in the development of health-care foods or medical products for improving learning and memory. And the method has wide practical application value.

Description

Technical Field

[0001] The present invention relates to the technical field of food engineering, and particularly relates to a carrier liposome of a plant extract capable of improving learning and memory ability, a preparation method thereof, and an application thereof. Background Art

[0002] With the increase in the global population and the extension of lifespan, the problem of aging has become increasingly serious. Aging is often accompanied by cognitive, memory, and learning ability dysfunctions, brain injuries, etc. In severe cases, it is manifested as neurological diseases such as Alzheimer's disease and Parkinson's disease. Ginkgo biloba preparations are natural active substances extracted from Ginkgo biloba leaves. Their main components can be divided into flavonoids and lactones. Their effects in treating cardiovascular and cerebrovascular diseases, improving cerebral blood circulation, and promoting brain cell metabolism have been verified in large numbers. For example, the health food for improving human immunity and promoting wisdom and brain health and its preparation method disclosed in Patent CN101356975B verify the effects of ginkgo flavonoids in increasing cerebral blood flow and promoting wisdom through the addition of Ginkgo biloba extract. In addition, some studies have pointed out that the mechanism of action of ginkgo flavonoids (Gk) may be that it can increase the levels of acetylcholine neurotransmitters and monoamine neurotransmitters in the brain tissue, increase the activity of superoxide dismutase in the brain tissue, thereby scavenging free radicals, and ultimately reducing apoptosis of brain tissue cells.

[0003] As the main active ingredient of Hedysarum polybotrys Hand.-Mazz., modern pharmacological studies have shown that Hedysarum polybotrys polysaccharide (HPS) has effects such as immune regulation, antioxidant, anti-inflammatory, anti-tumor, body protection, prevention and treatment of diabetic complications, and anti-osteoporosis. Research shows that the mechanism of action of Hedysarum polybotrys polysaccharide may be related to reducing the damage of free radicals to brain cell DNA, enhancing the body's antioxidant capacity, maintaining the integrity of hippocampal nerve cells and brain tissue structure, and improving neuronal and brain tissue neurodegeneration. For example, the application of Hedysarum polybotrys and Hedysarum polybotrys polysaccharide in the preparation of drugs for improving learning and memory and treating Alzheimer's disease disclosed in Patent CN102018747A shows that Hedysarum polybotrys and Hedysarum polybotrys polysaccharide can protect SH-SY5Y cells from damage by Aβ25-35 and increase the survival of SH-SY5Y cells; through the Morris water maze experiment, it shows that Hedysarum polybotrys and Hedysarum polybotrys polysaccharide can improve the cognitive dysfunction of rapidly aging mice and Alzheimer's disease rats with hippocampal injection of Aβ.

[0004] However, there is currently no research or report on the synergistic effect of ginkgo flavonoids and astragalus polysaccharides. The reasons may be as follows: Although both of them can improve brain function, their specific mechanisms of action are different. It is possible that due to the mutual competition of the two nutrients for binding sites, the combined use of the two may actually lead to a decrease in the effect. On the other hand, ginkgo flavonoids and astragalus polysaccharides themselves have problems of poor solubility and low bioavailability. Among them, ginkgo flavonoids are liposoluble, while astragalus polysaccharides are water-soluble. The difference in their solubility increases the technical difficulty of transporting these two components simultaneously, making it difficult to pass through the intestinal barrier and even more impossible to target and enrich in the brain, which greatly limits their development and application in food, health products, and pharmaceuticals. Summary of the Invention

[0005] The present invention aims to overcome the defects in the prior art that the food and drugs using ginkgo flavonoids or astragalus polysaccharides alone have poor effects in improving learning and cognitive functions, and it is difficult to target and enrich ginkgo flavonoids and astragalus polysaccharides in the brain when used synergistically. The present invention provides a ginkgo flavonoid-astragalus polysaccharide dual-loaded liposome, its preparation method, and application to overcome the above defects.

[0006] To achieve the above object of the present invention, the present invention is realized through the following technical solutions: In the first aspect, the present invention discloses a ginkgo flavonoid-astragalus polysaccharide dual-loaded liposome, which is composed of a core liposome and an outer modifier modified on the surface of the core liposome through electrostatic interaction; The core liposome is composed of a lipid coating and a water-soluble nutrient embedded in the lipid coating; The lipid coating includes lipid excipients and liposoluble nutrients; The liposoluble nutrient includes ginkgo flavonoids; The water-soluble nutrient includes astragalus polysaccharides; The outer modifier includes a food-derived modifier and activated vitamin B12 chemically grafted on the food-derived modifier.

[0007] Ginkgo flavonoids are the main active components in ginkgo leaf extract, with the functions of antioxidation, anti-inflammation, and improvement of blood circulation; Hedysari polysaccharide is a polysaccharide substance extracted from Hedysarum polybotrys, with the functions of immune regulation, anti-fatigue, and antioxidation. When the two nutrients act on the brain simultaneously, ginkgo flavonoids dilate cerebral blood vessels, increase cerebral blood flow, and enhance the oxygen supply and metabolic substrate delivery of neurons by inhibiting platelet-activating factor and enhancing nitric oxide release; at the same time, Hedysari polysaccharide can reduce the damage of chronic inflammation to hippocampal neurons, up-regulate the expression of brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF), and both nutrients have antioxidation functions. Under the above combined actions, the improvement of the brain's learning and cognitive functions can be achieved. Therefore, to verify and exert the synergistic effect of ginkgo flavonoids and Hedysari polysaccharide, it is necessary to ensure that the release time and location of the two in the body are as consistent as possible to achieve the best effect. In the present invention, liposomes are used as the carriers for transporting the two nutrients. Liposomes are an artificial membrane. When amphiphilic molecules such as phospholipids are dispersed in an aqueous phase, the hydrophobic ends of the phospholipids aggregate together to avoid the aqueous phase, while the hydrophilic heads are exposed to the aqueous phase, forming a cell-like structure-closed vesicle with a phospholipid bilayer membrane, and its diameter ranges from 25 to 1000 nm. Due to the different solubilities of ginkgo flavonoids and Hedysari polysaccharide, in the present invention, the lipophilic nutrient is first made into a lipid-coated body with lipid excipients, and then the water-soluble nutrient is embedded by the lipid-coated body, so as to achieve the dual transportation of ginkgo flavonoids and Hedysari polysaccharide. In practice, the dual-transport liposomes of the present invention can be prepared according to the following ratio: lipophilic nutrient 1-3 mg / mL, water-soluble nutrient 1-3 mg / mL, lipid excipient 11-33 mg / mL, and the content ratio of lipophilic nutrient to water-soluble nutrient is preferably 1:1.

[0008] However, since ginkgo flavonoids and astragalus polysaccharides are respectively embedded in the lipid phase and the aqueous phase, when the liposomes of the present invention are orally administered, due to the change of body temperature in vivo, it is difficult for the two nutrients to be released simultaneously at the same position. On the one hand, the astragalus polysaccharides in the aqueous phase are extremely easy to leak rapidly in the initial stage. At this time, the ginkgo flavonoids are still slowly released in the lipid phase, resulting in a burst release effect, which may cause too high local concentration or premature metabolism. On the other hand, the membrane fluidity of the lipid-coated body encapsulating ginkgo flavonoids becomes stronger at body temperature. If the ginkgo flavonoids are released in advance, short-term blood flow overload may exacerbate oxidative stress, and the astragalus polysaccharides do not scavenge free radicals in time, leading to neuronal damage. Therefore, through multiple experiments, the present invention uses a food-derived modification material grafted with activated vitamin B12 (VB12) to modify the liposomes, which not only greatly improves the thermal stability of the liposomes, but also improves the small intestine absorption rate of the two nutrients. After the VB12-modified liposomes are endocytosed by target cells, the two drugs can be synchronously released into the cells through the low pH environment of lysosomes or enzyme-triggered membrane rupture, avoiding the problem of asynchronous release in vitro. In addition, VB12 can specifically bind to the receptor (intrinsic factor of the small intestine IF) expressed on the epithelial cells of the ileal region of the small intestine, thereby enhancing the absorption of liposomes in the small intestine. In practice, the volume ratio of the core liposome to the outer modifier is preferably 1:1-4, more preferably 1:3.

[0009] Furthermore, the food-derived modification material is a polysaccharide modifier. To ensure the stable delivery of liposomes in vivo, the present invention uses a polysaccharide outer modifier to modify liposomes, thereby improving their environmental stability, and using a polysaccharide outer modifier can ensure their biosafety.

[0010] Furthermore, the polysaccharide modifier is chitosan (CS). Chitosan has excellent biological functions and can undergo chemical modification reactions. In practice, first, VB12 is activated and grafted onto CS to obtain a VB12-CS solution as the outer modifier; then, through electrostatic interaction, the positively charged outer modifier is modified on the outer surface of the negatively charged core liposome, thereby realizing the surface modification of the dual-carrier liposome. After subsequent experimental verification, chitosan grafted with vitamin B12 can significantly improve the thermal stability of the two nutrients. After heating for 2 h, the particle size of the liposomes has no obvious change. In practice, the outer modifier can be prepared according to the following ratio: VB12 0.4-0.6 mg / mL, chitosan 2-8 mg / mL, and the solvent is 1% - 1.5% acetic acid solution.

[0011] Furthermore, the lipid excipients are soybean phospholipid and cholesterol.

[0012] Furthermore, the mass ratio of soybean phospholipid to cholesterol in the lipid excipient is 6:1, and the mass ratio of ginkgo flavonoids to the lipid excipient is between 1:5 and 1:7. Through subsequent experiments, it was found that when the above conditions are met, the encapsulation efficiency of ginkgo flavonoids or astragalus polysaccharides is relatively balanced, ensuring that both nutrients can be stably encapsulated in liposomes, which is more conducive to large-scale industrial production.

[0013] In a second aspect, the present invention also discloses a method for preparing a ginkgo flavonoid-astragalus polysaccharide dual-carrier liposome, which is characterized by comprising the following steps: S1. Dissolve the fat-soluble nutrient containing ginkgo flavonoids and the lipid excipient in ethanol, fully dissolve and mix them, and then remove the ethanol therein to form a lipid-coated body; S2. Dissolve the water-soluble nutrient containing astragalus polysaccharides in a buffer solution, and use the buffer solution to wash the lipid film to obtain a core liposome with a fixed volume; S3. Activate vitamin B12, and chemically graft the activated vitamin B12 onto the food-derived modification material to obtain an outer modifier; S4. Mix the outer modifier with the core liposome and subject it to microfluidic treatment to obtain the ginkgo flavonoid-astragalus polysaccharide dual-carrier liposome.

[0014] In practice, in step S2, ethanol can be removed by evacuating with a rotary evaporator. In practice, if a rotary evaporator is used to remove ethanol, the rotary evaporation time is preferably 10-20 min. In step S3, phosphate buffer solution (PBS) can be used as the buffer solution for washing the film, preferably 5-10 mM, pH 6.5-7.5, the film washing time is preferably 1-2 h, and the stirring temperature is 40-50 °C.

[0015] Furthermore, the step of activating vitamin B12 in step S3 is as follows: dissolve vitamin B12 in anhydrous dimethyl sulfoxide containing succinic anhydride and 4-dimethylaminopyridine, and perform rotary evaporation treatment to obtain activated vitamin B12.

[0016] Furthermore, in step S3, the food-derived modification material is first subjected to microfluidic treatment, and then the activated vitamin B12 is chemically grafted onto the food-derived modification material to obtain an outer modifier. Since many food-derived modification materials have high viscosities, the food-derived modification material is first subjected to microfluidic treatment to flatten the molecular structure of the modification material, so that more VB12 can be grafted onto the food-derived modification material. The pressure value of the microfluidic treatment is preferably 100 MPa.

[0017] In a third aspect, the present invention also discloses the application of the ginkgo flavonoid-astragalus polysaccharide dual-carrier liposome in the preparation of a health food for improving learning and memory.

[0018] Fourthly, the present invention also discloses the application of ginkgo flavonoids - astragalus polysaccharide dual - delivery liposomes in the preparation of drugs for treating Alzheimer's disease.

[0019] Therefore, the present invention has the following beneficial effects: (1) By using liposomes that can simultaneously encapsulate lipophilic ginkgo flavonoids and water - soluble astragalus polysaccharides as carriers, the present invention overcomes the defect of poor solubility of ginkgo flavonoids and astragalus polysaccharides, providing conditions for them to pass through the intestinal barrier and accumulate in the brain. (2) Through the modification of liposomes with polysaccharide outer modifiers grafted with vitamin B12, the present invention not only improves the thermal stability of the above - mentioned two types of nutrients, but also VB12 can specifically bind to the receptors expressed on the epithelial cells of the ileal region of the small intestine, increasing the small intestine absorption efficiency of the two nutrients and improving the bioavailability. (3) Through animal models, the present invention verifies that ginkgo flavonoids and astragalus polysaccharides can increase the concentration of brain - derived neurotrophic factors, and have an obvious improvement on the learning and memory function of mice, verifying the synergistic effect of the two nutrients in improving learning and memory ability. (4) The liposomes and outer modifiers of the present invention are all food - grade raw materials. The liposomes of the present invention can also reduce the poor taste of ginkgo flavonoids and astragalus polysaccharides, and are more suitable for the production practice of related foods and health products. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a process flow chart of the preparation of ginkgo flavonoids - astragalus polysaccharide dual - delivery liposomes according to the embodiments of the present invention.

[0021] Figure 2 It is a particle size distribution diagram of the dual - delivery liposomes of the embodiments and comparative examples of the present invention.

[0022] Figure 3 It is a comparison diagram of the zeta potential of the dual - delivery liposomes of the embodiments and comparative examples of the present invention.

[0023] Figure 4 It is a comparison diagram of the encapsulation efficiency of ginkgo flavonoids and astragalus polysaccharides in the embodiments and comparative examples of the present invention.

[0024] Figure 5 It is a diagram of the appearance change of liposomes during the 2 - hour heating of the dual - delivery liposomes of the present invention at 100 °C.

[0025] Figure 6 It is a schematic diagram of the particle size change of liposomes during the 2 - hour heating of the dual - delivery liposomes of the present invention at 100 °C.

[0026] Figure 7 It is a schematic diagram of the zeta potential change of liposomes during the 2 - hour heating of the dual - delivery liposomes of the present invention at 100 °C.

[0027] Figure 8 Schematic diagram of the experiment on the improvement of learning and cognitive abilities of D-galactose-induced aging memory impairment mice by the dual-loaded liposomes of the present invention.

[0028] Figure 9 Schematic diagram of the experimental results of the intestinal absorption of the dual-loaded liposomes of the present invention.

[0029] Figure 10 Schematic diagram of the behavioral test results of the experiment on the improvement of learning and cognitive abilities of D-galactose-induced aging memory impairment mice by the dual-loaded liposomes of the present invention.

[0030] Figure 11 Schematic diagram of the effects of the dual-loaded liposomes of the present invention on the contents and mRNA expression levels of glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) in the brain tissues of mice with decreased learning and memory functions. Detailed implementation manners

[0031] The following further describes the present invention in conjunction with specific embodiments. Those of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following descriptions are generally only a part of the embodiments of the present invention, rather than all of the embodiments. Therefore, all other embodiments obtained by those of ordinary skill in the art without creative efforts based on the embodiments of the present invention shall fall within the scope of protection of the present invention. In the following text, the core lipid without surface modification is called O-L; the ginkgo flavonoid-polysaccharide double-loaded liposome of the present invention is called M-L.

[0032] Example 1: (1) Preparation of unmodified core liposomes O-L: Soybean phospholipids and cholesterol were mixed at a ratio of 6:1 (w / w). Subsequently, ginkgo flavonoids and the soybean phospholipid-cholesterol mixture were dissolved in ethanol at a ratio of 1:6 (w / w) to obtain a mixed suspension. The mixed suspension was placed in an ultrasonic instrument (40 kHz, 200 W, 30 min) for treatment, and then ethanol was removed by rotary evaporation at 45°C (80 rpm, 15 min) to form a lipid film. The polysaccharide of Hedysarum polybotrys was dissolved in 100 mL of PBS buffer with a concentration of 10 M and a pH of 7.4, and the buffer was slowly added to the lipid film. After washing the film, it was treated with microfluidics at 120 MP for 2 cycles to obtain ginkgo flavonoid-polysaccharide double-loaded liposomes (O-L); the volume of ethanol used was generally 50-100 mL / g based on the mass of soybean phospholipids. (2) Preparation of the outer modifier: VB12 was dissolved in anhydrous dimethyl sulfoxide containing succinic anhydride (SA) and 4-dimethylaminopyridine and then rotary evaporated. Subsequently, 0.6 g of chitosan was dissolved in 1% acetic acid solution and hydrated with the activated VB12 for 24 h to obtain the outer modifier VB12-CS. (3) Preparation of modified liposomes M-L: The O-L obtained in step (1) and the outer layer modifier obtained in step (2) were stirred and mixed evenly at a volume ratio of 1:1, and finally obtained modified liposomes (M-L) after being treated by dynamic high-pressure microfluidization (120 MPa) twice, and stored at 4°C.

[0033] Comparative Example 1: (1) Preparation of unmodified core liposomes O-L: Soybean phospholipids and cholesterol were mixed at a ratio of 6:1 (w / w). Subsequently, ginkgo flavonoids and the soybean phospholipid-cholesterol mixture were dissolved in ethanol at a ratio of 1:4 (w / w) to obtain a mixed suspension. The mixed suspension was placed in an ultrasonic instrument (40 kHz, 200 W, 30 min) for treatment, and then ethanol was removed by rotary evaporation at 45°C (80 rpm, 15 min) to form a lipid film. Astragalus polysaccharide was dissolved in 100 mL of PBS buffer with a concentration of 10 M and a pH of 7.4, and the buffer was slowly added to the lipid film. After washing the film, it was treated with a microfluidizer at 120 MP for 2 cycles to obtain ginkgo flavonoid-astragalus polysaccharide dual-loaded liposomes (O-L); the volume of ethanol used was generally 50-100 mL / g based on the mass of soybean phospholipids. (2) Preparation of the outer layer modifier: VB12 was dissolved in anhydrous dimethyl sulfoxide containing succinic anhydride (SA) and 4-dimethylaminopyridine and then rotary evaporated. Subsequently, 0.6 g of chitosan was dissolved in 1% acetic acid solution and hydrated with the activated VB12 for 24 h to obtain the outer layer modifier VB12-CS. (3) Preparation of modified liposomes M-L: The O-L obtained in step (1) and the outer layer modifier obtained in step (2) were stirred and mixed evenly at a volume ratio of 1:1, and finally obtained modified liposomes (M-L) after being treated by dynamic high-pressure microfluidization (120 MPa) twice, and stored at 4°C.

[0034] Comparative Example 2: (1) Preparation of unmodified core liposomes O-L: Soybean phospholipids and cholesterol were mixed at a ratio of 6:1 (w / w). Subsequently, ginkgo flavonoids and the soybean phospholipid-cholesterol mixture were dissolved in ethanol at a ratio of 1:8 (w / w) to obtain a mixed suspension. The mixed suspension was placed in an ultrasonic instrument (40 kHz, 200 W, 30 min) for treatment, and then ethanol was removed by rotary evaporation at 45°C (80 rpm, 15 min) to form a lipid film. Astragalus polysaccharide was dissolved in 100 mL of PBS buffer with a concentration of 10 M and a pH of 7.4, and the buffer was slowly added to the lipid film. After washing the film, it was treated with a microfluidizer at 120 MP for 2 cycles to obtain ginkgo flavonoid-astragalus polysaccharide dual-loaded liposomes (O-L); the volume of ethanol used was generally 50-100 mL / g based on the mass of soybean phospholipids. (2) Preparation of the outer modifier: Dissolve VB12 in anhydrous dimethyl sulfoxide containing succinic anhydride (SA) and 4-dimethylaminopyridine, and then perform rotary evaporation. Subsequently, dissolve 0.6 g of chitosan in 1% acetic acid solution and hydrate it with the activated VB12 for 24 h to obtain the outer modifier VB12-CS. (3) Preparation of the modified liposome M-L: Stir and mix the O-L obtained in step (1) and the outer modifier obtained in step (2) at a volume ratio of 1:1, and finally obtain the modified liposome (M-L) after 2 times of dynamic high-pressure microfluidization (120 MPa) treatment, and store it at 4 °C.

[0035] Example 1 is the preferred embodiment of the present invention, and the process flow chart is as Figure 1 shown. In Comparative Example 2 and Comparative Example 3, the ratio of ginkgo flavonoids to lipid excipients was changed, and the particle size distribution, zeta potential, and encapsulation efficiency of Example 1, Comparative Example 2, and Comparative Example 3 were tested. As Figure 2 shown, at different ratios of nutrients to the soybean phospholipid-cholesterol mixture, the particle sizes of the prepared ginkgo flavonoid-polysaccharide double-loaded liposomes of Astragalus membranaceus were slightly different, and the particle sizes of the liposomes were uniform. As Figure 3 shown, there was no significant difference in the zeta potential among Example 1, Comparative Example 2, and Comparative Example 3. As Figure 4 shown, when the content of lipid excipients is too high or too low, the encapsulation efficiency of the two nutrients will decrease. Therefore, when the mass ratio of ginkgo flavonoids to lipid excipients is between 1:5 and 1:7, the encapsulation efficiencies of ginkgo flavonoids and polysaccharide of Astragalus membranaceus can be maintained at a high level, which is suitable for subsequent large-scale production.

[0036] To test the thermal stability of the double-loaded liposome of the present invention, a thermal stability experiment was carried out: Heat the ginkgo flavonoid-polysaccharide double-loaded liposome (O-L) and the modified liposome (M-L) prepared in Example 1 at 100 °C for 2 h, and measure the particle size and zeta potential values at 5 min, 30 min, 1 h, 1.5 h, and 2 h respectively. The appearance change of the liposome during the 2 h heating is as Figure 5 shown, the particle size change of the liposome during the 2 h heating is as Figure 6 shown, and the zeta potential change of the liposome during the 2 h heating is as Figure 7 shown. It can be seen that the appearance of the modified liposome (M-L) changes very little after 2 h of heating, and the particle size and zeta potential values are more stable, showing excellent thermal stability.

[0037] To further verify the role of the double-loaded liposome of the present invention in improving learning and cognitive functions, an animal model experiment was carried out: As Figure 8As shown in the figure, mice were divided into 6 groups, namely COL: blank control group (intragastric administration of PBS + injection of normal saline), DGM: model control group (intragastric administration of PBS + injection of D-gal normal saline), POS: positive control group (intragastric administration of piracetam + injection of D-gal normal saline), G-H: free nutrient group (intragastric administration of free ginkgo flavonoids - astragalus polysaccharide mixture + injection of D-gal normal saline), O-L: unmodified liposome group (intragastric administration of ginkgo flavonoids - astragalus polysaccharide dual-loaded liposome + injection of D-gal normal saline), M-L: modified liposome group (intragastric administration of ginkgo flavonoids - astragalus polysaccharide dual-loaded liposome modified with vitamin B12-coupled chitosan graft + injection of D-gal normal saline). Mice were intragastrically administered and injected for 56 days, followed by 18 days of behavioral testing. After fasting for 24 hours, blood was collected from the mouse eyeballs and the mice were sacrificed by decapitation. Brain tissues were taken to detect key indicators.

[0038] The absorption effect of the dual-loaded liposome of the present invention in the small intestine of mice is as Figure 9 shown, where (a) the mRNA content of intestinal transporter ABCB1 in mice; (b) the mRNA content of intestinal transporter C2L2 in mice. It can be seen from the figure that the mRNA contents of intestinal transporters ABCB1 and C2L2 in the M-L group intragastrically administered with ginkgo flavonoids - astragalus polysaccharide dual-loaded liposome modified with VB12-coupled chitosan graft are the highest, indicating that M-L has a better absorption and transport effect.

[0039] The results of the behavioral tests are as Figure 10 shown, including (a) the escape latency of directional navigation; (b) the number of times passing through the platform; (c) the step-down latency; (d) the schematic diagram of the number of step-down errors (5 min). In the Morris water maze test, the escape latency of the model control group (DGM) was significantly longer than that of the blank control group (COL), the O-L group was shorter than the DGM group but longer than the free nutrient group (G-H), and the escape latency of the M-L group was shorter than that of the O-L group ( Figure 10 a). The number of times the DGM group passed through the original platform was lower than that of the COL group ( Figure 10 b). In the step-down test, the escape latencies of the rats in the COL group, O-L group, and M-L group were shorter than that of the DGM group ( Figure 10 c), and the number of learning and memory errors was significantly reduced ( Figure 10 d). Therefore, both O-L and M-L liposomes have the effect of improving the learning and memory abilities of D-galactose-induced senile model mice, but the effect of M-L is better and is close to that of the positive drug on the market.

[0040] To verify the mechanism of action of the dual-loaded liposome of the present invention, the contents and mRNA expression levels of glial cell line-derived neurotrophic factor GDNF and brain-derived neurotrophic factor BDNF in the brain tissues of mice with decreased learning and memory function were detected: The experimental results are asFigure 11 as shown, including (a) the content of glial cell line-derived neurotrophic factor (GDNF) in mouse brain tissue; (b) the content of brain-derived neurotrophic factor (BDNF) in mouse brain tissue; (c) the mRNA expression level of glial cell line-derived neurotrophic factor (GDNF) in mouse brain tissue; (d) the schematic diagram of the mRNA expression level of brain-derived neurotrophic factor (BDNF) in mouse brain tissue. According to the results, compared with the DGM model group, the contents of neurotrophic factors GDNF and BDNF in the COL group, POS group, O-L group, and M-L group were higher. The expression levels of GDNF and BDNF in the brain tissues of mice in the POS group and O-L group were close, with no significant difference, and the expression level in the M-L group was the highest. The results of the mRNA level expression were consistent with the results of the content measured by the kit. The mRNA levels of neurotrophic factors GDNF and BDNF in mice in the M-L group were the highest, verifying the excellent effect of the ginkgo flavonoids-hongqi polysaccharide dual-loaded liposomes of the present invention.

[0041] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A Ginkgo flavonoids-Herbstilbene polysaccharide dual-carrying liposome, characterized by: It consists of a core liposome and an outer layer modified on the surface of the core liposome by electrostatic interaction; The core liposome is composed of a lipid coating body and a water-soluble nutrient embedded in the lipid coating body; The lipid coating body includes lipid auxiliary materials and fat-soluble nutrients; The fat-soluble nutrients include ginkgo flavonoids; The water-soluble nutrients include spatholobi polysaccharide; The outer layer modification includes a food-derived modification material and activated vitamin B12 chemically grafted onto the food-derived modification material.

2. The Ginkgo flavonoids-Herbstilbene polysaccharide dual-carrier liposome according to claim 1, characterized in that: The food-derived modified material is a polysaccharide modified substance.

3. The Ginkgo flavonoids-Herbstilbene polysaccharide dual-carrier liposome according to claim 2, characterized in that: The polysaccharide modification is chitosan.

4. The Ginkgo flavonoids-Herbstilbene polysaccharide dual-carrier liposome according to claim 1, characterized in that: The lipid auxiliary materials are soybean lecithin and cholesterol.

5. The Ginkgo flavonoids-Herbstilbene polysaccharide dual-carrier liposome according to claim 4, characterized in that: The mass ratio of soybean lecithin to cholesterol in the lipid auxiliary material is 6:1, and the mass ratio of ginkgo flavonoids to the lipid auxiliary material is between 1:5-7.

6. A method for preparing the Ginkgo flavonoids-Herbstilbene polysaccharide dual-carrier liposomes as claimed in any one of claims 1 to 5, characterized in that: The steps include: S1, dissolving the fat-soluble nutrients containing ginkgo flavonoids and lipid excipients in ethanol, fully dissolving and mixing, and then removing the ethanol to form a lipid coating body; S2, dissolving the water-soluble nutrients containing spatholobi polysaccharide in a buffer solution, washing the lipid film with the buffer solution, and fixing the volume to obtain core liposomes; S3, activating vitamin B12, and chemically grafting the activated vitamin B12 onto the food-derived modification material to obtain an outer layer modification; S4, mixing the outer layer modification material with the core liposome, and subjecting the mixture to microfluidization treatment to obtain the Ginkgo flavonoids-Scutellaria rubra polysaccharide dual-carrying liposome.

7. The method for preparing the Ginkgo flavonoids-Herbstilbene polysaccharide dual-carrier liposome according to claim 6, characterized in that: The step of activating vitamin B12 in step S3 is: dissolving vitamin B12 in anhydrous dimethyl sulfoxide containing succinic anhydride and 4-dimethylaminopyridine, and subjecting the solution to rotary evaporation to obtain activated vitamin B12.

8. The method for preparing the Ginkgo flavonoids-Herbstilbene polysaccharide dual-carrier liposome according to claim 6, characterized in that: In the step S3, the food-derived modified material is first subjected to microfluidization treatment, and then the activated vitamin B12 is chemically grafted onto the food-derived modified material to obtain an outer layer modified product.

9. Use of the Ginkgo flavonoids-Herbaceae polysaccharide dual-carrying liposomes according to any one of claims 1 to 5 in the preparation of health food for improving learning and memory.

10. Use of the Ginkgo flavonoids-Herbstilbene polysaccharide dual-carrying liposomes according to any one of claims 1 to 5 in the preparation of drugs for treating Alzheimer's disease.

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