Self-microemulsion formulations of Vietnamese camellia seed oil, self-microemulsion formulations of piperine using Vietnamese camellia seed oil as the oil phase, their preparation methods and applications

Abstract

This invention relates to a self-microemulsion formulation of Vietnamese camellia seed oil, a self-microemulsion formulation of piperine using Vietnamese camellia seed oil as the oil phase, and their preparation methods and applications. This invention prepares the optimal formulation of the Vietnamese camellia seed oil self-microemulsion formulation and the piperine self-microemulsion formulation using Vietnamese camellia seed oil as the oil phase through formulation screening and optimization. In vitro cell experiments and in vivo pharmacodynamic experiments show that the Vietnamese camellia seed oil self-microemulsion formulation has neuroprotective activity, can alleviate neuronal damage in the brain regions of Parkinson's disease model rats, can also improve motor dysfunction in Parkinson's disease model rats, and upregulate the levels of DA, DOPAC, and HVA in the striatum of the injured side of the Parkinson's disease model rats. Furthermore, when used in combination with piperine, it enhances the ameliorative effect of piperine on Parkinson's disease, exhibiting a good synergistic effect and providing a new treatment method for Parkinson's disease.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and specifically relates to a self-microemulsion preparation of Vietnamese camellia seed oil, a self-microemulsion preparation of piperine using Vietnamese camellia seed oil as the oil phase, and their preparation methods and applications. Background Technology

[0002] Parkinson's disease (PD) is the second leading cause of neurodegenerative disease worldwide, characterized primarily by the degeneration and loss of dopaminergic neurons in the substantia nigra pars compacta and the deposition of Lewy bodies within neurons. As an age-related disease, the prevalence of PD gradually increases with age. The clinical manifestations of PD are mainly motor symptoms such as resting tremor, muscle rigidity, bradykinesia, and postural instability, and non-motor symptoms such as cognitive impairment, mood disorders, and olfactory dysfunction may also occur. PD not only severely impacts the physical and mental health of patients but also imposes a significant economic burden on their families and society. Dopamine replacement therapy remains the core treatment strategy for PD; however, due to the severe peripheral metabolism of levodopa (L-DOPA), long-term, high-dose use of L-DOPA can trigger a series of adverse reactions. Therefore, finding scientific, rational, and effective drug treatments for Parkinson's disease remains a hot research topic.

[0003] Piperine (PIP) is an amide alkaloid isolated from plants such as *Piper nigrum* L. and *Piper longum* L., belonging to the Piperaceae family. Piperine possesses a wide range of pharmacological activities, including anti-inflammatory, antioxidant, analgesic, neuroprotective, antitumor, and antibacterial effects. In recent years, studies have shown that piperine has potential therapeutic value for various neurodegenerative diseases. In models of Alzheimer's disease, Parkinson's disease, Huntington's disease, and epilepsy, piperine can improve behavioral deficits, reduce β-amyloid protein deposition and α-synuclein aggregation, making its research on anti-neurodegenerative diseases show good clinical potential. However, piperine molecules are relatively low in polarity, slightly soluble in water, and have low oral bioavailability, limiting its efficacy in central nervous system diseases.

[0004] Camellia oleifera, a plant belonging to the genus Camellia in the family Theaceae, is a high-quality edible oilseed plant unique to my country. The fatty oil obtained by pressing mature Camellia oleifera seeds is called camellia seed oil, which has an unsaturated fatty acid content as high as 90% and is also rich in flavonoids, tea polyphenols, squalene, tocopherols, and other substances beneficial to the human body. Modern pharmacological studies have shown that camellia seed oil has anti-inflammatory, antioxidant, and gut microbiota-regulating effects, making it of significant application value. However, there are currently no reports on the use of camellia seed oil to intervene in Parkinson's disease (PD).

[0005] Self-microemulsifying drug delivery systems (SMEDDS) are thermodynamically stable solid or liquid formulations composed of a drug, an oil phase, an emulsifier, and a co-emulsifier. Compared to other lipid carriers such as solid lipid nanoparticles and liposomes, SMEDDS offer a simpler and more efficient preparation process, facilitating large-scale industrial production. After oral administration, SMEDDS formulations spontaneously form nanoemulsions ≤100 nm in size through gastrointestinal motility, enabling rapid drug diffusion and distribution within the gastrointestinal tract while reducing gastrointestinal irritation and improving drug stability. Numerous studies have demonstrated that SMEDDS can effectively improve the in vitro dissolution and release and oral bioavailability of poorly soluble drugs, enhancing oral absorption efficiency and thus resulting in better efficacy. However, the preparation of self-microemulsion drug delivery systems faces the following challenges: (1) difficult formulation screening, requiring consideration of solubility, emulsification efficiency, and compatibility between the oil phase and co-emulsifiers or emulsifiers, resulting in a narrow selection range; slight fluctuations in the oil / emulsifier / co-emulsifier ratio may lead to failure to form an emulsion, excessive particle size, or stratification; (2) poor stability; and (3) limited drug loading capacity. Therefore, developing a safe, stable, and effective self-microemulsion formulation for the treatment of Parkinson's disease has significant clinical importance and application prospects. Summary of the Invention

[0006] In view of the shortcomings and deficiencies of the existing technology, the first objective of the present invention is to provide a self-microemulsion preparation of Vietnamese camellia seed oil, wherein the self-microemulsion preparation of Vietnamese camellia seed oil is prepared with Vietnamese camellia seed oil as the oil phase and active ingredient, with polyoxyethylene hydrogenated castor oil as the emulsifier and polyethylene glycol 400 as the co-emulsifier.

[0007] The second objective of this invention is to provide a method for preparing a self-microemulsion formulation of Vietnamese camellia seed oil. The method involves weighing emulsifier and co-emulsifier according to the prescribed amount, mixing them evenly to obtain a mixture a, adding Vietnamese camellia seed oil dropwise into the mixture a, stirring, and obtaining a self-microemulsion formulation of Vietnamese camellia seed oil. As a preferred embodiment of the present invention, the amount of Vietnamese camellia seed oil added is controlled at 10% to 30%, and the mass ratio of emulsifier to co-emulsifier is controlled at 1:1 to 3:1.

[0008] As a preferred embodiment of the present invention, the amount of Vietnamese camellia seed oil is 3.64 g; the amount of polyoxyethylene hydrogenated castor oil is 11.95 g; and the amount of polyethylene glycol 400 is 4.41 g.

[0009] As a preferred embodiment of the present invention, the temperature is 200~400 r·min -1 Stir evenly for 3-5 minutes under the specified conditions.

[0010] The self-microemulsion preparation of Vietnamese camellia seed oil provided by this invention has neuroprotective activity, which can reduce the damage of nerve cells in the brain region of Parkinson's disease model rats. It can also improve the motor dysfunction of Parkinson's disease model rats, upregulate the content of dopamine (DA) and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the striatum of the damaged side of Parkinson's disease model rats, and alleviate the typical pathological damage of Parkinson's disease. Therefore, it can be used in the preparation of drugs for the treatment of Parkinson's disease.

[0011] The third objective of this invention is to provide a piperine self-microemulsion formulation using Vietnamese camellia seed oil as the oil phase. The piperine self-microemulsion formulation is prepared by loading piperine into a Vietnamese camellia seed oil self-microemulsion. The piperine self-microemulsion formulation is clear and transparent in appearance, uniformly transparent after emulsification, with a light blue opalescence, and has good stability.

[0012] The fourth objective of this invention is to provide a method for preparing a piperine self-microemulsion formulation using Vietnamese camellia seed oil as the oil phase. The method involves first preparing a Vietnamese camellia seed oil self-microemulsion formulation, then placing it in a vial, adding piperine raw material, and dissolving it by ultrasonication to obtain a transparent and homogeneous solution, which is the piperine self-microemulsion formulation using Vietnamese camellia seed oil as the oil phase.

[0013] As a preferred embodiment of the present invention, the mass ratio of piperine to Vietnamese camellia seed oil self-microemulsion preparation is (0.075-0.3):20.

[0014] As a preferred embodiment of the present invention, the ultrasonic temperature is 30~40℃ and the ultrasonic time is 10~30 min.

[0015] The self-microemulsion drug delivery system with Vietnamese camellia seed oil as the oil phase provided by this invention, when combined with piperine, can effectively enhance the therapeutic effect and neuroprotective effect of piperine against Parkinson's disease. Therefore, the piperine self-microemulsion preparation with Vietnamese camellia seed oil as the oil phase can also be used in the preparation of drugs for treating Parkinson's disease.

[0016] The present invention has the following advantages and beneficial effects: (1) Through in vitro neuroprotective activity studies, this invention found that the self-microemulsion preparation of Vietnamese camellia seed oil has a protective effect on SH-SY5Y cells damaged by 6-OHDA at a concentration of 2.66 mg / mL, proving that the self-microemulsion preparation of Vietnamese camellia seed oil has certain neuroprotective activity, which can reduce nerve cell apoptosis and maintain cell survival. In addition, the prepared piperine self-microemulsion preparation also has certain neuroprotective activity, and the effect is significantly better than that of the self-microemulsion preparation of Vietnamese camellia seed oil.

[0017] (2) By rationally selecting the oil phase, emulsifier, and co-emulsifier, and by rationally controlling the dosage of the formulation, the present invention enables the prepared piperine self-microemulsion formulation with Vietnamese camellia seed oil as the oil phase to have good drug loading, small particle size, good stability, and reduced drug degradation. At the same time, compared with a single nanoemulsion system, the piperine self-microemulsion formulation with Vietnamese camellia seed oil as the oil phase obtained by the present invention has the advantages of low energy consumption, high efficiency, and ease of preparation and production during the emulsification process.

[0018] (3) The present invention uses Vietnamese camellia seed oil as the oil phase and relies on a self-emulsifying drug delivery system to load piperine to prepare a piperine self-microemulsion formulation, which solves the problem of poor water solubility of piperine and improves drug solubility; in addition, since the self-microemulsion can quickly emulsify into tiny droplets when it comes into contact with water, it is beneficial to promote in vivo absorption, thereby improving the bioavailability of piperine.

[0019] (4) The piperine self-microemulsion preparation prepared in this invention uses Vietnamese camellia seed oil as the self-microemulsion oil phase matrix and as the active ingredient, which works synergistically with piperine to effectively improve the intervention efficacy of Parkinson's disease.

[0020] (5) The pharmacodynamic experimental results of this invention show that the self-microemulsion preparation of Vietnamese camellia seed oil can effectively improve the horizontal movement distance and duration, as well as the limb coordination and motor ability of Parkinson's disease model rats, enhance forelimb strength, and alleviate motor dysfunction in model rats. In addition, compared with the single self-microemulsion preparation of Vietnamese camellia seed oil, the prepared piperine self-microemulsion preparation has a more significant effect on the overall improvement of motor function in Parkinson's disease rats. The pharmacodynamic effect of this preparation is dose-dependent, and the therapeutic effect gradually increases with the increase of the dosage. Among them, the high-dose group has a better effect on improving the horizontal movement distance and duration of rats than the positive control group. At the same time, this group is better than the piperine group in improving the limb coordination and overall motor ability of rats.

[0021] (6) The neurotransmitter detection results of this invention show that the self-microemulsion preparation of Vietnamese camellia seed oil can significantly upregulate the content of dopamine (DA) and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the striatum of the injured side of Parkinson's disease model rats. PD model rats suffer from damage to dopaminergic neurons, resulting in a large depletion of striatal DA neurotransmitters, which in turn leads to motor dysfunction. This preparation can effectively increase the level of dopamine neurotransmitters in the brain, improve the disorder of the dopaminergic nervous system, reshape the neurotransmitter balance in the brain, and alleviate the typical pathological damage of Parkinson's disease. This result confirms that it has a clear anti-Parkinson's disease efficacy. In addition, compared with the single self-microemulsion preparation of Vietnamese camellia seed oil, the prepared piperine self-microemulsion preparation has a more significant improvement effect. At the same piperine dosage, the DA content in the injured side of the brain of rats in the PIP-CO-SMEDDS-H group was significantly increased compared with the piperine-only group, which is considered to be due to the synergistic effect of Vietnamese camellia seed oil and piperine in the self-microemulsion preparation.

[0022] (7) The self-microemulsion preparation of Vietnamese camellia seed oil provided by the present invention also played a certain role in improving the condensation of neuronal cell nuclei in the substantia nigra and striatum of PD rats, and could reduce the damage of neuronal cells in the brain region of Parkinson's disease model rats and inhibit neuronal apoptosis and degeneration. Compared with the single self-microemulsion preparation of Vietnamese camellia seed oil, the prepared piperine self-microemulsion preparation had a more significant improvement effect. The improvement effect of the PIP-CO-SMEDDS-H group was better than that of the piperine group under the same dosage. Attached Figure Description

[0023] Figure 1 It uses Vietnamese camellia seed oil as the oil phase, RH40 as the emulsifier, and anhydrous ethanol, PEG200 or PEG400 as co-emulsifiers. m The pseudo-ternary phase diagram is drawn when the ratio is 2:1; in the diagram, A uses anhydrous ethanol as a co-emulsifier; B uses PEG200 as a co-emulsifier; and C uses PEG400 as a co-emulsifier. Figure 2 It uses Vietnamese camellia seed oil as the oil phase, PEG400 as a co-emulsifier, and Tween 80, Tween 20, or RH40 as emulsifiers. m The pseudo-ternary phase diagram is drawn when the ratio is 2:1; in the diagram, A uses Tween 80 as emulsifier; B uses Tween 20 as emulsifier; and C uses RH 40 as emulsifier. Figure 3 It is a pseudo-ternary phase diagram of the Vietnamese camellia seed oil-RH40-PEG400 system; Figure 4 This is a three-dimensional effect diagram of particle size, PDI, and Zeta potential of a self-microemulsion formulation of Vietnamese camellia seed oil; in the diagram, A: particle size; B: PDI; C: Zeta potential; Figure 5These are transmission electron microscopy (TEM) images of self-microemulsions, with scale bars indicating 100 nm. In the image, A is a TEM image of self-microemulsion of Vietnamese camellia seed oil; B is a TEM image of self-microemulsion of piperine with Vietnamese camellia seed oil as the oil phase. Figure 6 This is a particle size distribution diagram of piperine self-microemulsion formulation with Vietnamese camellia seed oil as the oil phase; Figure 7 The zeta potential diagram of piperine self-microemulsion formulation with Vietnamese camellia seed oil as the oil phase; Figure 8 Figure 1 shows the stability study results of piperine self-microemulsion formulation with Vietnamese camellia seed oil as the oil phase; in the figure, A represents the effect of dilution factor on the particle size and PDI of the drug-loaded self-microemulsion; B represents the 24-hour stability study results. Figure 9 The study investigated the effect of 6-OHDA-induced SH-SY5Y cell survival; in the figure, A: piperine; B: piperine self-microemulsion formulation using Vietnamese camellia seed oil as the oil phase; C: Vietnamese camellia seed oil self-microemulsion formulation; compared with the Control group. ### p<0.001; compared with the 6-OHDA group, * p<0.05, ** p<0.01, *** p<0.001; Figure 10 This study investigated the effects of self-microemulsion formulations of Vietnamese camellia seed oil and piperine self-microemulsion formulations using Vietnamese camellia seed oil as the oil phase on the behavior of rats with Parkinson's disease. In the figure, A represents the horizontal movement distance in the open field; B represents the horizontal movement time in the open field; C represents the dwell time on the rotarod; and D represents the forelimb strength of the rats. Compared with the sham-operated group... # p<0.05, ## p<0.01, ### p<0.001; compared with the model group, * p<0.05, ** p<0.01, *** p<0.001; Figure 11 The study investigated the effects of self-microemulsion formulations of Vietnamese camellia seed oil and piperine self-microemulsion formulations using Vietnamese camellia seed oil as the oil phase on neurotransmitter levels in the striatum of the injured side in Parkinson's disease rats. The figure shows the effects compared to the sham-operated group. ## p<0.01, ### p<0.001; compared with the model group, * p<0.05, ** p<0.01, *** p<0.001; compared with the piperine group, $$$ p<0.001; Figure 12These are representative HE staining images of neurons in the substantia nigra pars compacta and striatum of Parkinson's disease rats treated with self-microemulsion formulations of Vietnamese camellia seed oil and piperine self-microemulsion formulations with Vietnamese camellia seed oil as the oil phase. Figure 13 The study investigated the effects of self-microemulsion formulations of Vietnamese camellia seed oil and piperine self-microemulsion formulations using Vietnamese camellia seed oil as the oil phase on the levels of TH and α-Syn proteins in the substantia nigra of the injured side in 6-OHDA rats. The figure shows the effects compared to the sham-operated group. ### p<0.001; compared with the model group, * p<0.05, ** p<0.01, *** p<0.001; compared with the piperine group, $ p < 0.05. Detailed Implementation

[0024] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0025] Unless otherwise specified, all materials and reagents used in the following experiments were commercially available. Specifically, piperine was prepared by the laboratory of the Department of Traditional Chinese Medicine Resources and Chemistry, School of Traditional Chinese Medicine, Capital Medical University (purity higher than 96%), and was also commercially available (CAS No.: 94-62-2); Vietnamese camellia seed oil was provided by Hainan Academy of Agricultural Sciences; Tween 20 was purchased from Beijing Solarbio Technology Co., Ltd.; Tween 80 and Span 80 were purchased from Shanghai Maclean Biotechnology Co., Ltd.; polyoxyethylene hydrogenated castor oil (RH40) was purchased from Shanghai Aladdin Biotechnology Co., Ltd.; anhydrous ethanol was purchased from Beijing Modern Oriental Technology Development Co., Ltd.; and polyethylene glycol 200 (PEG200) and polyethylene glycol 400 (PEG400) were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0026] Example 1

[0027] Preparation method of self-microemulsion formulation of Vietnamese camellia seed oil: Weigh out the emulsifier and co-emulsifier according to the prescription and mix them evenly to obtain mixture a. Add Vietnamese camellia seed oil to mixture a and stir at 200~400 r·min. -1 Stir evenly for 3-5 minutes under the specified conditions (in this example, at 400 r·min). -1 Under uniform stirring conditions for 3 min, a blank self-microemulsion preparation with Vietnamese camellia seed oil as the oil phase was obtained, namely, the Vietnamese camellia seed oil self-microemulsion preparation.

[0028] Specifically, in this embodiment, the prescription dosage is as follows: Vietnamese camellia seed oil: 3.64 g; emulsifier RH40, dosage: 11.95 g; co-emulsifier PEG400, dosage: 4.41 g, and the total system mass is 20 g.

[0029] Example 2

[0030] Preparation method of piperine self-microemulsion formulation using Vietnamese camellia seed oil as the oil phase: Weigh out the emulsifier and co-emulsifier according to the prescription and mix them evenly to obtain mixture a. Add Vietnamese camellia seed oil to mixture a and stir at 200~400 r·min. -1 Stir evenly for 3-5 minutes under the specified conditions (in this example, at 400 r·min). -1 Under uniform stirring conditions for 3 min, a blank self-microemulsion preparation with Vietnamese camellia seed oil as the oil phase was obtained, namely, the Vietnamese camellia seed oil self-microemulsion preparation.

[0031] Place the blank self-microemulsion formulation in a 50 mL vial, add piperine raw material, and sonicate to dissolve it. The sonication temperature is 30-40℃ (in this example, the temperature is controlled at around 35℃), and the sonication time is 10-30 min (in this example, sonication is 30 min). A transparent and homogeneous solution is obtained, which is the piperine self-microemulsion formulation with Vietnamese camellia seed oil as the oil phase.

[0032] Specifically, in this embodiment, the total mass of the blank self-microemulsion formulation system is 20 g, and the blank self-microemulsion formulation described in Example 1 is used, with a piperine addition of 0.075 g.

[0033] Example 3

[0034] The difference from Example 2 is that the amount of piperine added is 0.15 g.

[0035] Example 4

[0036] The difference from Example 2 is that the amount of piperine added is 0.30 g.

[0037] In this embodiment, the particle size of the piperine self-microemulsion formulation with Vietnamese camellia seed oil as the oil phase was determined. The specific determination method was as follows: 0.1 g of the piperine self-microemulsion formulation with Vietnamese camellia seed oil as the oil phase was weighed and diluted with 100 times the amount of distilled water (i.e., 10 mL). The particle size and PDI were determined using a laser particle size analyzer. Each sample was measured in parallel 3 times and the average value was taken.

[0038] In this embodiment, the Zeta potential of the piperine self-microemulsion preparation with Vietnamese camellia seed oil as the oil phase was determined. The specific determination method was as follows: 0.1g of the piperine self-microemulsion preparation with Vietnamese camellia seed oil as the oil phase was weighed and diluted with 100 times the amount of distilled water (i.e., 10mL). The Zeta potential was measured using a Zeta potential meter. Each sample was measured in parallel 3 times and the average value was taken.

[0039] It should be noted that, in this embodiment, the preparation method of the self-microemulsion formulation of Vietnamese camellia seed oil and the preparation method of the self-microemulsion formulation of piperine using Vietnamese camellia seed oil as the oil phase are the optimal solutions determined by the inventors after extensive experiments, repeated explorations, comprehensive summarization and analysis of various experimental data and phenomena, and systematic summarization of experimental conclusions. The following is a detailed introduction to the optimization experiment of the formulation of the self-microemulsion formulation of Vietnamese camellia seed oil: 1. Screening of prescription reagents: 1.1 Compatibility Study: Take 1 g of Vietnamese camellia seed oil and emulsifier mixed at a ratio of 1:9 to 5:5. Take 0.1 g of the mixture of Vietnamese camellia seed oil and emulsifier and add it dropwise to 10 mL of pure water at a rotation speed of 200 r·min. -1 The morphology of the formed microemulsions was observed, and the evaluation criteria are shown in Table 1. The results are shown in Table 2. Table 1 Compatibility Evaluation Criteria grade Emulsification time Dispersion state and appearance A ≤1 min The solution is clear and transparent, with a slight bluish-white opalescence. B ≤1 min The solution is bluish-white and opalescent. C ≤2 min Bright white opaque emulsion D ≥2 min grayish-white or oily emulsion E >2 min Emulsification is difficult, with oil droplets or layering. Table 2. Results of the compatibility study between Vietnamese camellia seed oil and emulsifiers

[0040] As shown in Table 2, Span80 has a very poor emulsifying effect and is difficult to form a uniform and stable microemulsion with Vietnamese camellia seed oil. Among the other three emulsifiers, RH40 has a better emulsifying effect than Tween80 and Tween20. Therefore, RH40 was selected as the emulsifier to investigate the selection of co-emulsifiers. When screening emulsifiers and co-emulsifiers in the future, their emulsifying ability will be the main consideration.

[0041] 1.2 Screening of co-emulsifiers: The oil phase was determined to be Vietnamese camellia seed oil, the emulsifier was RH40, and the co-emulsifier was PEG400, PEG200, or anhydrous ethanol. The total mass was fixed at 0.1 g before adding water, and the ratio of emulsifier to co-emulsifier was K. m The raw materials were weighed with a mass ratio of 2:1 for the emulsifier / co-emulsifier mixture to the oil phase at mass ratios of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1, and the self-microemulsion formulation of Vietnamese camellia seed oil was prepared according to the method described in Example 1.

[0042] Vietnamese camellia seed oil microemulsion was placed on a magnetic stirrer, and distilled water at 37°C was slowly added dropwise until a clear, transparent solution was formed. The amounts of oil phase, emulsifier, co-emulsifier, and aqueous phase used at the final formation of the nanoemulsion were recorded. A pseudo-ternary phase diagram was plotted using Origin 2025 software; see [link to Origin software]. Figure 1 .

[0043] Experiments have shown that, with a stationary oil phase, emulsifier, and K... m Under the condition of 2, PEG400 as a co-emulsifier can form microemulsions when the ratio of oil phase to mixed emulsifier is 1:9 to 3:7, and the microemulsion region is larger than that of the other two. Therefore, PEG400 is selected as the co-emulsifier.

[0044] 1.3. Screening of emulsifiers: The oil phase was determined to be Vietnamese camellia seed oil, the co-emulsifier was PEG400, and the emulsifier was Tween80, Tween20, or RH40. The total mass before adding water was fixed at 0.1 g, and the ratio of emulsifier to co-emulsifier was K. m The raw materials were weighed with a mass ratio of 2:1 for the emulsifier / co-emulsifier mixture to the oil phase at mass ratios of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1, and the self-microemulsion formulation of Vietnamese camellia seed oil was prepared according to the method described in Example 1.

[0045] Vietnamese camellia seed oil microemulsion was placed on a magnetic stirrer, and distilled water at 37°C was slowly added dropwise until a clear, transparent solution was formed. The amounts of oil phase, emulsifier, co-emulsifier, and aqueous phase used at the final formation of the nanoemulsion were recorded. A pseudo-ternary phase diagram was plotted using Origin 2025 software; see [link to Origin software]. Figure 2 .

[0046] The results showed that RH40 formed the largest microemulsion area. Tween80 and Tween20 could only form a uniform and transparent microemulsion when the ratio of oil phase and mixed emulsifier was 1:9. At other ratios, they formed a milky white emulsion. Therefore, RH40 was selected as the emulsifier for piperine self-microemulsion formulation with Vietnamese camellia seed oil as the oil phase.

[0047] 1.4. Determination of the dosage and composition range of excipients: Before adding water, the total mass is fixed at 1 g, and the ratio of RH40 to PEG400 is K. m The raw materials were weighed with values ​​(weighed by mass ratio) of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1. The emulsifier / co-emulsifier mixture and Vietnamese camellia seed oil were weighed with mass ratios of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1. The self-microemulsion formulation of Vietnamese camellia seed oil (blank self-microemulsion formulation) was prepared according to the method described in Example 1.

[0048] The self-microemulsion formulation of Vietnamese camellia seed oil was placed on a magnetic stirrer, and distilled water at 37°C was slowly added dropwise. The formulations that ultimately formed the evaluation criteria (see Table 1) A and B states were recorded, along with the corresponding oil phase and the ratio of emulsifier to co-emulsifier. A pseudo-ternary phase diagram was drawn using Origin 2025 software. Figure 3 .

[0049] The results showed that a good, homogeneous, and clear microemulsion solution could be obtained within the oil phase range of 10-30%. Considering that the content of emulsifier should not be too high, K was set as follows: m The value range is 1:1 to 3:1.

[0050] 2. Determination of the optimal prescription and prescription validation: 2.1. Design for Stars - Response Surface Methodology for Optimizing Prescriptions: In the preparation of self-microemulsion formulations, the oil phase ratio and K m The values ​​have a significant impact on the particle size and zeta potential of self-microemulsion formulations; therefore, X1 (oil phase ratio) and X2 (K) were selected. m Two influencing factors (value) were considered, with particle size, PDI, and Zeta potential of the self-microemulsion formulation as evaluation indicators. An oil phase ratio of 10%–30% was selected, and K... m The values ​​ranged from 1:1 to 3:1. A two-factor, five-level star point design method was used to study the oil-phase ratio and K of self-microemulsion formulations of Vietnamese camellia seed oil. m The values ​​were further optimized, and the mass settings for each component are shown in Table 3.

[0051] Table 3. Factors and Levels of Star Design

[0052] The total mass of the Vietnamese camellia seed oil self-microemulsion formulation system was 1000 mg. Raw materials were weighed according to the formula in Table 4, and the formulation was prepared according to the method in Example 1. The particle size, PDI, and Zeta potential of the formulation emulsion were measured, and the results are shown in Table 4. Response surface methodology was performed using Design-Expert 13 software to obtain the three-dimensional response surface plot of the dependent variable (see...). Figure 4 ), thus obtaining the optimal prescription.

[0053] Table 4. Prescription Design and Results Serial Number <![CDATA[X1(oil%)]]> <![CDATA[X2(K m )]]> Particle size (nm) PDI Zeta (mV) 1 20.00 2.000 29.43 0.199 -30.78 2 10.00 2.000 21.30 0.058 -21.03 3 20.00 1.000 41.74 0.194 -27.40 4 27.07 2.707 45.71 0.227 -42.10 5 27.07 1.293 69.06 0.218 -34.10 6 20.00 2.000 28.09 0.200 -32.35 7 30.00 2.000 57.16 0.224 -39.10 8 20.00 2.000 29.41 0.212 -32.25 9 12.93 1.293 24.49 0.079 -16.72 10 20.00 2.000 25.69 0.166 -30.43 11 20.00 2.000 25.77 0.187 -29.53 12 20.00 3.000 27.67 0.189 -40.29 13 12.93 2.707 21.80 0.101 -33.39 The optimal prescription obtained from the simulation: Vietnamese camellia seed oil: 18.19%, K m Value: 2.707:1, that is, the total mass of the blank self-microemulsion formulation (Vietnamese camellia seed oil self-microemulsion formulation) system with Vietnamese camellia seed oil as the oil phase is 1000 mg: Vietnamese camellia seed oil: 181.9 mg, RH40: 597.4 mg, PEG400: 220.7 mg.

[0054] The self-microemulsion formulation of Vietnamese camellia seed oil prepared according to the above formula has a predicted particle size of 24.22 nm, a predicted PDI of 0.176, and a predicted Zeta potential of -36.59 mV.

[0055] 2.2 Prescription Verification Weigh the ingredients according to the following prescription: The total mass of the blank self-microemulsion formulation system using Vietnamese camellia seed oil as the oil phase was 1000 mg. Vietnamese Camellia Seed Oil: 181.9 mg RH40: 597.4 mg PEG400: 220.7 mg Three batches of self-microemulsion formulations of Vietnamese camellia seed oil were prepared according to the method described in Example 1, and the particle size, PDI, and Zeta potential of the self-microemulsion formulations were measured. The results are shown in Table 5. The relative error between the experimental and predicted values ​​was verified to be <10%, indicating that the optimized model can predict particle size, PDI, and Zeta potential.

[0056] Table 5. Predicted and Measured Values ​​of Response Surface Optimization Formulations for Self-Microemulsion Preparations of Camellia oleifera Seed Oil from Vietnam index Predicted value Measured value relative error % Particle size (nm) 24.22 25.51±0.16 5.33% PDI 0.176 0.173±0.003 1.73% Zeta potential (mV) -36.59 -37.38±1.53 2.11% Furthermore, in this embodiment, the physicochemical properties of the piperine self-microemulsion formulation using Vietnamese camellia seed oil as the oil phase are studied, and the specific details are as follows: (1) Appearance: Self-microemulsion formulations were prepared according to the methods in Example 1 (blank self-microemulsion) and Example 4 (drug-loaded self-microemulsion), respectively. An appropriate amount of the self-microemulsion formulation was added to pure water and fully emulsified using a magnetic stirrer. A small amount of the emulsified microemulsion was placed on a copper grid covered with a support film. After fixing for 10 min, excess liquid was blotted from the edge of the copper grid with a small piece of filter paper. The microemulsion was stained with 2% phosphotungstic acid negative staining solution for 5 min, excess staining was blotted, and the microemulsion was allowed to dry naturally before observing its morphology under a transmission electron microscope. Figure 5 It is evident that the prepared self-microemulsion formulation is spherical in shape, uniform in size, and with virtually no adhesion between the nanoparticles.

[0057] (2) Particle size and PDI analysis: Three parallel preparations of piperine self-microemulsion formulations using Vietnamese camellia seed oil as the oil phase were prepared. 0.1 g of each piperine self-microemulsion was diluted 100 times with distilled water at 37℃. The particle size was measured, and the results are shown below. Figure 6 The average particle size was (34.14±0.22) nm, and the PDI was (0.138±0.011).

[0058] (3) Investigation of Zeta potential Three parallel preparations of piperine self-microemulsion formulations using Vietnamese camellia seed oil as the oil phase were prepared. Each formulation was diluted 100-fold with distilled water at 37°C. The zeta potential of the self-microemulsion formulations was measured, and the results are shown below. Figure 7 The average Zeta potential was (-25.20±1.57) mV.

[0059] (4) Stability study of piperine self-microemulsion formulation using Vietnamese camellia seed oil as the oil phase: 1) Effect of dilution factor on particle size: 0.1 g of the piperine self-microemulsion preparation with Vietnamese camellia seed oil as the oil phase, as described in Example 4, was diluted 50 times, 100 times, and 200 times with distilled water at 37℃, respectively. The particle size and PDI were determined, and the results are shown in the figure. Figure 8 The particle size of the self-microemulsion showed little variation at different dilution factors, indicating that the dilution factor had almost no effect on the particle size; while the PDI values ​​were all less than 0.2, indicating that the system was uniformly distributed at different dilution factors.

[0060] 2) Short-term stability assessment: 0.1 g of the piperine self-microemulsion formulation from Example 4, using Vietnamese camellia seed oil as the oil phase, was diluted 100 times with distilled water at 37°C and sealed in a vial at 25°C. Particle size and PDI were measured at 0, 2, 4, 6, 8, 12, and 24 hours. The results are as follows: Figure 8 Over time, the particle size and PDI of the drug-loaded self-microemulsion showed relatively small changes, indicating that the self-microemulsion formulation has good stability.

[0061] Example 5: In vitro neuroprotective activity study Materials: Human neuroblastoma cell line SH-SY5Y, purchased from the National Biomedical Cell Line Resource Center (BMCR). Drugs included the self-microemulsion preparation of Vietnamese camellia seed oil prepared in Example 1 (i.e., the blank self-microemulsion preparation with Vietnamese camellia seed oil as the oil phase, CO-SMEDDS) and the piperine self-microemulsion preparation with Vietnamese camellia seed oil as the oil phase prepared in Example 4 (PIP-CO-SMEDDS); piperine (PIP); 6-hydroxydopamine hydrobromide (6-OHDA) (Sigma-Aldrich, USA); DMEM high-glucose culture medium (Shanghai Xiaopeng Biotechnology Co., Ltd.); 0.25% trypsin (containing EDTA) (Gibco, USA); fetal bovine serum (FBS) (Mengma Tianjin Biotechnology Co., Ltd.); penicillin-streptomycin (10,000 U / mL) (Gibco, USA); thiazolyl blue (MTT) (Beijing Solarbio Science & Technology Co., Ltd.); and cell-grade dimethyl sulfoxide (DMSO) (Sigma-Aldrich, USA).

[0062] Methods: The MTT assay was used to investigate the intervention effects of different formulations on 6-OHDA-induced damage to SH-SY5Y cells. Logarithmically growing cells were digested with trypsin containing 0.25% EDTA, and the reaction was terminated by adding serum-containing medium. The supernatant was removed by centrifugation. 2 mL of complete culture medium was added, and the cells were mixed thoroughly by pipetting. Platelet counts were performed using a platelet counter. SH-SY5Y cells were counted at a rate of 2.5 × 10⁶ cells per well. 4 Cells were seeded in 96-well plates and cultured in an incubator (37°C, 5% CO2) for 24 h. When the cells reached 70% confluence, the culture medium was discarded, and complete culture medium containing 6-OHDA (50 μM) was added to establish the cell model for 24 h. A series of drug concentrations (5-80 μg / mL for piperine in the PIP group and PIP-CO-SMEDDS group, and 0.33-5.33 mg / mL for the CO-SMEEDS group, based on the concentration of Vietnamese camellia seed oil microemulsion) were applied to the 6-OHDA-damaged SH-SY5Y cell model for 24 h. Cell viability was determined using the MTT assay. Each group was divided into 3 replicates, and the experiment was repeated 3 times.

[0063] Results: After 24 hours of treatment with PIP, CO-SMEDDS, and PIP-CO-SMEDDS on 6-OHDA-damaged SH-SY5Y cells, the cell viability was as follows: Figure 9As shown in the figure, compared with the model group, cell survival rates were significantly increased after administration of PIP and PIP-CO-SMEDDS (p < 0.05, p < 0.01). Notably, CO-SMEDDS at a concentration of 2.66 mg / mL had a protective effect on SH-SY5Y cells damaged by 6-OHDA (p < 0.05), indicating that the self-microemulsion formulation of Vietnamese camellia seed oil has certain neuroprotective activity.

[0064] Example 6: Therapeutic effect of 6-OHDA on 6-OHDA-induced Parkinson's disease rats Materials: Male Sprague Dawley (SD) rats, weighing 180–220 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Drugs included the self-microemulsion formulation of Vietnamese camellia seed oil prepared in Example 1 (i.e., the blank self-microemulsion formulation with Vietnamese camellia seed oil as the oil phase (CO-SMEDDS)), and the piperine self-microemulsion formulations with Vietnamese camellia seed oil as the oil phase prepared in Examples 2–4 (PIP-CO-SMEDDS); piperine; levodopa / carbidopa tablets (Madopar) (Shanghai Roche Pharmaceuticals Co., Ltd., specification: 0.25 g * 40 tablets); 6-hydroxydopamine hydrobromide (6-OHDA, Sigma-Aldrich, USA); CMC-Na (Sinopharm Chemical Reagent Co., Ltd.). The method is as follows: (1) Preparation of Parkinson's disease model rats: Rats were acclimatized for one week before modeling and were subjected to behavioral tests before surgery. Rats without rotational behavior were selected for model preparation. After anesthesia, the rats were fixed in a horizontal position on a stereotaxic instrument. After the head was prepared and disinfected, the head skin was cut open to fully expose the anterior fontanelle. According to the rat brain stereotaxic atlas by Paxinos et al., three coordinate points of the left striatum were determined: the first point was 0.7 mm anterior to the anterior fontanelle, 2.6 mm to the left of the sagittal line, and 6.0 mm subdurally; the second point was 0.7 mm anterior to the anterior fontanelle, 2.6 mm to the left of the sagittal line, and 5.5 mm subdurally; the third point was 0.7 mm anterior to the anterior fontanelle, 2.6 mm to the left of the sagittal line, and 5.0 mm subdurally. Using a microsyringe, 1.0 μL of 6-OHDA solution (4 mg / mL, dissolved in physiological saline containing 0.02% ascorbic acid) was slowly injected at each coordinate point at a rate of 1.0 μL / min. After the injection, the skin incision was sutured and penicillin powder was applied topically to prevent infection. The same surgical procedure was performed on the sham-operated group rats, injecting an equal volume of physiological saline containing 0.02% ascorbic acid into the left striatum.

[0065] (2) Animal grouping and administration: Seventy-two SD rats were randomly divided into eight groups, including the sham operation group (Sham), the model group (Model), the piperine group (PIP), the blank self-microemulsion group (CO-SMEDDS), the low-dose group of Vietnamese camellia seed oil-piperine self-microemulsion (PIP-CO-SMEDDS-L), the medium-dose group (PIP-CO-SMEDDS-M), the high-dose group (PIP-CO-SMEDDS-H), and the positive control drug Madopar group (Madopar), with nine rats in each group. Administration was started by gavage once daily for 42 consecutive days, beginning five weeks after the modeling surgery.

[0066] The dosage for each group is as follows: Sham group: administered an aqueous solution containing 0.5% CMC-Na.

[0067] Model group: given an aqueous solution containing 0.5% CMC-Na.

[0068] PIP group: Prepared with an aqueous solution containing 0.5% CMC-Na, the single dose is 20 mg / kg / day of piperine.

[0069] CO-SMEDDS group: Vietnamese camellia seed oil self-microemulsion formulation, i.e., blank self-microemulsion with Vietnamese camellia seed oil as the oil phase, with a single dose of approximately 240 mg / kg / day of Vietnamese camellia seed oil.

[0070] PIP-CO-SMEDDS-L group: Piperine self-microemulsion low-dose group, using the piperine self-microemulsion preparation prepared in Example 2 with Vietnamese camellia seed oil as the oil phase. The single dose was approximately 5 mg / kg / day of piperine and 240 mg / kg / day of Vietnamese camellia seed oil.

[0071] PIP-CO-SMEDDS-M group: drug-loaded self-microemulsion medium-dose group, the piperine self-microemulsion preparation prepared in Example 3 with Vietnamese camellia seed oil as the oil phase, the single dose is about 10 mg / kg / day of piperine and 240 mg / kg / day of Vietnamese camellia seed oil.

[0072] PIP-CO-SMEDDS-H group: drug-loaded self-microemulsion medium-dose group, the piperine self-microemulsion preparation prepared in Example 4 with Vietnamese camellia seed oil as the oil phase, the single dose is about 20 mg / kg / day of piperine and 240 mg / kg / day of Vietnamese camellia seed oil.

[0073] Madopar group: Prepared with an aqueous solution containing 0.5% CMC-Na, the single dose was 50 mg / kg / day.

[0074] (3) Behavioral testing: Open field test: The spontaneous activity of rats was assessed at 5 weeks post-modeling (before drug administration), 8 weeks post-modeling (3 weeks post-drug administration), and 11 weeks post-modeling (6 weeks post-drug administration). Before the experiment, the animals were allowed to acclimatize in the testing room for at least 10 minutes. The extent of free exploration by the rats within 5 minutes was tracked and recorded using a digital camera, including the horizontal movement distance and duration. Each rat was tested individually, and the open field test chamber was kept clean before each test.

[0075] The rotarod test was conducted at weeks 5, 8, and 11 post-modeling to evaluate limb coordination and motor skills in rats. Before the test, rats adapted to the rotarod at a low speed of 5 rpm for 10 seconds. The rotarod was then accelerated from 5 rpm to 20 rpm over 120 seconds. The dwell time on the rotarod was recorded. Each rat was tested three times, with at least 30 minutes between each test to avoid stress and fatigue and to obtain reliable data. The average of the three tests was taken.

[0076] Grip strength test: The grip strength test was conducted at weeks 5, 8, and 11 after modeling to detect changes in the rat's forelimb strength. The rat's tail was gently grasped, and it was slowly guided towards a T-shaped bar until its forelimbs gripped the bar. The rat was then slowly pulled horizontally until it could no longer grip the bar, and the instantaneous grip strength at the moment of release was recorded.

[0077] (4) Determination of monoamine neurotransmitter content in the striatum Six rats were used in each group, and 20 mg of the striatum from the injured side was accurately weighed for the determination of monoamine neurotransmitter content. Sample preparation methods: Sample preparation solution A was a 0.4 M perchloric acid solution, and sample preparation solution B was a buffer salt solution (containing 20 mM potassium citrate, 300 mM dipotassium hydrogen phosphate, and 2 mM EDTA·2Na). 200 μL of pre-chilled sample preparation solution A was added to a centrifuge tube, vortexed, and then ground in a ball mill. After grinding, the tube was placed in an ice bath in the dark for 1 h, followed by centrifugation at 12000 rpm for 20 min at 4 ℃. The supernatant was transferred to a new centrifuge tube, and pre-chilled sample preparation solution B was added at a ratio of 1:2 (v / v). The mixture was vortexed and placed in an ice bath in the dark for another 1 h. After centrifugation at 12000 rpm for 20 min (4 ℃), the supernatant was filtered through a membrane for analysis.

[0078] The content of monoamine neurotransmitters in the striatum was determined by high-performance liquid chromatography with a tandem electrochemical detector (HPLC-ECD). The mobile phase conditions were methanol (A) and buffer salt solution (B; containing 50 mM citric acid, 0.2 mM sodium octanesulfonate, and 0.1 mM EDTA·2Na) (A:B = 8:92, v / v), the flow rate was 0.8 mL / min, the column temperature was 30 °C, the detection voltage was 0.6 V, and the injection volume was 10 µL.

[0079] (5) HE staining to observe pathological changes in the substantia nigra and striatum of rats Brain tissue from rats in each group was collected, fixed in 4% paraformaldehyde, and then dehydrated, embedded, sectioned in paraffin, baked, dewaxed, stained with hematoxylin and eosin (HE), and mounted with neutral resin. The pathological changes of the substantia nigra and striatum in the brain tissue of rats in each group were observed under a microscope.

[0080] (6) Western blotting method was used to determine the expression of TH and α-Syn proteins in the substantia nigra of rat brain. Approximately 20 mg of rat substantia nigra tissue from the injured side was weighed and added to 300 µL of lysis buffer (950 µL RIPA lysis buffer + 10 µL PMSF + 20 µL 50× protease inhibitor + 20 µL 50× phosphatase inhibitor). The mixture was homogenized on ice, and the protein concentration was determined by BCA assay followed by high-temperature denaturation. Protein samples were separated by SDS-PAGE, transferred to a PVDF membrane, blocked with 5% skim milk powder at room temperature for 2 h, and then incubated overnight at 4°C with TH, α-Syn, and GAPDH primary antibody solutions. The next day, the membrane was incubated with secondary antibody solution at room temperature for 2 h. After washing, chemiluminescence solution was added evenly, and the membrane was automatically exposed. The gray values ​​of the sample bands were analyzed using ImageJ software. The ratio of the gray values ​​of the target protein band to the internal control band was calculated, using the GAPDH band as an internal control.

[0081] The results are as follows: (1) Effects on the behavior of rats with Parkinson's disease The open field test primarily assessed the spontaneous activity of rats. Compared to the sham-operated group, the model group exhibited significant motor dysfunction, with significantly reduced horizontal movement distance and duration (p<0.01). Furthermore, the spontaneous activity ability of the model group rats showed a continuous downward trend with the progression of the disease. Compared to the model group, after 3 and 6 weeks of drug intervention, the horizontal movement distance and duration (p<0.05) of PD rats in each drug-treated group were significantly improved. The effect of PIP-CO-SMEDDS on improving the spontaneous activity ability of rats showed a dose-dependent increase, with the high-dose PIP-CO-SMEDDS group showing the best improvement in spontaneous activity.

[0082] The rotarod test was used to evaluate the limb coordination and motor ability of rats. Compared with the sham-operated group, the retention time on the rotarod in 6-OHDA-induced PD rats was significantly reduced (p<0.001). After 3 and 6 weeks of administration, the retention time on the rotarod in PD rats in all administration groups was significantly improved. Among them, the high-dose PIP-CO-SMEDDS group was more effective than the piperine group in improving the motor ability of rats.

[0083] The grip strength test was used to detect changes in forelimb strength in rats. Compared with the sham-operated group, the forelimb muscle strength of rats in the model group was significantly reduced (p<0.05). Compared with the model group, after 3 weeks and 6 weeks of drug administration, all drug administration groups significantly improved forelimb muscle strength in PD rats (p<0.05). The experimental results are shown in […]. Figure 10 .

[0084] (2) Effects on monoamine neurotransmitters in the striatum of Parkinson's disease rats The content of monoamine neurotransmitters in the striatum of rats in each group was determined by HPLC-ECD. The results showed that compared with the sham-operated group, the levels of DA, DOPAC, and HVA in the striatum of the injured side of rats in the model group were significantly lower (p < 0.001); while the levels of DA, DOPAC, and HVA in the striatum of the injured side of rats in each treatment group were significantly higher than those in the model group (p < 0.05). Compared with the model group, the levels of DA, DOPAC, and HVA in the striatum of the injured side of rats in the CO-SMEDS group were significantly higher, indicating that the self-microemulsion of Vietnamese camellia seed oil has a certain anti-PD effect. At the same piperine dosage, the DA content in the injured side of rats in the PIP-CO-SMEDDS-H group was significantly higher than that in the piperine-only group (p < 0.001), indicating the synergistic effect of the self-microemulsion preparation on piperine. It is considered that the Vietnamese camellia seed oil in the self-microemulsion preparation and piperine have a synergistic effect. The experimental results are shown in […]. Figure 11 .

[0085] (3) Effects on the substantia nigra and striatum of Parkinson's disease rats HE staining results showed that, compared with the normal group, the model group rats exhibited pyknosis and abnormal morphology in the substantia nigra pars compacta and striatum neurons. However, administration of piperine and a self-microemulsion formulation of piperine using Vietnamese camellia seed oil as the oil phase significantly reduced the pyknosis of neurons in the substantia nigra pars compacta and striatum of PD rats, with the PIP-CO-SMEDDS-H group showing a better improvement than the piperine group at the same dosage. Furthermore, the CO-SMEDS group also showed some improvement in the pyknosis of neurons in the substantia nigra and striatum of PD rats. The experimental results are shown in […]. Figure 12 .

[0086] (4) Effects on the levels of TH and α-Syn proteins in the substantia nigra of the brain of rats with Parkinson's disease The intervention effect of the self-microemulsion formulation on the levels of TH and α-Syn proteins in the substantia nigra of the brain on the injured side of 6-OHDA-induced PD rats was determined by Western blotting. The results showed that compared with the sham-operated group, the level of TH protein in the substantia nigra of the injured side of the model group was significantly decreased (p < 0.001), while the expression level of α-Syn protein was significantly increased (p < 0.001). Piperine alone and high doses of PIP-CO-SMEDDS significantly increased the level of TH protein (PIP group, p < 0.01; PIP-CO-SMEDDS-H group, p < 0.001) and decreased the expression level of α-Syn in the substantia nigra of rats (PIP group, p < 0.05; PIP-CO-SMEDDS-H group, p < 0.001). Furthermore, at the same piperine dose, the expression level of α-Syn in the substantia nigra of rats in the PIP-CO-SMEDDS-H group was significantly lower than that in the PIP group (p < 0.05). The experimental results are shown in […]. Figure 13 .

[0087] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A self-microemulsion formulation of Vietnamese camellia seed oil, characterized in that, The self-microemulsion formulation of Vietnamese camellia seed oil is prepared using Vietnamese camellia seed oil as the oil phase and active ingredient, polyoxyethylene hydrogenated castor oil as the emulsifier, and polyethylene glycol 400 as the co-emulsifier.

2. The method for preparing a self-microemulsion formulation of Vietnamese camellia seed oil according to claim 1, characterized in that, The method involves weighing the emulsifier and co-emulsifier according to the prescription amount, mixing them evenly to obtain mixture a, adding Vietnamese camellia seed oil dropwise into mixture a, stirring, and obtaining a self-microemulsion preparation of Vietnamese camellia seed oil; the amount of Vietnamese camellia seed oil added is controlled at 10%~30%, and the mass ratio of emulsifier to co-emulsifier is controlled at 1:1~3:

1.

3. The method for preparing a self-microemulsion formulation of Vietnamese camellia seed oil according to claim 2, characterized in that, Vietnamese camellia seed oil: 3.64 g; polyoxyethylene hydrogenated castor oil: 11.95 g; polyethylene glycol 400: 4.41 g.

4. The method for preparing a self-microemulsion formulation of Vietnamese camellia seed oil according to claim 2, characterized in that, At 200~400 r·min -1 Stir evenly for 3-5 minutes under the specified conditions.

5. The self-microemulsion formulation of Vietnamese camellia seed oil prepared by the method according to any one of claims 2 to 4 is used in the preparation of a medicament for treating Parkinson's disease.

6. A self-microemulsion formulation of piperine using Vietnamese camellia seed oil as the oil phase, characterized in that, The piperine self-microemulsion formulation is prepared by loading piperine into a Vietnamese camellia seed oil self-microemulsion formulation prepared by the method described in any one of claims 2 to 4.

7. The method for preparing a piperine self-microemulsion formulation using Vietnamese camellia seed oil as the oil phase, as described in claim 6, is characterized in that... The method first prepares a self-microemulsion formulation of Vietnamese camellia seed oil according to any one of claims 2 to 4, then places it in a vial, adds piperine raw material, and dissolves it by ultrasonication to obtain a transparent and homogeneous solution, which is a self-microemulsion formulation of piperine with Vietnamese camellia seed oil as the oil phase.

8. The method for preparing a piperine self-microemulsion formulation using Vietnamese camellia seed oil as the oil phase according to claim 7, characterized in that, The mass ratio of piperine to Vietnamese camellia seed oil self-microemulsion preparation is (0.075-0.3):

20.

9. The method for preparing a piperine self-microemulsion formulation using Vietnamese camellia seed oil as the oil phase according to claim 7, characterized in that, The ultrasonic temperature is 30~40℃ and the ultrasonic time is 10~30 min.

10. The use of the piperine self-microemulsion preparation with Vietnamese camellia seed oil as the oil phase, prepared by the method according to any one of claims 7 to 9, in the preparation of a medicament for treating Parkinson's disease.

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