Bilobalide nano-emulsion as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicine, and particularly relates to bilobalide nano-emulsion and a preparation method and application thereof.The bilobalide nano-emulsion is prepared from, by mass, 0.1%-0.5% of bilobalide, 0.1%-1% of an oil phase, 1%-10% of an emulsifier, 1%-10% of a co-emulsifier and 78.5%-97.8% of a water phase, the nano-emulsion is used for preparing medicines for treating neurodegenerative diseases and cerebrovascular dementia, and solves the problems that bilobalide is poor in permeability and low in distribution in the brain due to drug-resistant protein mediated efflux.

Description

Technical Field

[0001] The invention belongs to the technical field of medicines, and in particular relates to a bilobalide nanoemulsion and a preparation method and application thereof. Background Art

[0002] Alzheimer's disease (AD) is the most common type of dementia and the third leading cause of death among the elderly. There are many drugs used to treat AD, such as donepezil and memantine. Compared with synthetic drugs, natural products have the characteristics of lower toxicity and fewer side effects. Bilobalide (BB) is the only sesquiterpene in Ginkgo biloba extract. BB can improve the pathological process and development of AD through multiple mechanisms. The specific molecular mechanisms include reducing the levels of Aβ40, Aβ42, TNF-α, IL-1β, IL-6 and p-STAT3 in brain tissue, and increasing the levels of synaptic proteins PSD95, Synapsin-1 and GluR1 (Translational psychiatry, 11(1):542, 2021); reducing the neuronal damage and apoptosis induced by Aβ25-35 in the frontal cortex and hippocampal CA1 region of rats (Pharmacology biochemistry and behavior, 106:77-84, 2013); reducing the production of Aβ and sAPPβ by participating in the PI3K pathway, inhibiting the activity of GSK3 by acting on the PI3K-GSK3β pathway, reducing the activity of cathepsin B and reducing the generation of Aβ (Neurochemical Research, 37(2): 298-306, 2012); enhancing the phosphorylation levels of BDNF and CREB in the mouse hippocampus, improving the proliferation and dendritic strength of hippocampal neuronal precursor cells (Journal of Alzheimer's disease, 18(4): 787-798, 2009), etc.

[0003] The preparation and application of bilobalide preparations currently provided in clinical practice include Chinese patent CN202110717103.X, which can effectively treat autism in children caused by hypoxic-ischemic brain damage by rationally selecting drug raw materials and scientifically proportioning them. In addition, Chinese patent CN109432117A discloses an external medicine for promoting postoperative skin wound healing and its use. The patented medicine has the effect of promoting the growth and healing of incision skin on postoperative skin wound healing, which is beneficial to the patient's postoperative recovery.

[0004] Chinese patent CN200510046141.8 discloses a composition containing ginkgolide A, ginkgolide B, ginkgolide C and bilobalide and its use in preparing drugs for treating Alzheimer's disease. Chinese patent CN201410227687.2 discloses a composition composed of bilobalide and any one or two of ginkgolide A and C and its use in preparing drugs for improving memory impairment, and its specific application includes preparing drugs for treating Alzheimer's disease or cerebrovascular dementia. However, current research involves multiple components and can only be achieved through multiple mechanisms. Since bilobalide belongs to Class III drugs (BCSⅢ) in biopharmaceutics, it has poor permeability and is difficult to penetrate the cell plasma membrane (Molecularpharmaceutics, 9(4):815-822, 2012, and bilobalide is also susceptible to efflux mediated by multidrug resistance protein 1 (MDR1) in the brain, resulting in a lower distribution of bilobalide in the brain (Journal of of Ethnopharmacology, 243:112098, 2019.), these reasons have greatly limited the development and utilization of BB preparations, so to date, no one has reported the therapeutic effect of bilobalide monotherapy on Alzheimer's disease (AD). Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a bilobalide nanoemulsion and a preparation method and application thereof.

[0006] This is achieved specifically through the following technical solutions:

[0007] The first purpose of the present invention provides: a bilobalide nanoemulsion, which is composed of the following components by mass percentage: 0.1%-0.5% bilobalide, 0.1%-1% oil phase, 1%-10% emulsifier, 1%-10% co-emulsifier, and 78.5%-97.8% water phase.

[0008] The oil phase is medium chain triglycerides.

[0009] The emulsifier is polysorbate 80 or polysorbate 20.

[0010] The co-emulsifier is polyethylene glycol.

[0011] Specifically, the bilobalide nanoemulsion consists of the following components by mass percentage: 0.1-0.5% bilobalide, 0.1-1% medium chain triglyceride, 0.5-5% polysorbate 80, 0.5-5% polysorbate 20, 1-10% polyethylene glycol 400, and 78.5-97.8% distilled water.

[0012] More specifically, the bilobalide nanoemulsion consists of the following ingredients by mass percentage: 0.2-0.4% bilobalide, 0.4-1% medium chain triglyceride, 1-4% polysorbate 80, 1-4% polysorbate 20, 5-10% polyethylene glycol 400 and 80.6-92.4% distilled water.

[0013] More specifically, the bilobalide nanoemulsion consists of the following components by mass percentage: 0.23% bilobalide, 0.46% medium chain triglyceride, 2.08% polysorbate 80, 2.08% polysorbate 20, 7.27% polyethylene glycol 400, and 87.88% distilled water.

[0014] In the present invention, bilobalide can reduce the damage of brain neurons in Alzheimer's disease, has anti-inflammatory and blood-brain barrier permeability regulating effects. Medium-chain triglycerides are oil phases, which have good solubility and can improve the loading rate and stability of drugs. Polysorbate 80 is an emulsifier used to promote the absorption and penetration of drugs. Polysorbate 20 is an emulsifier used to promote the absorption and penetration of drugs. When used in combination with polysorbate 80, the stability and emulsification effect of the emulsifier can be improved. Polyethylene glycol 400 is an auxiliary emulsifier used to increase the solubility of the emulsifier, reduce interfacial tension, and adjust the HLB value of the emulsifier. Distilled water is the water phase, which forms an interfacial film with the oil phase to encapsulate the drug under the synergistic effect of the emulsifier and the auxiliary emulsifier.

[0015] The second object of the present invention provides: a method for preparing the bilobalide nanoemulsion, comprising the following steps:

[0016] (1) mixing bilobalide, medium chain triglycerides, polysorbate 80, polysorbate 20, and polyethylene glycol 400, and dispersing them by ultrasonication at a temperature of 20-35° C.;

[0017] (2) adding distilled water to the mixture prepared in step (1), continuing ultrasonic dispersion, and finally filtering through a 0.22 μm filter head to obtain a nanoemulsion.

[0018] The third object of the present invention provides: the use of bilobalide in the preparation of drugs for treating neurodegenerative diseases and cerebrovascular dementia.

[0019] The medicine comprises bilobalide and a pharmaceutically acceptable auxiliary material or carrier.

[0020] The dosage form of the drug is an acceptable dosage form prepared by conventional pharmaceutical techniques, such as tablets, injections, oral solutions, and nanoemulsions.

[0021] Preferably, the drug is in the form of a nanoemulsion.

[0022] Specifically, the bilobalide nanoemulsion is used in the preparation of drugs for treating neurodegenerative diseases and cerebrovascular dementia.

[0023] The neurodegenerative diseases include Alzheimer's disease and Parkinson's disease.

[0024] The cerebrovascular dementia includes stroke and vascular dementia.

[0025] More specifically, the use of the bilobalide nanoemulsion in the preparation of drugs for treating Alzheimer's disease, Parkinson's disease, stroke, and vascular dementia

[0026] The bilobalide nanoemulsion can be prepared according to the method of the present invention, or according to a conventional production method in the pharmaceutical field.

[0027] Beneficial effects:

[0028] The biloba lactone nanoemulsion of the present invention belongs to a traditional Chinese medicine preparation, has obvious therapeutic effect, small side effects, and a wide range of indications, has the effects of reducing hippocampal neuron damage, reducing malondialdehyde content, inhibiting oxidative stress, increasing superoxide dismutase levels, reducing myeloperoxidase levels, increasing superoxide dismutase activity, improving spatial memory and spatial exploration ability of mice, and has a good protective effect on hippocampal damage caused by Alzheimer's disease.

[0029] The invention scientifically controls the dosage ratio of bilobalide, medium-chain triglyceride, polysorbate 80, polysorbate 20, polyethylene glycol 400 and distilled water, thereby solving the problem of low distribution of bilobalide in the brain due to poor permeability and susceptibility to efflux mediated by drug-resistant proteins.

[0030] The production method of the invention is simple and convenient, and can be prepared into pharmaceutically acceptable dosage forms, such as nanoemulsion, tablet, injection and oral solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Effects of different drugs on the total number of arms entered and the percentage of spontaneous alternation in AlCl3-induced AD mice (mean ± standard deviation); A: total number of mice entering the arms; B: percentage of spontaneous alternation;

[0032] Figure 2 The results of hematoxylin-eosin (HE) staining of mouse hippocampus; A: Group I; B: Group II; C: Group III; D: Group IV; E: Group V; F: Group VI;

[0033] Figure 3Determination of related indicators of mouse brain supernatant (mean ± standard deviation); A: superoxide dismutase activity; B: malondialdehyde content; C: nitric oxide content; D: myeloperoxidase activity; E: acetylcholinesterase activity; F: glutathione peroxidase activity;

[0034] Figure 4 It is a pseudo-ternary phase diagram of the emulsifier; wherein A: diethylene glycol monoethyl ether; B: polyethylene glycol 400; C: glycerol; D: caprylic acid capric acid polyethylene glycol glyceride;

[0035] Figure 5 The results of the influence of various factors on particle size and polydispersity index; A: bilobalide dosage (%); B: ultrasonic time (min); C: ultrasonic power (W); D: oil phase-mixed emulsifier ratio (%); E: mixed emulsifier-co-emulsifier ratio; F: water phase dosage (%);

[0036] Figure 6 : Response surface diagrams and contour diagrams of each factor. A: AB interaction response surface diagram; B: AC interaction response surface diagram; C: BC interaction response surface diagram; D: AB interaction contour diagram; E: AC interaction contour diagram; F: BC interaction contour diagram;

[0037] Figure 7 This is the cumulative release of bilobalide from bilobalide nanoemulsion under different pH conditions. DETAILED DESCRIPTION

[0038] The specific embodiments of the present invention are further described in detail below, but the present invention is not limited to these embodiments, and any improvement or substitution based on the basic spirit of the present embodiment still falls within the scope of protection required by the claims of the present invention.

[0039] Example 1

[0040] A method for preparing bilobalide nanoemulsion comprises the following steps:

[0041] 1. Weigh the following drugs: bilobalide 0.05 g, medium chain triglyceride 0.1 g, polysorbate 80 0.45 g, polysorbate 200.45 g, polyethylene glycol 400 1.57 g, and distilled water 18.99 g.

[0042] 2. Take bilobalide, medium-chain triglycerides, polysorbate 80, polysorbate 20, and polyethylene glycol 400, mix them, and ultrasonicate them at 25°C for 10 minutes.

[0043] 3. Add distilled water to the mixture prepared in step (2), continue ultrasonication for 20 minutes, and filter through a 0.22 μm filter to obtain a nanoemulsion.

[0044] Experimental Example 1

[0045] 1. Materials

[0046] 1.1 Drug bilobalide (provided by the Biochemical Engineering Center of Guizhou University)

[0047] 1.2 Animals: Kunming white mice, purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd.

[0048] 2. Methods and Results

[0049] Effects on AlCl3-induced AD mice

[0050] 48 mice, weighing 17-19g, were taken and divided into 6 groups on average: After the mice adapted for 5 days, all mice were randomly divided into 6 groups, namely Groups Ⅰ-Ⅵ. Group Ⅰ was the normal group and did not receive any treatment. Group Ⅱ was the model group, and the animals were gavaged with 170mg / kg / day of AlCl3 solution every day for 15 days. Group Ⅲ was the positive drug group, which was the same as Group Ⅰ. On this basis, 0.65mg / kg / day of donepezil was gavaged every half an hour for 15 days. Group Ⅳ was the bilobalide group, which was the same as Group Ⅰ. On this basis, 10mg / kg / day of bilobalide was gavaged every half an hour for 15 days. Group Ⅴ was the bilobalide nanoemulsion low-dose group, which was the same as Group Ⅰ. On this basis, 5mg / kg / day of nanoemulsion was gavaged every half an hour. Group VI was the high-dose group of bilobalide nanoemulsion, which was the same as Group I. On this basis, the mice were gavaged with 10 mg / kg / day of nanoemulsion every half an hour (as shown in Table 1).

[0051] A 5-day Y-maze experiment was conducted from day 10 to day 15. During the experiment, mice were placed at the end of an arm and allowed to explore freely for 5 minutes. Each mouse was trained once a day, and the number and order of the mice entering different arms were recorded. When the mice entered different arms three times in succession, it was recorded as a spontaneous alternation. The number of spontaneous alternations within 5 minutes was counted, and the percentage of spontaneous alternation (%) was used to evaluate the spatial exploration ability and memory ability of the mice.

[0052] Spontaneous alternation percentage (%) = spontaneous alternation times / (total arm entry times - 2)*100%

[0053] Table 1. Grouping of mice

[0054]

[0055]

[0056] Depend on Figure 1It can be seen that there was no significant difference in the total number of arm insertions between the model group and each treatment group compared with the normal group. However, compared with the normal group, the percentage of spontaneous alternation in the model group was significantly decreased (P<0.001), and compared with the model group, the percentage of spontaneous alternation in each treatment group was significantly increased. Among them, the positive drug treatment group (P<0.001) and the high-dose group of bilobalide nanoemulsion (P<0.001) both significantly improved the spatial exploration and spatial memory abilities of mice. The above results indicate that: compared with the model group, both doses of the present invention can effectively improve the spatial memory and spatial exploration abilities of AD mice (P<0.001).

[0057] HE staining( Figure 2 ) results showed that compared with the normal group, neuron cell body shrinkage, alkaline staining and severe neuron damage could be observed in the model group; the damage in the bilobalide group and the low-dose group of bilobalide nanoemulsion was slightly reduced, while the damage in the high-dose group of bilobalide nanoemulsion was less severe; the positive drug treatment group was basically close to the normal group, with the least damage.

[0058] As can be seen from the above figure, compared with the normal control group, neuron damage was obvious in the model group, while compared with the model group, both doses of the present invention could effectively improve the damage of hippocampal neurons in mice.

[0059] As Figure 3 shown, compared with the normal group, the superoxide dismutase activity in the model group was decreased (P<0.001), the malondialdehyde content was increased (P<0.001), the nitric oxide content was increased (P<0.001), the myeloperoxidase content was increased (P<0.001), the glutathione peroxidase activity was decreased (P<0.001), and the acetylcholinesterase activity was increased (P<0.001); compared with the model group, the low-dose group and the high-dose group of bilobalide nanoemulsion could significantly increase the superoxide dismutase activity (P<0.001), increase the glutathione peroxidase activity (P<0.05), decrease the malondialdehyde content (P<0.001), decrease the nitric oxide content (0.001<P<0.01, P<0.001), and decrease the acetylcholinesterase activity (P<0.001).

[0060] As can be seen from the above figure, the mechanism of the neuroprotective effect of bilobalide nanoemulsion may be related to reducing neuron damage, increasing the superoxide dismutase level, increasing the glutathione peroxidase level, decreasing the oxidative stress level, decreasing the myeloperoxidase level and decreasing the acetylcholinesterase level. Therefore, the present invention has an obvious neuroprotective effect on aluminum trichloride chemically induced Alzheimer's disease mice.

[0061] Example 2

[0062] A method for preparing bilobalide nanoemulsion comprises the following steps:

[0063] 1. Weigh the following drugs: bilobalide 0.10 g, medium chain triglyceride 0.10 g, polysorbate 800.5 g, polysorbate 200.5 g, polyethylene glycol 4001.00 g, distilled water 97.8 g;

[0064] 2. Take bilobalide, medium-chain triglycerides, polysorbate 80, polysorbate 20, and polyethylene glycol 400, mix them, and ultrasonicate them at 35°C for 10 minutes.

[0065] 3. Add distilled water to the mixture prepared in step (2), continue ultrasonication for 20 minutes, and filter through a 0.22 μm filter to obtain a nanoemulsion.

[0066] Example 3

[0067] A method for preparing bilobalide nanoemulsion comprises the following steps:

[0068] 1. Weigh the following drugs: bilobalide 0.50 g, medium chain triglyceride 1.00 g, polysorbate 80 5.00 g, polysorbate 20 5.00 g, polyethylene glycol 400 10.00 g, distilled water 78.5 g;

[0069] 2. Take bilobalide, medium-chain triglycerides, polysorbate 80, polysorbate 20, and polyethylene glycol 400, mix them, and ultrasonicate them at 20°C for 10 minutes.

[0070] 3. Add distilled water to the mixture prepared in step (2), continue ultrasonication for 20 minutes, and filter through a 0.22 μm filter to obtain a nanoemulsion.

[0071] Experimental Example 2

[0072] 1. Experimental method: The oil phase was determined to be medium-chain triglycerides. Without the addition of an auxiliary emulsifier, the ratio of the oil phase to the emulsifier, i.e., 1:9, 2:8, 3:7, and 4:6, was used to evaluate the emulsification effect based on appearance and stability. The evaluation was divided into five levels: A>B>C>D>E. The higher the evaluation, the better the emulsification effect of the nanoemulsion (as shown in Table 1). Based on the emulsification effect, the emulsifier was determined to be a mixed emulsifier (Tween 80: Tween 40). In addition, the auxiliary emulsifier was determined to be polyethylene glycol 400 based on the pseudo-ternary phase diagram. Secondly, based on the preliminary experiment, the control variable method was used to examine the effects of different levels of BB dosage (%), ultrasonic power (W), ultrasonic time (min), oil phase-emulsifier ratio, emulsifier-auxiliary emulsifier system ratio, and water phase dosage (%) on the particle size and polydispersity index of the nanoemulsion. According to the single factor experimental results, SPSS ANOVA single factor variance analysis was used to determine the influence of different levels of each factor on the particle size and polydispersity index of nanoemulsion, and the factors with significant levels were selected for the next step of response surface optimization. According to the above results, based on the Box-Benhnken (BBD) experimental design principle, with ultrasonic power (W), mixed emulsifier-co-emulsifier ratio (%), and water phase dosage (%) as independent variables, particle size (Y1) and polydispersity index PDI (Y2) as response values, a 3-factor 3-level response surface analysis experiment was designed. The ANOVA variance analysis response surface results showed that when Y1 was the response value, the model fit was high (R2=0.9959) and the model was significant (P<0.0001), and the lack of fit item was not significant (P>0.05), indicating that the established model was reliable and had a good fit. In addition, when the polydispersity index was used as the response value, the model fit was poor compared with the particle size (R2=0.7180) and the model significance was low (P=0.0220), so the particle size (Y1) was determined as the response value.

[0073] 2. Results:

[0074] 2.1 Investigation results of oil phase, emulsifier and co-emulsifier

[0075] Table 1 Emulsifier evaluation table

[0076]

[0077] Table 2 Solubility of BB in oil phase (mean ± SD)

[0078]

[0079] Table 3 Compatibility results of Labrafac Lipophile WL 1349 and single emulsifier

[0080]

[0081]

[0082] Table 4 Investigation results of composite emulsifiers

[0083]

[0084]

[0085] As shown in Table 2, BB has the highest solubility in the oil phase MCT. The oil phase with higher solubility can improve the loading rate and stability of the drug, so MCT is selected as the oil phase of the nanoemulsion. The compatibility results of MCT and a single emulsifier are shown in Table 3. When MCT: Tween80 is 2:8 or MCT: Tween 20 is 1:9, the highest emulsification grade A can be achieved. Considering that the composite emulsifier has better emulsification effect and stability, the composite emulsifier was investigated. As shown in Table 4, when Tween 80: Tween 20 is 1:1, the emulsification is rapid, the solution is clear and transparent with a light blue opalescence, so Tween80: Tween 20 (1:1) is selected as the composite emulsifier of BB nanoemulsion.

[0086] Next, the choice of co-emulsifier was further determined. The pseudo-ternary phase diagram results are as follows Figure 4 As shown. The area size of the nanoemulsion formed by different co-emulsifiers is HP, when PEG400 is selected as the co-emulsifier, the emulsification area is the largest and the emulsification effect is the best. WL 1349 was used as the oil phase, Tween 80:Tween 20 (1:1) was used as a mixed emulsifier, and PEG400 was used as a co-emulsifier.

[0087] 2.2 Results of single factor investigation and central composite design-response surface methodology to optimize prescription

[0088] The particle size and polydispersity index were determined as follows: Figure 5 As shown, next, SPSS ANOVA variance analysis was used to determine the significance of the results. As shown in Table 5, the significance level of ultrasonic power, emulsifier-co-emulsifier ratio, and water phase dosage was high, that is, the effects on particle size and polydispersity index were significant (P<0.01), and the optimization was of great significance. Therefore, ultrasonic power, emulsifier-co-emulsifier ratio, and water phase dosage were used as three factors of the response surface to perform central point design-response surface method optimization to obtain the optimal prescription.

[0089] According to the results of the single factor investigation, the ultrasonic power, emulsifier-co-emulsifier ratio, and water phase dosage were used as independent variables of Box-Benhnken (BBD), and the particle size and polydispersity index were used as response values. The star point design-response surface method was used to optimize the prescription. The star point design factor level table is shown in Table 6, and the response surface experimental design and results are shown in Table 7. Design Expert13 was used to fit the response surface results. When the particle size was used as the response value, the quadratic regression equation obtained by fitting was: Y = -14415.54 + 2.11A + 4.70B + 334.13C + 0.00AB - 0.00AC - 0.04BC - 0.00A 2 -0.01B 2 -1.97C 2 As shown in Table 8, the model fit is relatively high (R 2 =0.9959) and the model was significant (P<0.0001), while the lack of fit term was not significant (P>0.05), indicating that the established model was reliable and had a good fit. In addition, when the polydispersity index was used as the response value, the model fit was poorer than the particle size (R 2 =0.7180) and the model significance was low (P=0.0220) (Table 9), so the particle size (Y1) was determined as the response value to predict the optimal prescription. The three-dimensional graph of the response surface and the two-dimensional graph of the contour line are shown in Figure 9. Figure 6 As shown, the results of pairwise interaction show that the contour lines of AB and BC are dense and elliptical at the edges, indicating that the ultrasonic power and the mixed emulsifier-emulsifier ratio, the mixed emulsifier-emulsifier ratio and the amount of water phase interact significantly, with P values ​​of 0.0236 and 0.0003, respectively, both less than 0.05; while the contour lines of AC are sparse at the edges, indicating that the interaction between ultrasonic power and the amount of water phase is not significant, with a P value greater than 0.05; as can be seen from the 3D graph, the response surfaces of AB and BC are steeper than AC, indicating that AB and BC interact significantly and have a significant effect on the particle size, which is consistent with the results of variance analysis.

[0090] Table 5 SPSS ANOVA variance analysis results

[0091]

[0092] Table 6 Star point design factor level table

[0093]

[0094]

[0095] Table 7 Response surface experimental design and results (mean ± standard deviation, n = 6)

[0096]

[0097] Table 8 Results of variance analysis of BBD design (with particle size as response value)

[0098]

[0099]

[0100] Table 9 Results of variance analysis of BBD design (with polydispersity index as response value)

[0101] source sum of squares Degrees of Freedom Mean Square F-number P-value Significance Model 0.0145 6 0.0024 4.24 0.0220 * A 0.0045 1 0.0045 7.86 0.0187 * B 0.0040 1 0.0040 7.09 0.0238 * C 0.0001 1 0.0001 0.1143 0.7423 # AB 0.0001 1 0.0001 0.1395 0.7166 # AC 0.0001 1 0.0001 0.2172 0.6511 # BC 0.0063 1 0.0063 11.00 0.0078 * Residual 0.0057 10 0.0006 Lack of Fit 0.0054 6 0.0009 11.17 0.0176 * Pure Error 0.0003 4 0.0001 Total deviation 0.0202 16

[0102] 2.3 Model Validation Results

[0103] Three batches of BB-NEs were prepared by the optimal recipe predicted by the BBD experiment. The measured particle sizes are shown in Table 10 , which are very close to the predicted particle size of 32.61 nm and RSE < ± 5 ( Table 10 ), indicating that the constructed model has good prediction and can be used for the optimization of the preparation process of BB-NEs.

[0104] Table 10 Verification experimental results (mean ± standard deviation, n = 3)

[0105] Experiment number Particle size (nm) RSE(%) 202405304 33.36±0.28 2.30 202405305 33.10±0.36 1.49 202405306 33.51±0.18 2.76

[0106] Experimental Example 3

[0107] 1. Experimental method: Accurately measure an appropriate amount of BB-NEs and place it in a treated dialysis bag. After all bubbles are exhausted, tie the two ends tightly. Place the dialysis bag in 100 mL of pH 1.2 hydrochloric acid buffer (simulated gastric acid) and pH 6.8 and pH 7.4 phosphate buffers. Oscillate in a water bath at 100 r / min at 37°C. Take samples at 0 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 24 h, and 30 h, respectively, inject the samples into a high performance liquid chromatography (HPLC), record the peak area, calculate the cumulative release, and draw the cumulative release curve.

[0108] 2. Results:

[0109] Figure 7 The cumulative release of bilobalide from bilobalide nanoemulsion under different pH conditions; under pH=1.2, the drug release rate is very fast, and the release amount reaches about 90% in about 5 hours, and then tends to be stable. Under pH=6.8 and pH=7.4, the drug release rate is very slow, and the release amount is less than 10% in 30 hours. This shows that the drug is released faster in an acidic environment (pH=1.2), and slower in a neutral or slightly alkaline environment (pH=6.8 and pH=7.4). This release characteristic may be related to the chemical properties of the drug, the formulation form, and its solubility under different pH conditions.

Claims

1. A bilobalide nanoemulsion, characterized in that: The invention is composed of the following components by mass percentage: 0.1%-0.5% of bilobalide, 0.1%-1% of oil phase, 1%-10% of emulsifier, 1%-10% of auxiliary emulsifier and 78.5%-97.8% of water phase.

2. A bilobalide nanoemulsion as claimed in claim 1, characterized in that, The invention is composed of the following components by mass percentage: 0.1-0.5% of bilobalide, 0.1-1% of medium chain triglyceride, 0.5-5% of polysorbate 80, 0.5-5% of polysorbate 20, 1-10% of polyethylene glycol 400 and 78.5-97.8% of distilled water.

3. A bilobalide nanoemulsion as claimed in claim 2, characterized in that, The bilobalide nanoemulsion consists of the following components by mass percentage: 0.2-0.4% of bilobalide, 0.4-1% of medium chain triglyceride, 1-4% of polysorbate 80, 1-4% of polysorbate 20, 5-10% of polyethylene glycol 400 and 80.6-92.4% of distilled water.

4. A bilobalide nanoemulsion as claimed in claim 2 or 3, characterized in that, The bilobalide nanoemulsion consists of the following components by mass percentage: 0.23% bilobalide, 0.46% medium chain triglyceride, 2.08% polysorbate 80, 2.08% polysorbate 20, 7.27% polyethylene glycol 400, and 87.88% distilled water.

5. A method for preparing a bilobalide nanoemulsion as described in any one of claims 1 to 4, characterized in that, The steps include: (1) mixing bilobalide, medium chain triglycerides, polysorbate 80, polysorbate 20, and polyethylene glycol 400, and dispersing them by ultrasonication at a temperature of 20-35° C.; (2) adding distilled water to the mixture prepared in step (1), continuing ultrasonic dispersion, and finally filtering through a 0.22 μm filter head to obtain a nanoemulsion.

6. Use of a bilobalide nanoemulsion as claimed in any one of claims 1 to 4 or a bilobalide nanoemulsion prepared by the preparation method as claimed in claim 5 in preparing drugs for treating neurodegenerative diseases and cerebrovascular dementia.

7. The use according to claim 6, characterized in that The neurodegenerative diseases include Alzheimer's disease and Parkinson's disease.

8. The use according to claim 6, characterized in that The cerebrovascular dementia includes stroke and vascular dementia.

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