A compound plant exosome composition, a preparation method and application thereof in preparation of a medicine for treating Parkinson's disease
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- DOLAIMI BIOTECHNOLOGY (WUHAN) CO LTD
- Filing Date
- 2026-07-01
- Publication Date
- 2026-08-04
Smart Images
Figure CN122499247A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, specifically relating to a compound plant exosome composition, its preparation method, and its application in the preparation of drugs for treating Parkinson's disease. Background Technology
[0002] Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease, affecting more than 10 million people worldwide. Its pathological features mainly include the progressive loss of dopaminergic neurons in the substantia nigra pars compacta of the midbrain, the aggregation of α-synuclein in the remaining neurons to form Lewy bodies, and the resulting significant decrease in striatal dopamine levels.
[0003] Clinically, Parkinson's disease manifests not only as classic motor symptoms such as bradykinesia, resting tremor, rigidity, and postural instability, but also, and perhaps more importantly, as non-motor symptoms such as anxiety, depression, insomnia, and REM sleep behavior disorder, which often precede the onset of motor symptoms by years or even decades and persist throughout the disease's course. Epidemiological surveys show that approximately 30%-60% of Parkinson's patients experience anxiety or depression, and 70%-80% suffer from sleep disorders. These non-motor symptoms severely impact patients' quality of life and are major causes of disability and increased burden on caregivers.
[0004] Currently, first-line drugs for treating Parkinson's disease, such as levodopa and dopamine receptor agonists, can partially alleviate motor symptoms, but long-term use can easily lead to serious adverse reactions such as the "on-off" phenomenon, end-of-dose phenomenon, and dyskinesia. More challenging is that existing drugs are ineffective for non-motor symptoms such as insomnia, depression, and anxiety; some drugs may even induce or worsen psychiatric symptoms. Therefore, developing novel therapeutic drugs that can simultaneously intervene in both motor and non-motor symptoms and have a good safety profile is of significant clinical importance and has broad application prospects.
[0005] In recent years, plant-derived exosome-like nanovesicles (PELNs) have attracted widespread attention as a new direction for the modernization of traditional Chinese medicine research. PELNs are membrane-bound vesicles secreted by plant cells, with diameters ranging from 30 to 200 nm, carrying various bioactive molecules such as proteins, lipids, nucleic acids, and secondary metabolites. Compared with animal-derived exosomes, plant exosomes have advantages such as abundant sources, low preparation costs, lack of ethical controversy, and low immunogenicity. Existing studies have shown that plant exosomes from turmeric, grape, and lemon possess anti-inflammatory, antioxidant, and antitumor activities.
[0006] However, current research on plant exosomes primarily focuses on exosomes derived from single medicinal herbs or fruits, with no reports of extracting exosomes from multiple Chinese medicinal herbs possessing properties of "tonifying qi, nourishing yin, calming the mind, and promoting blood circulation" through scientific formulation. More importantly, during the decoction process of traditional Chinese medicine compound prescriptions, complex interactions occur between different medicinal components, potentially producing novel active substances or synergistic effects not present in single herbs. Whether these interactions during "co-decoction" affect the formation and function of exosomes, and how to utilize the blood-brain barrier-crossing properties of exosomes co-extracted from compound prescriptions to intervene in the non-motor symptoms of Parkinson's disease, are pressing technical problems that need to be solved in this field. Summary of the Invention
[0007] To address the problems existing in the prior art, this invention provides a compound plant exosome composition, a preparation method, and its application in the preparation of drugs for treating Parkinson's disease. The compound plant exosome composition can effectively alleviate non-motor symptoms such as anxiety, depression, and insomnia in Parkinson's disease model mice. Its effect is significantly better than that of traditional Chinese medicine decoctions and single-herb exosome physical mixtures, providing a new drug option for the treatment of Parkinson's disease, and in particular, providing an innovative intervention strategy for Parkinson's disease-related non-motor symptoms that have long lacked effective treatment methods.
[0008] This invention is achieved through the following technical solution: In a first aspect, the present invention provides a compound plant exosome composition, characterized in that the compound plant exosome composition is obtained by blending, extracting and separating six Chinese medicinal materials: Codonopsis pilosula, Ophiopogon japonicus, Schisandra chinensis, Ziziphus jujuba var. spinosa, Salvia miltiorrhiza, and Lycium barbarum.
[0009] Furthermore, the weight proportions of the Chinese medicinal materials are as follows: Codonopsis pilosula 24-45 parts, Ophiopogon japonicus 36-54 parts, Schisandra chinensis 10-20 parts, Ziziphus jujuba var. spinosa 21-45 parts, Salvia miltiorrhiza 24-42 parts, and Lycium barbarum 18-45 parts.
[0010] In a second aspect, the present invention provides a method for preparing the aforementioned compound plant exosome composition, comprising the following steps: (1) Mix six Chinese medicinal materials, namely Codonopsis pilosula, Ophiopogon japonicus, Schisandra chinensis, Ziziphus jujuba var. spinosa, Salvia miltiorrhiza, and Lycium barbarum, add phosphate buffer solution for cell wall breaking and homogenization treatment to obtain tissue homogenate; (2) The tissue homogenate in step (1) is centrifuged at 6000~10000r / min for 15~30min and the supernatant is collected; (3) The supernatant after centrifugation was subjected to ultrafiltration concentration, gel chromatography purification and sterilization in sequence to obtain the compound plant exosome composition.
[0011] Further, in step (1), after adding phosphate buffer and soaking for 20-50 minutes, the cell wall is broken and homogenized. The cell wall breaking and homogenization time is 1-2 minutes, and the pH of the phosphate buffer solution is 7.2-7.4. After the cell wall breaking and homogenization in step (1), a filter bag is used for preliminary filtration. The pore size of the filter bag is 100-200 μm.
[0012] Furthermore, in step (3), the ultrafiltration membrane used for concentration is a membrane module with a pore size of 50~150KD, and the target concentration volume is 5~20% of the volume before concentration.
[0013] Furthermore, the gel chromatography column described in step (3) uses Capto Core 700 packing material, the mobile phase is PBS buffer solution with pH 7.4, the flow rate of the mobile phase is controlled at 150~300 cm / h, and the sample loading volume is 5~20% of the column bed volume of the gel chromatography column.
[0014] Furthermore, in step (3), sterilization is performed by filtration using a 0.22 μm filter membrane.
[0015] In a third aspect, the present invention provides the use of the aforementioned compound plant exosome composition in the preparation of a drug for treating Parkinson's disease.
[0016] Furthermore, the compound plant exosome composition improves both motor and nonmotor symptoms of Parkinson's disease.
[0017] Furthermore, nonmotor symptoms of Parkinson's disease include insomnia, anxiety-like behavior, and depressive symptoms.
[0018] Compared with the prior art, the beneficial effects achieved by the present invention are as follows: (1) Synergistic Effect: This invention breaks through the traditional technical bias and uses a co-extraction method to obtain a compound plant exosome composition. The protective effect on nerve cells and the improvement effect on Parkinson's disease-related symptoms are significantly better than the simple superposition of exosomes from individual medicinal materials. The exosomes from the six medicinal materials carry different active ingredients and produce synergistic effects in targeting dopaminergic neurons and regulating neuroinflammation. Even if some exosomes from individual medicinal materials do not have significant neuroprotective activity when used alone, they can still achieve excellent results after co-extraction.
[0019] (2) Dual symptom intervention: The compound plant exosome composition prepared in this invention has a significant effect on improving non-motor symptoms in Parkinson's disease model mice. It can effectively improve insomnia, alleviate anxiety-like behavior, and exert an antidepressant effect. At the same time, it also significantly improves Parkinson's disease-related motor dysfunction, achieving dual intervention on motor and non-motor symptoms of the disease.
[0020] (3) Scientific and reasonable compatibility: The compatibility of medicinal materials in this invention follows the theory of "principal, assistant, adjuvant, and guide" in traditional Chinese medicine, and is highly consistent with the functional characteristics of exosomes. The exosomes from which the principal, assistant, adjuvant, and guide herbs are derived respectively undertake functions such as immune regulation, calming the mind, and antioxidation. The six herbs work together to form a complete therapeutic system, ensuring the therapeutic effect.
[0021] (4) Advantages of process innovation: This invention uses a compound co-extraction process to prepare exosomes. Compared with the process of physical mixing after extraction of single herbs, it can better retain the interaction products between herbs. The resulting compound exosomes have better particle size uniformity, batch stability and biological activity. Attached Figure Description
[0022] Figure 1 The image shows the NTA detection results of the compound plant exosome composition prepared in Example 1; Figure 2 Transmission electron microscope image of the compound plant exosome composition prepared in Example 1; Figure 3 Zeta potential diagram of the compound plant exosome composition prepared in Example 1; Figure 4 The figures show the rotarod test results of the compound plant exosome compositions in Examples 1-3; Figure 5 The graph shows the test results of the rotator experiment for each experimental group; Figure 6 The diagram shows the test results of the elevated cross maze experiment for each experimental group; Figure 7 The graph shows the results of the forced swimming experiment for each experimental group; Figure 8 The graph shows the experimental test results of sleep incidence rate in each experimental group; Figure 9 The graph shows the results of the sleep duration experiment for each experimental group. Detailed Implementation
[0023] The present invention is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specifically described in the following examples are generally performed under conventional conditions or as recommended by the manufacturer.
[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. All reagents and materials used in this invention are readily available through conventional means, and unless otherwise specified, they shall be used in accordance with conventional methods in the art or as per the product instructions.
[0025] Example 1 Preparation of compound plant exosome composition: Codonopsis pilosula, Ophiopogon japonicus, Schisandra chinensis, Ziziphus jujuba var. spinosa, Salvia miltiorrhiza, and Lycium barbarum were identified as meeting the relevant requirements of the Pharmacopoeia of the People's Republic of China (2020 edition). The specific preparation method is as follows: (1) Mix 45g of Codonopsis pilosula, 36g of Ophiopogon japonicus, 18g of Schisandra chinensis, 45g of Ziziphus jujuba var. spinosa, 36g of Salvia miltiorrhiza, and 18g of Lycium barbarum, add them to 2L of phosphate buffer (PBS, pH 7.4), soak for 30min, and then homogenize for 2min using a cell wall breaker to obtain a compound herbal tissue homogenate. Filter the homogenate with a filter bag (pore size 150μm) to remove large impurities. (2) Centrifuge the tissue homogenate at 4℃ and 10000r / min for 20min and collect the supernatant; (3) The supernatant after centrifugation was concentrated by ultrafiltration. The ultrafiltration membrane was a membrane module with a pore size of 100KD. The volume after concentration was 1 / 10 of that before concentration. The concentrate was purified by gel chromatography column. The gel chromatography column was filled with Capto Core 700 packing material. The mobile phase was PBS buffer solution with pH 7.4. The flow rate of the mobile phase was controlled at 150 cm / h. The sample loading volume was 10% of the column bed volume. The exosome enrichment solution was filtered through a 0.22 μm sterile filter membrane to obtain the compound plant exosome composition.
[0026] The compound plant exosome composition prepared in Example 1 was detected using a nanoparticle tracking analyzer (NTA), and the results are as follows: Figure 1 As shown, the particle concentration of the obtained exosome suspension was approximately 4.7 × 10⁻⁶. 10 The particle size distribution was 137.2 ± 9.2 nm, with an average particle size of 137.2 ± 9.2 nm. Transmission electron microscopy images are shown below. Figure 2 As shown, the exosomes exhibit typical cup-shaped or spherical vesicle structures with uniform particle size distribution; the zeta potential detection results are as follows. Figure 3 As shown, its potential value is stably distributed in the range of -20 to 30 mV, the particle surface is negatively charged and has good colloidal dispersion stability.
[0027] Example 2 The raw material formula for preparing the compound plant exosome composition in Example 2 is as follows: Codonopsis pilosula 45g, Ophiopogon japonicus 54g, Schisandra chinensis 18g, Ziziphus jujuba var. spinosa 30g, Salvia miltiorrhiza 24g, and Lycium barbarum 27g. The preparation method of the compound plant exosome composition is the same as that in Example 1.
[0028] Example 3 The raw material formula for preparing the compound plant exosome composition in Example 3 is as follows: Codonopsis pilosula 24g, Ophiopogon japonicus 48g, Schisandra chinensis 18g, Ziziphus jujuba var. spinosa 21g, Salvia miltiorrhiza 42g, and Lycium barbarum 45g. The preparation method of the compound plant exosome composition is the same as that in Example 1.
[0029] Comparative Example 1 Comparative Example 1: Exosomes were extracted from 45g Codonopsis pilosula, 36g Ophiopogon japonicus, 18g Schisandra chinensis, 45g Ziziphus jujuba var. spinosa, 36g Salvia miltiorrhiza, and 18g Lycium barbarum using the method described in Example 1. Exosomes from Codonopsis pilosula, Ophiopogon japonicus, Schisandra chinensis, Ziziphus jujuba var. spinosa, Salvia miltiorrhiza, and Lycium barbarum were obtained. The six exosomes were then mixed to obtain physically mixed exosomes.
[0030] Comparative Example 2 Preparation of traditional decoctions.
[0031] (1) 45g of Codonopsis pilosula, 36g of Ophiopogon japonicus, 18g of Schisandra chinensis, 45g of Ziziphus jujuba var. spinosa, 36g of Salvia miltiorrhiza, and 18g of Lycium barbarum. Weigh out the above six Chinese medicinal herbs according to their weight. Cut the two types of hard rhizomes, Codonopsis pilosula and Salvia miltiorrhiza, into slices 2-5mm thick. Ophiopogon japonicus, Schisandra chinensis, Ziziphus jujuba var. spinosa, and Lycium barbarum can be used directly as raw slices without cutting.
[0032] (2) Mix all the medicinal materials and put them into a ceramic pot. Add room temperature drinking water, and add water 2cm above the surface of the medicinal materials. Soak in room temperature cold water for 90 minutes until the medicinal materials are completely moistened and there is no dry core.
[0033] (3) Cover the clay pot and heat it over a high flame until the liquid boils. Then turn to a low flame and simmer for 40 minutes. Filter the liquid while it is still hot using double-layered degreased medical gauze and collect the first decoction.
[0034] (4) Add room temperature drinking water to the dregs, with the liquid level 1 cm above the surface of the dregs. Bring to a boil again over high heat, then simmer over low heat for 25 minutes. Filter through gauze in the same way to obtain the second decoction.
[0035] (5) Combine the two filtrates and stir well. The total volume of the final combined filtrate is about 200 mL of compound traditional decoction.
[0036] Example 4 Pharmacodynamic evaluation of a mouse model of Parkinson's disease 1. Experimental animals and grouping SPF-grade male C57BL / 6J mice, 8-10 weeks old, weighing 22-25 g. Mice were housed in a constant temperature (22 ± 2℃), constant humidity (50 ± 10%), 12-hour light / dark cycle environment with free access to food and water; A subacute Parkinson's disease mouse model was established using MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). Model establishment method: Mice were intraperitoneally injected with MPTP (30 mg / kg / d) for 5 consecutive days.
[0037] Experimental timeline: Days 1-5, MPTP intraperitoneal injection for modeling; Days 6-18, drug treatment; Days 21-25, behavioral experiments.
[0038] The mice were randomly divided into the following groups (n=12 per group): 1) Blank control group (Control): Intraperitoneal injection of an equal volume of normal saline, followed by tail vein injection of an equal volume of PBS; 2) Model group: MPTP was injected intraperitoneally to establish the model, followed by an equal volume of PBS injected via the tail vein; 3) Levodopa positive drug group (L-DOPA): MPTP was injected intraperitoneally to establish the model, followed by oral administration of levodopa (25 mg / kg + benserazide 6.25 mg / kg) once a day for 13 consecutive days (days 6-18). 4) Traditional decoction group (Comparative Example 2): MPTP was injected intraperitoneally to establish the model, followed by oral administration of the same amount of traditional decoction (concentrated by boiling to a concentration of 0.5 g / mL, oral administration volume 10 mL / kg), once a day for 13 consecutive days (days 6-18). 5) Physically mixed exosome group (Mix-Exo, Comparative Example 1): MPTP was injected intraperitoneally to establish the model, followed by tail vein injection (total exosome volume same as in Example 1, 3.0 × 10⁻⁶). 10 Particles / each), once every other day (on days 6, 8, 10, 12, 14, 16, and 18), for a total of 7 times; 6) Low-dose compound exosome group (Com-Exo-L, the compound plant exosome composition prepared in Example 1): MPTP was injected intraperitoneally to establish the model, followed by tail vein injection of the compound exosomes prepared in Example 1 (1.0 × 10⁻⁶). 10 Particles / each), once every other day (on days 6, 8, 10, 12, 14, 16, and 18), for a total of 7 times; 7) Medium-dose group of compound exosomes (Com-Exo-M, compound plant exosome composition prepared in Example 1): MPTP was injected intraperitoneally to establish the model, followed by tail vein injection of compound plant exosomes prepared in Example 1 (3.0 × 10⁻⁶). 10 Particles / each), once every other day (on days 6, 8, 10, 12, 14, 16, and 18), for a total of 7 times; 8) High-dose compound exosome group (Com-Exo-H, compound plant exosome composition prepared in Example 1): MPTP was injected intraperitoneally to establish the model, followed by tail vein injection of compound plant exosomes prepared in Example 1 (1.0 × 10⁻⁶). 11 Particles / each), once every other day (on days 6, 8, 10, 12, 14, 16, and 18), for a total of 7 times; 9) Mid-dose group in Example 2: MPTP was injected intraperitoneally to establish the model, followed by tail vein injection of the compound plant exosomes prepared in Example 2 (3.0 × 10⁻⁶). 10 Particles / each), once every other day (on days 6, 8, 10, 12, 14, 16, and 18), for a total of 7 times. 10) Mid-dose group in Example 3: MPTP was injected intraperitoneally to establish the model, followed by tail vein injection of the compound plant exosomes prepared in Example 3 (3.0 × 10⁻⁶). 10 Particles / each), once every other day (on days 6, 8, 10, 12, 14, 16, and 18), for a total of 7 times. It should be noted that the exosome group adopted an alternate-day dosing regimen based on previous pharmacokinetic studies showing that exosomes have a relatively long half-life in vivo (approximately 24-36 hours), while the traditional decoction group adopted a daily dosing regimen based on the conventional administration method of traditional Chinese medicine decoctions. Both dosing regimens underwent preliminary optimization to ensure comparisons were conducted under their respective optimal dosing conditions.
[0039] 2. Behavioral experiments All of the following behavioral experiments were conducted on days 3-7 (i.e., days 21-25) after the end of drug administration, and all experiments were conducted under double-blind conditions.
[0040] 1) Motor function evaluation The rotarod test is a classic method for evaluating motor coordination in a mouse model of Parkinson's disease. The rotarod test was conducted on day 3 after drug administration (day 21 of the experiment). Before the experiment, all mice underwent three consecutive days of training (days 19-21, rotation speed 5 rpm, gradually increasing to 20 rpm), three times a day, with 30-minute intervals between each training session. During the formal experiment, the rotarod was initially set to 5 rpm, and then uniformly accelerated to 40 rpm over 5 minutes. Mice were placed on the rotarod, and the time from the start to the fall (latency period) was recorded. Each mouse was measured three times, with 15-minute intervals between each measurement, and the average value was taken as the final result.
[0041] Behavioral experiments were conducted on the low-dose, medium-dose, and high-dose groups of the compound exosomes, as well as the medium-dose groups in Example 2 and Example 3. The results of the rotarod experiment are shown in the figure below. Figure 4 As shown; the experimental results showed that the low-dose group of compound exosomes (1.0×10) 10 At a dose of 1.0 × 10⁻⁶ particles / animal, some behavioral indicators showed a trend of improvement, but the improvement did not reach statistical significance. 11 Effects of the low-dose group (particles / piece) compared to the medium-dose group (3.0×10⁻⁶ particles / piece) 10There was no significant difference in the number of particles / each (p>0.05); among the compound plant exosomes prepared in Examples 1, 2, and 3, Example 1 showed better results. Therefore, in this invention, the medium-dose group of compound exosomes (3.0×10⁻⁶) was selected. 10 The main data presented below is represented by particles (each particle).
[0042] The results of the rotator experiment for each experimental group are as follows: Figure 5 As shown in the figure, the latency of mice in the model group on the rotarod was significantly shortened to 118.6 ± 15.3 seconds, only 24.4% of that in the blank control group (486.2 ± 38.5 seconds). The latency in the levodopa positive drug group recovered to 342.7 ± 31.2 seconds, but some mice exhibited abnormal involuntary movements. The latency in the traditional decoction group was 168.4 ± 22.7 seconds, showing limited improvement. The latency in the physical mixed exosome group was 256.3 ± 28.4 seconds, significantly better than the model group (p<0.05). The latency in the medium-dose compound exosome group (Com-Exo-M) mice was prolonged to 385.6 ± 42.3 seconds, significantly better than not only the model group (p<0.01) and the traditional decoction group (p<0.01), but also significantly better than the physical mixed exosome group (p<0.05). In addition, except for the levodopa positive drug group, no abnormal involuntary movements or stereotyped limb twisting were observed in the traditional decoction group, the physically mixed exosome group, or the various doses of compound exosome groups. This result indicates that the compound plant exosome composition prepared in this invention has a significant protective effect against MPTP-induced motor dysfunction, and the synergistic effect brought about by the compound co-extraction process is superior to that of simple physical mixing.
[0043] 2) Evaluation of anxiety-like behaviors On day 4 after the end of drug administration (day 22 of the experiment), the elevated cross maze test was conducted. The elevated cross maze consisted of two open arms (30 cm × 5 cm), two closed arms (30 cm × 5 cm × 15 cm), and a central platform (5 cm × 5 cm), 50 cm above the ground. During the experiment, mice were placed facing the open arms on the central platform, and the following indicators were recorded within 5 minutes: the number of times they entered the open arms and the time spent in the open arms. The percentage of time spent in the open arms was calculated as (open arm time / total time spent in the open arms × 100%). Higher percentages of the number of times they entered the open arms and the percentage of time spent in the open arms indicated lower anxiety levels in the mice. The elevated cross maze test results for each experimental group are shown below. Figure 6As shown in the figure, the model group mice exhibited typical anxiety-like behavior, with an open-arm dwell time percentage of only 8.3 ± 2.1%, significantly lower than the blank control group (32.6 ± 4.5%, p<0.01). The open-arm dwell time percentage in the traditional decoction group was 12.4 ± 3.1%, and in the physically mixed exosome group it was 18.7 ± 3.6%, both statistically different from the model group (8.3 ± 2.1%) (p<0.05), indicating that physically mixed exosomes have a certain ameliorative effect on anxiety-like behavior. The open-arm dwell time percentage in the medium-dose compound exosome group was 27.5 ± 4.2%, significantly better than the physically mixed exosome group (p<0.05), and closer to the blank control group (32.6 ± 4.5%), confirming the unique advantages of the co-extraction process in anti-anxiety. The positive control group (14.5 ± 3.8%) showed no statistically significant difference compared to the model group (p>0.05), indicating that the compound plant exosome composition prepared in this invention is more effective than levodopa in improving anxiety-like behavior.
[0044] 3) Evaluation of depressive-like behaviors The forced swimming test was conducted on day 5 after the end of drug administration (day 23 of the experiment). The forced swimming test is a classic method for evaluating depressive-like behavior in rodents. The experiment was conducted in a transparent cylindrical container (25 cm high, 15 cm in diameter), with a water depth of 15 cm and a water temperature of 23-25℃. Mice were placed in the water and allowed to acclimatize for 2 minutes. The immobility time for the following 4 minutes was recorded (immobility was defined as the mouse ceasing active struggling and only exhibiting the slightest limb movements required to keep its head above water). The results of the forced swimming test for each experimental group are as follows: Figure 7 As shown in the figure, the immobility time of mice in the blank control group was 62.3 ± 9.8 seconds, while that in the model group was significantly prolonged to 168.5 ± 14.2 seconds, exhibiting typical "behavioral despair." The immobility time in the traditional decoction group was 142.6 ± 15.3 seconds, slightly improved compared to the model group but without statistical significance. The immobility time in the physically mixed exosome group was 118.4 ± 12.7 seconds, significantly better than the model group (p<0.05). The immobility time in the medium-dose compound exosome group (Com-Exo-M) was significantly shortened to 78.6 ± 10.5 seconds, showing a highly significant difference compared to the model group (p<0.01), but no statistical difference compared to the blank control group (p>0.05). The antidepressant effect of the compound exosome group was significantly better than that of the physically mixed exosome group (p<0.05), indicating that the compound co-extraction process has unique advantages in antidepressant effects.
[0045] 4) Evaluation of insomnia symptoms To evaluate the effect of the compound plant exosome composition of this invention on Parkinson's disease-related insomnia, we used a sodium pentobarbital-induced sleep experiment. This experiment consisted of two sub-experiments: Sleep incidence experiment: On day 25 (7 days after the end of drug administration), mice in each group (n=10) were intraperitoneally injected with a subthreshold dose of sodium pentobarbital (25 mg / kg). Immediately after injection, the mice were placed in a quiet environment, and observation was conducted for 30 minutes to determine if they were asleep. Sleep was defined as the absence of the righting reflex for more than 30 seconds. The sleep incidence rate of each group was recorded, and the results are as follows: Figure 8 As shown, the sleep incidence rate in the model group mice was only 10% (1 / 10), indicating that MPTP treatment severely disrupted the sleep-wake rhythm in mice. The sleep incidence rate in the traditional decoction group was 30% (3 / 10). The sleep incidence rate in the physically mixed exosome group was 40% (4 / 10). The sleep incidence rate in the medium-dose group of the compound exosomes of this invention reached 70% (7 / 10), which is quite close to that of the blank control group (90%, 9 / 10).
[0046] Sleep duration experiment: On day 8 (day 26) after the end of drug administration, mice in each group (n = 10) were intraperitoneally injected with a suprathreshold dose of sodium pentobarbital (45 mg / kg). The time from the disappearance of the righting reflex to its recovery (sleep duration) was recorded, and the results are as follows: Figure 9 As shown in the figure, the sleep duration of mice in the model group was only 18.6 ± 4.3 min, significantly shorter than that of the blank control group (58.4 ± 6.1 min, p<0.01). The sleep duration of the traditional decoction group was 26.3 ± 5.8 min. The sleep duration of the physical mixed exosome group was 38.2 ± 6.5 min, significantly better than that of the model group (p<0.05). The sleep duration of the medium-dose compound exosome group (Com-Exo-M) was significantly prolonged to 52.7 ± 7.8 min, which was highly significant compared with the model group (p<0.01), but not statistically different from the blank control group (p>0.05).
[0047] In summary, the compound plant exosome composition of the present invention has a significant effect on improving Parkinson's disease-related sleep disorders, and its effect is significantly better than that of traditional decoctions and physically mixed exosomes.
[0048] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention are included within the protection scope of the present invention.
Claims
1. A compound plant exosome composition, characterized in that, The compound plant exosome composition is obtained by blending, extracting and separating six Chinese medicinal materials: Codonopsis pilosula, Ophiopogon japonicus, Schisandra chinensis, Ziziphus jujuba var. spinosa, Salvia miltiorrhiza and Lycium barbarum.
2. The compound plant exosome composition according to claim 1, characterized in that, The weight proportions of the Chinese medicinal materials are as follows: Codonopsis pilosula 24-45 parts, Ophiopogon japonicus 36-54 parts, Schisandra chinensis 10-20 parts, Ziziphus jujuba var. spinosa 21-45 parts, Salvia miltiorrhiza 24-42 parts, and Lycium barbarum 18-45 parts.
3. A method for preparing the compound plant exosome composition according to claim 1 or 2, characterized in that, Includes the following steps: (1) Mix six Chinese medicinal materials, namely Codonopsis pilosula, Ophiopogon japonicus, Schisandra chinensis, Ziziphus jujuba var. spinosa, Salvia miltiorrhiza, and Lycium barbarum, add phosphate buffer solution for cell wall breaking and homogenization treatment to obtain tissue homogenate; (2) The tissue homogenate in step (1) is centrifuged at 6000~10000r / min for 15~30min and the supernatant is collected; (3) The supernatant after centrifugation was subjected to ultrafiltration concentration, gel chromatography purification and sterilization in sequence to obtain the compound plant exosome composition.
4. The method for preparing the compound plant exosome composition according to claim 3, characterized in that, After adding phosphate buffer in step (1) and soaking for 20-50 minutes, the cell wall is broken and homogenized. The cell wall breaking and homogenization time is 1-2 minutes, and the pH of the phosphate buffer solution is 7.2-7.
4. After the cell wall breaking and homogenization in step (1), a filter bag is used for preliminary filtration. The pore size of the filter bag is 100-200μm.
5. The method for preparing the compound plant exosome composition according to claim 3, characterized in that, In step (3), the ultrafiltration membrane used for concentration is a membrane module with a pore size of 50~150KD, and the target concentration volume is 5~20% of the volume before concentration.
6. The method for preparing the compound plant exosome composition according to claim 3, characterized in that, The gel chromatography column described in step (3) uses Capto Core 700 packing material, the mobile phase is PBS buffer solution with pH 7.4, the flow rate of the mobile phase is controlled at 150~300 cm / h, and the sample loading volume is 5~20% of the gel chromatography column bed volume.
7. The method for preparing the compound plant exosome composition according to claim 3, characterized in that, In step (3), sterilization is performed by filtration using a 0.22 μm filter membrane.
8. The use of a compound plant exosome composition according to claim 1 or 2, or a compound plant exosome composition prepared by any one of claims 3 to 7, in the preparation of a drug for treating Parkinson's disease.
9. The application according to claim 8, characterized in that, The compound plant exosome composition described above improves motor and nonmotor symptoms of Parkinson's disease.
10. The application according to claim 9, characterized in that, Nonmotor symptoms of Parkinson's disease include insomnia, anxiety-like behavior, and depressive symptoms.