Acorus tatarinowii oil-ginsenoside Rg1 liposome as well as preparation method and application thereof

Abstract

The invention belongs to the field of traditional Chinese medicine modernized preparations, and adopts a liposome drug carrier to prepare ginsenoside Rg1 and rhizoma acori graminei oil into the rhizoma acori graminei oil-ginsenoside Rg1 liposome (VOA-Rg1-LP) which is small in particle size, uniform in distribution, high in bioavailability and stable. The lipidosome realizes brain delivery of the ginsenoside Rg1 by virtue of self passive brain targeting and combination with the rhizoma acori graminei oil, so that the brain targeting release amount of the ginsenoside Rg1 is remarkably increased. Compared with a single drug, the liposome has the advantages that the release rate of the ginsenoside Rg1 and the grassleaf sweelflag rhizome oil is delayed, the slow release effect is achieved, and meanwhile, the drug effect of the grassleaf sweelflag rhizome oil and the ginsenoside Rg1 on the Alzheimer disease is effectively improved.

Description

Technical Field

[0001] The present invention belongs to the field of modernized Chinese medicine preparations, and specifically relates to the field of nano preparations and novel drug preparations for nervous system diseases. Technical Background

[0002] As the trend of population aging becomes increasingly serious, brain diseases such as Alzheimer's disease (AD), Parkinson's disease, and epilepsy have seriously endangered human health. However, the existence of the blood-brain barrier prevents drugs from entering the brain to exert their therapeutic effects. The delivery efficiency of traditional drug delivery systems is limited, and many drugs are limited in the treatment of brain diseases due to their poor water solubility, short biological half-life, and low bioavailability. How to promote drug penetration through the blood-brain barrier is the key to preventing and treating brain diseases, and it is also the difficulty in designing brain-targeted drug delivery systems.

[0003] Ginsenoside Rg1 is the main active ingredient of ginseng, which has the effects of promoting hippocampal neurogenesis, improving neural plasticity, enhancing learning and memory, anti-aging, anti-fatigue, and improving immunity. It is widely used in the prevention and treatment of neurodegenerative diseases such as AD. However, ginsenoside Rg1 is easily soluble in water and has a very low blood-brain barrier permeability, which seriously limits its wide application in the treatment of neurodegenerative diseases.

[0004] The Chinese herbal medicine Acorus tatarinowii has the effects of opening the mind and eliminating phlegm, refreshing the mind and improving intelligence, removing dampness and stimulating appetite, and is often used as a herbal medicine in Chinese herbal compound prescriptions. Modern pharmacological studies have shown that the active ingredients of Acorus tatarinowii volatile oil have the effects of clearing the mind and improving intelligence, protecting neurons, improving memory and improving cognitive function. However, unlike ginsenoside Rg1 which is easily soluble in water, Acorus tatarinowii volatile oil (VOA) is highly lipophilic, and VOA is difficult to combine with ginsenoside Rg1 in conventional preparations.

[0005] Due to the limitation of blood-brain barrier, it is difficult to effectively deliver therapeutic drugs such as Rg1 to the brain through traditional preparation forms and administration routes. Therefore, it is urgent to prepare brain-targeted preparations with different properties of ginsenoside Rg1 and active ingredients of Acorus tatarinowii volatile oil to achieve the purpose of effectively treating Alzheimer's disease through brain-targeted delivery. Summary of the invention

[0006] The object of the present invention is to provide a calamus oil-ginsenoside Rg1 liposome (VOA-Rg1-LP) and a preparation method thereof. The liposome is administered via the nasal route, and the effective ingredients of calamus oil contained therein can be directly absorbed into the brain through the nasal mucosa. The VOA-Rg1-LP liposome effectively improves the drug loading, encapsulation rate and bioavailability of calamus oil and ginsenoside Rg1, and has a sustained release effect, which can better exert the efficacy of calamus oil and ginsenoside Rg1 in treating brain diseases.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The invention firstly provides an Acorus calamus oil-ginsenoside Rg1 liposome. The liposome is prepared from the following raw materials: cholesterol, phospholipids, Acorus calamus oil and ginsenoside Rg1.

[0009] In a preferred embodiment of the present invention, the mass ratio of cholesterol to phospholipid is 1:4-1:7; preferably, the mass ratio of cholesterol to phospholipid is 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:7.

[0010] In a preferred embodiment of the present invention, Acorus calamus oil and ginsenoside Rg1, calculated by the mass of ginsenoside Rg1 and the volume of Acorus calamus oil, ginsenoside: Acorus calamus oil = 1g: (0.375-2)mL; preferably, the volume ratio is 1g: 0.375mL, 1g: 0.5625mL, 1g: 0.75mL, 1g: 0.818mL, 1g: 1mL, 1g: 1.5mL, 1g: 2mL.

[0011] In a preferred embodiment of the present invention, based on the total mass of cholesterol and phospholipids and the total mass of Acorus calamus oil and ginsenoside Rg1, m (胆固醇与磷脂) :m (石菖蒲油与人参皂苷Rg1) It is 1:1-1:7; preferably, the mass ratio is 1:1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.2, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7.

[0012] In a preferred embodiment of the present invention, the phospholipid is one or more of hydrogenated soybean lecithin, egg yolk lecithin, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine, and distearoylphosphatidylcholine.

[0013] Secondly, the present invention provides a method for preparing calamus oil-ginsenoside Rg1 liposomes, comprising the following steps:

[0014] Dissolve cholesterol, phospholipids and Acorus calamus oil in anhydrous ethanol to obtain an anhydrous ethanol solution;

[0015] Dissolve ginsenoside Rg1 in ultrapure water, dropwise add the anhydrous ethanol solution into the ultrapure water containing ginsenoside Rg1, evaporate the anhydrous ethanol, and obtain a suspension;

[0016] The suspension is subjected to ultrasonic treatment to obtain the Acorus calamus oil-ginsenoside Rg1 liposome.

[0017] In a preferred embodiment of the present invention, cholesterol and phospholipids are first dissolved in anhydrous ethanol to obtain an anhydrous ethanol solution; and then Acorus calamus oil is added to obtain an ethanol solution containing Acorus calamus oil;

[0018] In a preferred embodiment of the present invention, ginsenoside Rg1 is dissolved in ultrapure water, and the solution is continuously stirred while heating, and the anhydrous ethanol solution is added dropwise to the ultrapure water / ultrapure water containing ginsenoside Rg1, and the anhydrous ethanol is continuously stirred to completely evaporate, thereby obtaining a suspension;

[0019] In a preferred embodiment of the present invention, the ginsenoside Rg1 is dissolved in ultrapure water, and the mass concentration of ginsenoside Rg1 in the obtained pure water solution is 0.12%-0.32%; preferably, the mass concentration of ginsenoside Rg1 is 0.12%, 0.16%, or 0.24%.

[0020] In a preferred embodiment of the present invention, the amount of anhydrous ethanol by volume is 80-87 mL / g compared to the total mass of cholesterol and phospholipids.

[0021] In a preferred embodiment of the present invention, the dripping is performed by using a syringe at a speed of 0.8 mL per second; the speed of the continuous stirring is 800 r / min; the heating temperature is 40°C; and the heating time and the continuous stirring time are both 1 hour.

[0022] Furthermore, the continuous stirring device is a digital display constant temperature magnetic stirrer.

[0023] In a preferred embodiment of the present invention, the ultrasonic time is 25 minutes and the ultrasonic power is 100W.

[0024] In a preferred embodiment of the present invention, after the ultrasonic treatment, filtration can be performed using a filter membrane to obtain the Acorus calamus oil-ginsenoside Rg1 liposome; the filter membrane includes a 0.22 μm microporous filter membrane.

[0025] The calamus oil-ginsenoside Rg1 liposome prepared by the invention is a nano drug-carrying system with light blue opalescence.

[0026] In addition, the present invention also provides a pharmaceutical preparation, which comprises the aforementioned Acorus calamus oil-ginsenoside Rg1 liposome.

[0027] In a preferred embodiment of the present invention, the pharmaceutical preparation is a nasal preparation, an oral preparation, an injectable preparation, a sustained-release preparation, or the like.

[0028] In addition, the present invention provides a use of Acorus calamus oil-ginsenoside Rg1 liposome in preparing a drug for preventing and / or treating Alzheimer's disease.

[0029] The beneficial effects of the present invention are as follows: the calamus oil-ginsenoside Rg1 liposomes and the preparation method thereof provided by the present invention, by rationally configuring the component contents of lecithin, calamus oil, ginsenoside Rg1 and cholesterol and optimizing the preparation process, successfully achieve the encapsulation of the hydrophilic drug Rg1 and the hydrophobic drug calamus volatile oil in the liposomes at the same time. The liposomes can directly act on the nasal cavity, be absorbed into the brain by the nasal mucosa, significantly improve the bioavailability and brain entry of calamus oil and ginsenoside Rg1, and have good brain targeting; compared with a single solution, it has a better sustained release effect. Moreover, the calamus oil-ginsenoside Rg1 liposomes prepared by the present invention show good therapeutic effects in the AD cell model constructed by PC12 cells.

[0030] Compared with existing technologies:

[0031] 1. The liposomes prepared by the method of the present invention improve the dispersibility and stability of the Acorus calamus oil-ginsenoside Rg1 liposomes, and the particle size of the liposomes is less than 100 nm, which can be better absorbed by the nasal mucosa, thereby increasing the content entering the brain and having good brain targeting.

[0032] 2. The encapsulation rate of the Acorus calamus oil-ginsenoside Rg1 liposome prepared by the present invention is higher than that of the existing ginsenoside Rg1 liposome (Zhou Hongwei et al. Optimization of the preparation process of ginsenoside Rg1 liposome by central composite design-response surface methodology [J]. Information on Traditional Chinese Medicine, 2011, 28(04): 38-41.), thereby improving the utilization rate of raw materials in the preparation process. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The particle size distribution and potential diagram of the Acorus calamus oil-ginsenoside Rg1 liposome of the present invention, wherein: Figure 1 A is the particle size distribution diagram, Figure 1 B is the potential diagram;

[0034] Figure 2 This is a transmission electron microscopy (TEM) image of the Acorus calamus oil-ginsenoside Rg1 liposome of the present invention;

[0035] Figure 3 The in vitro release curve of the Acorus calamus oil-ginsenoside Rg1 liposome of the present invention;

[0036] Figure 4 The fluorescence uptake diagram of Acorus calamus oil-ginsenoside Rg1 liposomes using rhodamine B as a fluorescent probe in 16HBE and PC12 cells, where: Figure 4 A is the result of 16HBE cell uptake for 1 h; Figure 4 B is the result of PC12 cell uptake for 1 h; Figure 4 C is the result of 16HBE cell uptake for 2 h; Figure 4 B is the result of PC12 cell uptake for 2 h;

[0037] Figure 5 The effect of Acorus calamus oil-ginsenoside Rg1 liposome on the levels of inflammatory factors and oxidative factors, among which, Figure 5 A is the effect on ROS; Figure 5 B is the effect on SOD; Figure 5 C is the effect on MDA; Figure 5 D is the effect on TNF-α; Figure 5 E is the effect on IL-β; Figure 5 F is the effect on IL-18 expression level;

[0038] Figure 6 It is the content of brain tissue at different time points after nasal administration of Acorus calamus oil-ginsenoside Rg1 liposome;

[0039] Figure 7 This is the appearance of the Acorus calamus oil-ginsenoside Rg1 liposome prepared by the present invention. DETAILED DESCRIPTION

[0040] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] Example 1 Single factor experiment to investigate liposome preparation conditions

[0042] In this example, the liposomes of Acorus calamus oil-ginsenoside Rg1 were prepared by ethanol injection method and were freshly prepared before the experiment. The preparation steps are as follows:

[0043] 1) firstly weighing lecithin, cholesterol and calamus oil according to the formula amount and dissolving them in the formula amount of anhydrous ethanol to obtain an anhydrous ethanol solution containing calamus oil;

[0044] 2) Place the ultrapure water in the formula amount in a beaker, heat it to 40°C on a magnetic stirrer, maintain the magnetic stirring speed at 800r / min, slowly drip the lipid material and the anhydrous ethanol solution of the drug into the aqueous phase with a syringe, continue stirring and keep heating for 1h, pay attention to observe the complete evaporation of anhydrous ethanol to obtain a suspension;

[0045] 3) After ultrasonicating the suspension for 25 minutes, the Acorus calamus oil-ginsenoside Rg1 liposomes were obtained, and the liposomes were transferred to a 10 mL centrifuge tube for storage.

[0046] The same method was used in anhydrous ethanol without adding calamus oil to obtain ginsenoside Rg1 liposomes; the same method was used in ultrapure water without adding ginsenoside Rg1 to obtain calamus oil liposomes.

[0047] During the liposome preparation process, the shear stirring temperature, rotation speed, ultrasonic time and dosage were investigated separately. Among them, the membrane material ratio refers to the mass ratio of cholesterol to phospholipid. The results are shown in Table 1:

[0048] Table 1 Average particle size and PDI of Acorus calamus oil-ginsenoside Rg1 liposomes at different factor levels

[0049]

[0050]

[0051] Based on the results of the investigation, the nanoliposomes of the present invention can be prepared under various parameters, and the amount of raw material calamus oil, the amount of ginsenoside Rg1 and the membrane material ratio (the mass ratio of cholesterol to phospholipid) are used as factors for response surface optimization. Other optimal conditions are: stirring temperature of 40°C, dripping rate of 0.8mL / min, rotation speed of 1000r / min, and ultrasonic time of 25min.

[0052] Example 2 Response surface optimization experiment to investigate liposome composition

[0053] In this example, the liposomes of Acorus calamus oil-ginsenoside Rg1 were prepared by ethanol injection method and were freshly prepared before the experiment. The preparation steps are as follows:

[0054] 1) First, weigh lecithin, cholesterol, and Acorus calamus oil according to the formula amount and dissolve them in anhydrous ethanol in the formula amount to obtain an anhydrous ethanol solution;

[0055] 2) Place the ultrapure water in a beaker, heat it to 40°C on a magnetic stirrer, maintain the magnetic stirring speed at 1000r / min, slowly drip the lipid material and the anhydrous ethanol solution of the drug into the aqueous phase with a syringe, continue stirring and keep heating for 1h, pay attention to observe the complete evaporation of anhydrous ethanol to obtain a suspension;

[0056] 3) After ultrasonicating the suspension for 25 minutes, the Acorus calamus oil-ginsenoside Rg1 liposomes were obtained, and the liposomes were transferred to a 10 mL centrifuge tube for storage.

[0057] Based on the results of single-factor experiments, the formulation was optimized to obtain stable Acorus calamus oil-ginsenoside Rg1 liposomes with a particle size less than 100 nm. The particle size, PDI and encapsulation efficiency were used as evaluation indicators, and the ratio of phospholipids and cholesterol, and the amount of Acorus calamus oil and ginsenoside Rg1 added were used as response surface optimization factors. The results are shown in Table 2.

[0058] Example 3 Encapsulation efficiency determination

[0059] The encapsulation efficiency of the Acorus calamus oil-ginsenoside Rg1 liposome prepared according to the formula and dosage given by the Design Expert software in Example 2 was measured. An appropriate amount of Acorus calamus oil-ginsenoside Rg1 liposome was added to a centrifuge tube, centrifuged at 13000r / min for 30min, and the supernatant was taken for Rg1 liquid phase test analysis to obtain the free Rg1 content (W1); at the same time, an appropriate amount of Acorus calamus oil-ginsenoside Rg1 liposome was measured and placed in the inner tube of an ultrafiltration centrifuge tube (100KD), centrifuged at 4 000rpm for 10min, and the obtained filtrate was further centrifuged at 12 000rpm for 10min, and the supernatant was taken for liquid phase analysis to obtain the free Acorus calamus oil content (W2). Another appropriate amount of Acorus calamus oil-ginsenoside Rg1 liposome solution was measured, and an appropriate amount of methanol was added for ultrasonic demulsification. After centrifugation, liquid phase testing and analysis were performed to obtain the total Acorus calamus oil content (W3) and total Rg1 content (W4) in the Acorus calamus oil-ginsenoside Rg1 liposome.

[0060] The encapsulation efficiency (EE%) is calculated as follows:

[0061] EE Rg1 (%) = (W4-W1) / W4×100%

[0062] EE VOA (%) = (W3-W2) / W3×100%

[0063] Among them, EE Rg1 : Rg1 encapsulation efficiency, EE VOA : Encapsulation efficiency of Acorus calamus oil, W1: content of free Rg1; W2: content of free Acorus calamus oil; W3: content of total Acorus calamus oil in Acorus calamus oil-ginsenoside Rg1 liposomes; W4: content of total Rg1 in Acorus calamus oil-ginsenoside Rg1 liposomes.

[0064] The experimental design and results are shown in Table 2:

[0065] Table 2 Experimental design and results

[0066]

[0067] Example 4 Characterization of Acorus calamus oil-ginsenoside Rg1 liposomes

[0068] The Acorus calamus oil-ginsenoside Rg1 liposome prepared by the present invention was measured by a laser particle size analyzer. The results are as follows: Figure 1 As shown in A and B, respectively, the nanoemulsion prepared according to Example 2 has an average particle size of 95.13 nm, a polydispersity index (PDI) of 0.227, and a Zeta potential of -16.50 mV.

[0069] The morphology of nanoemulsion was observed by transmission electron microscopy (TEM). Figure 2 As shown, the obtained liposomes are regular spherical particles, the particles are evenly distributed, and there is no obvious adhesion between them.

[0070] Example 5 In vitro release of Acorus calamus oil-ginsenoside Rg1 lipid

[0071] The following is an in vitro release test of the effective ingredient ginsenoside Rg1 with a high content in the Acorus calamus oil-ginsenoside Rg1 liposome prepared by the present invention, and the above test results are compared with the in vitro release rate of the Acorus calamus oil-ginsenoside Rg1 mixture. The specific test method is as follows:

[0072] Appropriate amounts of calamus oil-ginsenoside Rg1 liposomes and an equal amount of Rg1 and calamus oil mixed solution were measured and placed in pretreated dialysis bags, each placed in 200 mL of artificial nasal fluid dissolution medium, and released in vitro at 37°C and 100 rpm / min. 0.5 mL of dialysate was taken at different time points, and an equal volume of new release medium was added at the same time. The release solution was filtered with a 0.22 μm microporous membrane and the content of ginsenoside Rg1 was determined by ultra-performance liquid chromatography (UPLC). The test results are shown in Figure 3 as shown in .

[0073] from Figure 3 It can be seen that when ginsenoside Rg1 reaches the same cumulative release rate, the release time of ginsenoside Rg1 in the Acorus calamus oil-ginsenoside Rg1 liposome prepared by the present invention is significantly prolonged. In addition, within the same release time, the cumulative release rate of the prepared Acorus calamus oil-ginsenoside Rg1 liposome is significantly lower than that of the mixed solution of Rg1 and Acorus calamus oil. The above results show that Acorus calamus oil-ginsenoside Rg1 liposome has a better sustained release effect.

[0074] Example 6 Preparation of Acorus calamus oil-ginsenoside Rg1 liposomes loaded with rhodamine B fluorescent probe

[0075] Liposomes were prepared according to the method of Example 2, wherein the membrane material ratio was 6:1, the amount of calamus oil added was 16 μL, and the amount of ginsenoside Rg1 added was 24 mg. A certain amount of rhodamine B was added to the aqueous phase to prepare calamus oil-ginsenoside Rg1 liposomes and ginsenoside Rg1 liposomes loaded with fluorescent probes.

[0076] Example 7 Cellular Uptake of Acorus Calamus Oil-Ginsenoside Rg1 Liposomes

[0077] (1) Grouping and drug administration

[0078] Human bronchial epithelial cells (16HBE) and rat adrenal pheochromocytoma cells (PC12) in logarithmic growth phase were inoculated in 12-well plates, and the experiments were divided into normal control group, rhodamine B aqueous solution group, ginsenoside Rg1 liposome group containing rhodamine B, and calamus oil-ginsenoside Rg1 liposome group containing rhodamine B. After the cells adhered to the wall, the corresponding drug solution was added, and after incubation for 1h and 2h, they were washed three times with cold PBS, fixed with 4% paraformaldehyde for 15min, then washed with PBS, added with DAPI dye solution and incubated for 20min, and finally washed with PBS and placed under a fluorescence inverted microscope to observe the fluorescence of rhodamine B in each group of cells.

[0079] (2) Cellular uptake results

[0080] Microscopic observation revealed that free rhodamine B could hardly enter 16HBE cells, while liposome-encapsulated rhodamine B could be taken up by cells, and the amount of uptake increased with time, but was lower than that of VOA-Rg1-LP liposomes containing Acorus calamus oil. The uptake results of PC12 cells were basically consistent with those of 16HBE cells. Figure 4 shown. Figure 4 Middle: Rhodamine B solution: Rhodamine B, Acorus calamus oil-ginsenoside Rg1 liposomes with Rhodamine B as probe: Rhodamine B-VOA-Rg1-LP, Ginsenoside Rg1 liposomes with Rhodamine B as probe: Rhodamine B-Rg1-LP.

[0081] It can be seen from this that VOA-Rg1-LP liposomes can be absorbed by nasal mucosal cells and enter the brain, and Acorus calamus oil can effectively promote ginsenoside Rg1 liposomes to pass through the nasal mucosa, thereby increasing the amount entering the brain.

[0082] Example 8 Pharmacodynamic effects of Acorus calamus oil-ginsenoside Rg1 liposomes on AD model cells

[0083] (1) Aβ 25-35 Inducing PC12 cells to construct an in vitro AD cell model

[0084] PC12 cells in the logarithmic growth phase were inoculated in 6-well plates and cultured. After the cells adhered to the wall, 20 μM Aβ 25-35 The solution was incubated for 4 h to construct an in vitro AD cell model.

[0085] (2) Grouping and drug administration

[0086] The experiment was divided into normal group, model group, ginsenoside Rg1-Acorus calamus oil mixture group, ginsenoside Rg1 solution group, Acorus calamus oil solution group, ginsenoside Rg1 liposome group, Acorus calamus oil liposome group and Acorus calamus oil-ginsenoside Rg1 liposome group. After the corresponding drugs were incubated with cells for 24 hours, the cell supernatant and cells were collected, and the levels of reactive oxygen species (ROS), malondialdehyde (MDA), interleukin-18 (IL-18), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and superoxide dismutase (SOD) in the cells were detected by ELISA kits.

[0087] (3) ELISA test results

[0088] ELISA test results showed that compared with the normal group, the levels of ROS, MDA, IL-18, TNF-α and IL-1β in the model group were significantly increased, and the level of SOD was significantly decreased, indicating that Aβ 25-35 The AD model was successfully established by induction. Compared with the model group, the levels of ROS, MDA, IL-18, TNF-α and IL-1β in each drug group were reduced, and the SOD level showed an upward trend. The therapeutic effect of the Acorus calamus oil-ginsenoside Rg1 liposome group was significantly better than that of the free drug group, Acorus calamus oil liposome group and ginsenoside Rg1 liposome group. Figure 5 shown. Figure 5 In the experiment, control group: Control, AD model group: Model, physical mixture of Acorus calamus oil and ginsenoside Rg1: Physical mixture, Acorus calamus oil-ginsenoside Rg1 liposomes: VOA-Rg1-LP, ginsenoside Rg1 liposomes: Rg1-LP, Acorus calamus oil liposomes: VOA-LP.

[0089] The Jin Zhengjun Q value calculation method was used to evaluate the drug combination effect in the Acorus calamus oil-ginsenoside Rg1 liposomes. The formula is Q = Ea + b / (Ea + Eb-Ea × Eb), where Ea + b is the level of biochemical indicators in the combined drug treatment group, and Ea and Eb are the levels of biochemical indicators when drug a and drug b are treated separately. If the Q value is 0.85-1.15, it means that the two drugs are simply added together, if Q>1.15, it means that the two drugs have a synergistic effect, and if Q<0.85, it means that the two drugs have an antagonistic effect. The results of the calculation of the Jin Zhengjun formula for the Acorus calamus oil-ginsenoside Rg1 liposomes are shown in Table 3. It can be seen that the Acorus calamus oil-ginsenoside Rg1 liposomes have a good therapeutic effect on AD, and Acorus calamus oil and ginsenoside Rg1 have a synergistic effect.

[0090] Table 3 Levels of inflammatory factors and oxidative factors in each group after drug treatment

[0091]

[0092]

[0093] Example 9 Study on the distribution of Acorus calamus oil-ginsenoside Rg1 liposomes in rat brain

[0094] The Acorus calamus oil-ginsenoside Rg1 liposomes prepared in Example 2 were injected into SD rats via the nasal cavity and tail vein, respectively, and their distribution in the brain of SD rats was observed.

[0095] (1) Experimental animal grouping

[0096] A total of 84 SD rats were randomly divided into intranasal administration (in.) group of Acorus calamus oil-ginsenoside Rg1 liposome, intranasal administration group of ginsenoside Rg1 liposome, intranasal administration group of Acorus calamus oil-ginsenoside Rg1 mixture, and intravenous injection (iv.) group of Acorus calamus oil-ginsenoside Rg1 liposome, with 21 rats in each group.

[0097] (2) Dosage method and sample collection

[0098] The corresponding drugs were administered to SD rats in each group through the nasal cavity or tail vein, and the dosage of ginsenoside Rg1 in each group of preparations was ensured to be 1 mg / kg. The rats were anesthetized with sodium pentobarbital at 5, 15, 30, 60, 120, 240, and 480 minutes after administration, and the brain tissues of each group were separated after being killed and stored at -80°C for testing. First, 0.3 g of brain tissue of rats in each group was accurately weighed and added to 1 mL of normal saline, and homogenized with a high-speed tissue homogenizer to obtain brain tissue homogenate. Accurately aspirate 90 μL of the homogenate, vortex for 3 minutes, add 1 mL of methanol, vortex for 5 minutes, and centrifuge at 3500 rpm in a low-speed centrifuge for 10 minutes. Take the supernatant and blow dry it in a nitrogen blower, add 100 μL of methanol to dissolve it, vortex for 5 minutes, centrifuge it at 12000 rpm in a high-speed centrifuge for 10 minutes, transfer the supernatant into a liquid phase injection vial, and use UPLC for content detection.

[0099] (3) Results of brain distribution measurement

[0100] Test results such as Figure 6As shown, the Cmax in the brain tissue of the Acorus calamus oil-ginsenoside Rg1 mixture group was 0.08 μg / g, the Cmax in the ginsenoside Rg1 liposome group was 1.05 μg / g, the Cmax in the Acorus calamus oil-ginsenoside Rg1 liposome nasal administration group was 1.17 μg / g, and the Cmax in the Acorus calamus oil-ginsenoside Rg1 liposome tail vein injection group was 0.74 μg / g. It can be seen that the nasal administration of Acorus calamus oil-ginsenoside Rg1 liposomes effectively promoted the entry of the drug ginsenoside Rg1 into the brain. Figure 6 In the trial, the Acorus calamus oil-ginsenoside Rg1 liposome nasal administration group was VOA-Rg1-LP in., the ginsenoside Rg1 liposome nasal administration group was Rg1-LPin., the Acorus calamus oil-ginsenoside Rg1 mixture nasal administration group was Mixture in., and the Acorus calamus oil-ginsenoside Rg1 liposome tail vein injection group was VOA-Rg1-LP iv.

[0101] The calamus oil-ginsenoside Rg1 liposome prepared by the present invention has a light blue opalescence. Figure 7 shown.

[0102] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A calamus oil-ginsenoside Rg1 liposome, characterized in that: The liposome is prepared from the following raw materials: cholesterol, phospholipids, calamus oil and ginsenoside Rg1.

2. The Acorus calamus oil-ginsenoside Rg1 liposome according to claim 1, characterized in that: In terms of mass ratio, cholesterol:phospholipid = 1:4-1:

7.

3. The Acorus calamus oil-ginsenoside Rg1 liposome according to claim 1, characterized in that: Calculated by the mass of ginsenoside Rg1 and the volume of Acorus calamus oil, ginsenoside Rg1:Acorus calamus oil=1 g:(0.375-2) mL.

4. The Acorus calamus oil-ginsenoside Rg1 liposome according to claim 1, characterized in that: Based on the total mass of cholesterol and phospholipids and the total mass of Acorus calamus oil and ginsenoside Rg1, m (胆固醇与磷脂) :m (石菖蒲油与人参皂苷Rg1) =1:1-1:

7.

5. The Acorus calamus oil-ginsenoside Rg1 liposome according to claim 1, characterized in that: The phospholipid is one or more of hydrogenated soybean lecithin, egg yolk lecithin, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine, and distearoylphosphatidylcholine.

6. The method for preparing the Acorus calamus oil-ginsenoside Rg1 liposome according to any one of claims 1 to 5, characterized in that: The process steps include: Dissolve cholesterol, phospholipids and Acorus calamus oil in anhydrous ethanol to obtain an anhydrous ethanol solution; Dissolve ginsenoside Rg1 in ultrapure water, dropwise add the anhydrous ethanol solution into the ultrapure water containing ginsenoside Rg1, evaporate the anhydrous ethanol, and obtain a suspension; The suspension is subjected to ultrasonic treatment to obtain the Acorus calamus oil-ginsenoside Rg1 liposome.

7. The preparation method according to claim 6, characterized in that: Compared to the total mass of cholesterol and phospholipids, the amount of anhydrous ethanol used by volume is 80-87 mL / g.

8. The preparation method according to claim 6, characterized in that: The ginsenoside Rg1 is dissolved in ultrapure water, and the mass concentration of the ginsenoside Rg1 in the obtained pure water solution is 0.12% to 0.32%.

9. A pharmaceutical preparation, characterized in that The pharmaceutical preparation comprises the Acorus calamus oil-ginsenoside Rg1 liposome according to any one of claims 1 to 5.

10. Use of the Acorus calamus oil-ginsenoside Rg1 liposome according to any one of claims 1 to 5 in the preparation of a medicament for preventing and / or treating Alzheimer's disease.

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